BIOT 1 Accelerated Development of Processes for the Integrated, Straight-Through Purification of Biopharmaceuticals Laura Crowel

Home , Autochem, Kluyveromyces marxianus

BIOT 1

Accelerated development of processes for the integrated, straight-through purification of biopharmaceuticals

Laura Crowell1, [email protected], Sergio Rodriguez1, Kerry R. Love1, Steven M. Cramer2, John C. Love1. (1) Koch Institute at MIT, Cambridge, Massachusetts, United States (2) Ricketts Bldg, Rensselaer Polytechnic Inst, Troy, New York, United States

Processes for the manufacturing of biopharmaceuticals typically vary widely in the type of and number of unit operations, particularly for molecules other than monoclonal antibodies. This leads to custom facility designs and unique strategies for process development for every new molecule. To enable flexible, multi-product manufacturing facilities and to reduce the speed to clinic for new molecules, streamlined manufacturing processes and associated strategies for process development are needed. We have developed a bench-scale, integrated and automated manufacturing platform capable of rapidly producing a variety of pre-clinical quality recombinant proteins. This system utilizes straight-through purification, where the eluate of one column is loaded directly onto the next column without any conditioning (pH or salt changes), removing the need for hold tanks or additional unit operations and significantly reducing processing time, buffer requirements, and manufacturing footprint. Development and optimization of straight-through chromatographic processes is a challenge, however, as conventional high throughput screening methods optimize each chromatographic step independently, with little consideration towards the connectivity of steps. Here, we demonstrate a method for the development and optimization of fully integrated, multi-column processes for straight-through purification. Resin selection was performed using an in-silico tool for the prediction of straight-through purification processes based on a one-time characterization of host-cell proteins combined with the chromatographic behavior of the product. A two-step optimization was then conducted to maximize yield while minimizing process- and product-related impurities. This optimization included a series of range finding experiments on each individual column, similar to conventional screening, followed by the development of a model for the fully integrated, multi-column process using design of experiments (DoE). We use these methods to produce pre-clinical quality products in as few as six weeks after obtaining the product sequence. Further, we present a method for predicting the optimal operating conditions for a new molecule within the same class based on its biophysical characteristics. Rapid process development techniques for integrated processes, such as those presented here, could enable the use of multi-product manufacturing facilities and reduce the speed to clinic for new molecules.

BIOT 2

Looking beyond dimensionless numbers for a robust continuous virus inactivation process Nidhi Thite1, [email protected], Mehdi Ghodbane2, Jessica R. Molek1, Joseph Boyer2. (1) Downstream Process Development, Glaxo Smith Kline, King of Prussia, Pennsylvania, United States (2) GSK, King Of Prussia, Pennsylvania, United States

In recent years, continuous Virus Inactivation (cVI) has attracted significant attention in the biopharmaceutical industry due to the growing interest in end-to-end continuous downstream processing. Introduction of cVI allows continuous downstream operation while reducing footprint, operational handling and improved control. When converting to a continuous unit operation, a tightly controlled residence time element to effectively inactivate viruses and reduce the risk of aggregation is needed. The system must be designed with sufficient robustness to handle changes to flowrate, titer, and pH along with short pauses. It’s essential to have the narrowest possible RTD to allow for the widest operating range. A Coiled Flow Inverter (CFI) is used to create a narrow Residence Time Distribution (RTD) to ensure material remains at low pH for sufficient time required for patient safety. Previous work in the area of CFI design has primarily been guided by two dimensionless parameters; Dean’s number and Modified Torsion number. However, other aspects like number of turns around a coil, redundant sensors and interaction between variables are not captured in its entirety with this method. This work explores the effect of different variables including flowrate, tube diameter, coil diameter, number of turns around a coil and the presence of in-line sensors on relative RTD. Using multivariant experimental design an empirical model was created to significantly reduce the RTD of the reactor compared to the use of the dimensionless parameters alone.

BIOT 3

Removal of host-cell proteins and mAb aggregates using cation-exchange multi- column displacement chromatography

Ohnmar Khanal2, Vijesh Kumar1, [email protected], Abraham M. Lenhoff3. (1) Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) University of Delaware, Newark, Delaware, United States (3) Univ of Delaware, Newark, Delaware, United States

Host-cell proteins (HCPs) and monoclonal antibody (mAb) aggregates are mostly removed during Protein A affinity and anion-exchange chromatography in downstream platform processes for mAbs. The remaining trace amounts of HCPs and aggregates are, however, difficult to remove in a typical cation-exchange (CEX) chromatography polishing step. HCP removal in CEX has not been studied in detail and depends largely on the pH of the wash buffers and the choice of resin chemistry. Aggregate clearance is achieved mainly by peak cutting and peak pooling in gradient elution, leading to yield loss. In the present work, we demonstrate the use of multi-column displacement chromatography for removal of HCPs and mAb aggregates. HCPs can be placed into two broad categories based on their isoelectric points (pI): HCPs with pI values higher than that of the mAb will flow through during product loading, while HCPs with pI values higher than that of the mAb will bind more strongly and displace the mAb. The displacement profiles of HCP versus mAb samples were constructed using analysis of elution fractions at different axial positions in the column. Based on ELISA analysis and capacity calculations for HCPs, it was found out that a column of bed height ~ 15 % of the main column prior to the main column would be required to displace the mAb sufficiently so as to obtain less than 10 ppm of HCP in the final elution pool. In a similar way, mAb aggregates, owing to their higher binding affinity, also showed displacement of monomer on the CEX column. The displacement profile was obtained by analyzing fractions of breakthrough load on a single column at different pH values. The optimal displacement condition was obtained at a pH where the mAb did not bind too weakly or too strongly to the column. To this end, a multi-column set-up was used to obtain product with less than 0.2% aggregate and 90% yield. These multi-column displacement methods can easily be incorporated into a continuous chromatography system to obtain product of high purity without compromising product recovery.

BIOT 4

Effects of filtration flux on the clearance of minute virus of mice

Sumith R. Wickramasinghe2, [email protected], Rong Fan1, FNU Namila1, Dharmesh Kanani3, Mi Jin3, Xianghong Qian1. (1) Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas, United States (2) Chemical Engineering, University of Arkansas, Fayetteville, Arkansas, United States (3) Downstream Biologics Process Development, Teva Pharmaceuticals, West Chester, Pennsylvania, United States

Viral safety is one of the major concerns in the production of mammalian cell and plasma derived biotherapeutic products. Demonstration of virus clearance is required by the regulatory agencies. Virus filtrationis a size exclusion-based virus clearance methodand is conducted industrially in dead-end mode. It is often operated in a batch mode under constant pressure. Membrane performance is shown to be affected by feed condition (pH, ionic strength and feed buffer components) as well as the operation condition (flux, pressure, time). During continuous bioprocessing, constant flux is a more desirable operation mode. Here the effects of flux operated under constant flux filtration mode and the solution condition on virus retention are systematically investigated for the filtration of monoclonal antibodies spiked with minute virus of mice (MVM) using three commercially available virus filters. Surprisingly, virus breakthrough was observed for two of the three filters when low constant flux operation was performed for a relatively longer periods of time. The interplay between virus loading on the filter, flux, membrane fouling and filtration time on virus retention will be discussed.

BIOT 5

Holistic data and informatics infrastructure for continuous biopharmaceutical manufacturing Patrick Pohlhaus, [email protected], Ashley Reeder, Sushmitha Krishnan, Timin Hadi, Jessica R. Molek. GlaxoSmithKline, King of Prussia, Pennsylvania, United States

In the pursuit of a continuous biopharmaceutical drug substance manufacturing paradigm it is important to not only consider the physical design of equipment but to also consider the supporting data and informatics infrastructure. Indeed, the data collected from manufacturing operations must be complete, available and fully contextualized in order to link processing conditions to corresponding output material quality. Such an informatics system will greatly facilitate process optimization and accelerate the goal of producing material manufactured via continuous processing that is of comparable quality to that obtained from traditional batch processing. This presentation will focus on the design considerations for a robust data and informatics infrastructure to maximize data extraction and value from GlaxoSmithKline’s (GSK) Integrated Drug Substance Manufacturing (IDSM) system. This comprehensive environment was optimized to facilitate both real time and secondary uses of data generated from a manufacturing system comprising 21 programmable logic controllers, 1000+ process sensors, eight spectrometers, nine automated at-line sampling points, and tens of thousands of configuration parameters. A topology was devised to direct the flow of data from various sources on the manufacturing system to primary repositories, secondary repositories and real time visualizations. Major systems to achieve the data flow were surveyed, down selected, tested and ultimately selected for the informatics architecture. The finalized structure makes use of software packages that meet the specific needs of continuous biopharmaceutical manufacturing and have demonstrated value and reliability in the pharmaceutical industry. Collectively, these tools facilitate the holistic capture and analysis of real time and historical process, spectral, online, at-line and model- calculated data types.

BIOT 6

Replacing process characterization studies by applying mechanistic model simulations: Smart way to accelerating BLA submission?

Gang Wang1, [email protected], David Saleh1, Federico Rischawy2, Simon Kluters1, Joey Studts1. (1) Late Stage Downstream Process Development GmbH & Co KG, Boehringer Ingelheim GmbH&Co. KG, Biberach, Germany (2) Early Stage Bioprocess Development, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Baden-Württemberg, Germany

On the way to submitting a biologics license application (BLA), there are thousands of lab hours required. The largest and most critical work package in late stage downstream process (DSP) development are process characterization studies (PCS). With the PCS results, one should be able to identify the critical process parameters (CPPs) and to define the proven acceptable ranges of the input parameters (PARs), with the final goal of designing a robust manufacturing process that is able to deliver a drug with constant quality. Currently, the industrial standard is to carry out lab scale experiments with multivariate process parameter adjustments and to use response surface models to approximate the univariate influences of process parameters on the critical quality attributes (CQAs). This practice still requires a large number of wet lab experiments. Furthermore, a gap between different scales and low resolution of statistical models used due to multivariate experimental design would deliver unreliable results. In the presented work, we investigated the feasibility of using mechanistic modeling to identify CPPs and PARs of the polishing chromatography step of an on-going late stage project in DSP. A calibrated and validated mechanistic model was used to predict PCS experiments carried out in the wet-lab. With this knowledge, dependencies between potential CPPs and CQAs were determined and the PARs could be defined using this mechanistic model.

BIOT 7

Process economic modeling of continuous, integrated biologics manufacturing

Aude Iwaniec1, [email protected], John H. Welsh1, Spyridon Gerontas5, Brian Keith4, Louise Taylor3, Martyn Hulley2, Tibor Nagy4, Saranky Sivapathasundram1. (1) Pall Biotech, Portsmouth, United Kingdom (2) Astrazeneca, Cambridge, United Kingdom (3) CPI, Darlington, United Kingdom (4) FUJIFILM Diosynth Biotechnologies, Billingham, United Kingdom (5) Allergan Biologics Ltd., Liverpool, United Kingdom

Biosimilars and patent expiry are forcing the biopharma industry to find new ways to maintain competitiveness by ensuring affordability, quality, and delivery performance. Despite great improvements in upstream processing efficiency, higher titers create downstream processing bottlenecks and facility fit issues: Equipment reaches its physical and capacity limits thereby increasing processing time, material consumption and overall cost. Continuous processes have been proposed as a solution to many of these issues as they offer higher productivity while reducing cycle times, buffer/resin usage and required footprint.

Process modeling can be used to develop and support the adoption of continuous and integrated downstream technologies by predicting and comparing process performance. More specifically, economic modeling is powerful for initial screening and detailed evaluation of process economics, resulting in process outcomes, footprint and an estimation of continuous processes’ benefits. A cost of good analysis software is used to model and compare the cost of three corresponding batch and continuous processes for the Continuous Processing Consortium which is developing and operating an automated multi-product platform for mAb downstream processing.

This presentation provides (a) the approach and tools to batch and continuous process modeling, (b) models, CoGs results and comparison of 3 different batch and corresponding continuous mAb downstream processes - including cost breakdown, Gantt chart, and sensitivity analysis, and (c) strengths and weaknesses of models BIOT 8

Process development for continuous flow-through polishing purification for mAb processing

Sandeep Mora, [email protected], Junyan Zhang, Meghan Higson, Jeffrey Barna. BioContinuum™ Platform , MilliporeSigma, Cambridge, Massachusetts, United States

Over the last several years, the biopharmaceutical industry has had a significant focus on connected and continuous processing to improve both process economics and plant utilization. As opposed to the traditional polishing trains comprised of bind-elute chromatography operations, an all flow-through polishing train easily enables connected and continuous processing while simultaneously improving process economics, flexibility, and productivity. Leveraging commercially available and novel prototype chemistries and devices, we investigated how a properly designed flow-through polishing train can be used to meet the stringent demands expected for mAb polishing purification. A streamlined methodology will be described with two monoclonal antibody molecules to investigate the performance of individual units as well as synergies between technologies. For both individual technologies and connected processes, results will be discussed on their ability to meet purity and yield targets robustly. Finally, we will show how leveraging the integrated combination of unit operations can result in improved performance over the standard batch, segregated processing paradigm, including the economic benefits of the flow-through polishing toolbox using BioSolve modeling.

BIOT 9

Evaluation of single-use TFF Technology for purification of activated polysaccharides used in conjugate vaccine manufacturing

Pouria Motevalian1, [email protected], Jonathan Steen2, Janine De Leon1, Verl Sriskanda3, Ivette Carino1, Amarnauth Prashad1. (1) BioProcess Research and Development, Pfizer Inc., Andover, Massachusetts, United States (2) MilliporeSigma, Bedford, Massachusetts, United States (3) Analytical Research and Development, Pfizer Inc., Andover, Massachusetts, United States

Highly effective vaccines against diseases caused by N. meningitidis, S. pneumoniae, and H. influenza can be produced by chemical conjugation of the capsular polysaccharide from these bacteria to an immunogenic protein such as tetanus toxoid, CRM197 or diphtheria toxoid. The conjugation process typically involves a polysaccharide activation step in preparation for covalent linkage to the immunogenic protein. Tangential Flow Filtration (TFF) is widely used for buffer exchange and purification of activated and conjugated polysaccharides against reaction byproducts or residuals.

Over the past decade, single-use TFF technologies have emerged to reduce system preparation time, promote fast and flexible product change over, and ultimately shorten process development and manufacturing time/cost. The primary objective of this work was to evaluate a novel, gamma-sterilized Pellicon® single-use TFF capsule against traditional reusable Pellicon® cassettes with respect to performance, flush requirement, product recovery, and ease-of-use; whereas the secondary objective was to assess the scalability of Pellicon® TFF cassettes and capsule for the purification of activated polysaccharides. In particular, the performance of a recently developed Pellicon® single- use TFF capsule was compared against the traditional Pellicon® cassette by assessing TFF process performance (such as flux, residuals clearance, and yield) and post- purification product attributes (such as concentration and mass-weighted average molecular weight). Good scaling was shown by comparing process performance and product attributes across different scales and formats (88 cm2 vs 1100 cm2 cassettes and 1000 cm2 capsule). Similar TFF process performance and post-purification product attributes were observed for the single-use capsule compared to the reusable TFF cassettes. In addition, the capsule requires a smaller flush than the cassette, and it is easier to use since it does not require a compression holder or pre-sanitization. The results provide insight into the application of the single-use TFF capsule and scalability of TFF processes for the purification of activated polysaccharides used in conjugate vaccine manufacturing.

BIOT 10

Enabling high concentration monoclonal antibody drug substance generation by ultrafiltration/diafiltration with process strategies to minimize particulate formation

Jessica Hung, [email protected], Shing Fung Lam, Dongyoun Jang, Hasin Feroz, Swarnim Ranjan, Kyle Skillings, Melissa Holstein, Sanchayita Ghose. Biologics Process Development, Bristol Myers Squibb, Devens, Massachusetts, United States

During the final stages of ultrafiltration/diafiltration (UF/DF) for generating high concentration monoclonal antibody drug substance (DS), high inlet pressures and low fluxes can become process limiting. The large volume reduction from load to final DS may also translate to long process times if the UF/DF system is not sized or configured optimally. High solution turbidity and particulate burden in the DS may also pose challenges for the subsequent final filtration step. The objective of this study was to develop alternative UF/DF configurations or methods to reduce process time, pressure challenges, and final DS particulate burden (turbidity). A secondary objective was to investigate the risk factors that promote particulate formation during UF/DF in order to guide the development of alternate UF/DF strategies. Risk factors were chosen around understanding the impact of shear exposure, flow characteristics, interfacial effects and protein concentration. UF/DF strategies were in turn developed to understand and mitigate the impact of particulate formation and loading strategy on UF/DF process attributes in order to overcome the low flux and high pressure drops that limit the manufacturing of high concentration DS. Strategies were identified to minimize process times as well as reduce particulate formation. Both shorter process times and reduced particulate burden were found to help reduce the pressure increase during UF2, which in turn can increase the capability of UF2 for generating higher concentration drug substance.

BIOT 11

Developing a simple automation platform to increase throughput for tangential flow ultrafiltration process development

Michael Homsy, [email protected], Michael Winters, Matthew P. Watson, Joseph G. Joyce. Merck & Co. , West Point, Pennsylvania, United States

Increasing productivity in process development can be achieved using small-scale models and automation. Breakthroughs in miniaturization and automation have facilitated high throughput (HT) process development for many downstream unit operations. For example, HT chromatography experiments are now routinely conducted at the microliter scale using small-scale columns with automated liquid handlers. Unfortunately, miniaturization of tangential flow filtration (TFF) systems is challenging due to larger liquid volume requirements. Recognizing challenges with miniaturizing TFF, an alternative strategy leveraging automation has emerged to increase throughput of TFF development. Various commercially available TFF systems now offer full automation capabilities with the ability to run several experiments simultaneously. Procurement of a commercial, automated TFF system, however, can be expensive and may require extensive customization. This work demonstrates an inexpensive and flexible automated TFF system that has been developed at Merck as a platform that can use existing lab equipment. Arduino microcontrollers were programmed to control equipment to run automated concentration and diafiltration functions. A LabVIEW application was designed to connect to multiple Arduinos wirelessly via Bluetooth for data acquisition and display and to initiate automation functions remotely. This remote operation capability along with built-in safety features are especially desirable for work involving potentially harmful biological agents and toxic chemicals. Implementing this system has allowed multiple small-scale experimental arms to be run simultaneously with minimal operator involvement. This presentation will describe features of the automated TFF system and will highlight a range of experiments the automated TFF system has been used for, including ultrafiltration development of a live virus vaccine. Although this approach has so far been used exclusively for ultrafiltration experiments, this technology is highly adaptable and can be used for any activity or unit operation that requires integration of different lab scale instruments.

BIOT 12

Development of isoporous block copolymer membranes for virus filtration: Effects of structural and chemical features on throughput, LRV, and robustness Marty Siwak, [email protected]. Terapore Technologies, South San Fransisco, California, United States

Virus filtration remains one of the most variable process steps in downstream mAb or protein purification. It is also a dominant expendable cost for DSP. Existing commercial VF membranes are based fundamentally on an extension of ultrafiltration technology. To achieve acceptable LRV values, this conventional technology leads to sacrifices in capacity due to broad pore size distributions. In contrast, ISOBLOCK self-assembling technology results in filters with periodic, ordered pore structures, enabling high throughput while maintaining high virus retention. Four key membrane properties can influence both capacity and virus LRV: pore size distribution, membrane asymmetry, surface chemistry, and membrane thickness. Data will be presented to show the effects of these properties and ultimately how they fit into the design of a downstream VF process.

BIOT 13

Effects of solution condition and process interruption on the clearance of minute virus of mice during virus filtration of protein therapeutics

Xianghong Qian1, [email protected], FNU Namila1, Steven J. Traylor2, Tung Nguyen2, Nripen Singh3, Sumith R. Wickramasinghe4. (1) Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas, United States (2) Biologics Process Development, Bristol-Myers Squibb, Hopkinton, Massachusetts, United States (3) Process Development, Bristol Myers Squibb, Acton, Massachusetts, United States (4) Chemical Engineering, University of Arkansas, Fayetteville, Arkansas, United States

Viral safety is one of the major concerns in the production of mammalian cell and plasma derived biotherapeutic products. A demonstration of virus clearance is required by the regulatory agencies. Virus filtration can provide a robust removal of virus. Virus filtration unit operation has been integrated into many downstream purification processes. Here the effects of solution condition and process interruption on virus retention during virus filtration are systematically investigated for the filtration of Fc- fusion protein and monoclonal antibodies spiked with minute virus of mice (MVM) using three commercial parvovirus filters. Our results indicate that virus breakthrough occurs more frequently at a later stage of filtration due to the increased virus loading resulting in the repartitioning of the virus particles at the solid-liquid interface. Pressure release from process interruption also leads to more severe virus breakthrough due to the back diffusion and redistribution of the virus particles in the membrane pores. Our study shows that virus breakthrough is complex, but strongly filter and solution condition dependent. Surprisingly, virus breakthrough appears to be most severe under the relatively low membrane fouling condition for one of the commercial filters investigated.

BIOT 14

Low flux impacts on virus filtration: Devil in the details Daniel Strauss, [email protected], Alice Butler, Naokatsu Hirotomi. Asahi Kasei Bioprocess America, Glenview, Illinois, United States

Virus filtration provides highly robust virus removal for many different virus types over wide ranges of operating conditions. One operating parameter that has been identified as potentially impacting viral clearance is transmembrane pressure. Using Planova 20N filters, we have previously shown that effective parvovirus removal can be expected under typical operating pressures of 0.5 to 1.0 bar but operating at lower pressures can impact parvovirus clearance under certain solution conditions. This phenomenon is hypothesized to occur due to low flow rates, which allow for diffusion of particles that would otherwise be trapped by hydrostatic forces. However, such a mechanism raises the question as to whether other mechanisms of flow rate reduction will also impact viral clearance. In this presentation, we discuss how other feedstock conditions, such as high product concentration and high aggregate content, can also reduce flow rate. Especially, we show that the mechanism by which flow reduction occurs can have different impacts on viral clearance capabilities.

BIOT 15

Fouling and yield during sterile filtration of virus-like particle vaccines

Neil Taylor1, [email protected], Wanli (Justin) Ma2, Adam Kristopeit2, Sheng-ching Wang2, Andrew L. Zydney1. (1) Dept of Chemical Engineering, Penn State University, University Park, Pennsylvania, United States (2) Vaccine Process Development, Merck & Co, Inc., West Point, Pennsylvania, United States

There is growing interest in the development of new classes of vaccines to protect against the spread of dangerous infectious diseases. Many of these new vaccines are based on virus-like particles that are in the 100 – 400 nm size range. This significantly complicates the use of sterile filtration to insure sterility of the final vaccine product. This study evaluated the performance characteristics of four commercially available, sterile- filters using a model nanoparticle system, consisting of polystyrene latex spheres, to simulate the behavior of virus-like particle vaccines. Experiments investigated the effect of filtrate flux, particle concentration, and operating mode on membrane fouling and particle yield. Confocal microscopy was used to directly visualize the location of particle capture within the depth of the different sterile filters. Surprisingly, the sterile filters were able to pass particles of 200 nm or greater in diameter under appropriate operating conditions. For example, one filter achieved more than 75% yield of 330 nm particles (average size determined by dynamic light scattering) with a loading capacity greater than 1000 L/m2 at a flux of 300 LMH. The particle yield was independent of filtrate flux at values above 200 LMH during constant flux filtration experiments but decreased at a flux of 100 LMH. The particle yield and fouling behavior varied significantly for filters with different asymmetry and pore morphology. Although these results would need to be confirmed with actual biological samples, our work provides important insights into the factors controlling the transmission of relatively large particles through commercially available sterile filters. BIOT 16

Challenges applying traditional crystallization approaches to monoclonal antibodies

Michael Rauscher, [email protected], Lauren Rockwell, Sunitha Kandula, Jennifer Pollard. Merck & Co., Summit, New Jersey, United States

Crystallization has a long history of being used for isolation and purification in small molecule processing, due to its economic advantages and operational ease. For biological molecules, the use of crystallization has often been limited to structure determination using x-ray diffraction. However, if robust conditions for protein batch crystallization could be identified, it could confer a lot of the same advantages to a large molecule downstream process, including formulation options. Protein crystallization conditions are typically identified using vapor diffusion screens, which can be translated to batch reactions using a DOE approach. These empirical results provide an assessment of process robustness, but not full process understanding. Although this effectively identifies operating ranges, a more fundamental study of the particular crystallization via solubility curves and the resulting phase diagram could better inform the process design. Targeting a specific protein concentration vs. a specific precipitant concentration would allow identification of the metastable zone, where crystal nuclei can form, and the labile zone, where crystal nuclei can grow, in addition to ineffective regions where the product will be soluble or precipitate. Generating these data via small molecule crystallization techniques can leverage established workflows, but present challenges due to protein properties. Traditional crystallization experimentation relies on understanding the phase equilibria and manipulating the solubility of the molecule, via temperature or the addition of an anti-solvent. These fundamental experiments utilize a supersaturated solution, typically produced by adding solids to a fixed amount of solvent at high temperature. For proteins, solubility depends on multiple factors, including concentration and type of buffer salt, pH, and excipients. Therefore, different methods are required to bring the solution to supersaturation. Work was done testing these approaches on an automated liquid handler, starting with both crystalline and high concentration liquid forms of the antibody.

BIOT 17

Seeding strategies for the selective crystallisation of model proteins and monoclonal antibodies

Xiaoyu Li2, Huaiyu Yang2, Wenqiam Chen2, Ian Rosbottom2,1, [email protected], Jerry Y. Heng2. (1) Chemical Engineering, University of Leeds, London, United Kingdom (2) Chemical Engineering, Imperial College London, London, United Kingdom The recognition of the selective nature of biopharmaceutical drugs and the advances in biotechnology have led to over 246 approved products with cumulative revenues reaching $140 billion. Upstream biomanufacture of proteins has advanced, such that the time and expense bottleneck in biopharmaceutics lies in the chromatographic purification steps. Crystallisation is a more economic and energy efficient alternative purification step, producing crystalline proteins with higher purity and stability. However, this technology is yet to mature to manufacturing scale and robustness. Here, we report our optimised crystallisation, additive and seeding strategies to crystallise model proteins and also a monoclonal antibody (mAb, Anti CD-20), and additionally demonstrate selective crystallisation of target proteins from a binary mixture. Mesoporous silica nanoparticles (NPs) are shown to be effective heterogenous nucleants for the selective crystallisation of lysozyme from a binary mixture of lysozyme and thaumatin. From hanging drop solution conditions which can crystallise both proteins, the ‘hard template’ SBA-15 NPs are found to not only promote the nucleation of lysozyme, but also suppress the crystallisation of thaumatin. These conditions are also effectively scaled up to the ml level. Further to this, the effectiveness of graphene oxides and DNA polymeric additives are also examined as seeds for protein crystallisation promotion(1). For anti-CD20, our optimisation strategies has resulted in the first ever sufficiently crystalline particle for X-ray diffraction investigation, proving the existence of the monoclonal antibody in the crystal structure. This investigation also reveals the sulphate anion as the ideal precipitant ion for the reproduceable crystallisation of this material. Here, we report on the use of several templating strategies for the crystallisation of proteins demonstrating the feasibility, on both model protein systems and a mAb, for selective crystallisation for downstream separations.

BIOT 18

Understanding the synchronization and the improved productivity of an automated semi-continuous fermentation process (self-cycling fermentation) at the transcriptomic level

Yusheng Tan, [email protected], Roman V. Agustin, Dominic Sauvageau. Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada

Industrial fermentation provides a wide variety of bioproducts (such as food, biofuels, pharmaceuticals, cosmetics, etc.) based on the biocatalysts. While batch reaction (BR) is the most commonly used mode of operation in industrial fermentation, it requires considerable downtime for harvesting, sterilization, and clean-up. Also, lag phases are often significant in conventional BR, reducing overall productivity. Self-Cycling Fermentation (SCF), an advanced automated semi-continuous fermentation approach, has shown significant advantages over BR in terms of yield and productivity in many microbial systems. Here we show how growing a yeast engineered to overproduce shikimic acid under SCF led to a two-fold increase in product yield and a four-fold increase in specific productivity (shikimic acid produced per cell per hour), compared with BR operation. In order to understand the cellular mechanisms leading to these increases, we performed transcriptomic analysis, through RNA-Seq and real-time PCR techniques of the yeast growing under BR and SCF. Preliminary results indicate that a large number of genes related to cell cycle and DNA replication were prominently upregulated during the early stages of SCF cycles, inferring significant synchronization achieved by the cell population. Further, numerous genes related to oxidative phosphorylation and the TCA cycle were significantly up-regulated in the late stages of SCF cycles, which may contribute to the increased shikimic acid productivity through enhanced power generation. These results highlight the features of SCF operation at the transcriptomic level, enhance the understanding of the process, and promote the commercialization of advanced semi-continuous processes in industry.

BIOT 19

Bioreactor engineering for a type 3 secretion system powered protein production process

Samuel Leach, [email protected], Danielle Ercek. Chemical and Biological Engineering, Northwestern University, Chicago, Illinois, United States

The revenue from protein products manufactured by microbial fermentation eclipsed $50 billion in 2015, which includes pharmaceutical products such as proinsulin and enzymes for food production and agriculture. While bacteria are advantageous as protein production hosts because of their tractability and high titer, the difficulty and expense of downstream purification prevents the market from growing. When proteins are expressed intracellularly in bacteria, product recovery typically requires lysis, protein refolding, and multiple purification steps, which must be carefully optimized for each product. A solution to this problem is to export the product from the cytoplasm to the extracellular space. Salmonella enterica natively employ a Type 3 Secretion System (T3SS) for exporting proteins outside the cell, making it a promising target for this purpose. Previously, we have shown that heterologous proteins can be exported through the T3SS at titers up to 400mg/L, but secretion titer is sensitive to many environmental factors which are critical to scale up. Therefore, determining how bioreactor operating conditions impact T3SS secretion titer is necessary for its future use as a protein production platform. We explored how oxygen transfer, pH, glucose feed rate, and growth rate affect secretion titer through cultivation in a sparged stirred tank bioreactor. In this talk, I will present the results of these optimizations, including the identification of a stir speed and air flow rate regime which maximizes titer, and the observation that a slower cellular growth rate increases secretion rate. These results provide insight into the optimal operating conditions to maximize secreted protein, furthering the development of a protein production platform based on the S. enterica T3SS.

BIOT 20 Metabolic engineering of non-model yeast Cutaneotrichosporon oleaginosus for valorization lignin and lignin-derived aromatics

Allison Yaguchi1, Stephen Lee1, Michael Spagnuolo2, Mark A. Blenner3, [email protected]. (1) Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States (2) Clemson University, Clemson, South Carolina, United States (3) Chemical Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

Cutaneotrichosporon oleaginosus, previously known as Cryptococcus curvatus, is a non-model oleaginous yeast known for its ability to metabolize many alternative substrates, including xylose, and toxic lignocellulosic hydrolysate inhibitors such as 5- hydroxymethylfurfural and furfural. We discovered C. oleaginosus also tolerates and metabolizes lignin-derived aromatics, highlighting this organisms’ potential to utilize all components of lignocellulosic biomass. C. oleaginosus is able to fully metabolize phenol, 4-hydroxybenzoic acid (pHBA), and resorcinol as sole carbon sources, as well as co-utilization with glucose and xylose. When buffered, cells also metabolize p- coumarate robustly and to a lesser degree ferulate and syringate as sole carbon sources. In a simple batch flask culture, C. oleaginosus is able to accumulate nearly 50% of its biomass as lipids while utilizing pHBA, p-coumarate, and resorcinol. Optimizing fed-batch feeding strategies increased lipid accumulation to over 69%, the second highest value in literature for this organism to date. Transcriptomic analyses were used to elucidate aromatic metabolic pathways in C. oleaginosus and improve the existing genome annotation significantly. Biochemical analysis suggests ortho ring cleavage is used throughout the aromatic metabolic pathways. We also recently demonstrated C. oleaginosus metabolizes monomers released from alkaline pretreated cornstover lignin, and the presence of lignin is not inhibitory to growth. With such an exceptionally desirable natural phenotype, this yeast could become a preferred host for oleochemical production if novel synthetic biology tools are developed. We identified both strong constitutive and phenolic-regulated promoters to drive expression of heterologous or native genes. In parallel, we are developing higher efficiency transformation methods for increasing the pace of engineering efforts. Overall, our work establishes C. oleaginosus as a promising platform to robustly convert all components of lignocellulosic biomass into novel high-value oleochemicals.

BIOT 21

Turning pollution into products through commercial scale gas fermentation

Michael Koepke, [email protected]. LanzaTech, Skokie, Illinois, United States

Rapid population growth and climate change are posing some of the most urgent challenges to mankind and have intensified the need for low-cost manufacturing of fuels, chemical-building blocks, materials and food from sustainable resources.

Gas fermentation using autotrophic microorganisms offers a sustainable path to these products from a range of local, highly abundant, waste and low-cost resources. LanzaTech has pioneered a gas fermentation process using anaerobic acetogenic microbes capable of fixing carbon oxides.

While 10 years ago, acetogens were considered genetically inaccessible and mass- transfer of gases was considered a major scale up hurdle, LanzaTech has since developed a suite of synthetic biology tools successfully scaled up the process from the laboratory bench to full commercial scale. In May 2018, LanzaTech successfully started up a world-first commercial scale (48k MTA) gas fermentation plant using emissions from the steel making process as feedstock for ethanol production.

The technology has been demonstrated with a diverse range of additional low-cost feedstocks including waste gases from other industrial sources (e.g., processing plants or refineries) or syngas generated from any biomass resource (e.g., unsorted and non- recyclable municipal solid waste, agricultural waste, or organic industrial waste) and additional plants are under construction.

In order to maximize the value that can be added to the array of gas resources that the process can use as an input, LanzaTech has established a unique biofoundry that enables automated strain engineering of anaerobic organisms and strain screening in context of flammable and toxic CO and H2 gases. Through this platform, LanzaTech has demonstrated direct production over 50 different products from gas. Several of these products have been scaled up to pilot scale and developed for commercial rollout.

BIOT 22

Leveraging transposon technology to accelerate the development process of microbial cell factories for high-level production of bio-based chemicals

Zengyi Shao, [email protected], Yuxin Zhao. Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States

Achieving high-level production in a heterologous host requires the introduction of a long pathway and stable expression of the involved genes. For the latter, genome integration is a better choice than plasmid expression, especially considering that many non-model microbial hosts do not have readily usable plasmids. The question is which locus in the genome could favor gene integration and expression. Genome is organized into the accessible euchromatin regions and tightly packed heterochromatin regions. Genes located in the heterochromatins are normally silent because they cannot be accessed by transcriptional machinery. Unfortunately, for the majority of the microbial hosts, how their genomes are packed is not known. To address this issue, we leverage the cut-and-paste feature of the transposon system to insert the heterologous pathway flanked by the two terminal transposon recognition sequences randomly to the highly accessible genomic regions. Using the 14-kb shikimate pathway as an example, we identified genomic hotspots favoring pathway integration and increased the production of shikimate from 1.8 g/L (plasmid-based production) to 6.2 g/L in one-step. More importantly, this strategy also efficiently reduced false positives (caused by the integration of only the selection marker instead of the pathway) and improved the efficiency of identifying top-performing variants from 15% to 97%. The two biggest challenges of engineering these non-model microbes are (1) the lack of genetic manipulation tools and (2) the missing knowledge of genotype-phenotype correlation, which constrain the efficiency of further strain development to improve the productivity. The transposon may hop into the genes that negatively impact the production of the target compound. Therefore, disruption of their functions will lead to increased product yields. Currently, we are expanding this success to achieve rapid strain engineering in three species to produce nutraceuticals and biopolymer precursors. Demonstrating the general applicability of our strain engineering solution will push the frontier of the biochemical engineering field with a radical technology to accelerate strain engineering for other interesting products.

BIOT 23

Universal tools informed by functional genomics for constructing microbial cell factories

Joseph Brady, [email protected], Melody Tan, Charlie Whittaker, Neil Dalvie, Kerry R. Love, John C. Love. Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

Construction of efficient cell factories often requires engineering of multi-circuit pathways for synthesis of novel compounds and improved cell productivity. Genome- level screens can identify genes and pathways that enhance the production of desired chemicals, fuels, or biologics. While modern gene editing tools like CRISPR/Cas9 enable precise genome editing, where to integrate new genes and how best to modulate expression remain challenging, especially in non-model organisms. The AltHost Consortium, an MIT-industry partnership, seeks to engineer microbial hosts to be fast, low-cost, and highly productive alternatives for biomanufacturing. Informed by functional genomics, we present tools for modifying non-model organisms for a desired phenotype. First, we demonstrate a framework informed by ATAC-Seq and RNA-Seq for identifying optimal landing sites for heterologous expression cassettes. Second, we present a tool based on the Kozak sequence for tuning protein expression to a target level without promoter engineering. We demonstrate the utility of these tools by engineering the yeast P. pastoris to be a highly productive cell factory for quality biologics. By leveraging functional genomics, we believe our approach can be extended to develop these tools in almost any microbial host.

BIOT 24

Direct production of fatty alcohols from glucose using engineered strains of Yarrowia lipolytica Lauren T. Cordova, [email protected], Jonathan Butler, Hal S. Alper. The University of Texas at Austin, Austin, Texas, United States

Fatty alcohols are important industrial oleochemicals with broad applications and a growing market. As such, we sought to engineer Yarrowia lipolytica to serve as a renewable source of fatty alcohols (specifically hexadecanol, heptadecanol, octadecanol, oleyl alcohol) directly from glucose. Through screening four fatty acyl-CoA reductase (FAR) enzyme variants across two engineered background strains, we identified that the previously characterized MhFAR enabled the highest production. The use of two strains demonstrated the close relationship between the fatty acid distribution and corresponding fatty alcohol production profile. Strain engineering, fed-batch flask cultivation, and dodecane extractive fermentation improved the fatty alcohol titer to 1.5 g/L. Scale-up of this strain in a (non-extractive) 2L bioreactor led to 5.8 g/L total fatty alcohols at an average yield of 36 mg/g glucose with a maximum productivity of 39 mg/L hr. These parameters represent the highest fatty alcohol production in Y. lipolytica. Finally, we utilized this fatty alcohol reductase to generate a customized fatty alcohol, linolenyl alcohol, from α-linolenic acid. Overall, this work demonstrates Y. lipolytica is a robust chassis for diverse fatty alcohol production and highlights the capacity to obtain high titers and yields from a purely minimal media formulation directly from glucose without the need for complex additives.

BIOT 25

Shikimate pathway refactoring in the fast-growing yeast Kluyveromyces marxianus drives high titer production of 2-phenylethanol

Mengwan Li1, [email protected], Xuye Lang2, Nancy A. Da Silva3, Ian R. Wheeldon4. (1) Chemical and Environmental Engineering, University of California, Riverside, Riverside, California, United States (2) Chemical Engineering, UC Riverside, Riverside, California, United States (3) University of California, Irvine, California, United States (4) A242 Bourns Hall, UC, Riverside, Riverside, California, United States

The non-conventional yeast Kluyveromyces marxianus is one of the fastest growing eukaryotes, is thermotolerant to temperatures upward of 50°C, and has the capacity to assimilate a wide range of C5 and C6 sugars. These traits make K. marxianus an attractive host for the industrial production of biochemicals. In previous strain screening experiments, we have also identified a strong phenotype of short and medium chain ester production, including the production of ethyl acetate in grams per liter quantities, and the synthesis of isoamyl and phenylethyl acetate in 10s of miligrams per liter quantities. These acetate esters are produced via an alcohol acetyltransferase (Atf), which condenses acetyl-CoA with the relevant primary alcohol. Knockout of this function leads to the accumulation of the alcohol precursors and represents a valuable starting for their biochemical production. In this work, we focus on refactoring the Shikimate pathway for 2-phenylethanol production, an important precursor in the fragrance industry with market values upwards of $1,000/kg. To do so, we first developed a one- step markerless multigene integration system that can effectively integrate three unique expression cassettes in a single round of strain engineering. We used this new technique to rapidly create a 27-member strain library that varied the expression of Shikimate pathway genes ARO4, ARO7, and PHA2. This refactoring experiment identified an engineered strain with a five-fold increase in 2-phenylethanol production and demonstrated new capabilities in the rapid engineering of K. marxianus.

BIOT 26

Implementation of CRISPR-Cas9 system in Geobacter sulfurreducens to enable ammonium generation from nitrogen gas

Mark Poole1,3, [email protected], Amy Grunden2, Douglas F. Call1. (1) Civil, Construction, and Environmental Engineering, North Carolina State University, Raleigh, North Carolina, United States (2) Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, United States (3) Biological & Agricultural Engineering, North Carolina State University, Raleigh, North Carolina, United States

+ Nitrogen gas (N2) conversion to ammonium (NH4 ) is an essential, but cost- and energy- expensive process, with the industrial Haber-Bosch process constituting the major form of artificial N2 fixation implemented today. Biological nitrogen fixation (BNF) is a more efficient process that has seen significant barriers to commercial implementation. Geobacter sulfurreducens is a free-living N2-fixing soil bacterium that can also respire on electrodes. Recent evidence from our group has indicated that the metabolic rate of G. sulfurreducens while respiring in microbial electrochemical cells (MECs) is correlated + to N2 fixation. However, NH4 is not secreted from cells due to tight regulatory control. The CRISPR-dCas9 system is a robust method for offsetting undesirable regulatory control. In this study, the CRISPR-Cas9/dCas9 system was implemented for the first time in G. sulfurreduencs. As evidence for this new approach, we first targeted acetate utilization in G. sulfurreducens via acetate kinase. Next, we targeted a protein that + inhibits the function of nitrogenase in response to high extracellular NH4 (DraT). In this study, DraT is knocked down with CRISPR-dCas9 to ensure continued function of + nitrogenase in the presence of high extracellular NH4 . Lastly, the knockdown mutants were run under N2-fixation conditions in MECs to determine their ability to secrete and + accumulate NH4 .

BIOT 27

Economics of intensive/continuous downstream manufacturing

Paul A. Sinclair, [email protected]. Biopharm Services Ltd, Chesham, Buckinghamshire, United Kingdom

Within Bioprocessing there is a keen interest in the intensification of BioPharm manufacturing. The key drivers are assumed to be improved flexibility, reduced risk and reduced cost. It is an evolving area and key to gaining insight is the analysis of the downstream requirements from upstream. Advanced bioprocess models are used to understand the drivers and resource requirements of different modes of intensified downstream (DSP) operation. Using as the basis Biophorum technology roadmap models the three areas addressed are. Connected processing: Batch operations that are linked directly without product hold points. Continuous processing: Material flows between unit operations, two scenarios are considered partial (some batch) and fully continuous operation. Solution management: How this is impacted by connected and continuous processing. By considering the total resource requirements the models have provided insights into the impact of new modes of operation and, how these are affected by scale of operation and the influence upstream feed condition(fed batch versus perfusion). Focusing on continuous manufacturing the economic benefits in terms of Cost of Goods (CoG) in the downstream are dependent on scale ranging between 0% to 22% savings for capacities up to 1800 kg/yr. of mAb production. Many of the benefits can be realized from partial integration of continuous technology. The models identify those areas where the new emergent continuous/connected technology can optimised further. At capacities of 900kg/yr. DSP costs are split 60% process and 40% solutions, the bulk of the costs are consumables (chromatography resins, filters etc.). This means that continuous technologies should be optimised for reduced resin usage, and that solution management is a key area to be addressed. Intensified processing can impact negatively on solution management and the options to reducing its impact through use of concentrates and buffer preparation skids is explored.

BIOT 28

Comparison of productivities and cost models for protein A capture matrices

Paul R. Randolph, [email protected]. Centocor, Malvern, Pennsylvania, United States

Advances in bioreactor technologies over the last decade have increased productivity and expression to levels where current capabilities of protein A resins are challenged. To study ways to increase capture productivity, reduced scale comparative evaluations of five different Protein A capture resins and 3 types of Protein A membranes for capturing monoclonal antibodies were performed. Dynamic binding capacity (DBC) was measured for each resin and membrane across a range of flow rates and fitted to hyperbolic functions. Resulting data were used to model productivity in various operational modes. Resin lifetime was estimated by measuring DBC after timed exposure to sodium hydroxide. Results of the analysis were used along with target process parameters to calculate expected operating costs for each resin type in various scenarios representing high volume commercial processing and low volume or infrequent processing, representing a clinical development scenario.

BIOT 29 Optimisation of continuous crystallisation to produce protein crystals of desirable physical properties: Case of lysozyme

Huaiyu Yang1, Wenqiam Chen1, Xiaoyu Li1, Shuaijun Shen1, Ian Rosbottom1,2, [email protected], Jerry Y. Heng1. (1) Chemical Engineering, Imperial College, London, United Kingdom (2) Chemical Engineering, University of Leeds, London, United Kingdom

Crystallization could be a cost effective and robust method to purify protein molecules in the pursuit of the next generation of biopharmaceutic therapies. However, the step from traditional small-scale high throughput screening to crystallizers which can produce high yields of pure crystalline protein material, is in its infancy. Here, we present a platform for continuous oscillatory flow crystallization (COFC) (Figure 1) and its application for the crystallization of lysozyme. A workflow is developed from µl screening experiments, to scaled up batch oscillatory flow crystallization (BOFC) and COFC experiments. The induction time for lysozyme is found to be inversely proportional to the initial protein concentration, with the same relationship found between the induction time and the frequency and amplitude of the oscillation. However, it is observed that the decrease in induction time comes at a cost to the crystal size and quality. In turn, the high shear environment from increased amplitude and frequency of the COFC can also damage the fragile protein crystals, resulting in the formation of more fine particles and an undesirable size distribution. This led to further investigation of slug flow mixing in the continuous crystalliser, where the crystal size and shapes are compared to the particles produced from the osciallatory flow reactor. Further work around optimization of the crystallization parameters and crystallizer geometry using the gCrystal platform to find the optimum scale up conditions are also discussed. The use of seeding and potential templating materials is also covered with a view to expanding the use of this crystallizer to less well characterized protein materials. It is demonstrated that purification of proteins can be achieved by crystallization in a continuous mode. This route offers the possibility to revolutionise downstream separations of biopharmaceuticals.

BIOT 30

Integrated continuous bioprocessing with CHO cells and ultra-high productivity for biologics manufacturing

Weichang Zhou, [email protected]. WuXi AppTec Co Ltd, Shanghai, China

Ultra-high productivity continuous bioprocesses have been developed for production of monoclonal antibodies, fusion proteins and bispecfic antibodies using CHO cells. These enable, for example, 2,000L disposable bioreactors to achieve comparable productivity as traditional 20,000L stainless bioreactors, and to significantly reduce manufacturing cost of goods. This process technology platform consists of continuous CHO cell culture and continuous direct product capture. Several case studies, which achieved a cell culture productivity of 2-3 g/L/day, and a similar or better purification yield of the traditional purification process, will be discussed to highlight advantages of this integrated continuous bioprocessing platform in terms of productivity gains and quality improvements.

BIOT 31

Protein A column sanitization: Enabling continuous antibody production for GMP manufacturing

Rob Piper, [email protected]. Just Biotherapeutics, Seattle, Washington, United States Continuous manufacturing of biologics offers the promise of lower capital outlay, improved productivity and reduced cost of goods. Continuous operation can be facilitated by extending the sterile envelope from the bioreactor to the capture step and into the downstream process through the use of sterile, single use components and equipment. However, chromatography columns remain a weak link since in-house packed columns are not sterile, and gamma irradiated columns are expensive and not widely available commercially. Here, we present a case study in the development and implementation of an inexpensive chemical sterilization process for in-house packed columns that permits extended perfusion durations and continuous capture. Initial introduction at the 500L scale was met with mixed success due to contaminations during two of four runs. To address this issue, we performed a series of experiments looking at several different Protein A resins, sanitant types and sanitization techniques. We found that the resin and sanitant can interact and impact column sanitization. With this information, we developed a technique to successfully sanitize process scale columns in the PD lab and for two 500L scale fully continuous production runs. A robust sanitization step will be a key step in enabling continuous manufacturing at process scale.

BIOT 32

Development and pilot scale implementation of a continuous viral inactivation strategy as part of a fully continuous mAb process

Chad Varner, [email protected], Michael J. Coolbaugh, Tarl Vetter, Emily Davenport, Kevin Brower. Purification Development, Sanofi, Framingham, Massachusetts, United States

Continuous manufacturing strategies often intensify downstream processes by connecting and automating batch operations. This mode of operation allows companies to increase facility utilization, decrease COGs, leverage prior/platform knowledge, and switch to single-use technologies. However, focusing on connecting existing batch processes may limit the ability to maximize the potential benefits of truly continuous bioprocessing. We have previously demonstrated a fully continuous mAb process from media to drug substance executed at the pilot scale for 25 days. In this talk, we will present a continuous viral inactivation strategy that can convert discrete protein A eluates (from continuous capture) into a constant stream of low-pH viral inactivated material, ready for process polishing. Several features of the continuous viral inactivation process will be discussed: implementing a novel control strategy to overcome detection response time limitations, active adaptation to changing volume and mass flow rates, product safety controls, single-use assembly, and challenges of implementation. This continuous VI strategy results in a consistent flow of material to be processed downstream; potentially opening the doors for downstream operations to move beyond connected, rapid-batch processing to novel processes not constrained to current technologies.

BIOT 33 Assessing in-line buffer conditioning for implementation and facility benefit for traditional and ICB manufacturing applications

Emily Davenport1, [email protected], Nathalie Frau2, Kevin Brower1. (1) Purification Development, Sanofi, Framingham, Massachusetts, United States (2) Business, Innovation, Integration, and PMO, Sanofi, Waltham, Massachusetts, United States

Buffer solutions are essential for all biologics processing, and preparation of these solutions can easily become a bottleneck for production, particularly in existing facilities. Process intensification of upstream operations creates larger demands for downstream processing, and thus, increases in buffer consumption. This has put strain on buffer preparation operations, often restricted by the facility’s capacity or footprint for buffer storage. In this work, in-line buffer conditioning (ILC) was assessed as a solution for improving manufacturing facilities’ capability, compared to traditional buffer preparation. ILC is defined as the simultaneous dilution and titration of single-component stock solutions. The systems of three different vendors were tested. Method development, control strategy, reproducibility, and accuracy of these systems were evaluated to determine the ease of use and feasibility of implementation. One system was tested to support an end-to-end, fully integrated downstream process (ICB) for a 100L bioreactor campaign of 45 days, reducing labor resources by 50%. For some facilities, pilot-scale intensified or ICB processes cannot be supported without the use of an ILC system. Here, we show the benefits of ILC implementation, particularly for companies with a platform downstream process. Results from this evaluation advise instrumentation selection and implementation strategy for existing and new facilities, both batch and ICB, based on a high-titer antibody model.

BIOT 34

Engineered multidimensional co-culture model for examining important bacteria: Macrophage interactions in inflammatory bowel disorders

Kimberly Wodzanowski1, [email protected], Catherine L. Grimes1,3, April M. Kloxin2,4. (1) Chemistry and Biochemistry, University of Delaware, Newark, Delaware, United States (2) Chemical Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (3) Biological Sciences, University of Delaware, Newark, Delaware, United States (4) Materials Science and Engineering, University of Delaware, Newark, Delaware, United States

The immune system is a complex network comprised of various cellular components that work together to protect the body from damage, with a key function of differentiating between pathogenic bacteria and the 39 trillion commensal bacteria that constitute our microbiome. Misrecognition of commensal bacteria has been linked to many inflammatory bowel disorders such as Crohn’s disease in humans yet remains mechanism of misrecognition remains unknown. Enabled by recent advances in molecular tools, fragments of the bacterial cell wall peptidoglycan have been identified that bind to pattern recognition receptors within macrophages, which activate a signaling cascade that turns on the immune response. Typically, these interactions are studied using two-dimensional invasion assays on tissue culture plastic plates; however, the dimensionality and stiffness of these traditional culture systems are not representative of the cell-microenvironment interactions that exist in vivo. Three- dimensional (3D) cell culture systems better simulate the native cellular environment and are used here to control key biochemical and mechanical properties of the cell microenvironment. We established an approach for the culture of human macrophages in three dimensions within well-defined, biomimetic hydrogels decorated with integrin binding peptides that mimic the natural in vivo environment and linked with cell- degradable sites for cell-driven migration and remodeling to probe macrophage response to bacterial invasion. With this approach, we have modeled the invasion with bacteria and their engulfment by macrophages, utilizing innovative chemical probes to label the cell walls of pathogenic bacteria, and assessed the resulting activation of macrophages using RT-qPCR and ELISA. These studies provide new insights into the interactions between host-pathogens, and this new method affords unique opportunities for examining how our immune system recognizes and misrecognizes bacteria toward a better understanding of the mechanisms of immune misrecognition diseases.

BIOT 35

Microbial-mediated transport of beneficial bacteria and agrochemicals

Christopher Hawxhurst1, [email protected], Jamie Micciulla2, Grant Bouchillon3, Kurt Ristoph4, Robert K. Prudhomme5, Daniel Gage2, Leslie M. Shor6. (1) Chemical Engineering, University of Connecticut, Storrs, Connecticut, United States (2) Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, United States (3) Environmental Engineering, University of Connecticut, Storrs, Connecticut, United States (4) Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States (5) Dept. of Chemical Biological Engineering, Princeton University, Princeton, New Jersey, United States (6) Chemical, Materials, and Bioengineering, University of Connecticut, Storrs Mansfield, Connecticut, United States

Protists can move readily through unsaturated soils and chemotact towards bacterial prey, which are abundant at growing tips of plant roots. Many protists species are indiscriminate eaters, capturing and ingesting bacteria-sized objects in the soil. Protists may therefore serve as transport vehicles for polymer-coated agrochemicals and introduced plant growth promoting rhizobacteria (PGPR): accumulating prey-sized particles, transporting the “payload” through soil, and targeting delivery to the growing root tips. We developed a transport assay comprised of an emulated soil micromodel to screen candidate soil protists for performance in taking up, transporting, and depositing fluorescent microspheres or polymer-encapsulated nanoparticles through the emulated physical structure of sandy loam soil. Next, we created a mesoscale transport assay containing real sandy loam soil and a live Medicago truncatula plant within a 3D-printed device, referred to here as “µ-rhizoslides”. Using the µ-rhizoslides, we studied the most promising protist candidate from the transport assay. We tracked position of fluorescent particles or fluorescent protein-expressing PGPR (bacteria) on a weekly basis in replicate µ-rhizoslides that either contained or did not contain live protists (Colpoda). We demonstrate inclusion of Colpoda facilitated transport of fluorescent particles and of bacteria along growing plant roots. µ-rhizoslides containing protists exhibited bacterial cells located further down plant roots compared with bacteria-only µ-rhizoslides. Even though protists are natural predators of the bacteria used in transport assays, the inclusion of protists still enhanced transport of bacteria to the distal region of plant roots: the portion most in need of colonization by PGPR. Facilitating transport and targeting delivery of valuable agrochemicals or bacteria to the tips of growing plant roots will greatly facilitate the implementation of more sustainable no-till farming practices, reduce overall agrochemical use, improve surface water quality, and may dramatically reduce or even reverse the net impact of the agriculture sector on global climate change.

BIOT 36

Towards microbiome engineering using highly selective bacteriocin lysostaphin and phage endolysin PlyPH

Amala Bhagwat, [email protected], Cynthia H. Collins, Jonathan S. Dordick. Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States

Development of novel selective antimicrobials is gaining momentum due to increased bacterial drug resistance, microbiome dysbiosis caused by broad-spectrum antibiotics and the need to understand and precisely engineer human and environmental microbiomes. Cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins represent a unique, highly specific catalytic repertoire hydrolyzing bacterial peptidoglycan. Owing to their exquisite binding and/or catalytic specificity and lower probability of gaining microbial resistance, they are not only emerging as promising alternative to antibiotics for therapy and infrastructural resilience applications but also potentially for microbiome engineering and remodeling via selective pathogen elimination. We have assessed the ability of two different lytic enzymes, bacteriocin lysostaphin (Lst) and endolysin PlyPH targeting skin pathogens Staphylococcus aureus and Bacillus cereus, respectively, to control a community (mixture or co-culture) of S. aureus and B. cereus with a skin commensal Micrococcus luteus. Lst (100 ug/mL) was highly effective and selective, causing ~ 3-log reduction in S. aureus, however, 100 ug/mL PlyPH selective but less active, resulting in ~1.5-log reduction in B. cereus in an actively growing co-culture. Lst and PlyPH were limited by proteases secreted during growth, as suggested by their increased activity in culture supernatants in the presence of a protease inhibitor cocktail, highlighting effect of bacterial metabolism on enzyme activity. Since Lst and PlyPH activity is governed by cell wall binding, analysis of their binding is critical for elucidating activity in nutrient-rich environments mimicking natural microbiomes. Lst and PlyPH binding was measured in four increasingly complex media by evaluating binding isotherms and real-time binding kinetics of their isolated fluorescently-tagged binding domains using both fluorescence and Surface Plasmon Resonance. Slower binding kinetics was observed with increased medium complexity, causing limited killing in nutrient rich media. In conclusion, we demonstrate utility of lytic enzymes and fundamental considerations to enhance their robustness for microbiome engineering.

BIOT 37

Engineering bacteria to program animal behavior and lipid metabolism

Baizhen Gao, Qing Sun, [email protected]. Chemical Engineering, Texas A&M University, College Station, Texas, United States

A mammalian host shares an intimate and lifelong partnership with resident microbial communities. Microbiota dysbiosis is associated with multiple metabolic, brain, and behavioral disorders including obesity, Clostridium difficile infection, inflammatory bowel disease, and autism. Restoration of gut bacteria including fecal microbiota transplant has been shown to reverse disease progression. With evidence supporting the bidirectional integrated gut-brain axis, microbiota has the potential to be used for behavioral and metabolism disorder disease therapy like obesity. However, there has been limited research on using engineered bacteria for animal behavior and lipid homeostasis interference. The goal of this research is to engineer bacteria to sense environmental stimuli, process the signal and respond by programming the behavior and metabolism of the model animal Caenorhabditis elegans, which is a nematode and has previously been used for microbiome-host interaction studies. We started with engineering bacteria to modulate C. elegans’ green fluorescence protein expression and fat storage. The goal is that chemical-inducible worm fat accumulation and behavior can be achieved through genetically modified gut bacteria without any genetic modifications of the animal host. Next, we built synthetic genetic circuits including “AND”, “OR” and “NOR” in bacteria to achieve programmable and sophisticated control of the worm phenotype and fat accumulation. The significance of this research lies in the study of microbiota-host interaction by adapting bacterial logic gates to animals and the potential of our platform for disease therapies including autism and obesity.

BIOT 38

Metabolite cross-feeding interactions in adipocyte-hepatocyte co-cultures elucidated with a novel 13C-metabolic flux analysis technique

Eleanor H. Oates, [email protected], Maciek R. Antoniewicz. University of Delaware, Newark, Delaware, United States

Metabolic disorders are multifactorial, caused by interactions of numerous environmental, lifestyle, and genetic variables. One such cause is the dysregulation of metabolic interactions between the body’s myriad of cell types responsible for maintaining normal physiology and energy balance. A key to characterizing and identifying novel therapeutic targets for these disorders is to gain a better understanding of the dynamic nature of metabolic interactions and determine how these can be monitored and controlled. Towards this end, we established a co-culture system between adipocytes and hepatocytes that can be used for identifying and quantifying metabolite cross-feeding. These interactions are of particular interest since failed cross- feeding has been directly linked to a cluster of disorders, including diabetes and obesity. Our co-culture system provides a convenient and high-throughput platform for investigating how system perturbations affect adipocyte-hepatocyte interactions and for fine-tuning experimental techniques so that they can be later translated into in vivo studies. In this presentation, we first introduce the adipocyte-hepatocyte co-culture system that we established. Second, we describe a novel approach we developed for co-culture 13C-metabolic flux analysis (13C-co-MFA) that can quantify changes in metabolite cross- feeding rates and individual cell population fluxes. To illustrate these techniques, we performed co-culture experiments with two global regulators, insulin and isoproterenol. The addition of insulin induced cells into a “fed-like” state of metabolism in which both hepatocytes and adipocytes consumed glucose and utilized it for glycogenesis and lipogenesis, respectively. The addition of isoproterenol, on the other hand, promoted a “fasted-like” state of metabolism in which adipocytes released metabolic precursors such as glycerol, lactate, and glutamine, that were then used by hepatocytes for gluconeogenesis. To quantify fluxes, we performed parallel 13C-tracer experiments with six different isotopic tracers and applied 13C-co-MFA to precisely determine metabolic fluxes in both cell types and cross-feeding rates for the metabolites that were identified to be exchanged between the two cell types.

BIOT 39

Connecting biology to electronics through a redox communication network of tyrosine, tyrosinase, and eumelanin

Eric VanArsdale3, [email protected], David Hornstrum1, Eunkyoung Kim2, Juliana Pitzer3, Gregory F. Payne2, William E. Bentley3. (1) KTH Royal Institute of Technology, Stockholm, Sweden (2) Univ of Maryland Biotech Inst, College Park, Maryland, United States (3) Fischell Dept of Bioengineering, University of Maryland, College Park, Maryland, United States

Systemic control of cellular physiology will allow for the design of synthetic electronic- biological communication networks, capable of coordinating physiology across large distances, disparate environments, and varied time scales. In order to achieve this connectivity, we have begun to develop cellular equivalents of network technology. To do this, we have engineered a cell-based co-culture system in which “sensor” and “producer” cells work together to generate a redox signal in response to molecular communication. The sensor cell population integrates molecular cues to regulate the expression of a surface-linked tyrosinase. Similarly, the producer cell population regulates the synthesis of the enzyme-substrate L-tyrosine, which is converted by the sensor cell population into the redox signal L-DOPA. In co-cultures, this system enables real-time electrochemical detection of molecular information by measuring the oxidative current of L-DOPA. This system was able to eavesdrop on cell-cell molecular communication, which we demonstrate by intercepting the quorum sensing signals of Pseudomonas aerugionosa. We have also adapted this system to receive electronic signals through “transmitter” cells that produce quorum sensing signals in response to electrochemically generated hydrogen peroxide. Finally, we have stored cellular and electronic information in cellularly-produced “hard drives” to store redox memory, through controlled oxidation and reduction of the catechol-quinone groups of eumelanin with redox mediators. The melanin memory units were capable of directing cellular signaling, either by attenuating or amplifying the oxidative signature of a mediator mixture. Together, these cellular units allow for regulated information exchange with biology to allow for rational, user-guided cellular communication and physiology.

BIOT 40

Integrated electrical-optical interface between neurons and bacteria reveals bidirectional communication and coupled responses to the contact

Vikas Trivedi1, [email protected], Todd C. Chappell1, Daniel A. Fuenmayor2, Sophia K. Jannetty3, Joan M. Lemire3, Katharine A. Bowers3, Brian P. Timko2, Nikhil U. Nair4, Michael Levin3, Celia Herrera-Rincon3. (1) Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, United States (2) Biomedical Engineering, Tufts University, Medford, Massachusetts, United States (3) Department of Biology and Allen Discovery Center, Tufts University, Medford, Massachusetts, United States (4) Chemical & Biological Engineering, Tufts University, Medford, Massachusetts, United States

The microbiota is continuously being recognized as an important component in the maintenance of health and has been correlated with onset and progress of disease. Some diseases that are of neurological origin are now being recognized as a neuronal response to microbial infection e.g. Alzheimer disease. In addition, microbial metabolites capable to modulate emotional and physiological state of organism via neurons have been identified. Recently it has been shown that microbes mediate species-specific and inter-species interaction via electrical communication like neurons. This begets an interesting question – can neurons and microorganism communicate in an intelligible manner via ions? Given the rich behavioral and molecular repertoire shared by neurons and bacteria, we sought to develop a novel and molecularly- tractable platform within which direct inter-kingdom communication could be demonstrated and dissected. Our multi-modal system which integrates optical and electrical signal detection, revealed that neurons respond to the presence of bacteria, and that this response depends on the bacterial species and density. Likewise, bacteria showed responses to neural presence and activity, as revealed by specific changes in their membrane potential and calcium dynamics. Our proof-of-principle data in this highly tractable platform reveal crosstalk mediated by electrical and chemical signals and illustrate a novel example of cross-kingdom communication between highly diverse cell types. The ability to eavesdrop on information passing between these two very different levels of biological organization will facilitate the understanding of brain- bacteria communication for diagnosis of neuronal or bacterial states in health and disease.

BIOT 41

Designing living therapeutics

Pamela Silver, [email protected]. Systems Biology, Harvard Medical School, Boston, Massachusetts, United States

We understand in detail how biological systems sense and respond to their environment. This is especially true for bacteria, their associated phages and other microbes. This deep understanding makes the engineering of microbes an optimum target for the design of living diagnostics and therapeutics. We have thus engineered bacteria and phages in the mammalian gut to sense signals of disease and to deliver therapies. Together, our work presents a platform for design principles that can be employed across microbiomes in general.

BIOT 42

Design and directed evolution of conformational antibodies specific for amyloid aggregates

Alec Desai1, [email protected], Matthew D. Smith1, Yulei Zhang1, Peter M. Tessier1,2,3. (1) Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States (2) Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States (3) Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States

Antibody specificity is arguably the most important and most challenging property to optimize during in vitro antibody selection and affinity maturation. We have sought to develop methods for both designing and sorting antibody libraries that maximize the specificity and affinity of selected antibody variants against insoluble, complex antigens (amyloid fibrils of the Alzheimer’s Aβ42 peptide). First, we have developed a novel library design method that samples wild-type residues and a small number of the most frequently occurring residues at multiple positions in antibody binding loops based on natural antibody diversity. Second, we have developed methods for isolating antibodies with high conformational specificity and low off-target binding by performing selections against the antigen (Aβ fibril) and closely related non-antigens (Aβ monomer) and polyspecificity reagents. By evaluating the relative enrichment of mutations during antibody selections, we find that most mutations that enhance antibody affinity compromise conformational specificity and that deep sequencing methods are critical to identify extremely rare sets of mutations that increase affinity while maintaining strict conformational and sequence specificity. We are currently testing our library design, sorting and bioinformatics methods to generate novel antibodies against a number of other complex antigens (e.g., tau and α-synuclein fibrils and oligomers). We expect that these approaches will improve the in vitro isolation of diverse types of antibodies with high specificity and other drug-like properties.

BIOT 43

Withdrawn

BIOT 44

Specificity profiling and high-resolution epitope mapping of challenging mAbs

Kyle M. Doolan, [email protected], David F. Tucker, Jonathan T. Sullivan, Duncan Huston-Paterson, Edgar Davidson, Ross Chambers, Benjamin J. Doranz, Joseph Rucker. Integral Molecular, Philadelphia, Pennsylvania, United States

Toxicity failures caused by off-target or unintended on-target liabilities are a leading cause of attrition in clinical and preclinical programs. Given the effort, cost, and technical limitations of traditional biopharmaceutical de-risking strategies, there is a need for a robust and scalable approach that can be rapidly applied to the discovery phases of therapeutic programs. To enable this, we have developed a Membrane Proteome Array (MPA) platform containing 5,300 human membrane proteins. Each membrane protein is expressed in live cells in their native conformation. We have tested hundreds of MAbs for specificity and off-target binding using the MPA. This presentation will give examples of how the MPA can be used to de-risk antibodies early in the development process. We will also discuss the mechanistic origins of off-target binding using our Shotgun Mutagenesis Epitope Mapping platform.

BIOT 45

Exploring linker composition to enhance biophysical properties of antibody fragments for cancer theranostics

Jeong Min Han, [email protected], Thomas J. Magliery. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, United States

Single-chain variable fragment (scFv) is the smallest functional unit of monoclonal antibody with a non-native peptide linker holding the variable domain together. Its small size allows bacterial expression making engineering easy, and further gains some pharmacokinetic advantages such as better tumor penetration and reduced immunogenicity. Besides, the engineered scFvs can be easily put into different formats (diabodies, bispecifics, conjugates) to tune the half-life and functions. However, despite the great potentials, scFvs and its derivatives have not made it into the clinic. Part of the problem is that nearly all scFvs have been engineered with flexible (Gly4Ser)n linkers, which are susceptible to proteolysis leading scFvs to poor stability. Yet there has been no comprehensive study on compositional effects of the linker. Here, we explored the linker composition to enhance biophysical properties of 3E8; 3E8 is a scFv that has strong affinity to a disaccharide, sialyl-Tn on Tumor-Associated Glycoprotein-72, which is present in 80% of adenocarcinomas but in healthy human tissues making it a great candidate for cancer targeting. First, sets of linker libraries in varying lengths and compositions were constructed and characterized to determine favorable linker trends in scFvs. The results showed stabilizing effects for conformationally constrained linkers. Based on this, a much broader sequence space was explored via combinatorial linker library and phage display, a high-throughput screening method. After the fifth round of biopanning, the library was deep sequenced by next-generation sequencing, and the hits appearing in repeat were further characterized to confirm stability enhancements. The results showed a significant improvement in expression level, thermal stability and proteolytic susceptibility with high frequency of conformationally constrained amino acids throughout the linker. Moving forward, we are currently working on generating a high-resolution structure to understand the stability change at the molecular level. Also, we constructed complementarity-determining region libraries to alter its specificity towards a series of cancer-related glycans to generate novel binders for cancers with poor prognosis. Taken together, this study provides valuable insights into scFv engineering that is currently lacking in order to stabilize scFvs and expand its utility for cancer theranostics.

BIOT 46

Characterization of natural bacteriocin IIa antimicrobial proteins to map sequence-function relationships

Daniel Tresnak, [email protected], Benjamin Hackel. Chemical Engineering and Materials Science, University of Minnesota - Twin Cities, Minneapolis, Minnesota, United States

Bacteriocin IIa antimicrobial proteins (AMPs) are a protein family characterized by small size (roughly 30-50 amino acids), simple protein structure, and strain-specific targeting of the mannose phosphotransferase systems (manPTSs) of various gram-positive bacteria. Given their high specificity, bacteriocin IIa AMPs are one potential alternative to broad-spectrum antibiotics for treating various bacterial infections, with vancomycin- resistant Enterococci (VRE) garnering particular interest. However, it is poorly understood how varying bacteriocin IIa protein sequence affects AMP potency and selectivity; thus, it is unclear how to best engineer these AMPs as therapeutics. Towards this goal, we are experimentally evaluating the Enterococcal inhibitory activity of 230 naturally occurring and rationally designed bacteriocin IIa AMPs and 900 random multi-mutants of six previously characterized bacteriocin IIa AMPs. We screened these two pools of AMPs expressed in L. lactis against six strains of Enterococcus and identified functional clones using high-throughput sequencing. We identified a total of 66 unique AMPs which displayed antimicrobial activity and are individually producing these AMPs to identify selective, potent molecules through quantification of the minimum inhibitory concentration across several Enterococcus strains. The activity data will then be mapped to the AMP and manPTS sequences to identify protein interactions which determine AMP selectivity and activity. The emerging data linking sequence and multi- strain potency will identify sequence-function relationships for informing future bacteriocin IIa rational design.

BIOT 47

Engineering peptide insertions in virus-like particles using systematic apparent fitness landscapes

Stephanie A. Robinson1,3, [email protected], Emily Hartman3, Matthew B. Francis2, Danielle Ercek1. (1) Chemical and Biological Engineering , Northwestern University, Chicago, Illinois, United States (2) University of California, Berkeley, California, United States (3) Chemical Biology, University of California, Berkeley, Berkeley, California, United States

Virus-like particles (VLPs) are nanoparticles made of non-infectious viral capsids. For example, the MS2 bacteriophage is a robust VLP and is studied as an imaging and drug delivery system. The MS2 VLP is stable for extended periods of time in heat, is high- yielding with simple recombinant expression, and can encapsulate artificial cargo, making the system ideal for a targeted drug-delivery vehicle. Recently, the Francis and Tullman-Ercek groups explored methods of engineering this capsid using protein engineering techniques such as generating an apparent fitness landscape. A fitness landscape generates a fitness score for mutants based on survivability of a genotype after selections such as: capsid assembly, thermostability, and acid sensitivity. This fitness landscape can inform on which residues are tolerated in the protein’s assembly and may reveal surprising structural properties. Here, the MS2 fitness landscape was used to identify a highly mutable region in the coat protein known as the FG loop. The mutability and unique structure of the FG loop was used to inform the generation of a tripeptide or (NNK)3 insertion library at the FG loop of the MS2 coat protein. These libraries show that the FG loop is particularly tolerable of residues disruptive to secondary structure, with high localizations of charge, and that can hydrogen bond. These effects dominate capsid assembly to such an extent that when these well- performing residues are grouped with extremely poorly performing residues, capsid assembly is still recovered. Ultimately, this work validates the utility of fitness landscapes in engineering VLPs, and illuminate how properties such as charge, flexibility, and the hydrogen bonding in the FG loop conserve capsid assembly, and expand the scope of peptide insertions which could be engineered into VLPs.

BIOT 48

Studying the transport of CPP-cargo protein fusions into Candida albicans using experiments and simulations Sayanee Adhikari1, [email protected], Mahdi Ghorbani1, Jeffery B. Klauda1, Amy J. Karlsson1,2. (1) Chemical and Biomolecular Engineering, University of Maryland, College Park, College Park, Maryland, United States (2) Fischell Department of Bioengineering, University of Maryland, College Park, College Park, Maryland, United States

Cell-penetrating peptides (CPPs) are a class of small peptides with the ability to cross membranes and carry biomolecules with them. We are exploring CPPs as vehicles for delivering biomolecular cargo into the opportunistic fungal pathogen, Candida albicans. We recombinantly produced the CPPs histatin 5 (Hst5) and MPG as fusions to green fluorescent protein (GFP) to explore the intracellular delivery of GFP into C. albicans using CPPs. MPG is a short amphipathic CPP that inserts readily into natural membranes. Hst5, which is found in human saliva, is a histidine-rich antimicrobial peptide that must cross the cell membrane to exert its fungicidal activity. We studied CPP-mediated translocation of GFP using flow cytometry to detect GFP inside cells. Fusion to MPG led to translocation of GFP into 38% of C. albicans cells, which was significantly higher than the 13% cells that exhibit translocation of GFP without a CPP (p ≤ 0.001). In contrast, Hst5 did not translocate GFP into cells, with only 5% of cells showing Hst5-GFP translocation. Our results show that MPG can be used to deliver GFP, while Hst5 is not as promising. To better understand the translocation of MPG and Hst5, we used molecular dynamics simulations to study their interaction with C. albicans cell membranes. Our simulation studies showed that MPG enters the membrane model readily, with the N-terminal residues initiating the entry. In contrast and consistent with experimental results, Hst5 fails to enter the membrane even after 400 ns. Using knowledge gained from simulations, we designed MPG variants with amino acid substitutions to improve translocation into C. albicans cells and are currently testing the variants for enhanced translocation efficiency. Our results highlight the utility of combining experiments and simulations to study the translocation of CPPs and to engineer CPPs for improved delivery of cargo into C. albicans cells.

BIOT 49

Genetically encoded 3,4-ethylenedioxythiophene (EDOT) functionality for fabrication of protein-based conductive polymers

Maiko Obana, [email protected], David A. Tirrell. MC 210-41, Caltech, Pasadena, California, United States

Conductive polymers are an important class of materials because of their unique electronic and optical properties. Unlike inorganic conductors, conductive polymers are easily functionalized and processed, and therefore, are widely utilized at the interface with biology (e.g. biosensors, cell culture, neural probes, drug delivery). By linking conductive polymers and proteins via genetically incorporated monomer units, we are developing protein-based conductive materials. Because the size and sequence of proteins are precisely controlled by gene expression, we can fine tune the design of the materials such as the number of monomer units in a protein and the chemical environment around the monomer. In the presentation, we will demonstrate genetic incorporation of a non-canonical amino acid (designated EDOT-Lys) bearing the 3,4-ethylenedioxythiophene (EDOT) group, which is subsequently polymerized to form conductive polymers. E. coli expressing mutants of M. mazei pyrrolysyl-tRNA synthetase (mmPylRS) revealed strong fluorescence from GFP as an indication of successful incorporation of EDOT-Lys. We will also present polymerization of the unstructured protein XTEN and small peptides through their pendant EDOT groups.

BIOT 50

Understanding fouling mechanisms of filters during the harvest of monoclonal antibody therapeutics

Stijn H. Koshari, [email protected], Kusum Solanki, Hong Zhang, Hiren D. Ardeshna, Antonio R. Ubiera, Robert Luo. Downstream Process Development, GlaxoSmithKline, King of Prussia, Pennsylvania, United States

The harvest step in a monoclonal antibody downstream purification process removes biomass, particulates, and other material from the cell culture to generate a product stream suitable for purification by liquid chromatography. In a widely employed, two- step harvest process, coarse solids and intact cells are first removed using a centrifuge, and the resulting centrate is further clarified by depth and membrane filtration in series. Conversely, direct depth filtration has the potential to greatly simplify the overall harvest process by directly loading the cell culture broth on high-capacity depth filters and consequently removing the need for centrifugation. We evaluated this potential of direct filtration for multiple biopharmaceutical assets to understand the underlying mechanisms that define fouling and the resulting capacity limitations in these filters. In this presentation, we show how each of the individual filters contributes to the overall capacity of the direct filtration train, and how the capacity is affected by characteristics of the cell culture broth and process parameters, including enhanced upstream processes such as high cell density perfusion. Our findings highlight the limitations of direct filtration when implemented in large-scale, high cell density facilities. Ultimately, this increased understanding of the fundamental principles that govern depth and membrane filtration can aid in moving from a trial-and-error to a more predictive and methodology-based approach for harvest development.

BIOT 51

Performance characteristics of hollow fiber microfilters for purification of monoclonal antibodies by precipitation

Zhao Li1, [email protected], Qin Gu2, Jon coffman5, Todd M. Przybycien3, Andrew L. Zydney4. (1) ChE, Penn State University, University Park, Pennsylvania, United States (2) CMU, Pitts, Pennsylvania, United States (3) Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States (4) Dept of Chemical Engineering, Penn State University, University Park, Pennsylvania, United States (5) AstraZeneca, Gaithersburg, Maryland, United States

Precipitation can be a highly effective step in the initial purification of monoclonal antibodies (mAb). High yield of the precipitates can be produced continuously in a tubular reactor using cross-linking and volume exclusion agents. A fully continuous precipitation process has been explored by integrating the precipitation step with hollow fiber microfiltration for dewatering and washing. The objective of this study was to investigate opportunities for increasing the performance of the hollow fiber microfiltration by increasing the critical flux/conversion and reducing the product loss due to redissolution of the antibody product. Experiments were performed using human serum Immunoglobulin G (IgG) as a model antibody with 0.2 µm pore size polyethersulfone membranes. IgG precipitates with >99% yield were produced through using combinations of zinc chloride and polyethylene glycol in a tubular precipitation reactor with residence time of approximately 30 s. Critical flux experiments were performed using a total recycle system, with the inlet feed flow rate and precipitate concentration varied to determine their effects on the critical flux. Washing experiments were performed using different IgG precipitate concentrations and wash buffer conditions. The overall conversion in the hollow fiber module could be increased by decreasing the feed flow rate, providing more effective washing of the protein product. Pre- concentration of the IgG gave precipitates that were more resistant to product redissolution, leading to an increase in product yield. These results provide important insights into opportunities for improving the design and operation of a precipitation – microfiltration process for the initial capture / purification of monoclonal antibody products.

BIOT 52

Strategies dealing with difficult to remove HCPs when operating with only flow- through polishing steps Greg Evangelist, [email protected]. Downstream Development , Biogen , Durham, North Carolina, United States

Process changes often occur when molecules transition from early to late phase development. To ensure that safety or efficacy will not be affected due to process changes, an analytical comparability assessment between the prior and new drug substance is carried out. Recently, an early phase mAb program transitioned to late stage development that included both a cell line change and an update to the downstream purification platform. The updated platform consisted of Protein A capture, followed by AEX and HIC polishing steps operated in flow through mode. As part of the comparability assessment LC/MS analysis of drug substance produced by the new process identified two HCPs that were not present in the early phase clinical lots. One of the HCPs was well known in the literature and did not pose a significant concern, while little to no information could be gleaned for the second HCP. To address removal of these HCPS whilst not making significant changes to the newly implemented purification process, a holistic approach was taken to their removal. Protein A wash studies, modulation of AEX operating conditions, and introduction of depth filtration technologies were explored. Of these approaches depth filters provided the best removal with minimal impact on yield and the downstream process. Removal of the two HCPs was achieved by adoption of depth filters, resulting in comparable drug substance product quality to the early stage material.

BIOT 53

Evaluation of caprylic acid precipitation as a tool to increase clearance of potentially critical host cell proteins

Frederik Rudolph, [email protected], Viktor Gross, Christopher Thamm, Joey Studts. Boehringer Ingelheim, Biberach an der Riss, Germany

HCPs are a highly diverse range of proteins. While some HCPs can be degradative to the product itself, others could induce an unwanted immune response compromising the safety and efficacy of the biologic. The majority of host-cell proteins are removed through multiple chromatography steps and filtrations; individual host-cell proteins can be more challenging and can be carried through even into bulk drug substance. Hence reducing them to the generally accepted level can be challenging. A promising strategy to achieve this goal is to integrate a precipitation step during harvest operations. In this talk, we discuss caprylic acid (CA) precipitation, as a promising tool to increase clearance of potential critical HCPs and therefore reduce the burden on the subsequent purification and filtration steps. Operational conditions (e.g. pH and caprylic acid concentration) were evaluated with respect to yield, impurity clearance (e.g. HCPs) and impact on product quality attributes (e.g. HMW, LMW, charge pattern). Precipitated cell culture pools were further purified to investigate the impact on following purification steps. In summary, 4 different IgGs were used for this study to demonstrate the applicability of this technique as valuable option for increasing impurity clearance in mAb processes.

BIOT 54

Purification process including cell separation using protein A magnetic beads

Veronique Chotteau1,2, [email protected], Nils Brechmann1,2, Kristofer Eriksson3. (1) Industrial Biotechnology - Cell Technology Group, KTH - Royal Institute of Technology , Stockholm, Sweden (2) AdBIOPRO, Competence Centre for Advanced BioProduction by Continuous Processing, Sweden, Stockholm, Sweden (3) R&D, Lab-on-a-Bead AB, Uppsala, Sweden

Magnetic bead-based purification from cell suspension has been used for decades for analytical purpose but their use in larger scale has not been possible. A purification process of mAb carried out with non-clarified cell suspension could provide significant savings of operation time and labor compared to the legacy procedure of cell clarification followed by column chromatography purification.

Recently, high capacity magnetic protein A agarose beads LOABeads able to support a purification process have become commercially available. To support such process, we have developed a magnetic separator with which the cell clarification and capture of antibody (mAb) can be performed in a single step. This was applied to harvest CHO cell fed-batch cultures in 20 L pilot scale. A preliminary mAb capture step process was first developed with a batch of 26 L clarified harvest using 1 L magnetic beads in the magnetic separator. Two fed-batch runs of 16 L working volume were then purified by direct application on the non-clarified cell suspension of these runs. A rapid mAb adsorption larger than 96.6% was observed within one hour. The yield of this step was 86% and provided a 16 times concentration factor. The mAb purity was similar to a protein A capture by chromatography column performed on clarified harvest however the host cell protein content (<10 ppm) was significantly lower. This study demonstrated the feasibility of magnetic bead-based purification in absence of cell clarification.

Figure: Pilot scale purification from non-clarified cell suspension. (A) Adsorption efficiency from 2 runs. (B) Elution profile (run B2). (C) Samples of the cell suspension (run B2) taken at 0, 5, 10, 15, 30, 60, and 120 min, centrifuged and analyzed by non-reduced SDS-PAGE (lanes 1 to 7), together with the purified and eluted mAb (lane 8).

BIOT 55

Affinity magnetic precipitation for antibody purification

Raquel dos Santos, Ana L. Carvalho, Ana C. Roque, [email protected]. Chemistry Department, UCIBIO, FCT-NOVA, Caparica, Portugal

Anything but conventional chromatography, also known as the ABC trend, is currently attracting interest to develop purification alternatives to packed-bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media alone, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new method combining precipitation and magnetic separation was developed and coined affinity magnetic precipitation. Precipitation screens based on the Hofmeister series and a commercial precipitation kit, were tested with magnetic particles for antibody capture from different sources. Two different antibodies sources were tested: (i) polyclonal antibodies from human serum plasma, and (ii) anti-TNFα mAb from clarified CHO cell supernatants. The study comprised the discovery of the best precipitant and precipitation conditions using magnetic particles functionalized with a synthetic ligand previously developed. The addition of particles was crucial to achieve higher antibody purity and recovery. Nonetheless, the optimization of the precipitant and precipitation conditions also revealed to be essential to minimize antibody loss and maximize its purity. The elution was always performed using PBS at pH 7.4. As such, this approach may lead not only to antibody capture but also conditioning and concentration in one single step. Contrary to Protein A affinity chromatography, in our studied system, the elution conditions are mild. The activity of the eluted mAb was assessed In terms of process performance, the hybrid system enabled 80% purity and 50% recovery of polyclonal antibodies from plasma, and 97% recovery yield and 99% purity from anti-TNFα mAb. The presence of aggregates and the biological activity of the eluted fractions were evaluated, showing homogeneous eluted samples with high biological activity. The potential to use a hybrid system based on precipitation and magnetic fishing with affinity towards protein purification enables its selective capture and concentration in a single step unit operation.

BIOT 56

Fouling phenomena during sterile filtration of glycoconjugates Parinaz Emami1, [email protected], Fatemeh Fallahianbijan1, Pouria Motevalian4, Brenda Carrillo-Conde3, Kelvin Reilly2, Andrew L. Zydney1. (1) Dept of Chemical Engineering, Penn State University, University Park, Pennsylvania, United States (2) Pfizer Inc, County Longford, Ireland (3) Pfizer Inc., Chesterfield, Missouri, United States (4) Pfizer Inc, Andover, Massachusetts, United States

In the past decades, there has been significant interest in the development of glycoconjugates for novel vaccines. Protein-polysaccharide conjugates have been developed for protection against diseases like meningitis, pneumonia, and otitis media. One of the challenges in the manufacture of these large biotherapeutics is in the sterile filtration step, with the high degree of membrane fouling leading to low capacity and potential yield lost. The overall objective of this study was to evaluate the fundamental phenomena governing transmission and fouling during sterile filtration of novel glycoconjugate vaccines.

Experiments were performed using several glycoconjugates provided by Pfizer, Inc. Sterile filtration data were obtained during both constant flux and constant pressure operation using sterile filters with different pore morphologies and chemistries including asymmetric polyethersulfone (PES) and homogeneous polyvinylidene fluoride (PVDF) membranes. The location of fouling within the different filters was examined by confocal microscopy using fluorescently labeled glycoconjugates.

Pressure data obtained during constant flux filtration showed a gradual increase in the transmembrane pressure (TMP) during the initial stage of filtration followed by a sharp rise in TMP as the membrane became more heavily fouled. A noticeable decline in transmission was observed for some of the glycoconjugates, typically corresponding to the time at which the sharp increase in TMP was observed. Confocal microscopy images showed that the glycoconjugates were captured primarily near the filter entrance, typically in a band penetrating several microns into the depth of the membrane. The volumetric capacity for most glycoconjugates was greater for the asymmetric PES membrane compared to that of the PVDF membrane, although the glyco-conjugate with the smallest hydrodynamic radius showed greater capacity with the PVDF membrane. The capacity during constant flux operation was also a function of the filtrate flux, with greater capacity typically achieved when operating at higher filtrate flux. These results provide important insights into the fouling phenomena observed during sterile filtration of glycoconjugate vaccines.

BIOT 57

Three dimensional quantification of depth and membrane filter structure based on high resolution X-ray imaging with flow based simulation

Thomas Francis F. Johnson1, [email protected], John H. Welsh2, Paul Shearing3, Daniel G. Bracewell4. (1) Biochemical Engineering, University College London, London London, United Kingdom (2) Pall Biotech, Portsmouth, Hampshire, United Kingdom (3) Chemical Engineering, University College London, London, London, United Kingdom (4) UCL Dept Biochemical Engr, London, United Kingdom

High resolution 3D imaging has been applied to produce detailed representations of membrane and depth filter structure to analyse geometric characteristics and simulate flow through porous networks. Four depth filters and five membrane filters were selected for respective micrometre and nanometre scale imaging using X-ray computed tomography, enabling visual and quantitative comparisons between materials and to established literature values using non-imaging techniques to validate accuracy of 3D datasets acquired.

Average pore size was the main basis of comparison to vendor-provided nominal retention, with other parameters such as porosity and surface area to volume ratio also evaluated. Variation within and between filters was also of interest, in particular the interface between the rejection and capacitance layers within a membrane that can also be symmetric or asymmetric. 3D simulation was also performed on these structures to quantify permeability and tortuosity through these materials to identify flow characteristics that could be related to confocal microscopy findings for both depth and membrane filters.

This study enabled nanometre scale locational comparison within and between filtration structures that enhances understanding of the degree of heterogeneity and how geometrical characteristics inside of membrane and depth filters are linked to function and performance.

BIOT 58

Redirecting metabolic flux via combinatorial multiplex CRISPRi-mediated repression for isopentenol production in E. coli

Taek Soon Lee1,2, [email protected], Tian Tian2. (1) Joint BioEnergy Institute, Emeryville, California, United States (2) Lawrence Berkeley National Laboratory, Berkeley, California, United States

CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to re-direct metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes in order to increase precursor availability in a heterologous isopentenol biosynthesis pathway. To identify genomic knockdown targets in competing pathways, we first designed a single-gRNA library with 15 individual targets, where 3 gRNA cassettes targeting gene asnA, prpE, gldA increased isopentenol titer by 18-24%. We then combined the 3 single-gRNA cassettes into two- or three- gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression. Our strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of target metabolite, while the repression level needs to be adjusted to balance the metabolic network and achieve the maximum titer improvement.

BIOT 59

Increasing ammonia (NH3) production in model diazotroph Azotobacter vinelandii using kinetic modeling for rational metabolic engineering of the biological nitrogen fixation (BNF) system

Anna M. Crumbley1, [email protected], Jeron Chen1, Susan Butler2, Michael Lee3, Ramon Gonzalez4. (1) Chemical and Biomolecular Engineering, Rice University, Houston, Texas, United States (2) Bioengineering, Rice University, Houston, Texas, United States (3) Biosciences, Rice University, Houston, Texas, United States (4) Chemical and Biomedical Engineering, University of South Florida, Houston, Texas, United States

Nitrogen is an essential building block in biological processes. However, most plants and animals cannot use it in its most abundant atmospheric form, diatomic nitrogen (N2) gas. Instead, these organisms rely on limiting sources of fixed nitrogen primarily obtained from nitrogen-fixing microorganisms or the industrial Haber-Bosch process. Due to anticipated rising food demands globally, in situ use and synthetic expression of bacterial enzymatic processes for N2 conversion are being pursued as alternative and environmentally-friendly generators of ammonia (NH3) for agricultural fertilizer and industrial applications. However, layers of fastidious environmental regulatory controls and the complexity of expressing the 19+ genes necessary for functionality in aerobic environments have challenged heterologous expression and limited efforts to optimize its function for strategic applications. To develop a more thorough understanding of the system for rational metabolic engineering of nitrogen fixation, we developed kinetic models of the nitrogenase expression system in the aerobic, free-living, model soil bacterium Azotobacter vinelandii. Using the kinetic models, we identified five new potential targets for engineering that increased NH3 production in silico. Employing synthetic biology strategies to validate the model results, in vivo data indicates that NH3 accumulation can be synthetically tuned using lac inducible overexpression of targeted genes to produce statistically significant amounts of NH3. These results suggest that kinetic models should play a role in identifying indirectly associated enzyme targets that can be optimized to maximize NH3 production, not only in model organism A. vinelandii but also more broadly in other agriculturally-relevant microorganism and synthetic in planta nitrogen-fixation processes.

BIOT 60

13C metabolic flux analysis and kinetic modeling of Clostridium thermocellum grown on cellobiose Charles Foster1, [email protected], Satyakam Dash2, Saratram Gopalakrishnan1, Shyam Srinivasan1, Costas D. Maranas1. (1) Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States (2) Penn State University, University Park, Pennsylvania, United States

Clostridium thermocellum is a promising candidate for consolidated bioprocessing due to its ability to consume and ferment cellobiose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that enzymatic, regulatory, and possibly thermodynamic bottlenecks that throttle back metabolic flow still exist. This work aims to reveal some of these factors by elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13C- metabolic flux analysis (13C-MFA) using three distinct datasets. Subsequently, using the resultant flux distribution in conjunction with batch fermentation process yield data for various mutant strains, we constructed an updated kinetic model of C. thermocellum core metabolism (i.e. k-ctherm101). During kinetic parameterization, we introduced a systematic method for identifying a minimal set of putative regulatory mechanisms that helps to achieve good model agreement with experimental data using the K-FIT kinetic parameterization algorithm. Flux elucidation results confirmed that that both pyruvate, phosphate dikinase and the malate shunt are pyruvate contributors. 13C labeling data interpreted by 13C-MFA alluded to serine generation via the mercaptopyruvate pathway. The pathway was shown to be thermodynamically feasible and candidate gene Clo1313_0176 was identified for encoding this activity in C. thermocellum. Statistical analysis on k-ctherm101 confirmed that the C. thermocellum-specific regulatory mechanisms on lactate dehydrogenase (activated by fructose 1,6-bisphosphate), alcohol dehydrogenase (inhibited by ethanol and NAD+), and pyruvate, phosphate dikinase (activated by ammonia) were essential to accurate training data recapitulation.

BIOT 61

Chemoinformatic-guided engineering of polyketide synthases

Amin Zargar1,2, [email protected], Ravi Lal2, Luis Valencia2, Jessica Wang2, Tyler Backman2, Pablo Cruz-Morales2, Ankita Kothari2, Miranda Werts2, Andrew Wong2, Constance Bailey2, Arthur Loubat2, Yuzhong Liu2, Veronica Benites2, Samantha Chang2, Amanda Hernandez2, Jesus Barajas2, Mitchell Thompson2, Carolina Barcelos2, Rasha Anayah2, Hector Garcia Martin2, Aindrila Mukhopadhyay2, Edward Baidoo2, Leonard Katz2, Jay D. Keasling3,2,1. (1) QB3, UC Berkeley, Berkeley, California, United States (2) Joint Bioenergy Institute, Lawrence Berkeley National Labs, Berkeley, California, United States (3) Depts Chem Biomole Eng Bioeng, Univ of California Berkeley, Emeryville, California, United States

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A central challenge in PKS design is replacing a partially reductive module with a fully reductive module through a reductive loop exchange, thereby generating a saturated β-carbon. In this work, we sought to establish an engineering strategy for reductive loop exchanges based on chemoinformatics, a field traditionally used in drug discovery. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). These results demonstrated that chemical similarity between the substrate of the donor loops and recipient LipPKS1 correlated with product titers. Consequently, we identified donor loops with substrates chemically similar to LipPKS1 for further reductive loop exchanges, and we observed a statistically significant correlation with production. Reductive loops with the highest chemical similarity resulted in production of branched, short-chain fatty acids reaching a titer of 165 mg/L in Streptomyces albus J1074. Collectively, our work formulizes a new chemoinformatic paradigm for de novo PKS biosynthesis which may accelerate the production of valuable bioproducts.

BIOT 62

Kinetic rationale for functional redundancy in fatty acid biosynthesis

Alexander Ruppe, Kathryn Mains, Jerome M. Fox, [email protected]. Chemical and Biological Engineering, University of Colorado, Boulder, Boulder, Colorado, United States

Cells build fatty acids with biocatalytic assembly lines in which a subset of structurally unique components (e.g., enzymes or catalytic centers) often exhibit overlapping activities. Although the reactions necessary for the construction of different varieties of fatty acids are well resolved, the biochemical advantage of multiple enzymes that carry out the same reaction remains poorly understood. Our group has developed a detailed kinetic model of the fatty acid synthase (FAS) of Escherichia coli and paired that model with a reconstituted in vitro system to examine the biochemical repercussions of functional redundancy in fatty acid synthesis. Analyses of different FAS compositions indicate that systems with partially redundant dehydratases and/or ketoacyl synthases enable broader control over competing biochemical objectives—the fraction of unsaturated fatty acids, the total concentration of fatty acids, and the average length of fatty acids—than FASs with individual multi-functional enzymes. Maximal production of unsaturated fatty acids, for example, requires a dehydratase that is not essential for their synthesis. Broadly, the results of this study supply a kinetic rationale for the retention of redundant FAS enzymes in microbial genomes and provide a framework for exploiting functional redundancy in the design of robust oleochemical producing microbes.

BIOT 63

One-step microbial synthesis of chondroitin sulfates using engineered Escherichia coli

Abinaya Badri1, [email protected], Robert J. Linhardt1, Mattheos Koffas2,1. (1) Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States (2) Center for Biotechnology and Interdisciplinar, Rensselaer Polytechnic Institute, Troy, New York, United States

Sulfated glycosaminoglycans (GAGs) like heparan sulfates and chondroitin sulfates constitute an essential component of the extracellular matrix in higher eukaryotes. Hence, they serve as important pharmaceuticals. Of these, chondroitin sulfates (CS) are majorly used in the treatment of osteoarthritis. CS is also used to improve liver function, lower blood sugar and inhibit tumor metastasis. It is also used in drug delivery, food thickeners and preservatives. CS is commercially extracted from porcine, bovine, chicken, fish and shark tissues. There is a growing interest in animal-free CS production world-wide. Among such efforts, we have engineered for the first time, single strains of Escherichia coli that have the capability to produce CS in a single scalable fermentation.

CS production involves biosynthesis and sulfation of chondroitin polymer. We previously engineered E. coli K4 to produce chondroitin by deleting the fructosyltransferase gene. However, expressing chondroitin sulfotransferase in this strain does not enable CS synthesis. In this work, we show that in addition to sulfotransferase expression, accumulation of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is crucial for CS synthesis. We employ different genetic constructs for accumulation of PAPS and expression of sulfotransferase to produce CS in E. coli. We also study the effect of different PAPS levels and polysaccharide transport efficiencies on sulfation and yield of CS from our engineered strains. This is the first report of animal-free one-pot, one-strain microbial CS synthesis. We propose that our animal-free product will also enable pharmaceutical and cell-culture applications, thus opening avenues to understand the importance of specific CS types in biological processes.

One-pot CS Synthesis Using Engineered Escherichia coli: Metabolic route in fermentation strain showing glucose derivatives polymerized, sulfated and subsequently transported out of the cell as chondroitin sulfate.

BIOT 64

Genetically encoded enzyme inhibitors for post-translational dynamic control of yeast metabolism

Christopher Gonzalez, [email protected], Cesar Carrasco-Lopez, Jose L. Avalos. Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States

The baker’s yeast Saccharomyces cerevisiae is one of the most widely-used microorganisms for industrial chemical production. However, economically-viable production of commodity chemicals with this organism is made challenging by the yeast’s overwhelming bias to produce ethanol. Because of the crabtree effect, fermentation is necessary for the yeast’s growth on glucose, making genetic knockouts of the ethanol pathway unfeasible for growth on industrially-relevant carbon sources. With dynamic control of the ethanol pathway, production of competing chemicals of interest could be improved without sacrificing biomass accumulation, by splitting the fermentation into a growth stage and a production stage. Most efforts to induce the switch from growth to production have focused on transcriptional regulation of the ethanol pathway; while transcriptional circuits can allow for high-fold changes in gene expression, they do not affect proteins already accumulated within cells, creating an inherent lag in circuit response. In this talk, I present a novel genetically-encoded post- translational inhibitor of a key enzyme in ethanol production, which we have biochemically and structurally characterized. The inhibitor exhibits low-nanomolar affinity for its target, with fast association on the order of 104 M-1s-1, and slow dissociation on the order of 10-5 s-1. The inhibitor shows potent, noncompetitive inhibition of its target, with one-to-one binding. Constitutive expression of the inhibitor in vivo shows up to a 10-fold reduction in the growth rate. Finally, inducible expression of the inhibitor shows improved production of chemicals of interest compared to purely transcriptional control of the ethanol pathway, demonstrating the advantage of post- translational regulation of metabolic pathways: with faster dynamic control, essential pathways can be more rapidly shut down, leaving a greater pool of substrate to be converted to products of interest.

BIOT 65

Multi-element metabolic flux analysis toward improved quantification of intracellular metabolism

Shriramprasad Venkatesan1, Claire Riordan1,2, Daniel Lugar1, Ganesh Sriram1, [email protected]. (1) University of Maryland, College Park, Maryland, United States (2) University College, Dublin, Dublin, Ireland

Isotope-assisted metabolic flux analysis (isotope MFA) is a methodology for the measurement of intracellular metabolic fluxes through a metabolic network. Isotope MFA proceeds by sequentially performing isotope labeling experiments, measuring the ensuing isotopomer patterns (usually by mass spectrometry) and using a mathematical model to interpret these patterns. Because isotope MFA directly measures how carbon flows and partitions in metabolic pathways, it complements and is a more direct gauge of metabolic activity than other ’omics methods such as transcriptomics or proteomics. Isotope MFA is a complex procedure due to the significant number of isotope measurements needed and the development of a mathematical model that relates isotopomers to fluxes. Accurate identifiability of fluxes in isotope MFA depends on the specific types of isotopically labeled substrates supplied to the cell culture under study. Previous studies have predominantly focused on carbon (13C) labels. This is natural because most metabolites have carbon backbones. However, there is potential for employing isotopes of other elements including 18O and 15N in isotope MFA. Here, we will present a computational and experimental evaluation of the potential of two types of 18O-labeled glucose (2-18O and 5-18O glucose) in isotope MFA. Our computational studies have revealed that these 18O-labeled varieties of glucose complement metabolic information available from 13C-labeled varieties including 1-13C and 6-13C glucose. We will present results demonstrating the merits of the 18O labels as well as the complementation between 18O and 13C labels. Furthermore, we will present experimental results on a microbial cell culture that evaluate whether the use of these labels can lead to quantification of metabolic fluxes with enhanced precision. Finally, we will also present the experimental and computational enhancements necessary for simultaneously measuring and processing isotopomers of multiple elements.

BIOT 66

PID control: Forgotten process parameter

Sarah W. Harcum1, [email protected], Kathryn Elliott1, Tom Caldwell1, Bradley Skelton1, Benjamin Synoground1, Cameron Schnabel1, Stephanie Klaubert2, Madison Williamson3. (1) Department of Bioengineering, Clemson University, Clemson, South Carolina, United States (2) Department of Chemical Engineering, Clemson University, Clemson, South Carolina, United States (3) Department of Biochemistry and Genetics, Clemson University, Clemson, South Carolina, United States

In order to gain a better understanding of Chinese hamster ovary (CHO) cell genome instability, the ambr® 250 bioreactor platform was adopted as the highly-controlled environment for this project. Development of the baseline and stress conditions for the genome instability studies has highlighted the need for control of critical process parameters, including the Proportional, Integral, and Derivative (PID) control loops. Process parameters or factors that are considered scale-independent, dissolved oxygen (DO) and pH, were determined to be sensitive to the PID values for the ambr® 250 process. Unlike larger bioreactor control loops that can use cascade settings to control DO utilizing agitation, air, and oxygen concurrently, the ambr® 250 requires individual levels of control that are used sequentially to change a particular process parameter. For example, the Level 1 for DO control could have PID values for the gas sparge (Air) 0.5 mL/min to 2.5 mL/min (P = 0.1, I = 500, D = 0). Once the DO drops below the setpoint, Level 2 would be called. The Level 2 default increases the Stir rate from 300 rpm to 600 rpm (P = 3.5, I = 250, D = 0). The consequence of having multiple levels is that each level needs to have the PID values tuned individually. For mammalian cell cultures Sartorius provides with the ambr® 250 platform with three default DO Levels and a set of upper and lower pH PID values. However, even with minor adjustments these PID values resulted in high variability in DO, were too slow to respond to antifoam additions at high-cell densities, and allowed the pH to drift away from the setpoint that resulted in lactate accumulation. After consulting with industrial ambr® 250 users and Sartorius field specialists, and tuning the PID for each level, DO and pH control loops PID values were developed that minimized uncontrolled oscillation and over- and under- shooting, as well. These improved PID control values can support CHO cell growth, reproducibly to over 47 million cells/mL in a fed-batch process. This work highlights that PID control loop values can profoundly affect culture outcomes, yet are often neglected and rarely mentioned in publications. BIOT 67

Incorporating Raman model development for bioreactor monitoring and glucose control into a platform process development workflow

Matthew Rehmann1, [email protected], Ziev Basson1, Gregory Lane2, Georgios Pyrgiotakis2, Nobel Vale2, Dhanuka Wasalathanthri1, Julia Ding1, Douglas Both2, Michael C. Borys1. (1) Global Product Development and Supply, Bristol-Myers Squibb, Devens, Massachusetts, United States (2) Engineering Technologies, Bristol- Myers Squibb, New Brunswick, New Jersey, United States

Raman spectroscopy enables real-time, in-line monitoring of upstream bioreactor process parameters such as glucose, lactate, and amino acid concentrations, and, within BMS, integrates with the existing automation infrastructure to control glucose levels during process development. Effective monitoring and control involves accurate collection of offline data and regression modeling to correlate spectra with measured values. While great progress has been made in recent years towards generalized Raman models, most models are still built on a cell line- and media-specific basis to ensure model accuracy. In this presentation, we outline an approach for generating Raman models for a new cell line during process development. This strategy primarily leverages data collected in the BMS platform process development or process characterization workflows, and requires few dedicated runs for Raman data collection, decreasing the resources needed to generate molecule-specific models. We compare the molecule-specific approach with generic models built from the same datasets, demonstrating comparable or improved model accuracy (i.e. lower standard error of prediction) for glucose, lactate, glutamate, and titer for 4 BMS-proprietary CHO cell lines. Finally, we conclude with successful application of these models for glucose and feed control.

BIOT 68

Control of glycosylation and titer in fed-batch monoclonal antibody production

Yu Luo1, [email protected], Devesh Radhakrishnan2, Evan Wells3, Deepro Banerjee3, Liqing Song3, Varghese Kurian1, Anne S. Robinson3, Babatunde Ogunnaike1. (1) Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) BioMarin Pharmaceutical Inc., Novato, California, United States (3) Chemical Engineering, Carnegie-Mellon University, Pittsburgh, Pennsylvania, United States

Therapeutic monoclonal antibodies (mAbs) are currently produced commercially in fed- batch cultures using media recipes and protocols based primarily on heuristics. The main production objectives are maximizing mAb titer (productivity requirement) and achieving desired glycan distributions (quality attribute) that result from glycosylation—a post-translational process that attaches sugar molecules (glycans) to antibodies. The glycan profile is a critical quality attribute of mAb manufacturing because it can either promote or inhibit a mAb drug's therapeutic effects. Yet, controlling glycosylation remains challenging because it is a complex, non-template driven process that takes place within the cell; the manipulated variables are far fewer than the controlled variables; the process is difficult to model due to its complexity; on-line sensors do not exist for characterizing glycans frequently enough for effective on-line control. Nonetheless, recent advances in both process modeling and glycan assays have improved the possibility of implementing model-based control of glycosylation in place of the current heuristics-based approach.

We present an overall model-based strategy for meeting production requirements in the manufacture of mAbs. The proposed multivariable cascade control system consists of two nested control loops. The outer loop—responsible for controlling productivity and product quality attributes—uses a model predictive controller (MPC) to determine the setpoints for the inner loop. The inner loop employs multiple PI controllers to maintain nutrients and operating parameters at desired levels in the cell culture. Due to infrequent glycan assays, the outer loop incorporates a state observer that uses sensor measurements and model predictions to reconstruct the complete glycan profile, which the MPC uses, along with the process model, to determine the desired control policies.

As a first step toward implementing on-line control of industrial fed-batch mAb manufacturing processes, we tested the complete control system in a MATLAB simulation and investigated a variety of industrially relevant process conditions under several operating sensor configurations (frequencies, qualities, and delays). Subsequently, we integrated the control system with the bioreactor through Open Platform Communications. The results offer insight into the optimal sensing strategy as well as the usefulness and limitations of the proposed control system.

BIOT 69

Advancement of process control using process analytical technology (PAT) in continuous biomanufacturing: Case study

Sobhana Sripada1, [email protected], Rajan Bhawnani1, Sushmitha Krishnan1, Aparajith Bhaskar2, Eduardo Lopez-Montero2, John Mack2, Jessica R. Molek1. (1) Biopharmaceutical Process Development, GlaxoSmithKline, Upper Merion, Pennsylvania, United States (2) Perceptive Engineering Limited, Daresbury, United Kingdom

Integrated continuous manufacturing is of growing interest in the biopharmaceutical industry due to process intensification and operational flexibility that can be achieved within a reduced manufacturing footprint. An effective control strategy for a fully- automated continuous process must ensure adequate control of critical quality attributes (CQA) while maintaining synchronous operation with other process stages. Process Analytical Technologies (PAT) facilitate product monitoring in real time and can enable implementing intuitive divert-to-waste strategies over long run times. Adopting feedback control in continuous processes can be challenging due to limited availability of PAT tools that can monitor a breadth of CQAs.

This work describes PAT development for inline protein concentration monitoring and its utilization for implementing Model Predictive Control (MPC) in a continuously operating Single-Pass Tangential Flow Filtration (SPTFF) system. Linking multiple inputs to multiple outputs proves beneficial in continuous operation by enabling control over product quality as well as process parameters. Maintaining target output concentration in SPTFF is a complex function of incoming feed concentration, flowrate, and membrane area. While PID loops can effectively control each input to its respective output, the complexity of SPTFF with multiple interrelated variables drives the need to introduce strategies that account for parameter interactions. The controller is hence trained to account for system dynamics and is utilized to control CQAs across disturbances.

BIOT 70

Binding of CHO host-cell proteins to monoclonal antibodies

Young Hoon Oh1, [email protected], Matthew Becker1, Kerri Mendola1, Leila Choe1, Kelvin H. Lee1, Abraham M. Lenhoff1, Yinges Yigzaw2, David J. Roush3. (1) Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Purification, Genentech, South San Francisco, California, United States (3) Process Development and Engineering, Merck, Sharp and Dohme, Colts Neck, New Jersey, United States

In the manufacture of biopharmaceuticals, individual host-cell proteins (HCP) may elude clearance and be present in measurable quantities in the final drug product, with the potential impact on the product quality and risk to patient safety. One cause of HCP co- purification is strong association with the product, such as a monoclonal antibody (mAb). We will present proteomic data, obtained by LC-MS, tracking HCPs in downstream processes for six different mAbs from two companies, showing the similarities and differences among the clearance patterns for different processes. In order to explore possible binding mechanisms and reasons for co-purification in these systems, a smaller group of CHO HCPs were expressed in prokaryotic hosts and purified. The dissociation constants, KD, for binding of these HCPs to the six mAbs studied above were measured; KD values in the tens of μM range were obtained for several HCP/mAb systems. The implications of the results for HCP clearance will be discussed.

BIOT 71

Process analytical technology to monitor phase transition in freeze-thaw processes of biopharmaceutical solution

Dennis Weber1, [email protected], Juergen Hubbuch1, Gerald Berghammer3, Volker Schoeberl2. (1) Institute of Engineering in Life Sciences Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology, Karlsruhe, Germany (2) Bilfinger Industrietechnik Salzburg GmbH, Schwetzingen, Germany (3) Research and Development, Bilfinger Industrietechnik Salzburg GmbH, Puch bei Hallein, Hallein, Austria

Freezing and thawing are essential steps in the manufacturing of biopharmaceutical products to overcome bottlenecks and to allow product shipment by increasing the product shelf life. Throughout a freezing process, the drug substance is exposed to different stresses, such as surface interactions and freeze concentration of formulation solutes. As a result, drug related impurities such as protein aggregates may be formed. The freeze concentration occurs because formulation solutes are excluded from crystallizing water molecules and concentrate towards the center of a freezing container. Additionally, the dense freeze concentrate sediments at the center of a freeze container resulting in a solute concentration gradient from the walls to the center and from the top to the bottom of a container in the frozen bulk. Depending on this concentration profile, the last point to freeze may vary in its position.

Currently, freeze-thaw processes are often monitored using thermocouples that allow local temperature measurements or one dimensional temperature analysis in case of parallel series of thermocouples. In order to describe the dynamic temperature profile throughout a freeze-thaw process, assumptions have to be made such as freezing parallel to the cooling surfaces. However, non-idealities like boundary effects and sedimentation of the freeze concentrate cause deviations from these estimates regarding freeze concentration, temperature profiles and the last point to freeze. In order to move towards in silico process modelling, improved process monitoring and understanding are necessary.

This work shows our current monitoring approach for freeze-thaw processes in a laboratory scale freeze-thaw device. In contrast to commercially available systems, our freeze container bottom is separately and actively tempered to minimize boundary effects. We investigated the freeze-thaw process at different temperatures for salt solutions by measuring spatially resolved temperature profiles with fiber optic temperature sensors. We used the data to establish a process analytical tool to monitor solid phase progression and actively control boundary conditions. Using this tool, variations in the last point to freeze position have been detected for different salt solution. Spectroscopy methods such as raman resonance have been used to cross validate the temperature based tool. The salt concentration profiles of the frozen bulk have been analyzed and correlated with the gathered data.

BIOT 72

Harnessing the power of a data-driven revolution to enable single-use and continuous processing technologies to meet the market need

Rene Reinbigler, [email protected]. Merck KGaA, Molsheim, France

There are several challenges associated with next generation processing for bio pharmaceutical production. The ambitious goals set by the customers and the overall market are being addressed through the combination of technology, bioprocess intensification and data driven insights. A comprehensive strategy is covering new modular hardware approach, standardized communication interface, smart data management and additional self- service cloud based on big data and machine learning. The combination of those capabilities drastically increases flexibility and automation enabling the biopharmaceutical Facility of the Future. On the intersection of technology and business there are new opportunities leveraging collaboration power of integrated ecosystem of vendors, service providers and customers. We are performing a set of pilot implementations where many of our assumptions and technologies are being tested and confirmed. In this session we will present the findings and concepts covering: Lab and Manufacturing sites connectivity – challenges and opportunities for OPCUA based, standardized connectivity and control integrating unit operations, consumables, operators and broader ecosystem Data management leveraging GMP data lake – what is the real value of advanced data management including ontology and taxonomy models covering end to end process definition? Advanced Analytics leveraging Machine Learning and AI – how ML and AI models can be used in GMP environment?

BIOT 73

Novel application of 2D-nanosheet and engineered DNA-binding proteins for specific gene detection

Moon-Soo Kim, [email protected]. Department of Chemistry, TCCW455, Western Kentucky University, Bowling Green, Kentucky, United States

DNA detection technologies play an important role in diagnostic applications in the areas of public health and biomedicine. Zinc finger proteins (ZFPs) are the most common DNA-binding domains and multiple ZF domains can be assembled to bind to any DNA sequence of interest. In this work, six finger ZFPs were engineered to bind to 18 base pairs of DNA within the stx2 gene that encodes for E. coli O157 shiga toxin and the tetM gene (tetracycline resistance gene) with high specificity. Here, we designed a novel sensing technology to detect ARGs in bacteria using a graphene oxide-based biosensor utilizing ZFPs. Our approach relies on the on and off effect of fluorescence signal in the absence and presence of target ARGs, respectively. Two-dimensional graphene oxide (GO) sheet possesses unique electronic, thermal, and mechanical properties. In this study, we take advantage of the quenching ability of GO to create a novel method for detecting the specific double-stranded DNA sequences within ARGs. Quantum dot-labeled ZFPs can be adsorbed onto GO via stacking interactions of aromatic and hydrophobic residues in conjunction with hydrogen bonding interaction between hydroxyl or carboxyl groups of GO and hydroxyl or amine groups of the protein. The fluorescence signal of QD-labeled ZFPs is quenched due to fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and GO when they are in close proximity. In the presence of target DNA, the bound protein-DNA complex is dissociated from the GO surface due to the conformational changes, thereby restoring the fluorescence signal. Here, we optimized GO concentrations used in the assay and QD-labeling on ZFPs and determined the sensitivity of our system. This study could bring new capabilities to molecular diagnostics and broaden the spectrum of GO applications in public health and clinical diagnostics.

BIOT 74

Withdrawn

BIOT 75

Novel application of graphene oxide-based biosensor utilizing CRISPR/dCas9 for detection of antibiotic resistant gene

Joel I. Omage1, [email protected], Wendy M. Cecil2, Moon-Soo Kim3. (1) Chemistry, Western kentucky University, Bowling Green, Kentucky, United States (2) Department of Chemistry , Western Kentucky University , Shepherdsville, Kentucky, United States (3) Department of Chemistry, TCCW455, Western Kentucky University, Bowling Green, Kentucky, United States

Antibiotic resistance is currently one of the biggest public health challenges globally. To fight this threat, we designed a rapid screening method to detect antibiotic resistance gene (ARG) in bacteria utilizing clusters of regularly interspaced short palindromic repeats (CRISPR) in complex with deactivated CRISPR associated proteins (dCas9) along with a 2-dimensional nano sheet graphene oxide (GO). Deactivated Cas9 (dCas9) is a mutated Cas9 protein that has lost its nuclease function. The dCas9 was used in complex with customized single guided RNA (sgRNA) for target DNA recognition since it was simple to design sgRNA. Our approach is based on the fluorescence quenching mechanism of GO in the absence and presence of target DNA, respectively. For this purpose, we used two-dimensional graphene oxide (GO) sheet for its unique quenching ability as a novel sensing platform. Alexa-labeled DNA-binding sgRNA/dCas9 was adsorbed onto the GO surface via the Cas9 antibody. In the absence of target DNA, fluorescence signal of labeled sgRNA/dCas9 will be quenched due to fluorescence resonance energy transfer (FRET) between Alexa fluorophore and GO when they are in close proximity. In the presence of target DNA, the bound dcas9/sgRNA-DNA complex is dissociated from GO, restoring the fluorescence signal. Using this technique, we can perform multiplex detection through various fluorescence signals produced from the differently labeled fluorophores per respective sgRNA.

BIOT 76

Low-cost, noninvasive dissolved CO2 measurement for biomedical/bioprocess applications

Joel Tyson, [email protected], Gabbi McLean, Benjamin Punshon-Smith, Vida Rahmarnejad, Samyukta Rao, Ritvind Suketana, Xudong Ge, Iordan V. Kostov, Leah M. Tolosa, Govind Rao. University of Maryland Baltimore County, Baltimore, Maryland, United States

The analysis of gases dissolved in the blood or in tissue culture is essential for monitoring the respiratory health of human patients and of cells in vitro. Despite the need for continuous sensors of dissolved gases in both venues, truly noninvasive and continuous measurement has not yet been realized. Barriers to diffusion of dissolved gasses are a major hurdle in appropriate sensor development. In transdermal measurement of dissolved blood gases, the skin slows the time to steady state diffusion of CO2 and O2. When measuring cell culture respiration, some kind of barrier may be necessary to preserve sterility. In both cases, undesirable sensing methods are used to overcome this, such as heating, offline sampling that disturbs the culture or needle sampling that uncomfortably pierces the skin. The Center for Advanced Sensor Technology has developed a method of completely non-invasive measurement of dissolved gasses by correlating the initial diffusion rates through these barriers to the amount in solution. Using gas permeable membranes as an interface between the diffusion barrier and the sensing element, which is kept impermeable, we find linear correlation between dissolved CO2 amounts in both cell culture medium and gas diffusing through skin-mimicking polymer. Each measurement is taken in under a minute. Design iterations were made with different optical sensing elements and varying sample chamber geometries. Increasing turbulent flow with serpentine sampler designs improved the amount of diffusion into the optical sensing path and can aid in the use of smaller, less expensive optical sensors. This system is applied to mammalian tissue culture to establish continuous dissolved CO2 measurements for future work in standardizing regenerative and organ-on-a-chip platforms. Continuing work is being done to improve the response of the sensing element for these applications, including miniaturization and developing a selective, graphene-based sensing element. BIOT 77

Distance tunable multiplexed sensing: Single molecule FRET approach

Anisa Kaur, [email protected], Soma Dhakal. Chemistry, Virginia Commonwealth University, Hanover, Virginia, United States

In recent years, simultaneous detection of multiple biomarkers has drawn much interest in the fields of bionanotechnology and clinical diagnostics. Recent studies have found a myriad of disease-related biomarkers spanning a wide variety of biomolecules and it has been shown that the accuracy of diagnosis increases significantly by measuring the level of multiple biomarkers instead of just one. Current popular methods of multiplexing, including microarrays, multiplexed polymerase chain reaction (PCR), and DNA barcodes often suffer from complex engineering and may yield only semiquantitative information. Although single molecule fluorescence resonance energy transfer (smFRET) is gaining popularity in multiplexed sensing as it can provide a sensitive and quantitative platform to tackle these limitations, it often requires complex labeling schemes and multicolor excitation sources. We have recently reported a multiplexed detection of three different DNA sequences using the toehold-mediated strand displacement process. This method requires only a single FRET pair for multiplexing, however, the limit of detection is ~200 pM. To expand the application of the method to detect micro RNA biomarkers that are present at low femtomolar (fM) concentration in biological samples, we have re-imagined our sensor design so that it does not require a toehold displacement process and enables a lower detection limit. Overall, we anticipate that our multiplexed method may find applications for high- confidence detection of biomarkers.

BIOT 78

Single molecule detection of a cancer susceptible gene kumar sapkota, [email protected], Soma Dhakal. Chemistry, Virginia Commonwealth University, Richmond, Virginia, United States

Mutation in p53 sequence, a cancer related gene, has been implicated for more than 50% of human cancer, thus selective and sensitive detection of p53 gene is of great significance in the early-stage diagnosis and targeted therapy of cancer. However, most of the ultrasensitive detection strategies reported so far either involves complex design or requires multi-step signal amplification. To address these challenges, we have designed a single-step FRET-based sensor that allows a sensitive detection of p53 by single-molecule counting. In addition to its simple design, this sensor allows zero- background and thus the detection limit is down to low femtomolar (fM) concentration without the need for target amplification. We also observed that this sensor is highly effective in discriminating single nucleotide polymorphisms (SNPs). Since simultaneous detection of different p53 mutants could be useful for screening genetic disorders, we are currently investigating the multiplexing ability of our sensor in order to simultaneously detect and quantify multiple targets.

BIOT 79

Lipid-functionalized single-walled carbon nanotubes as abiotic cell proxies to screen membrane-penetrating peptides for use in drug delivery and antimicrobials development

Nathaniel Kallmyer, [email protected], Nigel F. Reuel. Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States

Membrane-penetrating proteins play a key role in defense against foreign or competing microbes and have been used to design vehicles that can deliver therapeutics or combat antibiotic-resistant pathogens. The study of these naturally occurring proteins has led to the design of new, synthetic cell-penetrating peptides. These designer peptides have been used to deliver large molecules, such as GFP, into the cytoplasm and to lyse notoriously drug-resistant bacteria, such as S. aureus and P. aeruginosa. While these peptides demonstrate great potential, screening techniques to find and optimize their sequences are limited by the timescale of cell growth and require stringent safety protocols to prevent exposure of personnel to target pathogens. Herein, we demonstrate fluorescent single-walled carbon nanotube (SWNT) sensors as abiotic cell proxies to test penetrating peptides. These proxies are constructed by suspending SWNT in either phosphatidylcholine (PC) or lipopolysaccharide (LPS) to simulate the outer surface of an animal cell or Gram-negative bacterium respectively. Disruptions to this simulated cell surface, whether caused by surfactants, chaotropes, electrical potential gradients, or temperature changes, expose the SWNT surface to solvent and cause an immediate quenching of the fluorescent signal. When screening cell- penetrating peptides with these proxies, concentration thresholds are observed; below these thresholds, cell-penetrating peptides cause an increase to fluorescent signal (binding of the peptide to the SWNT membrane proxy), and above these thresholds, peptides cause a rapid decrease to signal (penetration of membrane proxy). The magnitude of the signal drop of PC-SWNT correlated inversely with the activation energy of peptide translocation. The observed concentration threshold similarly correlated inversely with published uptake efficiencies. A short response time (< 30 s) and reduced risk of pathogen exposure make this sensor a potentially valuable pre- screening tool for cell penetrating and antimicrobial peptide discovery and design.

BIOT 80

Engineering chemogenetically erasable reporter genes for background-free magnetic resonance imaging

Jason Yun1, [email protected], Charles Yue1, Audrey Chow2, Michelle Leong3, Arnab Mukherjee4. (1) Chemistry & Biochemistry, University of California, Santa Barbara, Santa Barbara, California, United States (2) Psychological & Brain Sciences, University of California, Santa Barbara, Santa Barbara, California, United States (3) Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, California, United States (4) Chemical Engineering, University of California, Santa Barbara, Santa Barbara, California, United States

A key challenge with improving safety and efficacy of gene and cell-based therapies is the inability to precisely locate and track these therapeutics in the context of living subjects. Optical genetic reporters provide the specificity needed for these technologies but cannot visualize deep-seated tissues in live animals. Reporter genes compatible with magnetic resonance imaging (MRI) achieve excellent depth penetration with a spatial resolution approaching 250 μm. However, detection of cells and gene expression using MRI reporters can be confounded by endogenous background contrast. To this end, we have developed an approach for background-free MRI based on chemogenetically degradable water channels known as aquaporins (AQP1). AQP1 was recently introduced as a metal-free MRI reporter that generates contrast by altering transmembrane water exchange. Here, we use small molecule-controlled degradation tags to conditionally degrade AQP1, thereby enabling on-demand control over AQP1 signal. To implement this technique, we acquire two sets of images: 1) an “on” image that includes the reporter 2) an “off” image after degrading the reporter. By digitally subtracting the “on” and “off” images, we can remove background contrast, enabling specific detection of AQP1 contrast. We engineered CHO cells to stably express AQP1 tagged with 3 distinct degradation sequences, namely DHFR, ER50, and FKBP12. Using diffusion weighted MRI, we quantified percent degradation as 73 ± 3%, 94 ± 5%, and 37 ± 11% (Fig 1a.) with half maximum degradation achieved in < 6 hours upon removing the cognate stabilizing ligand (Fig 1b.). Finally, we used AQP1-DHFR to demonstrate specific detection of AQP1 expressing cells, which could be visualized as an MRI “hot-spot” overlaid on a background-free MRI image (Fig 1c.) obtained by subtracting images with intact and degraded reporter signals.

Figure 1. Eraseable AQP1 as a genetically encoded reporter for background-free MRI. (a) Percent degradation of AQP1 using 3 unique degradation tags in the presence or absence of cognate ligand. Error bars represent S.E.M. of at least 4 biological replicates. (b) Response kinetics of ligand-dependent arrest of AQP1 degradation. (c) Background-free, hot-spot MRI image of CHO cell line expressing AQP1- DHFR.

BIOT 81

Improving aggregation rate predictions under low temperature storage conditions using an isochoric method

Diana Gomes1, [email protected], Susana Teixeira1, Juscelino Leao2, Miguel A. Rodrigues3, Christopher J. Roberts1. (1) University of Delaware, Newark, Delaware, United States (2) NIST, Gaithersburg, Maryland, United States (3) IST, Lisbon, Portugal

Storing proteins under low temperature (T) conditions is recommended as it favors the protein native state and slows aggregation events during months or even years (e.g. under refrigerated conditions). Accelerated stability testing is a standard approach to estimate the time required for protein degradation and is generally performed under “hot” T (HT) stress. For instance, the time required for protein monomer loss (Δm) within initial rate conditions, t80 (Δm ≤ 20% loss), is the most relevant for a commercial shelf- life prediction. Quantifying aggregation rates under sub-freezing T using an isochoric method (liquid state) has shown promising results as cold T (CT) and/or pressure (P) stress can also shift the thermodynamic equilibrium and increase the fractional unfolded population. Nevertheless, the combined effect of P vs CT conditions on protein denaturation have been poorly characterized. A model protein alpha-Chymotripsinogen A (aCgn) at neutral pH was chosen as a means to interpret simple folding transition systems under CT and high P and bridge the effectiveness of isochoric methods to protein stability predictions. In situ SANS measurements under CT and high P environments show that high-P effects can be reasonably disregarded for the present system. Similar findings were also observed for aCgn at lower pH conditions and Ovalbumin (Ova) at neutral pH. The partial conformational changes observed for Ova seem to be mainly due to the proximity to the cold denaturation T rather than a P effect. The results show that for aCgn at neutral pH the isochoric method can provide more realistic shelf life predictions with less extreme T extrapolations and negligible pressure induced denaturation.

BIOT 82

Monoclonal antibody glycan analysis using mass spectrometry

Sumit K. Singh, [email protected], Kelvin H. Lee. Chemical and biomolecular Engineering, University of Delaware, Newark, Delaware, United States

With the rapidly increasing use of monoclonal antibodies (mAbs) for clinical use, there is a growing demand for fast, efficient and reliable analytical techniques for comprehensive glycosylation analysis. Existing evidence shows that glycosylation in mAbs plays important role in their biological activity, physicochemical properties, and effector functions. Even small changes in linkage, position or site occupancy of glycans can adversely influence the effectivity of the products. Yet, the hallmark of mAb N- glycosylation is extensive heterogeneity associated with each glycosylation site. The current mass spectrometric approaches involve relatively complex workflows often performed off-line. Using a case example of NISTmAb, we attempted to investigate if this bottleneck could be overcome by acquiring the data using Waters Bioaccord LC-MS system which has features of automated calibration and method optimization. The performance of the system was benchmarked by comparing the data with the results obtained on Waters Synapt G2- si LC-MS system.

BIOT 83

Considerations in formulation development of a PEGylated protein

Lori Burton, [email protected]. Drug Product Science and Technology, Bristol- Myers Squibb, New Brunswick, New Jersey, United States

PEGylation is a process in which a polyethylene glycol (PEG) chain is covalently conjugated to an active therapeutic (often a peptide, small protein, or an antibody fragment) in order to enhance solubility, reduce immunogenicity and/or prolong circulating half-life in the body. For biotherapeutics, most of which must be administered by injection due to poor oral absorption, the reduction in dose frequency afforded by an extended in-vivo half-life can provide significant advantage in terms of patient convenience, especially for indications requiring chronic treatment. However, PEGylated proteins can also present a number of special challenges during formulation development. These challenges may include potential for chemical instability of the conjugate and, in case of subcutaneous products where injection volumes are limited, increased intrinsic viscosity, which can pose challenges for processing of drug substance and make injection through a fine-gauge needle difficult. In this presentation, we will discuss formulation development of a small PEGylated protein and strategies used to address stability- and viscosity-related challenges to enable a viable drug product suitable for subcutaneous use.

BIOT 84

Predicting antibody developability from molecular simulations and machine learning

PIN-KUANG LAI, [email protected], Bernhardt Trout. Chemical engineering, Massachusetts Institute of Technology, Quincy, Massachusetts, United States

After the discovery of target binding antibodies, the characteristics of developability such as high levels of aggregation, escalated viscosity and other stability issues assume critical importance. However, the design space for improving antibody stability is enormous because of the large size of proteins and the dependence of physiochemical properties on the formulation conditions. In this work, we implemented a combination of experimental and computational approaches to elucidate the key determinants for aggregation of 21 therapeutic monoclonal antibodies (mAbs). We have applied accelerated aggregation rate assessment, molecular dynamics simulations and a machine learning method for predicting antibody aggregation. The machine learning method combines features from sequence, amino acid properties, molecular descriptors (spatial aggregation propensity and spatial charge map) developed from our group. The contribution of different domains of antibodies is also included. With a rational feature selection, we extracted the most important features to classify and predict therapeutic mAbs with high and low aggregation rate. The cross-validation accuracy is 97% using only three features. The protocol proposed in this study has implications in training and predicting other stability properties with limited experimental data.

BIOT 85

Predicting protein-protein interactions of monoclonal antibodies at low and high concentrations using hybrid coarse grained models

Hassan Shahfar1,2, [email protected], Christopher J. Roberts1. (1) Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Physics and Astronomy, University of Delaware, Newark, Delaware, United States

Protein aggregation, poor solubility and viscosity are among the challenging factors for determining the biophysical properties of therapeutic protein formulations. It has been shown that protein-protein interactions (PPI) potentially have a great impact on the quality of these biophysical properties. To date, experimental characterization of PPI have demonstrated that these properties strongly depend on environmental conditions like pH, protein and salt concentrations and etc. However, experiments may be prohibitive due to availability of sufficient quantities of purified protein material, time and other resources. This motivates development of quantitative and semi-quantitative molecular models to predict PPI based on the protein sequence and environmental factors such as solution pH and ionic strength. Prior work showed that available coarse grained (CG) models were unable to do more than qualitatively predict strongly attractive PPI such as those measured using static light scattering (SLS) unless one “fit” the data in hindsight. We introduce a new hybrid coarse grained model which balances between domain-level models and all-atom models to be tractable to predict high- concentration behavior. Specifically, it focuses on the specific location of charged amino acids and therefore the particular charge distribution of MAb. To validate the model, the SLS profiles and concentration-dependent PPI of a series of monoclonal antibodies were tested. These MAbs have shown different experimental behaviors from net repulsive to strongly attractive interactions. The results show that this hybrid approach can capture strongly attractive PPI quantitatively or semi-quantitatively from only the knowledge of a homology model for three dimensional structure and amino acid sequence.

BIOT 86 High throughput potency characterization of vaccine antigen by affinity chromatography

Qin Yan, [email protected], Mandy Alger, Li Ma, Xianzhi Zhou, Jingning Li, Varnika Roy. Analytical Research Development (ARD), Technical Research Development (TRD), GlaxoSmithKline (GSK), Rockville, Maryland, United States

An antigen with multiple antigenic sites has been studied as a potential vaccine candidate. Antibodies against different epitopes were immobilized on the column to prepare affinity columns. Good stability of several such antibodies enabled the routine use of affinity chromatography on HPLC for characterization of the stabilities and binding activities of different antigenic sites. The procedure is high throughput and samples are injected directly without any sample preparation. The method can be used for both quantitation and binding activity of the antigen for in-process samples, final drug substance and drug product.

The performance of the affinity chromatography method is demonstrated by specificity, precision, linearity and accuracy. The method has been used to evaluate lot-to-lot consistency of antigen manufacture process. The method demonstrates stability indication capability, and has been used to support the stability study of both drug substance and drug product. The direct measurements of affinity chromatography are absolute %Bound and %Unbound, and they show strong correlation with relative %Potency by ELISA and %Unshiftable (the portion that doesn’t bind to the antibody) by Fabshift, respectively. It has also been used to measure antigen concentration in in- process samples.

BIOT 87

Apparent protein cloud point temperature determination using a low volume high- throughput cryogenic device in combination with automated imaging

Marieke Klijn1, [email protected], Anna K. Woell1, Juergen Hubbuch2. (1) Karlsruhe Institute of Technology, Karlsruhe, Germany (2) KIT, Karlsruhe, Germany

Biopharmaceutical product development requires a real-time storage period of at least 18 to 24 months in order to demonstrate its long-term stability. In the interest of reducing time and costs corresponding to such long-term storage experiments, there is a strong need to find short-term protein parameters that generate similar information on long-term physical protein stability. One of these short-term protein parameters that shows great promise is the protein cloud temperature (Tcloud). Tcloud is a known representative of protein-protein interactions, which play a dominant role in long-term physical protein stability. In addition to a shorter time frame to generate the desired stability information, material use and experimental effort should be minimized to obtain an efficient screening method. This can be achieved with a high-throughput and low volume approach. An experimental setup to determine Tcloud that incorporates all these desired characteristics is currently not available.

The presented work will show an experimental method to detect Tcloud in a low volume and high-throughput manner. This was achieved by combining an automated imaging system and a cryogenic device that is compatible with 96-well microtiter plates, leading to a sample volume of 24 μL. Image-based data evaluation was automated, which resulted in a full plate evaluation time in the order of magnitude of minutes. The performance of the experimental and analytical approach was evaluated via a robustness study and the obtained parameter to represent Tcloud was validated. Furthermore, the work presents a case study employing the developed method to investigate long-term physical protein stability in a shorter time frame. The combined results of this work display the applicability of the low volume high-throughput Tcloud detection method as part of a short-term analytical screening toolbox that optimizes the search for long-term stable protein formulations, thereby advancing short-term strategies for efficient formulation development.

BIOT 88

Orthogonal pre-use and post-use in situ integrity testing for single-use anion exchange chromatography

Jonathan F. Hester, [email protected], Daniel Lu. Separation and Purification Sciences Division, 3M, St. Paul, Minnesota, United States

Flow-through anion exchange (AEX) chromatography is frequently used in biopharmaceutical purification processes for the reduction of negatively charged host cell proteins and viral reduction as part of a validated viral clearance strategy. While AEX column chromatography is the technology most frequently used at commercial scale for electrostatic viral clearance, the introduction of single-use AEX technologies in recent years has illuminated the opportunity for reduced capital, regulatory, and operational costs. Physically resembling and operated like filters, single-use AEX devices feature improved specific capacity and enhanced flow rates compared with columns due to the replacement of diffusive kinetics with convective flow. These features create the potential for simpler operation, decreased processing times, and reduced buffer consumption leading to improved economics relative to columns. Additionally, their single-use nature obviates validation costs associated with cleaning and performance over repeat use cycles, including viral clearance performance. With respect to the deployment of single-use AEX devices in large-scale manufacturing, however, many investigators have noted the need for sensitive, in situ test methods, analogous to HETP and peak asymmetry studies conducted on packed columns, capable of detecting device defects that might result in reduced viral clearance. While the bubble point or pressure-hold type tests frequently used to test the integrity of filters can reduce the risk of undetected bypass due to mechanical defects, such tests alone would fail to detect defects in AEX capacity or in the density or uniformity of the AEX functional chemistry on the media. While a few in situ integrity tests enabling assessment of the chemical functionality of AEX single-use media have been published, they involve exotic analytes, require complex analytical procedures, and/or are only qualitative. In this talk, we present an orthogonal set of two non-destructive, in situ pre- use integrity tests and a companion post-use integrity test. When employed in combination, the tests characterize both the mechanical and chemico-functional integrity of a single-use AEX device, strongly reducing the risk of undetected viral clearance failures. Correlations between integrity test performance and bacteriophage clearance are presented in the context of a new family of biomimetic single-use AEX chromatography devices with robust viral clearance performance.

BIOT 89

Evaluating cleaning and re-use feasibility for an anion exchange membrane adsorber

Thomas Elich1, [email protected], Jaime De Souza1, Matthew Johnson2, Elizabeth M. Goodrich1, Mochao Zhao3. (1) MilliporeSigma, Burlington, Massachusetts, United States (2) MilliporeSigma, Bedford, Massachusetts, United States (3) MilliporeSigma, Burlington, Ontario, Canada

Many of today’s bioprocesses require flexible manufacturing strategies in order to maintain a diverse multiproduct facility. Membrane adsorber chromatography is an effective tool to improve manufacturing flexibility by enabling rapid mass transfer and eliminating the need for column packing. This technology also offers opportunities for increased purification productivity as compared to resin based technologies. While membrane adsorbers provide value as a single-use device, in some applications the ability to regenerate the device for multiple re-use cycles may be desired. In this work, a commercially available high capacity membrane adsorber featuring quaternary amine chemistry was evaluated for re-use feasibility in a monoclonal antibody polishing application. Results are presented to evaluate device performance over consecutive processing cycles with a focus on product yield, host cell protein purification, viral clearance, and device backpressure. Considerations for implementing membrane adsorber chromatography in both single-use and re-use applications are discussed.

BIOT 90

High-resolution purification of PEGylated biotherapeutics using membrane chromatography

Raja Ghosh1, [email protected], Guoqiang Chen1, John Pagano2, Deqiang Yu2, Sanchayita Ghose2, Zhengjian Li2. (1) McMaster Chemical Engr Dept, Hamilton, Ontario, Canada (2) Biologics Process Development, Bristol-Myers Squibb, Devens, Massachusetts, United States

Protein PEGylation is a proven and effective strategy for increasing the biological half- life of protein biotherapeutics. A PEGylated protein is also generally more stable and easier to formulate than the native from of a protein. In this presentation we discuss the purification of PEG-Biol-1, which is the mono-PEGylated form of a candidate therapeutic protein. While this molecule is PEGylated using a site-specific technique, some di/higher-PEGylated forms of the protein (HMW) are synthesized as by-products. Some unreacted protein and low molecular weight species (LMW) are also present after the reaction. Therefore, PEG-Biol-1 needs to be separated from both HMW and LMW species. The resolution obtained using resin based ion-exchange chromatography is flow rate dependent, with the resolution decreasing very significantly at higher flow rates. In this study, we examined membrane chromatography as a high-speed, high- resolution alternative to column based chromatography. The separation was carried out using a laterally-fed membrane chromatography (LFMC) device housing a stack of strong anion exchange Q membranes. Using this LFMC device, both speed and resolution of HMW/LMW separation from PEG-Biol-1 could be significantly increased. For instance, significantly higher resolution than that obtainable using an equivalent resin based column, could be obtained at five times the flow rate using the LFMC device. In addition to discussing the experimental results obtained from this study, we discuss the reasons for the superior performance of the LFMC device based on first principles and computational fluid dynamics (CFD).

BIOT 91

High throughput development of wash and elution conditions for new drug modalities: Back to first-principles approach

Brandon Coyle1, [email protected], Kelley Kearns1, Warren Kett2, Karol Lacki2. (1) Purification Process Development, Avitide, Lebanon, New Hampshire, United States (2) Avitide, Lebanon, New Hampshire, United States

As the biologics market matures, the complexity of molecules is increasing rapidly as companies search for new modalities beyond mAbs. These new modalities (e.g. recombinant proteins, enzymes, vaccines, Fc-fusions and virus-like particles) often have no platform purification and narrow stability windows, which can result in low process yields. In such situations, affinity resins can be explicitly designed to deliver capture performance equivalent to ProA resins, but operate within the permissible windows unique to each molecule. As an unfortunate consequence, a large downstream development burden results. This talk focuses on Avitide’s first principles approach to downstream development that is based on protein-protein interactions. By starting from first principles, we created a high throughput strategy to develop wash and elution conditions unique to each resin but that can be applied across all new drug modalities. This strategy has been used to successfully develop elution conditions for a variety of modalities across 20+ programs and has proven to be a useful tool across all of downstream development.

BIOT 92

Multimodal membrane adsorbers for clearance of minute virus of mice Xianghong Qian1, [email protected], Tanmoy Patra1, Shu-Ting Chen2, Sumith R. Wickramasinghe2. (1) Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas, United States (2) Chemical Engineering, University of Arkansas, Fayetteville, Arkansas, United States

During purification, manufacturers of mAbs and other therapeutic proteins must demonstrate reduction of 103-105or more virus particles than is estimated in a single dose equivalent of the unprocessed bulk. The level of virus clearance is calculated by summing the clearance obtained from individual unit operations having different mechanisms of action (e.g. size exclusion and ion exchange). Depending on the antibody titer, estimates of the number of virus particles in a single dose equivalent could be as high as 1010-1015retrovirus-like particles per mL. In addition, removal of adventitious virus using a model parvovirus is required. In recent years, several membrane adsorbers with high binding capacity have evolved for the removal of contaminants for the polishing steps during downstream purification of biologics. In particular, multimodal membrane adsorbers with tunable pore sizes and ligand properties have generated tremendous interest due to their many possible applications. Here the removal of Minute Virus of Mice (MVM), host cell proteins (HCPs) and DNA using multimodal membrane adsorber Puri™-MQ was studied. The membrane performance was evaluated over a range of feed and elution conditions. The effects of pH and HCP concentration on MVM clearance were investigated. Comparison with other membranes at similar conditions demonstrates that multimodal membranes are highly effective for virus clearance.

BIOT 93

New multimodal anion-exchange membranes for polishing of biologics

Joshua Osuofa1, [email protected], Jinxiang Zhou2, Scott M. Husson3. (1) Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States (2) Purilogics, LLC, Greenville, South Carolina, United States (3) Chemical Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

This presentation will highlight findings from a study using new multimodal membranes for high throughput polishing at elevated solution conductivities. Multimodal membranes combine Coulombic interactions, hydrophobic interactions and hydrogen bonding in binding bioprocess impurities, thereby enabling salt-tolerant performance under conditions where conventional ion-exchange media fail. In this study, we evaluated the performance of Purilogics’ multimodal anion-exchange membranes (PurexaTM-MQ) under a variety of industry relevant conditions. We will present dynamic binding capacity (DBC) measurements of bovine serum albumin (BSA), human immunoglobulin G (hIgG), and salmon sperm DNA (ssDNA) for a range of conductivities (0 -30 mS/cm), salt types, flowrates, and pH values (6-9). Using 1 mg/ml BSA, measured DBC values were greater than 90 mg/ml for the highest conductivities but showed a maximum of 130 mg/ml at around 6 mS/cm. DBC increased by 42% with a three-fold increase in BSA concentration. DBC values showed only 5% decrease over 40 cycles with regeneration. The membranes had exceptionally high ssDNA DBC of 50 mg/ml under high conductivity conditions, easily outperforming all other commercial products. Purilogics’ PurexaTM-MQ membranes can rapidly purify bioprocess pools without dilution or desalting of feed, and may also hold potential applications for DNA production in the gene therapy industry.

BIOT 94

Bridging high-throughput screening and mechanistic modeling for the development of multimodal chromatographic processes

Scott Altern1, [email protected], Jessica Yang2, Chris Williams2, John Welsh3, Jake Klockowski1, Steven M. Cramer1. (1) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States (2) Purification Development, Genentech, South San Francisco, California, United States (3) Biologics Process Development and Commercialization, Merck & Co., Inc., Kenilworth, New Jersey, United States

In recent years, high-throughput screening techniques have become more prevalent in the development of chromatographic processes. During initial development stages, it is often challenging to identify optimal combinations of resins and process conditions. Traditional trial-and-error based approaches require extensive experimentation to explore the available design space. High-throughput techniques have since been applied to expedite this time-consuming process. The introduction of robotic liquid handling systems, for instance, has greatly accelerated early stage development. Such capabilities have enabled fast screening of the design space to identify potentially useful process contenders. In addition to high-throughput screening, mechanistic modeling of chromatographic processes has also been shown to expedite early stage process development. Although process modeling can reduce experimental burden during later stages of development, the large amount of experiments necessary for model design presents a challenge for creating an integrated workflow. Since it is increasingly time consuming to model many processes within the design space, it is vital to identify top process candidates from high-throughput screens for further development. As such, an ideal workflow would combine high-throughput screening and mechanistic modeling to quickly develop a process of interest while minimizing the use of material.

In this paper we present an efficient workflow for combining high throughput screening and data generation with isotherm formalisms and column modeling to both accurately predict complex behavior in industrial multimodal chromatographic processes and to expedite process development. Examples are presented for both multimodal cation and anion exchange systems operated in bind-elute and flow-through modes of operation, respectively. Using this combined approach, we were able to capture the unique salt and pH dependent chromatographic behavior obtained for mAb preparative separations in multimodal chromatography. BIOT 95

Iterative mapping approach for identifying next generation multimodal chromatography resins: Chemical diversity, high throughput screening, and chromatographic modelling

Lalita Shekhawat, [email protected], Keyhan Esfandiarfard, Todd Markle, Eike Theel, Jean-Luc Maloisel, Gunnar Malmquist. R&D, GE Healthcare, Uppsala, Sweden

Biopharma is facing increased purification challenges due to increased molecular diversity, e.g. new ”multispecific” antibodies. Together with stronger requirements to remove product related impurities, this will make polishing more important in the future. Mixed-mode chromatography with multimodal ligands can provide unique selectivities and thus play a key role in the purification of complex feed streams. The goal of the present study is to develop an iterative framework for development and selection of new multimodal resins for future purification challenges. We have developed a chemically diverse virtual library of multimodal cation exchange ligands, to create a library based around the CaptoTM MMC resin, designed towards varied properties such as hydrophobicity, H-bonding, etc. A selected subset of the ligands was coupled to the Capto MMC ImpRes base matrix and evaluated chromatographically in a high throughput plate study of protein binding at low loading to assess chromatographic diversity. Additionally, the adsorption mechanisms of a monoclonal antibody (mAb) were studied and the preferential interaction adsorption model used to provide estimates of: (a) the total number of water molecules and counter salt ions released during mAb adsorption, and (b) hydrophobic contact area for various isocratic conditions. The separation resolution between the mAb and Fab fragments, as well as high molecular weight aggregates were studied using gradient elution. Promising new resins were identified and the modelling points to the importance of tuning the ligand hydrophobicity, but also highlights the role of other ligand-specific modes of interaction (e.g. π-π interactions). The improved performance of several new resins in separation applications will be discussed. The iterative approach proves to be a promising method for identifying new ligands for biopharmaceutical purification challenges.

BIOT 96

Prioritized detoxification of toxic metabolites by Escherichia coli

Kristin Adolfsen, Wen Kang Chou, Mark Brynildsen, [email protected]. Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States

Hydrogen peroxide (H2O2) and nitric oxide (NO) are toxic metabolites that immune cells use to attack pathogens, and these antimicrobials can be present at the same time in phagosomes. Using Escherichia coli as a model bacterium, we observed that simultaneous exposure leads to prioritized detoxification, where enzymatic removal of NO is stalled until H2O2 has been eliminated. This phenomenon is reminiscent of carbon catabolite repression, where preferred carbon sources are catabolized prior to less desirable substrates; however, H2O2 and NO are toxic, growth-inhibitory compounds, rather than growth-promoting nutrients. To understand why NO detoxification is delayed by H2O2, whereas H2O2 detoxification proceeds unimpeded, we confirmed that the effect depended on Hmp, which is the main NO detoxification enzyme, and used an approach that integrated computational modeling and experimentation to delineate and test potential mechanisms. Experimental results indicated that Hmp catalysis and NAD(P)H supply were not impaired by H2O2, whereas hmp transcription and translation were greatly diminished. Notably, a dependence of this phenomenon on transcriptional regulation parallels regulation of carbon catabolite repression. Together, these data suggest that bacteria regulate their detoxification of growth-inhibitory metabolites in a similar fashion as their consumption of growth-promoting substrates, which could have important ramifications for infectious disease, bioremediation, and biocatalysis from inhibitor-containing feedstocks.

BIOT 97

Mitochondrial compartmentalization enables high-specificity recursive alcohol production in Saccharomyces cerevisiae

Sarah K. Hammer1, [email protected], Jose L. Avalos1,2,3. (1) Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States (2) Andlinger Center for Energy and the Environment, Princeton University, Princeton, New Jersey, United States (3) Molecular Biology, Princeton University, Princeton, New Jersey, United States

2-ketoacids are one of four classes of compounds that undergo recursive carbon-chain elongation in nature, and are of particular interest due to their role as precursors to higher alcohols, which are valuable renewable fuel compounds. While expression of heterologous enzymes and protein engineering have been successfully employed in E. coli to increase the maximum number of iterative elongation cycles, it remains difficult to achieve specific production of longer-chain 2-ketoacids. Here, we demonstrate that mitochondrial compartmentalization in the yeast Saccharomyces cerevisiae can increase specificity of recursive pathways toward longer-chain products. We show that overexpression of the 2-ketoacid elongation enzymes in mitochondria of a four-carbon alcohol production strain boosts five-carbon alcohol production more than 13-fold. Total alcohol production also shifts from a predominately four-carbon alcohol mixture to one with more than 80% five-carbon alcohols. Reducing the loss of intermediates to competing pathways further increases specificity toward five-carbon alcohol production, in addition to achieving the highest titer reported for S. cerevisiae. This work establishes the ability of mitochondrial compartmentalization of recursive metabolic pathways to enhance specificity toward larger products while maintaining high titers, exemplifying the potential benefits of harnessing organelles to engineer recursive pathways for product specificity.

BIOT 98

Reaction compartmentalization leads to enhanced production of PHA in Yarrowia lipolytica

Michael Spagnuolo, [email protected], Meredith Bailey, Mark A. Blenner. Chemical Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

Making the most efficient use of the finite amount of carbon and energy present in feedstocks is a common goal for the microbial production of commodity chemicals, thus improving the yield of reactions and improving process economics. A well-studied method to improve reaction efficiency is to increase the local concentration of the reactants. To this end, we have focused on increasing the efficiency of reactions building off the beta-oxidation cycle of Yarrowia lipolytica as a means of improving production of the bioplastic polyhydroxyalkanoate (PHA). Local concentration changes are achieved by compartmentalizing key enzymes within the peroxisome, a membrane- enclosed organelle where beta-oxidation occurs. Import into this organelle is accomplished through recognition of some form of peroxisomal targeting signal (PTS) and mediated by a cascade of peroxisome-associated (PEX) enzymes. Manipulation of this tag and the associated enzymes resulted in a greater than three-fold increase in PHA production from the same carbon feed conditions. In an effort to further improve production, expansion of the total peroxisome volume was investigated through knockout and overexpression of specific PEX enzymes. Finally, improvements in carbon flux into the peroxisome were sought via traditional metabolic engineering strategies including carbon-sink knockouts, transporter manipulation, and pathway overexpression.

BIOT 99 Bacterial microcompartments as tunable nanobioreactors: Engineering cargo and pathway encapsulation

Taylor Nichols, [email protected], Danielle Ercek. Chemical and Biological Engineering, Northwestern University, Chicago, Illinois, United States

While metabolic engineering can enable the sustainable bioproduction of new materials, efforts are often impeded by pathway bottlenecks. To mitigate the effects of toxic or reactive intermediates and resource competition resulting from heterologous pathway incorporation, bacterial microcompartments (MCPs) have recently been considered for engineered compartmentalization in bacterial host organisms. MCPs are protein shells that form natively in certain bacteria to selectively encapsulate specific metabolic processes, functioning to sequester and colocalize pathway components to enhance flux. The use of MCPs as enclosed scaffolds for metabolic engineering applications requires the control of heterologous cargo encapsulation, with pathway selection designed to efficiently utilize the native benefits of MCP systems.

We use the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica LT2 as a scaffold for the encapsulation of heterologous cargo and pathway components. With this talk, I will detail how we developed a genomic integration platform to enable and control cargo encapsulation in Pdu MCPs. Specifically, we integrated different fluorescent reporter constructs into the operon that encodes the native Pdu MCP and subsequently assessed cargo expression and encapsulation levels. We were able to achieve encapsulation of multiple cargo, with different relative encapsulation levels observed. Importantly, the trends were reproducible and encapsulation efficiency was primarily determined by the targeting sequence and integration locus. Results from this platform have subsequently informed efforts to encapsulate an industrially-relevant non- native metabolic pathway within Pdu MCPs. This work established a genomic integration platform for heterologous cargo encapsulation in Pdu MCPs, further enabling their development as tunable nanobioreactors for the encapsulation of heterologous pathways.

BIOT 100

Engineering obligate mutualism in co-cultures of ammonium-secreting Azotobacter vinelandii and glucose-secreting Escherichia coli

Maciek R. Antoniewicz, [email protected], Camil Diaz. University of Delaware, Newark, Delaware, United States

Co-cultures are attractive microcosms for studying metabolic interactions in a controlled system. Co-cultures involving diazotrophs, or organisms capable of reducing atmospheric nitrogen into ammonia, are of particular interest sustainable alternatives to the Haber-Bosch process a source of nitrogen fertilizer.

While diazotrophs like Azotobacter vinelandii have successfully been engineered to produce ammonium aerobically, these strains also exhibit a high substrate demand. Indeed, we have previously shown that the majority (88-90%) of the carbon consumed by A. vinelandii is spent on respiration, ultimately to protect its nitrogenase from oxygen, rather than ATP production for fueling nitrogen fixation and other cellular processes. Thus, a major challenge in engineering stable co-cultures with nitrogen-fixers is to identify partners that can meet the high substrate demands of these diazotrophs while receiving limited nitrogen in return.

We investigated the performance of four, fully nitrogen self-sufficient co-cultures involving an ammonium-secreting strain of A. vinelandii paired with both wild-type and various engineered E. coli strains. 13C-metabolic flux analysis of these co-cultures unexpectedly revealed that A. vinelandii shares the majority of its fixed nitrogen. For example, even in commensalist pairings, E. coli constituted more than 60-75% of an exponentially growing co-culture population. Furthermore, using 13C-MFA, we identified additional cross-fed nutrients that are not otherwise secreted by either organism when grown in isolation.

Finally, in evolution studies, we were able to isolate a clone of one of our engineered E. coli strains that had gained the ability to continue producing glucose for A. vinelandii even under complete nitrogen starvation.

BIOT 101

Interspecies cell fusion facilitates the exchange of protein and other cellular material in a syntrophic clostridium co-culture

Kamil Charubin1, [email protected], Eleftherios T. Papoutsakis2. (1) Chemical Engineering, University of Delaware , Newark, Delaware, United States (2) Univ Delaware, Newark, Delaware, United States

In microbial fermentations at least 33% of the sugar substrate is lost as CO2 waste during the pyruvate decarboxylation, with the corresponding loss of electrons in the form of H2. To improve fermentation efficiencies we designed a synthetic and syntrophic co- culture of Clostridium acetobutylicum, which is capable of converting a variety of sugars into solvents, and C. ljungdahlii, an acetogen capable of fixing CO2 in the presence of H2. The co-culture was found to reach higher carbon recoveries and produce non-native products i.e. isopropanol and 2,3-butanediol. The physical contact between both strains was found to be crucial in maintaining the unique co-culture phenotype. The interspecies interaction led to a metabolite exchange, which allowed for the generation of non-native products, and a differential gene expression of the key enzymes in the co- culture. To investigate the interspecies interaction between C. acetobutylicum and C. ljungdahlii more closely we engineered both bacteria to express fluorescent proteins that do not require O2 for fluorophore activation, namely the FAST and Halo-Tag proteins. Correlative confocal and electron microscopy was used to study the timeframe and the extent of interactions between both strains in the co-culture. The co-culture of fluorescent clostridia showed exchange of fluorescent material between both species, indicating both species can exchange protein under co-culture conditions. The microscopy results were confirmed using flow cytometry to better understand the time frame of the interactions that occur in this synthetic syntrophy.

BIOT 102

Controlling pyocyanin synthesis in synthetic microbial consortia by regulating culture composition

Kristina Stephens, [email protected], Kayla Chun, Sally Wang, Eric VanArsdale, William E. Bentley. Fischell Dept of Bioengineering, University of Maryland, College Park, Maryland, United States

Interest in use of microbial consortia or co-cultures by metabolic engineers has expanded due to limitations of single populations. These limitations include high metabolic burden on cells and difficulty optimizing cells to carry out complex pathways. Co-cultures allow for division of labor and options for plug and play. Partitioning a product pathway by splitting it among different populations also provides opportunities to control synthesis of each section of the pathway by controlling culture composition. Importantly, this provides an additional layer of control (as opposed to transcriptional or translational regulation) in increasingly complex synthetic biology systems. However, these systems could benefit from methods for engineering cell-cell communication between strains in the co-culture, enabling the cells to work together autonomously. Quorum sensing (QS) systems are commonly used to engineer cell-cell communication, but there are limited numbers of orthogonal QS systems. Here, we show that E. coli can be engineered to produce pyocyanin as an additional molecular cue for engineering cell-cell communication. Previously, it has been shown that pyocyanin causes activation of the sox promoter in E. coli allowing for pyocyanin-mediated induction of a gene of interest. Interestingly, because the pathway for pyocyanin synthesis requires several genes, there are opportunities to split the pathway between different strains to allow for more complex behavior within the synthetic consortia. That is, we designed an E. coli co-culture for the synthesis of pyocyanin and then demonstrate that pyocyanin synthesis and cell response to pyocyanin is dependent on culture composition. Population A expresses P. aeruginosa genes phzA1-G1 to synthesize an intermediate product phenazine-1-carboxylic acid, while Population B expresses genes phzMS to synthesize pyocyanin. We show that by varying the ratio of Population A to Population B, we vary the amount of pyocyanin produced. Then, we show that by adding a third population, Population C, which contains a pyocyanin responsive promoter, we control activity in Population C based on the combined activity (and composition) of Populations A and B. We further explore methods for manipulating the dynamics of the co-culture system. We believe our strategy could be useful for many applications including for coordinating co-cultures that are synthesizing a product and for engineering cooperativity in synthetic biology systems.

BIOT 103

Analytical and process development challenges and opportunities in cell and gene therapy

Susan Abu-Absi, [email protected]. Pharmaceutical Development & Technology, bluebird bio, Cambridge, Massachusetts, United States

The manufacturing processes and analytical methods for engineered T-cell therapies (e.g. CAR-T) and autologous ex vivo gene therapies are complex. Cells are first collected from patients via apheresis, modified ex vivo with lentiviral vector (LVV) or other gene addition/gene editing methods, expanded in culture, and frozen as a living drug product. With the rapid growth of the cell and gene therapy field, the demand for LVV has increased exponentially. Current LVV production methods using transient transfection are robust but will ultimately be constrained volumetrically and temporally. To overcome these constraints, alternative production processes may be required to meet future demand. Autologous drug products present a tremendous opportunity for understanding critical quality attributes (CQAs), by correlation of drug product attributes with clinical results and via advanced cell characterization. Although most commercial manufacturers utilize a centralized manufacturing approach, there is a lot of interest in advancing manufacturing beyond decentralized manufacturing to point-of-care or even bedside manufacturing. This presentation will provide an overview of the analytical and process development challenges for cell and gene therapy and opportunities for advancing the technologies to enable these therapies to reach all patients who can benefit from them.

BIOT 104

Large scale production of cerebellar organoids from human pluripotent stem cells using the novel vertical-wheel single-use bioreactors

Teresa P. Silva1,2, Rui S. Luís2, Tiago G. Fernandes1, [email protected], Evguenia Bekman1,2,3, Carlos A. Rodrigues1, Sandra H. Vaz2,4, Leonilde M. Moreira1, Yas Hashimura5, Sunghoon Jung5, Brian Lee5, Maria Carmo-Fonseca2, Joaquim M. Cabral1. (1) iBB – Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal (2) Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal (3) The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Universidade de Lisboa, Lisbon, Portugal (4) Instituto de Farmacologia e Neurociências, Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal (5) PBS Biotech, Camarillo, California, United States

Human induced pluripotent stem cells (iPSCs) have great potential for disease modelling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to differentiate cerebellar neurons from pluripotent stem cells is still limited. Recently, we described the long-term culture of functional cerebellar neurons from human iPSCs, which were maintained in cultures for up to 145 days without the need for co-culturing when re-plated on laminin. Dendritic spine morphology analyses, calcium imaging, and electrophysiological evaluation strongly indicated an efficient maturation of cerebellar precursors, both at the individual cell level and at the level of network connectivity. Nevertheless, defining protocols that allow the production of large numbers of organoids and a high yield of mature neurons maintaining the 3D structure is still difficult. Here, we present a new approach for the reproducible and scalable generation of cerebellar organoids under chemically defined conditions by using the novel PBS Vertical-Wheel single-use bioreactors. RNA sequencing (RNA-seq) was performed to evaluate the global gene expression profiles for different time points of cerebellar differentiation in the dynamic system and compared with the static 3D cerebellar differentiation. Gene expression profiles showed no impact of agitation in the efficacy of cerebellar differentiation. Additionally, the data suggests that cerebellar organoids obtained in bioreactors induced a 3D microenvironment enriched in extracellular matrix when compared with static conditions. Furthermore, different types of functional cerebellar neurons, including Granule cells (PAX6+ and MAP2+) and Purkinje cells (Calbindin+) were readily identified in cryosections of the organoids generated from this dynamic culture system. Thus, we describe for the first time the continuous and scalable generation of human iPSC- derived neural organoids that display cerebellar identity, as well as the generation of functional cerebellar neurons that were maintained in suspension for as long as 3 months, without feeder layers. This culture system increased reproducibility between experiments and allowed large-scale production of cerebellar organoids, which is crucial for future applications in drug screening and toxicological testing, as well as to study the pathological pathways involved in cerebellar dysfunction.

BIOT 105

Effect of key cytokines on the growth of different T cell phenotypes

Canaan Coppola, [email protected], Brooks Hopkins, Zuyi Huang, William J. Kelly. Chemical Engineering, Villanova University, Villanova, Pennsylvania, United States

A significant fraction of CART batches fail every year due to slow Tcell growth rates during the expansion step of the process. Furthermore, the starting material for every batch differs in many ways including the distribution of phenotypes in the Tcell population, yet the final product specification is fixed. Tcell growth rates can be effected by levels of cytokines and different Tcell phenotypes respond differently to these growth factors. In this study, the growth rates of important CD4+ and CD8+ Tcell phenotypes (i.e. Tnaive, Teff, Tcm) were evaluated and modelled as a function of cytokine (Il2, Il7 and Il15) level. Cell health was assessed via an Annexin V-propidium iodide assay, and phenotype was determined via Flow Cytometry. The results indicate significant variation between the different Tcell phenotypes in terms of how their growth and health is affected by different levels of the different cytokines. Interestingly, all of the Tcell phenotypes in varying degrees exhibited a reduction in cell numbers in the first day or two following vial thaw and bead-induced activation. Immediately following that period, effector memory cells in general grew faster than the other phenotypes. Interestingly optimal growth rates were observed at intermediate cytokine levels, a result that is helpful in keeping process costs down. A fuzzy model employed effectively predicted how the Tcell growth rates varied with the different combinations of cytokines, and transcriptomics data helped to inform the model on metabolic activities relating to cell growth. Some of the experiments involved mixed culture of different Tcell phenotypes, to assess how interaction between Tcells effects their growth.

BIOT 106

Scalable adeno associated virus production process, from cell culture to purified bulk

Asa H. Hagner McWhirter, [email protected]. Bioprocess application, GE Healthcare, Uppsala, Uppsala, Sweden Adenovirus associated virus (AAV) vectors are increasingly used in clinical trials, evaluated for gene therapy applications. Manufacturing of safe and efficacious clinical- grade virus relies on a scalable and cost-effective production process. In this study, we have combined experimental work and process economy calculations, from AAV production in suspension cell culture to purified bulk product up to 10L scale. An efficient and scalable process for AAV production was developed by evaluation and optimization of each process step. We used AAV2 for process development, since this is the most used AAV vector in addition to serotype 5, 8, 9 and various synthetic capsid variants. AAV2 expressing the green fluorescent protein (GFP) was used for process development to facilitate analysis. First, suspension culture of HEK 293T cells adapted to serum-free medium and triple plasmid transfection were optimized using a DoE strategy for transfection efficiency and AAV2 production. The conditions were further developed for production in a single use stirred tank bioreactor system. Novel affinity and ion exchange based purification step alternatives were compared, including different chromatography resins and membrane formats. Finally, ion exchange chromatography and core bead technology were evaluated as an alternative to size exclusion chromatography for the polishing step before the formulation into storage buffer. Analytical methods for virus titer are challenging and depend on purity and quality of the sample. For vial genome titer qPCR was used and for total virus titer ELISA and HPLC methods were used. For infectious virus titer, we have used a flow cytometry-based assay counting GFP producing cells. Based on analytical data different downstream process alternatives were compared regarding load capacity, recovery, reduction of empty capsids and purity and we propose a robust and scalable process with a favorable process economy.

BIOT 107

Leveraging high-throughput strategies to accelerate AAV purification process development

Xiaotong Fu, [email protected], Asher Williams, Meisam Bakhshayeshirad. Gene Therapy, Biogen, Cambridge , Massachusetts, United States

The adeno-associated viral vector (AAV) has been broadly used as a delivery tool for gene therapy applications. Development of a robust purification process is essential in delivering high purity and potency AAV products to the clinic. The short clinical timelines and limitation of materials to support process development pose unique challenges when developing robust and scalable downstream purification processes.

Our approach in overcoming this challenge is to leverage high throughput (HTP) technology for purification process development. In this study, we demonstrated the capability of the TECAN automated liquid handling system to develop purification processes using robocolumns and self-packed resin plates. The purification performance and product quality achieved in the high throughput format was compared with that obtained in the bench scale column chromatography. The established high throughput strategies demonstrated good feasibility, significantly reduced material needs, and reduced development timelines. With further expansion and improvement, we envision that this HTP platform can be used for optimization and characterization of downstream purification processes used for manufacturing of gene therapy products.

BIOT 108

Development of AVIPureTM - AAV9: First affinity resin for AAV production that can be cleaned with sodium hydroxide enables high resin reusability and decreases annual resin costs for high scale production

Thomas C. Scanlon1, [email protected], Warren Kett2, Karol Lacki3. (1) Ligand Discovery, Avitide, Inc, Lebanon, New Hampshire, United States (2) CSO, Avitide, Inc, Lebanon, New Hampshire, United States (3) Technology Development, Avitide, Inc, Lebanon, New Hampshire, United States

Purification of recombinant AAV9 from cell lysates remains a technical challenge and an economic burden for commercial manufacturing of novel gene therapeutics. Currently available affinity resins for AAV9 purification are plagued by poor reduction in Host Cell Protein (HCP) and the inability to use NaOH solutions for Clean-In-Place (CIP). To support the rapidly-expanding gene therapy industry we described the development of AVIPureTM – AAV9, a novel affinity chromatography resin with ability to perform single- step purification of AAV9 from cell lysates with excellent yield and purity. Key distinguishing features are the ability to clean with NaOH and being based on an agarose bead for improved non-specific absorption. The large diameter agarose bead enables high flow rates and excellent resistance to fouling, important features when a typical purification cycle involves loading the column with up to 200 column volumes of host cell culture fluid. The order of magnitude improvement in resin lifetime promises to dramatically decrease resin costs for AAV programs requiring annual production of very large amounts of capsids. The affinity ligand is a novel peptide with exquisite specificity for rAAV9, binding the virus with picomolar affinity. A long, flexible spacer molecule attaches the ligand to the resin, enabling efficient capture of virus. Elution of several variants of AAV9 capsids elution was achieved in the range pH 4.5 – 3. Overall, AVIPureTM – AAV9 resin provides excellent yield, purity and throughput for single-step purification of rAAV9 and enables the use of sodium hydroxide as a cleaning agent for multiple cycles.

BIOT 109

Scalability and competitor benchmarking of anion exchange membrane chromatography devices for Adeno-Associated Virus purification using a DoE approach

Kurt Boenning, [email protected], Aydin Kavara, Julio Huato, Mark Schofield, Rene Gantier. Bioprocess R&D, Pall Corporation, Westborough, Massachusetts, United States Adeno-Associated Viruses (AAVs) are increasingly receiving approval for the in vivo treatment of acquired and inherited genetic diseases. However, purification challenges remain as AAV production typically results in a significant proportion of empty capsids which may invoke an immune response, without the ability to deliver the gene therapy payload. Here we propose a scalable purification process, based on anion exchange (AEX) membrane chromatography for final polishing and empty/full capsid separation. Despite widespread application of AEX membrane chromatography, there has been little published effort to understand the scalability of membrane devices when transitioning from filter plate experiments to flow capsules. A Design of Experiments (DoE) approach was implemented for the optimization of binding and elution conditions of AAVs via high throughput screening on Mustang Q filter plates. These conditions were transferred to Mustang Q capsules along with alternative membrane chromatography devices of comparable scale. Gradient and step elution experiments were carried out to understand the role of salt and additives in desorption of AAV from the membrane surface and the separation of empty and full capsids. In summary, a scalable and platform-ready AAV purification protocol was developed to facilitate the transition from Mustang Q filter plates to XT acrodiscs.

BIOT 110

Hyaluronic acid hydrogel platform to model dormancy in brain metastatic breast cancer cells in vitro

Akshay Narkhede, [email protected], James H. Crenshaw, Shreyas Rao. Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, Alabama, United States

Disseminated breast cancer cells (BCCs) lay dormant at the metastatic site resulting in metastatic relapse years after surgical resection of the primary tumor. Particularly, metastatic relapse in the brain marks the most advanced stage of the disease with a median survival period of only 4-9 months post-diagnosis. Dormant BCCs dynamically interact with the brain microenvironment which, in turn, mediates their phenotype. Specifically, the brain microenvironment provides mechanical (by modulating matrix stiffness) cues to the dormant BCCs. Herein, we model dormancy in BCCs via exploiting the mechanical cues provided by hyaluronic acid (HA) hydrogel representative of the brain extracellular matrix. MDA-MB-231Br and BT474Br were used as model metastatic cancer cells and were cultured on top of HA hydrogels. We observed that BCCs cultured on soft (~ 0.4 kPa) HA hydrogel were largely EdU and Ki67 negative suggesting that they were dormant whereas cells cultured on stiff (~ 4.5 kPa) HA hydrogel were proliferative as indicated by EdU and Ki67-positivity. Also, we observed nuclear localization of p21 and p27 (proteins associated with cancer dormancy) in cells cultured on soft hydrogel in contrast to their cytoplasmic localization in cells cultured on the stiff hydrogel. These results indicate that the hydrogel stiffness mediates dormancy in BCCs. We further investigated the role of focal adhesion kinases (FAK) in stiffness-mediated dormancy, wherein blocking FAK in cells cultured on stiff HA hydrogels resulted in decreased Ki-67 positivity, suggesting that FAK signaling, in part, mediates dormancy in BCCs. Further, we demonstrate that increasing cell density overrides the impact of soft hydrogel and partially revokes dormancy. Finally, we performed RNA sequencing to investigate the expression pattern of genes associated with dormancy in BCCs cultured on the soft hydrogel. This platform provides a tool to study microenvironmental regulation of dormancy, which may ultimately provide strategies to target dormant disease.

BIOT 111

3D co-culture model that mimic the role of hyaluronic acid on gastric cancer invasiveness

Sara Amorim1,2,3, [email protected], Diana Soares da Costa1,2, Iva Pashkuleva1,2, Celso Reis4,5,6, Rui L. Reis1,2,3, Ricardo Pires1,2,3. (1) 3B's Research Group, University of Minho, Guimarães, Portugal (2) ICVS/3B's Associated Laboratory, University of Minho, Braga, Braga, Portugal (3) The Discoveries Centre, Guimarães, Portugal (4) I3S, University of Porto, Porto, Porto, Portugal (5) Ipatimup, Porto, Portugal (6) Pathology and Oncology, University of Porto, Porto, Portugal

The tumour microenvironment is a complex niche of proteins, glycoproteins, glycosaminoglycans and different cell types. The glycosaminoglycan Hyaluronic Acid (HA) is one of the major constituents of the extracellular matrix (ECM) and its’ molecular weight (Mw) is linked to cancer cells behaviour, i.e. low Mws are associated to migration, whereas the high Mw HA have been linked to cellular latency and apoptosis. [1-3] In addition, it has been reported that the cancer invasive character is promoted by the crosstalk between cancer cells and mesenchymal stem cells (MSCs). In fact, MSCs induce Epithelial to Mesenchymal Transition (EMT) on cancer cells. In EMT cancer cells lose their polarity and intercellular junctions, with an upregulation of mesenchymal markers acquiring an enhanced migration character. [3,4] Herein, we developed a 3D co-culture model that recapitulates the tumour microenvironment. This model is based on a core-shell hydrogel, where the core can be tuned to present HA of specific Mws. We used this system to study the impact of HA Mw and the presence of MSCs in gastric cancer invasiveness. This was achieved using co-cultures of MKN45 cancer cells encapsulated in the core of the 3D system (alginate presenting HA of 6.4, 752 or 1500kDa) and bone marrow MSCs in the alginate-only shell. Under these experimental conditions we found a correlation between the HA’s Mw and cancer invasiveness: migration of MKN45 is promoted by short HA (6.4 kDa). In addition, the presence of MSCs promote EMT on MKN45 cancer cells, which was confirmed by the overexpression of the mesenchymal markers vimentin and n-cadherin. Furthermore, no EMT was detected on cancer cells cultured in the presence of MSCs conditioned media, showing that the MSCs-MKN45 crosstalk is necessary to promote the EMT on the MKN45 cancer cells. Our data clearly demonstrate that: 1) the size of HA Mw influences cancer cells migration and invasiveness, and 2) the EMT occurs in dependence of HA Mws’ and the physical crosstalk between MSCs and cancer cells.

BIOT 112

Biomanufacturing of GBM tumor microenvironment using a small scale bioreactor

Joe Park, [email protected]. Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, Alabama, United States

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor that originates from glioblastoma stem cells (GSCs). GSCs are able to create a tumor microenvironment (TME) and reside in the perivascular niche and hypoxic region where heterogeneity and cell to cell interaction exists. Many prior studies focused on modeling GBM using hydrogels, but these methods are limited to small scale with complex procedures and hardly represent complexity of GBM TME. Thus, there is a great need to develop a scalable platform for understanding of GBM TME and targeting GBM TME. Conventional organoid studies rely on growing them in serum-free media, resulting in sphere formation. This conventional method, however, often results in large random sphere aggregations and fails to control the size of the spheres. Here, we suggest a simple and more in vivo-like GBM culture method by optimizing the shear stress in a small scale bioreactor. We cultured patient-derived GBM cell lines with controlled size of GBM tumorsphere. GSCs grown under shear stress showed increase of Notch signals as well as pericytes markers which indicate characteristics of GSCs in perivascular niche. Further, GSCs grown under shear stress showed spatial increase of CD133 population. Collectively, GSC culture under optimized shear stress can model GSCs in perivascular niche including their microenvironment and GSCs expansion.

BIOT 113

Investigating glioblastoma stem cell behaviors in three-dimensional hyaluronic acid hydrogels

Pinaki Nakod, [email protected], Yonghyun Kim, Shreyas Rao. Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, Alabama, United States

Glioblastoma multiforme (GBM), an aggressive brain tumor, represents one of the most incurable human cancers due to its highly invasive nature. GBM tumors are highly heterogeneous that are composed of tumor cells and small portion of tumor initiating cells also known as glioblastoma stem cells (GSCs) that contribute to the therapeutic resistance and tumor recurrence following surgery. For improving the therapeutic efficiency, targeting GSCs and their surrounding microenvironment appears to be a promising approach. The tumor microenvironment provides physical, chemical and cellular cues that nurture the stemness properties of the GSCs and thus promote tumorigenesis, therapeutic resistance and tumor recurrence. A mechanistic understanding of the impact of these microenvironmental cues on GSCs is crucial in developing an efficient therapeutic approach. Here, we employed three dimensional (3D) hyaluronic acid (HA) hydrogels that allow incorporation of brain microenvironment cues to investigate GSC behaviors. In this study, U87 cell line and patient derived D456 cells were cultured adherently (in the presence of serum) and/or as suspension culture (serum-free) and were then encapsulated in HA hydrogels. We observed that all the seeded single cells expanded and formed spheres, and size of the spheres increased with time. Increasing the initial cell seeding density of cells influenced the sphere size distribution. Interestingly, clonal expansion of serum-free grown tumor cells in HA hydrogels was observed. Also, stemness marker expression of serum and/or serum- free grown cells was altered when cultured in HA hydrogels. Next, we hypothesized that the niche cells in the brain environment when cultured together with the GSCs enhance their stemness phenotype. To test this, we are currently investigating the impact of astrocytes and/or endothelial cells in our 3D HA model in different cell seeding densities. Overall, such biomimetic culture systems could further our understanding of microenvironmental regulation of GSC phenotypes.

BIOT 114

Responsive STING agonist nanovaccines for cancer immunotherapy

Guizhi Zhu, [email protected]. Pharmaceutics, Virginia Commonwealth University, Richmond, Virginia, United States Despite the recent breakthrough of immune checkpoint blockade (ICB) in cancer immunotherapy, the objective response rates (ORRs) to single ICB agents remain low, due to sparse preexisting target immune cells for ICB, heterogeneous immune checkpoint levels, and multi-tier immunosuppression. Rational combination of therapeutic vaccines and ICB can enhance ORRs while minimizing irAEs. Stimulator of interferon genes (STING) agonists are promising immunostimulators for cancer immunotherapy. STING is an intracellular receptor that senses cytosolic cyclic dinucleotides (CDNs) to induce the production of type I interferons (IFNs) and proinflammatory cytokines that activate immune cells such as CD8+ T cells and natural killer cells. However, the clinical translation of CDNs has been challenged by their susceptibility to enzymatic degradation or hydrolysis, poor pharmacokinetics, negative charges that hamper efficient cell uptake, and potential immune-related adverse events (irAEs) associated with systemic CDN dissemination. To address these challenges, we designed novel nanovaccines that deliver, protect, and conditionally release CDNs in target cells. We synthesized nanovaccines using polymers nanoparticle cores with surfaces of designer DNA that binds to CDNs under physiological pH but undergoes a conformational configuration to release CDNs in the acidic endosome. The nanoparticles protected CDNs from degradation. The resulting STING agonist nanovaccine was efficiently internalized by antigen-presenting cells and effectively release cargo CDN in acidic endolysosome. The released CDN eventually trafficked into the cytosol, allowing CDN to activate STING located on endoplasmic reticulum membrane. In dendritic cells (DCs), this nanovaccine elicited three integral signals for antigen presentation and T cell priming: upregulation of MHC-II (Signal 1), upregulation of co-stimulatory factors (Signal 2), and secretion of type I IFNs and proinflammatory cytokines (Signal 3), all of which outperformed that of the corresponding natural CDN and a gold-standard chemically-stabilized CDN counterpart. In a melanoma mouse model, intratumoral vaccination with these nanovaccines remodeled the immune milieu to be less immunosuppressive. Consequently, this nanovaccine dramatically inhibited the growth of an aggressive and poorly immunogenic melanoma in mice. These results demonstrate the potential of this CDN delivery system for cancer immunotherapy.

BIOT 115

SBE/AICHE/BIOT Wang award: Three continents, two biomanufacturing philosophies, multiple therapeutic modalities: Endless excitement of being a biochemical engineer

Konstantin Konstantinov, [email protected]. Codiak, Cambridge, Massachusetts, United States

Biochemical engineering offers diverse research opportunities for the biotechnology professional, either in academia or industry. This diversity is a unique advantage of our field, which becomes better appreciated as our careers progress, and we keep entering new and exciting research areas, often in an unpredictable way. This is how I feel about my own professional path, which began in the late 80s after a somewhat random, but very lucky choice. Looking back, it is amazing to see the large diversity of research topics I have worked on, spanning from single cell protein to exosomes, from simple batch to advanced continuous processing, from wild type bacteria to highly engineered human cells. Along with this diversity, there are certain fundamental trends, concepts and challenges that remain unchanged with time. My presentation will review some of these topics from a historical/personal perspective, including the ever-growing complexity of the new therapeutic modalities, the complex dynamics of transferring technology from academia to industry, the re-emergence of familiar concepts in highly advanced new forms and, not the least, the rapid advance of integrated continuous biomanufacturing. I will also share some experiences on the soft side of the job, including a few episodes involving Professor DIC Wang. Lastly, I will also share my thoughts of the future of biochemical engineering and what it will take for the new generation of biotech professionals to drive the technology to new heights. With time, the opportunities in the field will only get better. Biochemical engineering will continue to enrich our lives with joy and excitement, for which I will remain forever grateful.

BIOT 116

Interfacial study of biosurfactants in recombinant protein formulation for transdermal drug delivery applications

Subhabrata Das, [email protected], Evon bolessa, Courtney ODell, Celia Williams, Nandu Deorkar, Arvind Srivastava. Biopharma R&D, Avantor Performance Materials, Bridgewater, New Jersey, United States

Therapeutic Recombinant proteins are exposed to different kinds of stress conditions during production, storage, and shipping that can result in the formation of soluble and insoluble aggregates. The potential for intermolecular protein-protein interaction and subsequent increased probability of aggregation is observed at air-water interface where protein chains orient themselves to expose their hydrophobic pockets to increase their interaction with the aqueous surface. Non ionic surfactants like Polysorbate 80 and 20 are widely used in protein formulations to prevent agitation-induced aggregation because of its effectiveness at low concentrations, relatively low toxicity, ability to inhibit protein surface adsorption, aggregation under various processing conditions and act as a stabilizer against protein aggregation. Nevertheless, the alkyl polyoxyethylene side chains of Polysorbates undergo auto oxidation resulting in formation of hydroperoxides, side-chain cleavage, and eventual formation of short-chain acids, such as formic acid, which can affect the stability of the product. In this work, we have evaluated various biosurfactants and investigated their interfacial properties after having compared their performances with traditionally used Polysorbates for transdermal drug delivery application. Preliminary data suggests that the microbially derived Biosurfactants could be a better alternative of Polysorbates for proteins stabilization at the air-water interface over a diverse range of physicochemical properties, such as hydrophobicities, pKa’s and degrees of ionisation. This effect was attributed to the formation of a recombinant protein-biosurfactant interaction which influenced the diffusion profiles of the drug molecules.

BIOT 117

Protein-protein interactions and self association in antibody solutions via static light scattering, small angle X-ray scattering, and coarse-grained molecular dynamics simulations

Amjad Chowdhury1, [email protected], Keith P. Johnston2, Thomas Truskett3. (1) The University of Texas at Austin, Austin, Texas, United States (2) Univ of Texas, Austin, Texas, United States (3) Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, United States

Highly concentrated therapeutic monoclonal antibody (mAb) solutions are desired for subcutaneous delivery in patients. However, at these high concentrations, protein- protein interactions (PPI) including local, short-range attractions may cause prohibitively high viscosities and poor storage stability. The understanding of this behavior has been limited by the lack of characterization techniques that are applicable at these extreme conditions. Here, we determine the effects of pH and co-solute on the strength and geometry of short-range anisotropic attractions for both a mAb and a polyclonal bovine IgG by comparing static light scattering (SLS) and small angle x-ray scattering (SAXS) data to those computed in molecular dynamics simulations. The simulations utilize 12- bead models exhibiting a van der Waals attraction and additional short-ranged attractions between specific beads and quantify the distribution of reversible clusters in solution. Since our model captures key features of the protein shape, it can describe the experimental SAXS scattering profiles for solutions of 10-200 mg/mL protein with only a small (<1 kBT) variation in the model’s attraction strength. The apparent molecular weights calculated from SLS show reduced net attractions with the addition of NaCl and arginine. Structure factors (S(q)) from SAXS for mAb solutions with added NaCl exhibit an upturn at low q, which our modeling reveals is due to the formation of large reversible oligomers resulting from strong anisotropic attractions in the Fab and Fc regions. For the bovine IgG, reducing the pH below the pI or adding NaCl weaken short- range anisotropic attractions, but not enough to remove large reversible oligomers that raise viscosity. In contrast, for arginine added at pH 5.5, the scattering for both proteins can be approximated by a uniform attraction model exhibiting only monomers and dimers. The cluster size distributions are used to improve the understanding and prediction of experimental viscosities using an empirical viscosity model. The ability to discriminate between different interaction geometries and identify the presence of oligomers has great utility in designing and formulating low viscosity, stable mAb solutions.

BIOT 118

Fast dynamic surfactant reduces aggregation of biologics Joshua S. Katz, [email protected], Susan Jordan. Pharma Solutions, DuPont Nutrition and Biosciences, Wilmington, Delaware, United States

As the pharmaceutical industry shifts from small molecule drug to biologic drug development, new and unique formulation needs emerge. Issues relating to shelf- stability of biologic formulations remain a key challenge to new drug development for both the developed and developing world. N-myristoyl phenylalanine Jeffamine M1000 diamide (FM1000) is a new experimental surfactant that has been developed for stabilizing biopharmaceuticals. FM1000 exhibits an order of magnitude faster interfacial dynamics than conventional formulation excipients, leading to improved agitation stability. A three-fold drop in IgG aggregation rate was observed for IgG formulated with FM1000 compared to control surfactant formulations. In a study of abatacept aggregation, FM1000 minimized the monomer loss both in glass vials and in IV bags, even out-performing Poloxamer 188, the incumbent technology. FM1000 also reduced subvisible particle formation in a cetuximab model formulation. Coupling aggregation performance with fundamental surfactant interfacial dynamics and behavior will help better understand the underlying mechanisms of protein (de)stabilization, enabling the development of higher stability formulations.

BIOT 119

Core annular flows to enhance the injectability of high concentration drug formulations

Vishnu Jayaprakash, [email protected], Maxime Costalonga, Somayajulu Dhulipala, Kripa Varanasi. Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

Highly concentrated biologic drug formulations that can be delivered via subcutaneous injections offer tremendous benefits to global health, but they cannot be injected via commercial syringes and needles due to their high viscosities. Current approaches to solve this problem face several challenges ranging from cross contamination and high cost to needle clogging and protein inactivation. Here we report a simple method to enhance injectability using a core annular flow, where the transport of highly viscous drugs through a needle is made easier, thanks to co-axial lubrication by a less viscous fluid. We establish a phase diagram to ensure optimally lubricated core annular flow while minimizing the volume fraction of lubricant. This technique allows us to experimentally obtain up to a 7x reduction in injection pressure for the largest viscosity ratio that was tested, which is in agreement with theoretical predictions for eccentric core annular flows. Finally, we implement these findings into the design and fabrication of a double barreled syringe that significantly expands the range of injectable concentrations of several biologic formulations.

BIOT 120 Protein and virus formulation: Stepping stones toward thermal stability

Whitney Blocher McTigue1, Xue Mi3, Abigail Cabral4, Shari Traiger4, Jeanne A. Hardy5, Caryn Heldt3, Sarah L. Perry1,2, [email protected]. (1) Chemical Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, United States (2) Institute for Applied Life Sciences, University of Massachusetts Amherst, Amherst, Massachusetts, United States (3) Michigan Technological University, Houghton, Michigan, United States (4) Department of Biomedical Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, United States (5) 1021 Lederle Graduate Rsch Tower, UMass Amherst Chemistry Dept, Amherst, Massachusetts, United States

Maintaining the viability of vaccines and other therapeutic biomolecules presents a continued problem for relief programs and medicinal practice. The pharmaceutical industry’s critical reliance on refrigeration to maintain therapeutic efficacy results in almost half of vaccines being discarded annually due to breaks in the cold chain. To overcome this, we propose the use of polypeptide-based complex coacervation as a formulation strategy to concentrate and stabilize biomacromolecules. Complex coacervation is a liquid-liquid phase separation that utilizes electrostatics and entropy to encapsulate target moieties without the use of chemical crosslinking or harsh organic solvents. Complex coacervation enables the sequestration of therapeutic cargo with very high levels of partitioning and encapsulation efficiency. Despite the important role of charge, the use of two-polymer coacervates allows for the encapsulation of even weakly charged biologics at a variety of different conditions, even near their isoelectric point. We studied how variations in the overall composition, solution pH, and how the patterning of charges on the complexing polypeptides affected the partitioning and encapsulation of model proteins, including bovine serum albumin, hen egg white lysozyme, human hemoglobin, and the caspase family of enzymes. Furthermore, we extended our study to include the formulation of porcine parvovirus (PPV) and bovine viral diarrhea virus (BVDV) as model viruses. In particular, incorporation of PPV into complex coacervates resulted in a 100-fold increase in stability during accelerated aging studies at 60°C. This improvement in thermal stability strongly suggests the potential of coacervation-based strategies to decrease our reliance on the cold chain. These results provide a strong foundation toward creating thermally stable therapeutics using complex coacervation as a gentle, purely aqueous method of sequestering novel targets.

BIOT 121

Marvin Johnson award: Targeted therapeutic delivery; can the dream come true?

James R. Swartz, [email protected]. Stauffer III RM 113, Stanford Univ Dept Chem Eng, Stanford, California, United States

Paul Ehrlich proposed the “Magic Bullet” concept 120 years ago. We are still waiting - but there is hope. This talk will describe progress toward a multi-functional, protein based nanoparticle with the potential to realize Ehrlich’s dream. Perhaps more than any other field of endeavor, biochemical technology empowers an iterative progression in which process innovation is motivated by and then enables visionary product innovation. This is especially important for developing a magic bullet nanoparticle. A 2016 review reported the results of over 200 studies seeking to target therapeutics to tumors. The average delivery efficiency was 0.7%! Paradoxically, such studies tell us that our biggest enemy in this pursuit is the human immune system. It is very good at recognizing and destroying perceived garbage. Still, many viruses are all too effective at delivering their nucleic acid cargoes, and they can provide important design concepts and components. We have used the inner capsid from the hepatitis B virus as our starting point. Eight different types of mutations have been introduced while maintaining the ability of the 18kDa HepB core protein to fold and to assemble into a capsid composed of 240 monomers. Cell-free protein synthesis (CFPS) has been instrumental in producing the capsid protein as well as a targeting domain and an immune system avoidance signal. Non-natural amino acids are site specifically introduced to enable precise surface display of the latter two entities. Cargo loading with several hundred drug molecules or with proteins or nucleic acids is accomplished during capsid assembly. Conditional stabilization then retains the cargo and stabilizes the capsid until reducing conditions inside the targeted cells trigger cargo release. Throughout this development project, the flexibility offered by CFPS has been highly beneficial. Multiple new functionalities have been introduced into the virus-like particles (VLPs), and this has motivated several advances in CFPS technology. This iterative progression allowed these modular VLPs to become “smart” nanoparticles. The talk will describe advances in protein synthesis technology and in methods for producing precisely engineered protein-based nanoparticles. Such approaches can now be used for a variety of applications beyond therapeutic delivery. Finally, data from studies with cultured cells and mice will be used to illustrate VLP functionality and delivery performance.

BIOT 122

Utilizing multimodal chromatography and column modeling for antibody-drug conjugate purification

William R. Keller, [email protected], Michaela Wendeler. Purification Process Sciences, AstraZeneca, Gaithersburg, Maryland, United States

Antibody-drug conjugates (ADCs) are an exciting class of therapeutics currently receiving much interest. Their targeted delivery of cytotoxic agents (payloads) gives them a unique and promising position in oncology research. However, the conjugation processes can be very complex and often require removal of conjugation related impurities further downstream in the manufacturing process. Due to the relatively small size of the payloads and their small corresponding selectivity changes, removal of these impurities can be quite challenging using conventional chromatography resins. While hydrophobic interaction chromatography has shown some success separating such impurities, the potential of multimodal chromatography has been less explored. In this work, we present a methodology to identify and optimize suitable chromatographic conditions to remove undesired conjugation variants (i.e. unconjugated and under-conjugated species) using multimodal chromatography. First, filter plate binding and elution screening studies were carried out using several resins, salt types of varying kosmotropic strength, and pH values for both the impurities and products conjugated with two different payloads. These screening studies identified suitable separation conditions that were then carried forward to gradient elution column experiments. From the gradient studies, steric mass-action (SMA) model parameters were estimated for column simulation. The operating conditions were then optimized using the column model and the results were verified experimentally. The results presented here set forth a systematic screening and optimization protocol and demonstrate the utility of multimodal chromatography for ADC purification. Its implementation in process development can help to quickly overcome known challenges in bring these promising therapeutics to clinic.

BIOT 123

Fc gamma receptor as an affinity ligand for antibody glycovariant separation

Maria Livanos1, [email protected], Elizabeth Edwards1, Anja Krueger2, Stuart Haslam2, Anne Dell2, Daniel G. Bracewell1, [email protected]. (1) Biochemical Engineering, University College London, London, United Kingdom (2) Life Sciences, Imperial College London, London, United Kingdom

Biopharmaceuticals continue to grow as a fraction of pharmaceutical sales, the search for new products with increased efficacy often means the detailed glycobiology of the product and its interactions with the immune system are critical. Current products including antibodies and erythropoietin are heterogeneous in their glycosylation state, even though it is known that for example non-fucosylated antibodies are more potent in raising antibody-dependent cell cytotoxicity (ADCC) than fucosylated glycoforms. Current products using CHO cell lines express high levels of fucosylation. Processes capable of homogeneous glycosylation have therefore generated considerable interest and investment. These approaches have generally been upstream focused e.g. to knock out fut8 in CHO and create a fucosylation deficient host cell or engineer human glycosylation in yeast.

Here we propose to take a downstream processing based solution by designing affinity ligands capable of glycoform recognition for use in chromatography. This concept is based on the naturally occurring interaction between antibodies and immune system which is mediated by the Fc gamma receptor. This protein ligand has been used in affinity chromatography to perform both preparative separations and characterise the strength of the Fc gamma/Ab interactions. To be a useful basis for manufacturing such ligands need to have appropriate separation properties.

The wild-type Fc gamma 3a ligand has been shown to have selectivity between fucosylated and non-fucosylated antibody glycoforms. A cloning, expression and purification strategy for this Fc gamma affinity ligand is presented. Characterization of the ligand’s separation properties first required a strategy to create defined glycovariants of a monoclonal antibody product. This was done using an enzyme based strategy to remodel the antibody’s glycosylation and create homogeneous preparations e.g. non-fucosylated structures. The Fc gamma ligand and resulting glycoengineered antibodies were analysed by mass spectrometry. The affinity between the ligand and the defined antibody glycovariants were then characterized and resulting separation properties analysed.

BIOT 124

In silico optimization and robustness analysis of a hydrophobic interaction chromatography step for the purification of a monoclonal antibody

Christopher Gerberich, [email protected], Andre C. Dumetz, Gerald Terfloth. Downstream Process Development, GlaxoSmithKline, King of Prussia, Pennsylvania, United States

Optimization of a polishing chromatography step to robustly remove selected impurities can be a challenging task during the downstream process development of biopharmaceuticals. Since separation of impurities for these steps is driven by varying the salt concentration and pH of the mobile phase, the potential optimization space can be relatively large. Purely experimental development approaches focus the experimental work to obtain a satisfying result but they should not be expected to result in a process that is optimal in terms of productivity and robustness. This work seeks to employ mechanistic models to streamline the development process by using an evolutionary artificial intelligence algorithm to design optimal processes in silico after obtaining model parameters experimentally. A case study is presented for a hydrophobic interaction chromatography (HIC) step designed to separate monomer from aggregate. A mechanistic model was developed using the equilibrium-dispersive model along with a novel HIC isotherm to describe the binding kinetics of monomer and dimer. After fitting and validating the model, evolutionary AI was employed to design the optimal chromatographic sequence to maximize yield under purity and processing time constraints. The AI managed to make significant improvements in yield, processing time, and buffer consumption compared to the platform process. After designing the optimized process, its robustness was compared to the platform process using the Sobol method. The analysis shows that both processes are equally robust in terms of aggregate clearance, but the AI-designed process was significantly more robust in terms of yield. These results demonstrate the effectiveness of in silico process design and its advantage over the traditional, purely experimental development approaches currently used in the industry.

BIOT 125

Manufacturing difficult-to-purify monoclonal antibodies by dual-gradient cation exchange chromatography Diana Koulechova, [email protected]. Agenus, Berkeley, California, United States

Purification of monoclonal antibodies often includes cation exchange (CEX) chromatography as a critical polishing step. This step can be a manufacturability sticking point for low-pI antibodies. We present a case study of one such antibody, discovered to be highly resistant to aggregate resolution by standard methods. We show that it can be effectively purified of both product and process impurities by eluting with a dual salt- and pH-gradient during CEX. The method shows acceptable yield, employs standard buffers and resin, and requires no specialized equipment. Furthermore, we show that it can be optimized for facility fit and scalability, creating a reasonable at-scale process while maintaining acceptable yield, purity, and product quality.

BIOT 126

Case study: AEX resin age impact on chromatographic separation of fusion protein charge isoforms

Timothy Henderson, [email protected]. Bristol-Myers Squibb, Syracuse, New York, United States

In Imaged Capillary Isoelectric Focusing (iCIEF) method, a BMS molecules protein charge variants are separated using high voltage based on their isoelectric point (pI), the pH at which the glycoproteins have a net charge of zero. The distribution of the charge isoforms is primarily driven by the molecules sialic acid content with contributions from C-terminus lysine removal and asparagine deamidation. The protein molecules with more sialic acid moieties are acidic and therefore have an overall low pI compared to the protein molecules with less or no sialic acid groups.

The iCIEF method electropherogram profiles contains three main groups expressed as area percentages and distributed over a pH gradient of 4.22 to 6.14:

Group 1 (Acidic species(pI range 4.30-4.88), High degree of sialyation) Group 2 (Main group (pI range 4.88 -5.50) which represents the majority of charge variants observed in BMS-224818. The group is further subdivided into Group 2A expressed as percentage area ratio to a Reference Material (RM) and Major peaks (M1, M2, M3, M4, M5 and M6) expressed as ratios to a RM. Group 3 (Basic species (pI range 5.50 -6.05), Limited sialyation)

Process Monitoring for drug substance manufacturing processes at Bristol-Myers Squibb Company is performed to monitor process variation to ensure the parameters stay within the established control limits. During routine manufacturing of the BMS molecules shifts in the iCIEF Groups 2 and 3 were noticed beginning with the same drug substance Lot. This shift coincided with the retiring of expired resin and use of virgin resin in the AEX chromatography column. Through lab scale experimentation and statistical analysis, it was identified that AEX resin age had statistically significant effects on iCIEF Groups 2 and 3. As the column ages, the amount of low sialyated species decreases resulting in low iCIEF Group 3 and high iCIEF Group 2. Therefore, the inverse is also true. Low sialylated species are more predominant in lots manufactured using newer QFF resin resulting in lower iCIEF Group 2 and higher iCIEF Group 3.

BIOT 127

Development of a downstream purification toolbox for evaluating and mitigating PS80 degradation

Rachel L. Dyer1, [email protected], Nicholas DiGioia1, Alexandra Tsoras2, Yuanli Song1, Jie Chen1, Jay West1, Sohil Bhavsar1, Deborah Mohammed1, Mathura Raman1, Zhihua Liu1, Sanchayita Ghose1. (1) Bristol Myers Squibb, Devens, Massachusetts, United States (2) Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States

Polysorbate 80 (PS80) is one of the most commonly used surfactants in therapeutic protein formulations to prevent interface-induced protein adsorption, protein aggregation, or particulate formation. PS80 degradation may impact therapeutic protein quality and shelf life of the drug product. One of the PS80 degradation pathways is hydrolysis of the fatty acid ester bond by lipases, which is a type of host cell protein (HCP). The majority of HCPs are cleared in the downstream process, but a small population has shown to be problematic as they co-purify with the product of interest. In this work, we focused on downstream purification and explored the impact of downstream process conditions such as mobile phase and stationary phase properties on the clearance of problematic HCP populations and investigated the impact on PS80 degradation. To evaluate PS80 degradation, we applied multiple orthogonal assays and provided a mechanistic understanding by investigating conditions that impact lipase activity. The results show that, for multiple molecules, Protein A wash buffer, depth filtration, and certain polishing chromatography resins can remove problematic HCPs and reduce PS80 degradation. Based on this work, we were able to establish a downstream purification toolbox to enhance HCP removal and control PS80 degradation.

BIOT 128

Development of a robust and high throughput bispecific process which includes controlled fab arm exchange and hydrophobic interaction chromatography

Joseph A. Sakyiama1, [email protected], Amin Salehi2, Robert Ferrari1, Michael Wineburg1. (1) Active Pharmaceutical Ingredient Large Molecule, Janssen Pharmaceutical Company, Lower Gwynedd, Pennsylvania, United States (2) Building 1, Janssen Biotech, Malvern, Pennsylvania, United States Janssen has several Bispecific programs at various clinical phases. Some are Bispecific antibodies (BsAb) generated through a DuoBody® controlled Fab Arm Exchange (cFAE) process from different parental monoclonal antibodies. We have developed a highly efficient cFAE process, but residual parental contaminants remain and typically removed using bind-elute chromatography. Amongst the various options to remove residual parental mAbs, hydrophobic interaction chromatography (HIC) can be a powerful tool but generally has low capacity. This presentation explores the development of a robust and high throughput Bispecific process which employed cFAE and HIC to remove residual parental mAbs. The cFAE was robust for bispecific yield and non-reduce cSDS purity and high throughput. Development of the cFAE required an understanding of the chemistry and kinetic principles of the reaction to optimize critical parameters. The cFAE step is able to achieve bispecific yields of 93% with 35g/L IgG concentration and >97% non-reduced cSDS Bispecific purity. At this step, the sum of contaminant parental mAb levels are approximately 7%. The HIC had to be efficient at removing the parental residues and have high capacity. To optimize the HIC step, a strong understanding of the properties of different hydrophobic resins and the influence of salts on protein hydrophobicity were utilized. An HIC step capable of achieving 80g/L and over 99% bispecific purity was developed. Characterization data shows the BsAb is correctly assembled by analysis via peptide map, free sulfhydryl, intact mass, cIEF, and preserved binding activity of each parental arm.

BIOT 129

Evaluating the virus reduction potential of a mixed-mode chromatography media during early phase downstream development

Akunna Iheanacho2, [email protected], Coral Fulton2, Xuemei He1, William H. Rushton1, Irene Chen1, Louisa Vang1, Katelyn Pritchard1, Jenifer Dean2, Kaye Peden2, Sreyry Schaerdel2, Erin Reynolds2. (1) Bio-Rad Laboratories, Hercules, California, United States (2) Texcell North America, Inc., Frederick, Maryland, United States

To ensure the safety of biopharmaceutical products, downstream unit operations should be evaluated to determine their viral clearance capacity during process development. In this case study, we utilized a DoE approach to investigate the effects of buffer pH and conductivity on the removal of product and process-related impurities by a new mixed- mode chromatography media. Results from this study have offered insights on the interactions between this chromatography media and virus particles, as well as the design space for the removal of viral contaminants.

BIOT 130

Engineering TATA Box to create a promoter library to balance heterologous pathways in Y. lipolytica vijaydev ganesan, [email protected], William Burnette, Patrick Dayton, Mark A. Blenner. Chemical Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

Metabolic engineering requires precise control of gene expression to improve flux through target molecules. Oleaginous yeast Yarrowia lipolytica has been emerging as an industrial workhorse due to its high flux lipids biosynthetic pathways; however, fewer tools to control gene expression are available compared to model yeast S. cerevisiae. To date, most approaches to control gene expression use endogenous promoters or hybrid promoters. Both types of promoters have difficult to anticipate responses to different substrates and genetic contexts. Here, we develop a tunable expression system in Y. lipolytica by mutating the TATA Box region in different promoters. The 8bp TATA Box region was randomized through degenerate bases and promoter libraries created to express hrGFP. We used FACS to isolate TATA Box mutants resulting in different expression levels and validated individual mutants through RT-qPCR. The correlation between fluorescence intensity, relative transcriptional level and relative translational level is evaluated over time in the context of four commonly used yeast promoters TEF, TEF-Intron, EXP and POX3. To demonstrate the utility of finely tuned expression systems, we used the expression system to balance a multienzyme heterologous pathway for naringenin synthesis. This work generates a simple and reliable system for tuning the expression system in Y. lipolytica by modifying the TATA Box to balance a heterologous pathway

BIOT 131

BIOT Young Investigator Award: Using RNAs as a built-in sensors of cell biology

Lydia M. Contreras, [email protected]. University of Texas at Austin, Austin, Texas, United States

RNA has long been known to serve key roles in information flow of the central dogma, but its functional role in regulating gene expression across all life-forms, under varying environmental conditions, has become increasingly intriguing. From an application stance, our ongoing understanding of RNA functions, beyond their catalytic roles, have revolutionized our thinking of biotechnology, medicine, agriculture etc. Yet, deciphering underlying principles of structure-function relations that describe biochemical properties of RNAs remains a major challenge, hindering progress in our full comprehension of the wide biocatalytic roles that are suspected for these molecules. In part, this is due to the fact that mapping how the biochemistry and structural arrangements of RNAs give rise to their functions within the cell, in the context of complex cellular regulation and biological constraints, is not an easy task. In this talk, we will discuss current knowledge of RNAs as sensors and transducers and discuss our advancements in the development of quantitative, high-throughput methods that examine fundamental chemical paradigms of RNA function in vivo. We will focus in particular on the natural roles of RNAs as monitors of biomolecular processes that are particularly important during cellular stress responses. We will also discuss our work in this field and the new paradigms of RNA function that we are uncovering so that these molecules can be rationally redesigned for novel synthetic roles.

BIOT 132

Optogenetic circuits for dynamic control of Escherichia coli metabolism

Makoto A. Lalwani, [email protected], Jose L. Avalos. Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States

The ability to induce gene expression in Escherichia coli is a cornerstone of basic and applied research in the life sciences and biotechnology. Optogenetic regulation of gene expression offers additional capabilities such as easy tunability, reversibility, dynamic regulation of induction strength, and the ability to exert control without the expense and potential side effects of chemical inducers. Here, we develop optogenetic circuits for the bacterium Escherichia coli that induce gene expression in darkness and repress it under blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production compared to chemically induced controls in light-controlled fermentations that are scalable to at least lab-scale bioreactors. Furthermore, we show that our circuits can be used to control protein production with light. Our results show that light is a suitable alternative to chemical induction for microbial production of chemicals and proteins.

BIOT 133

Genomics and transcriptomics reveal genes involved in isobutanol tolerance and production in Saccharomyces cerevisiae

Sarah K. Hammer1, [email protected], Kouichi Kuroda4, José Montaño López1, Jose L. Avalos1,2,3. (1) Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States (2) Andlinger Center for Energy and the Environment, Princeton University, Princeton, New Jersey, United States (3) Molecular Biology, Princeton University, Princeton, New Jersey, United States (4) Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan

The four-carbon branched-chain higher alcohol, isobutanol, is an attractive advanced biofuel with fuel properties superior to ethanol. However, its toxicity to cells presents a major challenge when engineering microbes to produce it at economically competitive titers. In this work, we screened the Saccharomyces cerevisiae gene-deletion library to identify genes involved in isobutanol tolerance. We found that strains lacking genes encoding NADPH-generating enzymes in the pentose phosphate pathway are hypersensitive to isobutanol and other higher alcohols, but not to ethanol. In contrast, deletion of a transcriptional activator involved in nitrogen catabolite repression significantly increases yeast tolerance specifically to branched-chain higher alcohols. Transcriptomic data revealed that isobutanol prompts a nitrogen starvation response, which upregulates amino acid biosynthesis and nitrogen import while downregulating glycolysis, cell wall biogenesis, and membrane lipid biosynthesis, processes important for cell growth. Furthermore, we found that deletion of a single gene can disrupt this response, enhancing tolerance and boosting isobutanol production as much as 4.9-fold in engineered strains. This study illustrates how gene deletions can boost production by evading adaptive mechanisms to tolerate stress.

BIOT 134

Genetic devices for controlled gene expression in Clostridium

Nicholas R. Sandoval1, [email protected], Rochelle C. Joseph1, Nancy M. Kim2. (1) Chemical and Biomolecular Engineering, Tulane University, New Orleans, Louisiana, United States (2) BioInnovations PhD Program, Tulane University, New Orleans, Louisiana, United States

Control of gene expression on the transcription level is vital in synthetic biology for the construction of complex and sophisticated tools. To achieve such control, synthetic genetic parts must be developed that can enable such control. Although much recent progress has been made in transcription factor-based biosensors for workhorse organisms, non-model organisms such as Clostridium still lack tools for rapid and customizable gene expression control. Clostridium species are biotechnologically relevant, but do not have a large number of transcription regulation devices, reducing the speed of their engineering. In this work, we show two systems for transcriptional control in bacterial systems. First, we demonstrate a Cas12a-based CRISPRi gene repression system for the tuning of biosynthetic pathways. Cas12a CRISPR effector proteins are better suited for use in Clostridium due to the genus’ AT-rich genomes and the corresponding simple and T- rich protospacer adjacent motif. We demonstrate in Clostridium pasteurianum tunable repression based on proximity to regulation elements, strand, and number of targeted sites through reporter genes, transcription levels, and redistribution of carbon flux. Second, we demonstrate optimization of transcription factor (TF) DNA binding sites through both a rationale and high-throughput naïve approach (sort-seq). We engineer a set of engineered product-responsive transcription factor regulated promoters (biosensors) for real-time single cell monitoring of butanol accumulation. We demonstrate tunability of gene expression though altering the number, location, and sequence of the transcription factor binding sites. Additionally, we show transcription factor-operator binding energy plays a key factor in the tunability of such systems. Biophysical characteristics between the TF-operator are evaluated using surface plasmon resonance (SPR). This work can enable rational strategies to edit the dynamic range of transcription factor-based biosensors.

BIOT 135

Genetic tools for engineering Pseudomonas putida for the heterologous production of bacterial natural products Taylor B. Cook1, [email protected], Aditya Ailiani1, Brian Pfleger2. (1) Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States (2) Department of Chemical and Biological Enginee, University of Wisconsin Madison, Madison, Wisconsin, United States

Pseudomonas putida has shown promise as a heterologous host for producing polyketides and non-ribosomal peptides, natural products that commonly have medically-relevant bioactivities. Unfortunately, titers of polyketides and non-ribosomal peptides produced heterologously in P. putida are often low compared to native hosts. Recent reports were potentially hindered by poorly characterized genetic tools, such as weak synthetic promoters and ribosome binding sites and unstable replicative vectors. However, there has recently been substantial progress in the development of genetic tools for P. putida, including characterized plasmids, constitutive and inducible promoters, and several CRISPR-based genome-editing methods. To demonstrate the utility of our own CRISPR/Cas9-assisted genome-editing method for integrating heterologous gene clusters into P. putida, we have introduced a biosynthetic gene cluster (BGC) responsible for the synthesis of prodigiosin, a hybrid polyketide/non- ribosomal peptide with antibacterial and antitumor properties. Prodigiosin is a red pigment that is easily detectable and provides a facile screen for identifying mutants of P. putida with altered production of prodigiosin. We are actively screening for mutants that improve or abolish prodigiosin production to better understand how to engineer P. putida for the heterologous production of polyketides and non-ribosomal peptides.

BIOT 136

Enabling scalable, quality-by-design (QbD)-driven biomanufacturing of cell therapies: 10 year roadmap from the NSF ERC on cell manufacturing technologies (CMaT)

Krishnendu Roy, [email protected]. Georgia Tech, Atlanta, Georgia, United States

With the FDA approval of the first two cell therapies and several in the pipeline, therapeutic cells have rapidly emerged as a critical product platform in the biotech and pharma industry. However, for most cell-based therapeutics and regenerative medicine products little is known about their mechanisms of action (MoA), critical quality attributes (CQAs), and critical process parameters (CPPs) that could enable quality-driven manufacturing. In fact, understanding what “quality” means for a particular cell product and how to manufacture those cells with consistent and reproducible quality, at large scale, and at a cost significantly lower than the current approved products are the fundamental challenges facing the cell therapy field. The Center for Cell manufacturing Technologies (CMaT), and NSF-funded Engineering Research Center (ERC) is an eight university public-private consortium that is working closely with industry, academia, and government organizations to enable QbD driven manufacturing of cell therapies by developing new tools and technologies for CQA and CPP identification of complex cell products, real time monitoring of cell-manufacturing processes and products, rapid in- line or at-line analytics, feedback-controlled flexible bioprocessing, efficient supply chain management, scalable manufacturing of the most potent and safe cells, and the development of standards and best practices. In this talk I will provide an overview of our industry-led roadmap to achieve QbD driven cell manufacturing and show specific examples of projects and results from various CMaT funded projects in this area.

BIOT 137

T cells redirected with biepitopic and bispecific antibody mimic receptors for cancer immunotherapy

Rihe Liu, [email protected]. Eshelman School of Pharmacy, UNC Chapel Hill, Chapel Hill, North Carolina, United States

Tumors are inherently heterogeneous in antigen expression, and escape due to antigen loss remains one of the major limitations of targeted immunotherapy. Despite the remarkable clinical activity of adoptive therapy with chimeric antigen receptor (CAR) redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAA) may thus significantly improve the outcome of CAR-T cell therapies. To achieve this goal and overcome current drawbacks of the CAR technology in multiple targeting, we have engineered single domain antibody mimics to generate highly compact and stable ligands that, when combined with signaling molecules of T cells, redirect T cells with biepitopic or bispecific TAA recognition. We show that various classes of antibody mimics targeting EGFR and HER2 of ErbB receptor tyrosine kinases can be assembled into receptor molecules, which we call antibody mimic receptors (amR), and redirect T cells for biepitopic or bispecific TAA recognition in vitro and in vivo (Cancer Immunol Res. 2019;7(5):773-783).

BIOT 138

Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy

Lawrence A. Stern1, [email protected], Xin Yang1, Ignacio Moraga Gonzalez2,3,4, Brenda Aguilar1, Joseph R. Cohen1, Renate Starr1, Stephen J. Forman1, K Christopher Garcia2,3,4, Christine E. Brown1. (1) Beckman Research Institute, City of Hope, Duarte, California, United States (2) Howard Hughes Medical Institute, Stanford Univeristy School of Medicine, Stanford, California, United States (3) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, United States (4) Department of Structural Biology, Stanford University School of Medicine, Stanford, California, United States

Chimeric antigen receptor (CAR) design incorporates a binding moiety to direct T cell activation toward an individual cancer biomarker that a T cell would not normally recognize. Although antibody variable fragments are commonly used for their versatility in antigen recognition, naturally occurring ligands are also capable of directing specificity. One such ligand, IL13, has been incorporated as the binding moiety for CAR T cell therapy of IL13 receptor α2 (IL13Rα2) expressing glioblastoma multiforme (GBM). This IL13-CAR T cell therapy has shown notable safety and success in early stage clinical trials via local delivery. IL13Rα2 is an attractive target for many malignancies and has rare normal tissue expression, motivating application of systemic delivery of IL13-CAR T cells. However, IL13Rα1, the other receptor for IL13, has low level ubiquitous expression on normal tissue. Notably, IL13Rα1 and ILR4α, a high affinity receptor pair for IL13, are co-expressed in lung tissue. This normal tissue expression raises concerns for systemic application of IL13-CAR T cells including 1) impedance of CAR T cell trafficking, 2) early exhaustion of CAR T cells due to antigen experience on route to tumors, and 3) on-target off-tumor toxicity. Herein, we describe the engineering and investigation of CARs incorporating IL13 mutants (termed muteins) with markedly enhanced selectivity toward IL13Rα2-binding. Structure-guided mutagenesis of IL13 yielded two mutants that retain binding affinity for IL13Rα2 while diminishing affinity for IL13Rα1 >1,000-fold (termed C4 and D7). CARs incorporating these muteins direct T cell-mediated cytotoxicity against IL13Rα2-expressing tumors identically to IL13-CAR T cells both in vitro and in an orthotopic GBM model. Importantly, both muteins display significantly diminished T cell activity against IL13Rα1-overexpressing tumors in vitro, but only the C4 variant spares IL13Rα1 and IL4Rα co-expressing tumors in vitro and in vivo. T cell trafficking studies illuminate potential advantages of decreased IL13Rα1 interactions in the mutein-CAR context.

BIOT 139

DNA-RNA hybrid materials via dual enzyme polymerization for carrier-free therapeutic RNA delivery

Sangwoo Han, [email protected], Hyejin Kim, Jong Bum Lee. Chemical Engineering, University of Seoul, Seoul, Korea (the Republic of)

Development in nucleic acid nanotechnology has been attracted with their therapeutic functions and nontoxicity. As one of them, rolling circle replication (RCR) has been considered as a powerful tool to synthesize various nucleic acid-based architectures. We developed a novel enzymatic synthetic approach to produce cell-targetable DNA- RNA hybrid nanovectors. This material was generated by the dual enzyme polymerization, especially rolling circle amplification (RCA) for DNA and rolling circle transcription (RCT) for RNA with enhanced resistance to nuclease degradation compared to the natural DNA and RNA. DNA strands contains tumor-specific aptamer sequence and RNA strands includes the siRNA precursors resulting in the carrier-free tumor-targeted siRNA delivery. In further steps, DNA-supported RNA hydrogel embedding the therapeutic RNA particles was also fabricated. In previous studies, there are few reports in regard to RNA hydrogel due to the structural instability. To overcome this limitation, we employed dual polymerization resulting in macroscale RNA-based structure. This hydrogel is produced by self-assembly of DNA and RNA and their direct hybridization. This system is considered as a smart double-sustained release of RNA particles from hydrogel then siRNA from the particles. As a proof of concept, the hydrogel was applied to tumor-targeted green fluorescence protein (GFP) silencing assay by the double-releasing system in physiological condition. Taken together, our strategy for the synthesis of DNA-RNA hybrid materials has high potential to capacitate further exploration in gene and cancer therapy.

BIOT 140 Functionalized vitronectin peptide surfaces for stem cell differentiation towards the hepatic lineage

Kevin Ortiz-Rivera1, [email protected], Chun Wang3, Wei-Shou Hu2. (1) Biomedical Engineering, University of Minnesota-Twin Cities, Minneapolis, Minnesota, United States (2) Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota, United States (3) Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, United States

Human pluripotent stem cell (hPSC)-derived hepatocytes represent an unlimited source of cells for transplantation therapies, drug metabolism studies, and disease modeling. Current in-vitro hepatocyte differentiation protocols rely upon the use of undefined and animal derived cell adhesion substrates like Matrigel. To circumvent this challenge, defined human recombinant vitronectin and laminin have been used as substrates for directed-liver differentiation; however, they are susceptible to degradation, batch-to- batch variability, and high production cost. Synthetic peptides offer potential advantages due to their chemical stability and ability to be incorporated in different cell culture platforms beyond flat surfaces. In this study, we replaced Matrigel with a vitronectin- derived peptide (Ac-KGGPQVTRGDVFTMPC) as an adhesion substrate for human embryonic stem cells (hESCs) and their directed differentiation towards hepatocyte-like cells (HLCs) with simultaneous cell expansion. hESCs attached to the vitronectin (VN) peptide immobilized glass surfaces, proliferated, and expressed pluripotent markers after short term culture prior to hepatic differentiation. hESCs were successfully differentiated to endoderm cells, expanded, and further guided toward hepatocytes over 20 days following a stepwise differentiation protocol. Comparable level of expression of hepatocyte-specific transcripts, including albumin and alpha-1-antitrypsin (AAT), was observed between endoderm expanded HLCs on Matrigel and the VN peptide surface. Albumin and hepatocyte nuclear factor 4 alpha (HNF4a) protein expression was confirmed by immunofluorescence staining. Cells derived on the VN peptide surface exhibit characteristic morphological traits of hepatocytes derived in Matrigel such as polygonal shape and binucleation. In addition, functional studies revealed similar albumin secretion profile, drug-induced cytochrome P450 activity, and glycogen storage. Overall, we have demonstrated the application of vitronectin peptide modified surface as a completely synthetic and defined platform for the derivation of hepatocytes from hESC.

BIOT 141

Survival of aging CD264+ and CD264- populations of human bone marrow mesenchymal stem cells is independent of colony-forming efficiency

Sean Madsen1, Sean Jones2, Alan Tucker2, Margaret Giler1, Dyllan Muller1, Carson Discher1, Katie Russell1, Georgina Dobek2, Mimi Sammarco2, Bruce Bunnell2, Kim OConnor1, [email protected]. (1) Dept. of Chemical & Biomolecular Engineering, Tulane University, New Orleans, Louisiana, United States (2) School of Medicine, Tulane University, New Orleans, Louisiana, United States In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to non-engraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM-MSC) cultures. Sorted CD264+ hBM-MSCs from two age-matched donors have elevated b- galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264- hBM-MSCs. Counterintuitive to their aging phenotype, CD264+ hBM-MSCs exhibited comparable survival to matched CD264- hBM-MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony-forming efficiency. These findings have ramifications for the preparation of hBM-MSC therapies given the prevalence of aging CD264+ cells in hBM- MSC cultures and the popularity of colony-forming efficiency as a quality control metric in preclinical and clinical studies with MSCs.

BIOT 142

Characterization of the effect of extended K562 stimulation on natrual killer cell functionality for emergent off-the-shelf allogeneic therapy applications

Jennifer One1, [email protected], Frank Cichocki3, Wei-Shou Hu2, Samira Azarin2. (1) Biomedical Enginneering, University of Minnesota, Minneapolis, Minnesota, United States (2) Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, United States (3) Medicine Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, Minnesota, United States

Natural killer (NK) cells, which have both cytotoxic and cytolytic effector functions to kill cancer cells, are an ideal allogeneic therapy as they do not induce Graft-vs-Host disease and can be expanded by several orders of magnitude through multiple rounds of activation with K562 artificial antigen presenting cells. However, the changes that extended stimulation of NK cells might incur in cell cycle status, metabolism, and functionality necessary for production of sufficient cell quantities are not well understood and thus were the focus of this study. We observed that repeated activation allowed for 106-108 fold expansion, although a reduced growth rate was observed from approximately day 21 onwards, potentially signifying the presence of senescent cells in later stages of culture. Flow cytometry was used to further evaluate expression of various markers for exhaustion while functional cell cytotoxicity assays were used to determine the presence of important secreted factors for NK cell functionality. IncuCyte assays measured the rate of killing during the different growth phases. Together, this allowed us to explore the relationship between NK proliferative capacity and cytotoxic ability. RNA-sequencing analysis was used to explore activation-associated gene expression dynamics, while ATAC-sequencing was utilized to unveil the dynamic nature of epigenetic/transcriptional modifications during NK cell activation. Activation was found to be accompanied by an increase in oxygen consumption and upregulation of important metabolic pathways, demonstrating that metabolism is an important indicator of activation. Extensive characterization of prolonged K562 stimulation through transcriptome analysis, dynamics of chromatin accessibility, metabolic characterization, and phenotypic analysis of exhaustion and senescence allows us to evaluate the activation and expansion process of NK cells over continuous long-term culture. Insights into mechanisms underlying activation and expansion will provide a path to develop strategies for large-scale biomanufacturing while maintaining functionality, thus laying the groundwork as a potential off-the-shelf allogeneic cellular therapy.

BIOT 143

Harnessing expertise for creating smart automated image analysis: Training an AI for segmentation and classification of pleomorphic particles in nsTEM for gene therapy

Max Pihlström, [email protected], Saba Hussein Gore. Vironova AB, Stockholm, Sweden

Negative stain transmission electron microscopy (nsTEM) can provide an informative and unique insight into the characterization of viral gene delivery platforms. Traditionally, the analysis of such nsTEM images required an experienced microscopist to identify and categorize the particles of interest.

A neural network pre-trained on thousands of nsTEM images on a broad range of expert segmented viral morphologies is used as an adaptive backbone for segmentation and classification of new nsTEM material, specifically geared towards the gene and cell therapy field.

Using only a handful of image examples, a user can guide the pre-trained network and instantly obtain automatic and deterministic segmentation and classification of the new images.

Leveraging a solid backbone model, the network can adapt to both heterogeneous and pleomorphic new particles. This versatility enables investigatory analyses in a fast and continuous workflow. This is particularly useful when analyzing for example lentiviral vectors, where a pre-set definition guideline for detection is challenging.

This morphological characterization of gene therapy delivery platforms can support and speed up the drug development process.

BIOT 144

Development of a bioreactor system in production of cell-based hormone replacement therapy through multilayer cell encapsulation Kayla Thompson1, [email protected], Elizabeth Caraballo-Torrealba1, Sittadjody Sivanandane4, Emmanuel C. Opara4, Neil D. Danielson3, Jason Berberich2, Justin M. Saul1. (1) Chemical, Paper and Biomedical Engineering, Miami University, West Chester, Ohio, United States (2) Chem, Paper, Biomed Eng, 64 Eng Bldg, Miami University, Oxford, Ohio, United States (3) Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, United States (4) Wake Forest School of Medicine, Winston-Salem, North Carolina, United States

Osteoporosis, urogenital complications and other health risks are common in women who have a loss in ovarian hormones due to menopause or other causes of ovarian failure. Pharmacological hormone replacement therapy (pHRT) has been used for decades in women who have gone through menopause. However, in the past 20 years, pHRT use has decreased significantly due to concern over increased risk of heart disease and certain cancers. A variety of aspects may contribute to the risks associated with pHRT including age, the menopausal stage of the patient, the mode of delivery (e.g., transdermal versus oral), and delivered hormone dose. Another possible aspect is the presence of only one or two hormones (estrogen alone or in combination with progesterone) despite the complex cascade of hormones (e.g., inhibin, activin, testosterone) involved in the hypothalamus-pituitary-ovary (HPO) axis responsible for many aspects of a woman's physiological health. Our team has recently developed a cell-based hormone replacement therapy (cHRT) that can address several of these issues through a design in which two key ovarian cell types are in close proximity to each other as needed for ovarian hormone synthesis in native ovarian follicles. The cHRT constructs use a multilayer encapsulation technique to compartmentalize theca cells (TCs) and granulosa cells (GCs) to create a three-dimensional construct that mimics a native follicle structure. For these cHRT constructs, GCs are encapsulated in alginate, coated in poly-L-ornithine (PLO), then mixed with TCs in a 2nd layer of alginate. These biomimetic constructs have been shown to produce hormone concentrations similar to physiological levels both in vitro and in vivo. The implantation of such constructs, however, could be a regulatory challenge, so we are pursuing an ex vivo approach to production and characterization of the cell products. In this work, we are developing methods for the detection of ovarian hormones produced by the cHRT constructs by High Performance Liquid Chromatography with fluorescence detection (HPLC-FLD). Such techniques allow determination of ovarian hormone derivatives at low concentrations and that would be a better alternative to enzyme-linked immunosorbent assays.

BIOT 145

Understanding the effect of benzonase endonuclease on downstream purification of adeno-associated viral vector (AAV) manufacturing

Abhiram Arunkumar, [email protected], Blake Hotz, Nripen Singh. Technical Operations, Voyager Therapeutics, Cambridge, Massachusetts, United States Adeno-associated viruses (AAVs) are an emerging modality as promising vectors for gene therapy. The manufacturing process involves cell expansion, infection and subsequent purification processes, which involves adventitious viral inactivation, cell lysis, followed by several chromatography and ultrafiltration/Diafiltration steps. This work examined the use of benzonase® endonuclease in the clarified lysate for nucleic acid (DNA) digestion and its impact on downstream processes that involve affinity purification and polishing chromatography. The optimized conditions for benzonase® endonuclease were chosen to maximize the enzyme activity on DNA digestion and the effect of benzonase® treatment on the performance of the affinity step was studied in combination with several wash and elution conditions. The implications of using benzonase® treatment on the lifecycle of the affinity resin was also evaluated and the implications of this treatment step on impurities at the drug substance level was assessed. A guideline for an optimized process that balances AAV recovery, AAV purity, processing time and manufacturability is provided based on the outcomes of this work.

BIOT 146

Bridging of the synthetic chemistry, small molecules and biologics development and manufacturing strategies to deliver novel antibody drug conjugates

Engin Ayturk, [email protected], Tom Wagler, Michael Kaufman. Mersana Therapeutics, Shrewsbury, Massachusetts, United States

Mersana Therapeutics is a clinical-stage biopharmaceutical company with an innovative and differentiated antibody drug conjugates (ADCs) pipeline that is built upon novel linker platforms such as Dolaflexin® and Dolasynthen that deliver proprietary auristatin- based payloads via the state-of-the-art bioconjugation strategies. The unique hydrophilicity of the Dolaflexin platform enables the engineering of ADC therapies with high drug to antibody ratios (DAR) with an enhanced therapeutic window for the treatment of multiple cancers. In addition to managing a relatively complex supply chain architecture against aggressive timelines for process development, scale-up and technology transfer activities, dedicated CMC efforts led to the successful completion of several at-scale cGMP campaigns for the synthesis and manufacturing of key process intermediates. In order to deliver robust manufacturing processes with well-defined process controls in-place, we have conducted thorough product and process risk assessments to identify the development gaps and areas of focus to further process understanding and characterization scope, leveraging synergies between synthetic chemistry, small molecule and antibody production, and bioconjugation steps. This presentation will provide an overview of Mersana’s novel ADC platform and discuss the QbD-based process development and scale-up strategies via case studies focusing on purification and separation unit ops.

BIOT 147

How fast can we go? HPLC scouting system for accelerated method development of bispecific antibodies

James Martosella, [email protected], Gulnur A. Elove, Jessika S. Feliciano. BioTD Analytical Development, Janssen Research and Development, Malvern, Pennsylvania, United States

Bispecific monoclonal antibodies (bsAb) offer enhanced therapeutic efficacy over traditional monoclonal antibodies (mAb), but present unique analytical challenges related to their design and manufacturing strategy. As bispecifics production processes evolve, process development and comprehensive characterization studies are necessary to increase understanding and deliver a robust manufacturing process with desired product quality. Developing robust analytical methods is critical in the identification and quantitation of critical quality attributes and ensuring process consistency.

For bispecifics, the variety of unique manufacturing process brings challenges in the development of analytical methods not only to monitor the assembly process, but to subsequently separate the bispecific molecule from undesired impurities. For some bispecific molecules, the molecule assembly and subsequent removal of residual homodimer require sensitive and robust analytical methods to both quantify complete bsAb formation and to assess final bsAb purity.

In this work, we have evaluated a new HPLC Method Scouting System and Simulation Software in the development of analytical methods for bispecific molecules. Specifically, we have developed a rapid RP-HPLC in-process monitoring test for profiling the bispecific antibody and its potential fragments after manufacturing process assembly. This presentation will highlight the complete analytical design evaluation of the scouting system and simulation software in the development of analytical methods for profiling partially formed bispecific molecules. Advantages and limitations of both traditional and scouting HPLC systems will be discussed.

BIOT 148

Overcoming manufacturability challenges for bispecific antibodies through evolution of a platform process

Dane Grismer, [email protected], Yogender Gowtham, Srivatsan Gopalakrishnan, David Chang, Niket Bubna, Gautam Nayar, Sigma Mostafa. Process Development, KBI Biopharma, Research Triangle Park, North Carolina, United States

Bispecific antibodies present numerous challenges to the development of robust manufacturing processes. Low titers resulting from low cell line productivity, difficulties achieving product quality targets, and degradation of the antibody often lead to long development timelines and delays in delivering material to the clinic. As a contract development and manufacturing organization (CDMO), we have evolved a platform process over five years with more than 20 bispecific antibodies. Refinement of seed train, production, and harvest processes was undertaken for two families of bispecific molecules consisting of at least four molecules each, and for a wide array of other bispecific formats. Early implementation of the platform often required extensive screening and optimization studies to achieve acceptable performance, particularly for the production process. These adjustments to the process were necessary to achieve high titers and to modulate product quality targets such as homodimer levels. The introduction of remediation strategies in the harvest process has successfully prevented degradation of the product that occurred with multiple molecules. Strategies have also been introduced in the overall workflow to uncover the unique aspects and challenges for each new molecule early in the development timeline. Utilization of stable pools of clones provide an initial performance baseline and early testing of the harvest process. Conducting clone selection in parallel with other process development activities ensures that the final clone performs well with the final process. Overall, incremental improvements to the platform process have made it more robust and enabled development efforts to be more targeted for each new molecule.

BIOT 149

Crystallization of peptides: Case of glycine homopeptides

Mingxia Guo2, Ian Rosbottom2,1, [email protected], Jerry Y. Heng2. (1) Chemical Engineering, University of Leeds, London, United Kingdom (2) Chemical Engineering, Imperial College, London, United Kingdom

Amongst the biologic drugs family, peptides, as polymers of amino acids, play important roles in human physiology and are hence an important class of therapeutics. The unique selling point of peptides is that they are selective, which translates into them being efficacious, safe and tolerable for administration to humans. The economic bottleneck of peptide manufacture, analogous to many biomolecules, is the challenges associated with purification as chromatographic methods are most widely used. However, advances in crystallisation optimisation methods has resulted in this technique being a genuine alternative to chromatography for peptide purification. Here we present the nucleation kinetics, derived from induction time data, of a homologous series of mono-, di- and tri-glycine. The induction time is found to increase with increasing chain length of the peptide. However, the increase in the induction time between di- and tri-glycine is found to be much lower than between mono- and di- glycine. This is found to be due to the preferential nucleation of a tri-glycine di-hydrate structure, rather than pure tri-glycine. A single crystal of this form was isolated, where the coordinates of the crystal structure are solved for the first time. Molecular modelling examines the energetically favourable molecular conformation and intermolecular solute/solute and solute/solvent interactions, to further uncover the molecular mechanisms behind the experimental observations. In particular, the crystallisation of the tri-glycine hydrate is examined, with respect to why of the three molecules only the tri-glycine presents a hydrated structure. This study uncovers detailed molecular chemistry, which underpins the nucleation of peptides of different chain lengths, providing insight into how the self-assembly mechanism changes from amino acid, to peptide to protein. This information is fundamental to maturing crystallisation as a viable peptide and protein purification technique.

BIOT 150

Rapid downstream process development of an in-licensed non-platform protein

Timothy Tully, Benjamin Huffman, Caitlin Windsor, [email protected], Nicole Schonenbach, Joseph Silhavy. Pfizer, Inc., Chesterfield, Missouri, United States

A recent trend in drug development by established biotech companies has been the acquisition (in-licensing) of promising drug candidates. An established biotech company plays the role of providing the resources and expertise needed to bring this drug quickly to patients. But, based on the development experiences of the innovator company, process development of in-licensed drugs can be challenging.

This presentation will discuss the challenges and learnings from the rapid process optimization of an acquired early stage candidate to ensure Clinical trial material would meet Pfizer’s high quality standards. Increased complexity arose due to the stage of development when the molecule was acquired and the candidate design where platform techniques were not applicable.

Development was enabled by Pfizer’s high throughput screening lab to characterize the existing process as well as test alternative chromatography resins using batch binding and RoboColumns. Improvements were verified against the current process during lab and pilot scale confirmation runs. Optimization of this 4-column process was performed in 4 weeks and process changes were implemented immediately during the ongoing GMP manufacturing campaign.

BIOT 151

Noncovalent derivatization: Sustainable interfaces between molecules and between disciplines

John C. Warner1,2, [email protected]. (1) Warner Babcock Institute for Green Chemistry, Wilmington, Massachusetts, United States (2) Chemistry, Monash University, Clayton, Victoria, Australia

The defining text “Green Chemistry: Theory and Practice” and the 12 principles of Green Chemistry were published twenty years ago in 1998 by John Warner and Paul Anastas. There are now dozens of Green Chemistry textbooks, journals, conferences, university courses and degree programs around the world documenting molecular level mechanistic approaches to reducing or eliminating negative impacts of materials and products on human health and the environment. In the early days of Green Chemistry, Warner, as an industrial chemist, develop the concepts of Noncovalent Derivatization, a first-principles approach to designing multimolecular complexes to control chemical and physical properties. In 2014 he received the Perkin Medal (consider the highest honor in US Industrial Chemistry) for this work. This presentation will discuss Warner’s basic concepts of Green Chemistry and Noncovalent Derivatization. Examples of technologies developed and commercialized at the Warner Babcock Institute for Green Chemistry will be described.

BIOT 152

Development of a novel electrofuels platform for biochemical production from CO2 using synthetic designed proteins and non-growing cells

Nadim Massad1, Jonathan Preston2, Eskil Andersen2, Aleksandr Uvaydov2, Ronald L. Koder2, Scott Banta1, [email protected]. (1) Chemical Engineering, Columbia University, Fairfield, Connecticut, United States (2) Marshak Building Room 419, The City College of New York, New York, New York, United States

We are developing a new bioprocess for the production of biofuels from CO2 using renewable energy (Electrofuels). Autotrophic organisms frequently grow slowly, rely on the Calvin cycle for carbon fixation, and can be challenging to engineer for high yields and productivities. We are working to address these challenges by replacing the Calvin cycle with artificial photosynthetic proteins and designed metabolic pathways that will operate in non-growing bacteria cells suspended in photobioreactors. Renewable electricity will be used to power LEDs that specifically energize the artificial photosynthetic pathway in the stationary cells. This will allow for the efficient conversion of CO2 to biofuels and biochemicals such as glycerol We are developing a novel designed light harvesting complex that will split water and regenerate the ubiquitous biological cofactor NADH. At the same time, we have developed a synthetic metabolic pathway that utilizes NADH to reduce CO2 to formaldehyde and then the formaldehyde is ligated to create glycerol using another designed enzyme (formolase). Once installed in E. coli, the pathways will allow for light- powered NAD(H) regeneration which will power CO2 reduction to glycerol that is independent of the cellular metabolism. Thus the system will be operated in photobioreactors which will specifically energize the designed photosynthetic pathway in cells that are not growing. The most recent results of this promising new Electrofuels platform will be presented, including performance metrics for the newly designed photoprotein, pathway fluxes through the carbon fixation pathway, and protein engineering results on two key enzymes in the carbon fixation pathway (formaldehyde dehydrogenase and formolase).

BIOT 153

Is the environmental sustainability of upcoming continuous manufacturing processes better than the traditional processes?

Sri Madabhushi, [email protected], Jack Huang, Matthew Manahan, Nuno Pinto, Sianny Christanti, Henry Lin. Merck & Co., Inc., Kenilworth, New Jersey, United States

Biologics encompass a wide variety of therapeutics including monoclonal antibodies, fusion proteins, nanobodies, vaccines, and enzymes, among others. With the biologics market volume increasing at a rapid rate, there is a need to assess the efficiency of manufacturing processes to better understand their environmental impact. Based on the recommendation from ACS GCIPR Biopharma Focus group, Process Mass Intensity (PMI) can be used as the benchmarking metric to evaluate the material efficiencies of biologics manufacturing processes. With the rise of continuous biologics manufacturing processes, it is important to understand how the material efficiencies of these processes compare against the standard fed batch processes. Here, we calculated the PMI of a continuous manufacturing process and compared it to the PMI of traditional fed-batch processes for monoclonal antibodies (mAbs). Sensitivity analysis was performed to understand the differences and identify potential strategies to develop more efficient processes in terms of their environmental footprint. Furthermore, we compared the PMI of mAbs, vaccines and small molecules from a perspective of drug demand and dosage to gain insights into the practical implications of PMI.

BIOT 154

Sustainable bioprocessing for recovery of critical metal Indium

Astha Upadhyay, [email protected], Kyriakos Atmatzidis, Rouzbeh Tehrani. Temple University, Philadelphia, Pennsylvania, United States The environmental impact of indium mining from the mine tailings of zinc ore is colossal, and the usage of excessive chemicals has ecological footprints. Bioprocesses and green chemistry can be used to develop more sustainable and cleaner practices. Phytoextraction is an innovative technology to reduce the use of chemicals in the recovery of critical metals. The potential of hyperaccumulators as possible renewable feedstock to mediate the treatment of waste feedstock is evaluated in this study. Electronic waste is exploited as a source for energy-efficient and sustainable secondary production of these metals. Among the other critical metals that can be extracted from e-waste, Indium is the most commonly recovered one from Indium tin oxide (ITO) scrap. The initial analysis of the hyperaccumulator through Inductively coupled plasma mass spectrometry (ICP-MS) demonstrated 98% recovery of indium from the ITO feed solution, which was further validated by Energy Dispersive X-Ray Spectroscopy (EDX) analysis. Although the secondary production of critical metal using the chemical leaching process is well researched, there remains considerable uncertainty over the environmental footprint. Phytoextraction, on the other hand, eliminates the usage of extensive compounds for chemical leaching and promotes greener technology for the recovery of metals from e-waste. Further genetic research on the plant has the potential to intensify the hyperaccumulation. Life cycle assessment (LCA) reinforced the hypothesis that phytoextraction can be more energy-efficient and sustainable compared to the chemical leaching process. Further development of demonstrated process to a large-scale production will assist in achieving the eventual goal of a sustainable circular economy.

BIOT 155

Sustainable and functional tandem repeat protein fibers and films

Melik C. Demirel, [email protected]. ESM Dept, Penn State University, University Park, Pennsylvania, United States

Production of repetitive polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions have been an interest for the last couple of decades. Their molecular structure provides a rich architecture that can micro-phase-separate to form periodic nanostructures (e.g., lamellar and cylindrical repeating phases) with enhanced physicochemical properties via directed or natural evolution that often exceed those of conventional synthetic polymers. In this talk, we review programmable design, structure, and properties of functional fibers and films from squid-inspired tandem repeat proteins, with applications in advanced textiles.

BIOT 156

Deep learning to improve bacterial cell counting: Implementation of classification-type convolutional neural networks (CNN) Denis Tamiev1, [email protected], Paige Furman1, Nigel F. Reuel2. (1) BBMB, Iowa State University, Ames, Iowa, United States (2) CBE, Iowa State University, Ames, Iowa, United States

Accurate classification and counting of bacterial cells is important in various microbiome assessment experiments. Most common methods rely on flow cytometry or manual microscopy. Application of flow cytometers in bacterial cell typing is limited due to cell size (bacteria are up to 1000 times smaller than eukaryotes) leading to misclassification of multicellular clumps (biofilms), and cell debris for single cells. Automation of manual microscopy can be achieved with software. Most common software solutions for microscope image processing such as imageJ cannot accurately parse multicellular clumps into single cells or classify them into bacterial cell types (cocci, bacilli, spirilla, spores, etc). Recent progress in the field of big data and deep learning can be used to develop a classification-type Convolutional Neural Network (CNN) tool for bacterial cell counting. This presentation focuses on describing a custom CNN algorithm that identifies bacterial cell clusters on microscope images, and classifies them based on the number of cells found in each cluster (Figure 1A). Here, we developed a robust image preprocessing algorithm that improves the neural network’s confidence in classifying bacterial clusters by 13.96% (Figure 1D and E). This image preprocessing algorithm increases the size of the training database through image augmentation by 72 times (Figure 1B). Such augmentation allowed us to use a very small image dataset, and increased CNN’s accuracy by 13.83% (Figure 1C). In this work we also compare our algorithm to existing techniques and human counting performance. We conclude that classification type CNN can serve as a reliable tool for bacterial cell counting and sorting, and can be leveraged by groups that possess a simple camera-enabled microscope setup.

(A) Cell clusters. (B) Image preprocessing algorithms that normalize all cell clusters by size. Advanced rotation dataset was created by blending the bumper around the image clusters with the background, and rotating both native and inverted clusters by 10 degrees resulting in 72 rotationally augmented images. (D&E) Confidence of the CNN algorithm trained on the Null Bumper and Advanced Rotation datasets respectively (A-artifact; I-X cell clusters).

BIOT 157

Looking beyond GWAS: Identifying functional roles of SNPs using metabolic networks in Arabidopsis and Populus

Debolina Sarkar1, [email protected], Costas Maranas2. (1) Pennsylvania State University, University Park, Pennsylvania, United States (2) Penn State, University Park, Pennsylvania, United States

Genetic sources of phenotypic variation have been a focus of plant studies aimed at improving agricultural yield and understanding adaptive processes. Genome-wide association studies (GWAS) identify the genetic background behind a trait by examining the associations between phenotypes and single-nucleotide polymorphisms (SNPs). Although such studies are now common, these often lacks the power required to uncover the relatively small effect sizes conferred by most loci and tend to ignore the hierarchical nature of biological organization. An association between a genetic variant at a locus and a trait is also not directly informative with respect to the mechanisms that govern the variant being associated with the phenotype, making biological interpretation of the results a challenge. Here, we propose a complementary analysis (‘SNPeffect’) that offers putative genotype-to-phenotype mechanistic interpretations by integrating heterogeneous omics datasets with the biochemical knowledge encoded in metabolic models. SNPeffect was used to explain differential growth rate and metabolite accumulation in A. thaliana and P. trichocarpa accessions as the outcome of activating and inactivating SNPs present in the enzyme-coding regions of the genotypes. To this end, we also constructed a genome-scale metabolic model for Populus trichocarpa, first for a perennial woody tree. As expected, our results indicate that plant growth is a complex polygenic trait governed by carbon and energy partitioning. Growth-affecting SNPs were found to be primarily in amino-acid metabolism, glycolysis, TCA cycle, and energy metabolism. Faster-growing Arabidopsis genotypes were predicted to have higher fluxes through the protein metabolism pathways and employ the more energy efficient purine salvage pathway as opposed to de novo purine biosynthesis for generating AMP and GMP. In poplar, growth-determining SNPs with an upregulating role were found in genes belonging to cellulose and lignin biosynthesis, amino acid metabolism, and energy metabolism. This is in line with breeding strategies that target pathways governing carbon and energy partition. SNPeffect also identified putative growth-associated genes in both the species, distributed among pathways such as glycolysis, folate metabolism, and pyrimidine metabolism. These can serve as candidate genes for future studies. BIOT 158

Propagation of uncertainty improves the quality of microbiome data analysis

Jacob R. Price1, [email protected], Amy D. Willis2, Thea L. Whitman1. (1) Department of Soil Science, University of Wisconsin - Madison, Madison, Wisconsin, United States (2) Department of Biostatistics, University of Washington, Seattle, Washington, United States

The adoption of sequencing-based technologies to probe biological and ecological questions has become exceedingly common due to significant reductions in cost, improvements in the quality of the data produced, and the ability to efficiently and quickly characterize many samples at a time, as well as the concurrent development of numerous bioinformatics tools, software packages, and reference databases. While great advances have been made, there are a number of shortcomings that remain as barriers to microbiome analysis being a precise, replicable, and reproducible science.

One of these shortcomings results from how raw sequencing data are processed to create counts of observed microbial abundances. Typical microbiome analysis pipelines approach read-to-taxon assignment as a discrete classification problem, wherein each read is explicitly assigned to a single member of an operational taxonomic unit (OTU) set. These approaches treat the data as though there is zero uncertainty in the assignments once they are made and, thus, they overestimate the certainty we have in any given read-to-OTU assignment. This leads to the second major shortcoming: a lack of appropriate computational and statistical methods to interpret the OTU tables. In some cases, difficulties and imprecision arise from using analytical approaches which were not developed with the nuances of sequencing data in mind, such as the data being non-census and compositional in nature.

In this work, we illustrate a method for incorporating uncertainty into typical microbiome analyses in order to overcome these challenges via a new and fully integrated bioinformatics and statistical pipeline. We apply this pipeline to two disparate datasets (fire-affected soil and photobioreactor) and demonstrate results obtained from a collection of statistics and hypothesis tests commonly used in microbiome research, including several alpha- and beta- diversity measures, differential abundance testing, and estimates of relative abundance for use in community profiling. Quantifying and accounting for uncertainties in the generated results reduces the risk of false positives due to understated variability, and improves the sensitivity of the tests, while making microbial community analysis more replicable in the process.

BIOT 159

Extending BioSolve with multi-objective Bayesian optimization for automated tuning of upstream and downstream decisions in a mAb process Folarin B. Oyebolu1,2, [email protected], Richard Allmendinger1, Adriana G. Lopes2, Jonathan Shapiro3, Paul A. Sinclair2. (1) Alliance Manchester Business School, University of Manchester, Manchester, United Kingdom (2) Biopharm Services Ltd, Chesham, Buckinghamshire, United Kingdom (3) School of Computer Science, University of Manchester, Manchester, United Kingdom

BioSolve Process is the biopharmaceutical industry’s most powerful software for bioprocess analysis. It enables users to investigate a wide range of process scenarios and parameters, and make informed and targeted process design decisions. However, due to the complexity of the bioprocess models and the large decision space, these simulations can be labour- and time-intensive. This challenge motivates the use of optimisation techniques, such as Bayesian optimisation, that require only a few evaluations to arrive at near optimal solutions. BioSolve Process functionality has therefore been extended to incorporate a Bayesian optimisation tool that automates the selection of the most efficient process parameters, based on pre-defined user criteria. This optimisation tool is customised to tune a combination of categorical and discrete variables, subject to black-box constraints and conflicting objectives. Firstly, a case study based on optimising upstream decisions over a range of target throughputs for a traditional monoclonal antibody (mAb) process was used to assess the performance of the optimisation tool. The upstream decisions considered included choosing between stainless-steel and single-use bioreactors, the number of installed vessels, and how many bioreactors were pooled together for further downstream processing. Compared to manual full enumeration, the optimisation tool showed a substantial reduction in evaluations. A second case study incorporated downstream decisions. In particular, the optimisation tool was able to select between conventional or pre-packed chromatography columns, and from a set of Protein A chromatography resins on offer. Finally, the tool was used to estimate the set of trade-off solutions (Pareto front) for multi-objective problems for the entire mAb process train with the aim of minimising cost of goods (CoGs) in combination with capital expenditure, water usage, and process mass intensity (PMI). The results of these case studies identified scenarios in which certain process decisions were attractive. In addition, they demonstrated the efficiency of the optimisation tool in presenting users with good solutions to their decision problems through BioSolve Process.

BIOT 160

Standardized approach for multivariate analysis of bioreactor data from early development to commercialization

Lakshmi Cella1, [email protected], John S. Bowers2, Rohan Bhaumik1, Brian W. Kwan1, Josephine chiu1, Jonathan Cacciatore1, Gaurav Chauhan1. (1) Merck , Piscataway, New Jersey, United States (2) Merck, Kenilworth, New Jersey, United States Extensive amounts of data are generated starting from early development through commercialization of a biologics manufacturing process. Multivariate data analysis is a powerful technique which can be applied to this big data to further our process understanding. However, it is challenging to leverage this technique in the earliest stages such as clone selection and phase I process development, and throughout the development lifecycle. In this work we describe a methodology to perform multivariate data analysis at every stage of process development and use that information to inform continued activities. We also present a case study of its application. This standardized approach includes development of: (1) generic cell culture models used to monitor and compare the performance of different clones and phase I processes, (2) process-specific models based on phase III development to monitor and troubleshoot process characterization (PC) runs, (3) a scale-independent model built from PC and scale-down model qualification data to support scale-up tech transfer, (4) an enhanced scale-independent model using at-scale data for process performance qualification batch monitoring, and (5) a site-specific model for monitoring routine commercial manufacturing and beyond. Using this approach we will be leveraging data at every stage of process development and developing superior models for process monitoring and performance prediction.

BIOT 161

Use machine learning to predict the microbial community and system performance during bioelectrochemical desalination

Heyang Yuan1, [email protected], Ibrahim M. Abu-Reesh4, Brian Badgley3, Zhen He2, Shan Sun3. (1) Civil and Environmental Engineering, Temple University, Philadelphia, Pennsylvania, United States (2) University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, United States (3) Virginia Tech, Blacksburg, Virginia, United States (4) Qatar University, Doha, Qatar

Microbial desalination cells (MDCs) are an emerging concept for simultaneous water/wastewater treatment and energy recovery. The key to developing MDCs is to understand their fundamentals, such as the effects of salinity on system performance and the role of microbial community and functional dynamics. Herein, a tubular MDC was operated under a wide range of salt concentrations (0.05−4 M), and the salinity effects were comprehensively examined. The MDC generated higher current with higher salt concentrations in the desalination chamber. When fed with 4 M NaCl, the MDC achieve a current density of 300 A m−3 (anode volume), which was one of the highest among bioelectrochemical system studies. Community analysis and electrochemical measurements suggested that electrochemically active bacteria Pseudomonas and Acinetobacter transferred electrons extracellularly via electron shuttles, and the consequent ion migration led to high anode salinities and conductivity that favored their dominance. To simulate the microbial community dynamics, a Bayesian network modeling approach was developed. The networks were validated using Bray-Curtis similarity, root-mean square error, and a null model. A final Bayesian network based on the relative abundance of dominant taxa implied that the taxa putatively not capable of extracellular electron transfer (e.g., Bacteroidales and Clostridiales) might indirectly contribute to bioelectrochemical desalination. The Bayesian network was combined with logistic regressions to simulate current production and yielded the most accurate prediction at the order level. This study has for the first time used machine learning to simulate community dynamics in engineered bioprocesses, which presents a significant step toward predicting system performance.

BIOT 162

Biomaterial approaches for treatment of metastatic ovarian cancer

Samira Azarin, [email protected]. Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, United States

Ovarian cancer is typically diagnosed at advanced stages and consequently has poor clinical outcomes. Standard of care is surgical debulking followed by chemotherapy; while many patients initially respond to treatment, 20-30% of patients are resistant to the chemotherapy, as indicated by recurrence within six months of completing treatment. Various immune-based therapies are being explored as alternative therapies for ovarian cancer, including natural killer cells, chimeric antigen receptor T cells, and dendritic cell vaccines. However, these approaches require extensive ex vivomanipulation and expansion of cells. The combination of in vivo focal ablation of solid tumors with immune checkpoint blockade has shown promise in establishing an antigen-specific cytotoxic T cell response. As ovarian cancer is typically detected after it has spread throughout the abdominal cavity, ablation of the primary tumor is likely insufficient for establishing a therapeutically relevant response. To that end, we have developed biomaterials to achieve ablation of metastatic ovarian cancer cells. One such approach involves addition of metal components to polymeric biomaterial scaffolds that have previously been shown to recruit metastatic cells in order to facilitate non-invasive thermal ablation of infiltrating cells in the abdominal cavity. While this approach would be suitable for patients with disseminating tumor cells circulating throughout the abdominal cavity that can be captured by the scaffold, for patients with extensive metastatic nodules already established throughout the abdomen, we instead sought to deliver a biomaterial to those metastatic sites to facilitate ablation. To that end, we have also developed graphene-based biomaterials that bind to ovarian cancer spheroids. Incorporation of a sonosensitizer within the biomaterial enables killing of attached tumor cells via ultrasound treatment. Current efforts are focused on evaluating the immune response following ablation for both approaches and identifying strategies for local or systemic immune modulation to promote a robust cytotoxic T cell response.

BIOT 163

Engineering smart nanotechnologies to harness cytosolic immune surveillance machinery

John T. Wilson1,2,3, [email protected]. (1) Chemical & Biomolecular Engineering, Vanderbilt University, Nashville, Tennessee, United States (2) Vanderbilt- Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee, United States (3) Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, United States

Therapeutics that can harness the immense power and specificity of the immune system have enormous potential to improve human health. Cells of the immune system sense invading pathogens and/or pathology using a network of receptors that act cooperatively to enhance and shape the type and magnitude of an immune response. Hence, regulating the delivery of immunologic cues to the tissues, cells, and pathways of the immune system is fundamental to guiding immunological decision-making. While most therapeutic strategies to modulate immunity target cell surface or endo/lysosomal receptors, the cytosol is also rich in targets for immunomodulation. However, the development of therapeutics that harness these immune surveillance pathways is hindered by major drug delivery barriers, most notably inefficient delivery to cytosolic targets. This talk will focus on research in my laboratory focused on the molecular engineering of nanostructured, supramolecular polymeric materials that enhance intracellular delivery of immunomodulatory proteins, nucleic acids, and small molecules. I will discuss our efforts in developing pH-responsive, endosome-destabilizing “smart” materials for enhanced cytosolic delivery of antigens and immunostimulatory nucleic acids and the application of these approaches in cancer immunotherapy and vaccines for infectious disease. In particular, I will discuss our recent work in the following areas. First, I will describe the development and preclinical evaluation of polymersomes for efficient cytosolic delivery of cyclic dinucleotide STING agonists and describe how we are leveraging this technology to enhance responses to immunotherapy in multiple ways in a diversity of cancer types. Second, I will discuss the development of nanoparticle-based vaccines for installing tissue resident memory T cells at mucosal interfaces and the capacity of these materials to protect against respiratory infections. Collectively, these results highlight the potential to design and leverage self-assembled, pH-responsive materials to open access to cytosolic immune surveillance pathways with potential to lead to next generation immunotherapies and vaccines for a diversity of diseases.

BIOT 164

Enabling targeted cargo delivery to hematopoietic stem and progenitor cells with biomimetic membrane-wrapped nanoparticles

Jenna Harris, Samik Das, Eleftherios T. Papoutsakis, Emily Day, [email protected]. University of Delaware, Newark, Delaware, United States

Enabling targeted cargo delivery to hematopoietic stem and progenitor cells (HSPCs) is a long-standing problem whose solution would transform the treatment of diverse blood disorders. HSPCs are notoriously difficult to transfect, so there is a substantial need for a potent delivery system that will overcome biological barriers to efficiently deliver cargo to HSPCs. Here, we report the development of biomimetic membrane-wrapped NPs that meet this need.

The NPs we developed consist of poly(lactic-co-glycolic acid) (PLGA) cores, loaded with desired cargo, that are wrapped with megakaryocyte (Mk) cell-derived membranes that provide targeted recognition of HSPCs. Either hydrophobic (DiD fluorophores) or hydrophilic (small interfering RNA; siRNA) cargo can be loaded in MkNPs by synthesizing the PLGA cores through single- or double-emulsion solvent evaporation, respectively. These PLGA NPs can then be wrapped with Mk membranes by co- extruding them through a porous membrane. We confirmed successful wrapping by examining NP size, zeta potential, and morphology by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. Wrapped MkNPs are monodisperse and spherical, with diameter 10-20 nm larger than bare NPs and zeta potential matched to that of the source Mk membranes.

DiD-loaded MkNPs’ interaction with targeted HSPCs versus non-targeted HUVECs (human umbilical vein endothelial cells) and MSCs (mesenchymal stem cells) was evaluated by flow cytometry and confocal microscopy. HSPCs internalized MkNPs within 24 hr of incubationat a much higher rate than than the other two cell types, confirming the Mk membrane coating facilitates HSPC-specific binding.

To test delivery of functional cargo, we prepared MkNPs carrying siRNA targeting CD34 (siCD34) or negative control siRNA (siNeg). These were cultured with CD34+ HSPCs for 24, 48, 72, or 96 hr, then CD34 expression was analyzed by flow cytometry after exposing the samples to fluorophore-labeled anti-CD34 antibodies. MkNPs carrying siCD34 significantly decreased CD34 expression in HSPCs by >15% over 96 hr, while MkNPs carrying siNeg had no impact on CD34 expression.

In summary, MkNPs can be successfully loaded with and deliver either hydrophobic or hydrophilic cargo to HSPCs in vitro, while exhibiting minimal binding to non-targeted cell types. This exciting advance paves the way for further optimization and in vivoevaluation of MkNPs as HSPC-specific delivery vehicles.

BIOT 165

Evolution-guided design of pharmaceutical leads

Ankur Sarkar1, Edward Kim2, Akarawin Hongdusit1, Jerome M. Fox1, [email protected]. (1) Chemical and Biological Engineering, University of Colorado, Boulder, Boulder, Colorado, United States (2) Vevo, Inc., San Francisco, California, United States

Natural products and their derivatives represent a longstanding source of pharmaceuticals and medicinal preparations. These molecules—perhaps, as a result of their biological origin—tend to exhibit favorable pharmacological properties (e.g., bioavailability and “metabolite-likeness”) and can exert a striking variety of therapeutic effects (e.g., analgesic, antiviral, antineoplastic, anti-inflammatory, cytotoxic, immunosuppressive, and immunostimulatory). Recent advances in metabolic engineering and applied enzymology have suppled new approaches for the efficient biosynthesis and functionalization of known, pharmaceutically relevant natural products; complementary methods for the discovery and optimization new products with targeted, therapeutically relevant activities, however, remain poorly developed. In this presentation, I describe an approach for using microbial systems to evolve natural products that inhibit known drug targets. Our group has implemented this approach to build new inhibitors of protein tyrosine phosphatases (PTPs), a class of enzymes that has long eluded drug design, and we have characterized those inhibitors with detailed kinetic, biostructural, and in vitro cell studies. Our results provide evidence of convergent evolution in the biosynthesis of functional metabolites and illustrate how microbial systems can find surprising solutions to sophisticated biochemical objectives. The technology described in this presentation provides a new platform for the discovery and optimization of precisely targeted, readily synthesizable leads compounds.

BIOT 166

Surpassing thermodynamic, kinetic, and stability barriers to biocatalytic isomerization of galactose to tagatose Josef R. Bober, Nikhil U. Nair, [email protected]. Chemical & Biological Engineering, Tufts University, Medford, Massachusetts, United States

Bober & Nair (2019) Nature Communications

D-Tagatose is a rare ketohexose sugar with sweetness similar to that of sucrose. However, its glycemic index and caloric value is much lower because of low bioavailability, making it an attractive GRAS (generally regarded as safe) sugar substitute. Recent studies have also demonstrated that it is anti-hyperglycemic and prebiotic, which promotes gut health.

The enzyme L-arabinose isomerase (LAI) that isomerizes L-arabinose to L-ribulose can also isomerize D-galactose to D-tagatose. A variety of LAIs from different microorganisms have been isolated and have reported optimal activity at a range of temperatures and pH. Some of the limitations of tagatose biosynthesis using LAI that may be hindering commercial viability are, 1) unfavorable enzymatic kinetics since galactose is not the native substrate of LAI, 2) low enzyme stability, and 3) low equilibrium constant for galactose to tagatose isomerization.

Few previous reports have been successful at engineering enzymatic properties of LAI for industrial application; often addressing only one of the bottlenecks to productivity. To address the kinetic issue, several groups have used enzyme engineering methods to enhance activtity of LAI toward galactose. To counter low-stability issues, many groups have tested the utility of thermophilic enzymes. However, high reaction temperatures (≥ 80 °C) result in significant caramelization, which is undesirable. Finally, the thermodynamic limitations of isomerization arguably, the most recalcitrant issue since ΔG°rxn ≈ +1.2 kcal/mol.

There have currently been no studies that look to systematically analyze all three limitations – kinetic, thermodynamic, and enzyme stability. This work clearly demonstrates the presence of these three limitations and provides a novel approach to balance their advantages and limitations. We engineered the probiotic bacterium Lactobacillus plantarum as expression host in this approach that enabled ~50 % conversion of galactose to tagatose in 4 h (productivity of ~38 mmol tagatose L-1 h-1) ultimately reaching ~85 % conversion after 48 h at high galactose loading (300 mM) in batch culture. This is among the highest conversions and productivities reported to date for tagatose production using a mesophilic enzyme. Such an approach is expected to be applicable to other biocatalytic systems where similar trade-offs between kinetics, thermodynamics, and/or stability pose hurdles to process development.

BIOT 167

Ruggedized peptide-based receptors for biological sensing in military environments Matthew Coppock1, [email protected], Sanchao Liu1, Alexander Winton1, Wais Mojadedi2, An Ngo2. (1) CCDC Army Research Laboratory, Adelphi, Maryland, United States (2) Oak Ridge Associated Universities, Oak Ridge, Tennessee, United States

As the Army moves towards equipping the soldier with more advanced wearable sensing devices for real-time environmental, health, and performance monitoring, there is a significant need for the biological receptors integrated into such devices capable of consistent performance in multifaceted operational environments. The temperature and biological instability, long development times, and inconsistencies in production of monoclonal antibodies, the gold standard receptors for biological detection, has resulted in the advent of alternative antibody technologies to fill these technological gaps. Protein Catalyzed Capture (PCC) agent technology is capable of the bottom-up development of highly stable and tailorable receptors through iterative in situ ‘click’ chemistry cycles with one-bead-one-compound (OBOC) peptide libraries. Aside from the inherent thermal stability and binding performance comparable to, and oftentimes exceeding, monoclonal antibodies, the modularity of PCCs allows for easy integration into various detection platforms and assays. Capable of full receptor development in ~2 weeks, PCCs can fulfill the need for alternative antibodies by addressing critical gaps in adaptability, manufacturability, and stability. This broadly enabling technology has the capability of benefiting multiple Army entities in a wide range application spaces.

BIOT 168

Quantifying noncanonical amino acid incorporation events in yeast: Laying the groundwork for engineering more druglike proteins

Kelly A. Potts, Jessica T. Stieglitz, Ming Lei, James A. Van Deventer, [email protected]. Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, United States

Genetically encoding noncanonical amino acids (ncAAs) in proteins is a promising strategy for enhancing the properties of biological therapeutics. There are now tantalizing hints of the power of this approach in the literature, including antibody-drug conjugates and cyclic peptides with properties that would be difficult to access using other approaches. However, not all protein engineering platforms are yet compatible with ncAAs. This is especially true in the yeast S. cerevisiae, where the underdeveloped ncAA incorporation toolkit limits integration with powerful yeast display technology. One major barrier to effective integration is a lack of quantitative understanding of ncAA incorporation events. Here, we describe the evaluation of a number of reporters of ncAA incorporation efficiency and fidelity in response to the amber (TAG) codon in yeast that may help shed light on this question: single-fluorescent protein reporters, dual- fluorescent protein reporters, and cell surface displayed protein reporters. Across these varying reporter architectures, we found that flow cytometry-based evaluations with dual reporters yielded consistent quantitative trends across numerous incorporation conditions. In addition, using dual-fluorescent protein reporters, we found that ncAA incorporation efficiency and fidelity is influenced by both stop codon location and N- and C-terminal fluorescent protein identity. Finally, we investigated the feasibility of using several reporters to quantify the effects of single-gene knockouts on ncAA incorporation events. Both dual-fluorescent protein reporters and a modified “drop-in” version of yeast display revealed that several gene knockouts enhance ncAA incorporation efficiency without compromising fidelity. These findings highlight the important roles that quantitative reporter systems play in identifying high efficiency, high fidelity ncAA incorporation events, and suggest opportunities for improving ncAA insertion using genomic engineering. Using these reporters to facilitate the encoding of a diverse set of ncAAs in yeast is expected to set the stage for engineering more “druglike” proteins that leverage the broad set of chemistries present in noncanonical amino acid side chains.

BIOT 169

Development of a high-throughput cell screening toolkit for the directed evolution of glycosynthase enzymes for bespoke oligosaccharides synthesis using azido sugars

Chandra Kanth Bandi1, [email protected], Ayushi Agrawal2, Shishir P. Chundawat1. (1) Chemical and Biochemical engineering, Rutgers University, Piscataway, New Jersey, United States (2) Chemical and Biomolecular engineering, Clemson University, Clemson, South Carolina, United States

Glycans like oligosaccharides play a significant role in the regulation of biological systems which necessitates us to study their structure-function relationship and therefore develop techniques for synthesis of bespoke glycans. Development of chemoenzymatic routes for synthesis of designer glycans using glycosynthases has gained momentum in recent years. Glycosynthases (GSs) are a class of mutant glycosidase enzymes that can catalyze the formation of glycosidic linkages between two sugar moieties when provided with an activated sugar donor and appropriate acceptor group. Ever since a glycosyl hydrolase (GH) family 1 glucosidase was first mutated into an active glucosynthase, a multitude of other GH families have been engineered into glycosynthases by mutating the catalytic nucleophile acid residue in an ad hoc manner to small residues like alanine or glycine. However, the synthesis reactions catalyzed by GSs are highly inefficient and cannot be readily tweaked to alter substrate specificity. The currently available screening strategies are low throughput which limits the library size of mutant GSs to a few thousand constructs at any given time. Hence there is a dire need for a selection strategy that can selectively and efficiently screen improved GS variants from a sequence space of millions of possible variants. Here, we highlight results from our novel high-throughput screening method developed to rapidly screen glycosynthase mutants that utilize sugar azides as activated donor sugar substrates. This technique is based on using the click chemistry reaction to selectively tag the released glycosynthase by-product (an azide ion) and sort the mutant constructs using a fluorescent activated cell sorter. A GH29 fucosidase enzyme, which was previously engineered to function as a fucosynthase, was used as model enzyme to develop this screening method. This construct was subjected to random mutagenesis to create a large library and subsequently sorted using this screening method to isolate improved fucosynthase variants with higher specific activity for synthesis of bespoke fucosylated oligosaccharides. Lastly, we highlight how our screening strategy can be applied to evolve the substrate specificity of glycosynthase enzymes towards different acceptor or donor groups for synthesis of bespoke glycans like human milk oligosaccharides for commercial applications.

BIOT 170

Process development strategy for E. coli based cell-free protein synthesis reactions

Noelle Colant1, Jaime Teneb-Lobos1, Stephen Goldrick1, Stefanie Frank1, William Rosenberg2, Daniel G. Bracewell1, [email protected]. (1) Biochemical Engineering, University College London, London, United Kingdom (2) iQur, London, United Kingdom

Over the last decade, cell-free protein synthesis (CFPS) has been utilized as a production platform for antibodies, therapeutic proteins, and vaccine candidates. CFPS is advantageous as a production platform because reactions can achieve relatively high titers in a few hours and reactions have been demonstrated to scale linearly up to 100 L. CFPS reactions also tolerate conditions that are not typically attainable in traditional in vivo cultivations. The open nature of these in vitro systems allows for non- physiological conditions as well as the addition of components not naturally found in E. coli, like non-standard amino acids which can then be incorporated into proteins or chaperones and other agents for post-translational modifications.

CFPS process development strategies avoid the need for cell line development but must account for these differences. As CFPS reactions can be completed rapidly we were able to design a rational process development strategy that allows us to optimize titers in ~48 hours. The approach begins by examining the impact of the E. coli strain chosen for the extract, the formulation of the reaction mixture, and the optimization of the plasmid sequence on product titer. Using the system selected by this study we then investigated the key process parameters; temperature, pH of the reaction mixture, concentration of plasmid, percentage of extract in the reaction, and length of the reaction. This experimental design was then analyzed using multivariate data analysis (MVDA) to determine the reaction conditions that would maximize product titer.

The methodology was applied to two different products; hepatitis B core antigen (HBcAg) and superfolder green fluorescent protein (sfGFP). We found that the impact of the process parameters on titer is highly dependent on the product. The strain of the cell extract – in particular, whether or not the cells used for the extract had been induced during cultivation – was the most influential on product titer. Titers for sfGFP were improved in reactions with a non-induced BL21-StarTM (DE3) extract while an induced BL21-StarTM (DE3) extract enhanced HBcAg production. Following that, adjustments in temperature or pH of the reaction mixture were most influential and resulted in over a 40% increase in titer in some cases but again optima varied widely between the two products.

BIOT 171

Development of an integrated process for discovery and production of site- specific conjugated antibodies

Dante W. Romanini1, [email protected], Michal Achmatowicz2, Jim Falsey2, Bradley Herberich2. (1) M/S 29-2-B, Amgen, Thousand Oaks, California, United States (2) Mailstop 29-1-A, Amgen Inc, Thousand Oaks, California, United States

Numerous methods have been reported for the creation of site-specific antibody drug conjugates (ADC’s) in a research setting, but relatively few of those methods have been used to make investigational therapeutics for the clinic. One challenge for creating conjugated biologics is the complexity in developing a manufacturing process that provides adequate control of critical quality attributes while maintaining high yields and efficient use of plant resources. To support the advancement of a novel bispecific conjugated antibody, we developed an integrated conjugation/purification process that provides robust control over conjugation site, drug-to-antibody ratio, and impurities. Our process is a major improvement over the published literature. We introduced several improvements to overcome feed stream heterogeneity and provide better conjugation kinetics while reducing product-related impurities. All new steps were incorporated into the antibody purification process, eliminating the need for additional chromatography operations. The improved conjugation process was first used to accelerate drug discovery efforts, enabling a several-fold increase in synthesis and screening throughput. Once a clinical candidate was selected, we developed additional optimizations to enable technology transfer and consistent process control at large scale. The streamlined process can be performed in a standard biologics manufacturing facility in either a through-process or segmented mode, allowing for greater flexibility and efficient use of plant resources. We have demonstrated process and product consistency at scales spanning three orders of magnitude including multiple production batches to support early-phase development.

BIOT 172

3D-printed villi-on-a-chip with bioelectronics for drug development and manufacture

Chen-Yu Chen1, [email protected], Andrew Lamont1, Kimberly Lo1, Gregory F. Payne2, Ryan Sochol3, William E. Bentley4. (1) Bioengineering, University of Maryland, College Park, Maryland, United States (2) Univ of Maryland Biotech Inst, College Park, Maryland, United States (3) Mechanical Engineering, University of Maryland, College Park, Maryland, United States (4) Fischell Dept of Bioengineering, University of Maryland, College Park, Maryland, United States Gut dysbiosis (microbial imbalance) is known to influence the physiological function of the gut epithelium and is involved in several intestinal diseases and even mental disorders. Owing to their ability to host multiple human cell types and gut microbiota, synthetic in vitro models commonly referred to as “Organs-on-a-chip” (OOC) offer the capability to interrogate and modulate biological signaling at cellular length scales and in real time. Importantly, few systems recapitulate the spatial and chemical environments of the GI tract that are known to have a dramatic impact on the molecular signaling occurring in vivo. For example, oxygen tension and redox environments that extend from the crypts to the villous tips vary tremendously. To gain insight, we have developed a 3D-printed human villi-on-a-chip model that incorporates an internal capillary system that recreates lymphatic and circulatory conduits for molecular exchange. This capillary system provides for real-time monitoring of cellular permeability and redox signaling, and further recapitulates apical and basal transport in a structurally relevant model. The villus scaffold is assembled with ultra-high spatial resolution owing to a new two-photon direct laser writing (DLW) 3D printing system. The 3D fabrication strategy offers finely tuned parameters including pore-size, curvature, surface roughness, and channel size. Results are consistent with earlier efforts showing the upward migration of cells towards the villus tips. By coupling novel bioelectronic sensing modalities on adjacent “chips”, we can rapidly evaluate a chemical/biological landscape that is needed for the development of precision medicines. Conversely, owing to the integrated electronics, the device can also modulate the redox nature of the microenvironment, providing new insight in biological responses. That is, by advancing 3D printing configurations that faithfully recapitulate the geometric landscape and at the same time enable bidirectional regulation of the signaling events, we are in a better position to discover new insights on the complexities of the GI tract. These will serve to more rapidly understand disease progression and at the same time, facilitate the development of new therapies.

BIOT 173

Enabling chemical and structural biology with synthetic binding proteins

Shohei Koide, [email protected]. NYU School of Medicine, New York, New York, United States

The protein engineering community now has the capacity to rapidly create synthetic binding proteins with high potency and exquisite selectivity. Although its major focus remains on the development of biologic therapeutics, synthetic binding proteins have proven also to be transformative tools in mechanistic biomedical investigation. I will present applications of the Monobody technology, a synthetic binding protein system based on the fibronectin type III scaffold, to challenging questions in chemical biology and structural biology in which tailor-made Monobodies enable novel investigations and help gain new mechanistic insights.

BIOT 174 Perlman lecture: Base editing and prime editing: Chemistry on the genome without double-strand breaks

David R. Liu, [email protected]. Harvard Univ, Cambridge, Massachusetts, United States

Most genetic variants that contribute to diseaseare challenging to correct efficiently and without excess byproductsusing standard nuclease-based genome editing methods. In this lecture I describe the development of two approaches to precision genome editing that do not require double-strand DNA breaks, donor DNA templates, or HDR. Through a combination of protein engineering and protein evolution, we developed two classes of base editors (CBE and ABE), proteins that enable all four types of transition mutations (C to T, T to C, A to G, and G to A) to be efficiently and cleanly installed or corrected at target positions in genomic DNA without making double-strand DNA breaks. Base editing has been used by many laboratories around the world in a wide range of organisms. By integrating base editors with in vivodelivery strategies, we have addressed animal models of human genetic disease, in some cases with phenotypic rescue and lifespan extension. I will also describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed >175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutations without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to precisely insert various tags and epitopes into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing offers efficiency and product purity advantages over HDR, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle can correct most known pathogenic human genetic variants.

BIOT 175

B&B Wang award: Yarrowia lipolytica: Versatile microbial workhorse for expanding nature’s biosynthetic capacity

Peng Xu, [email protected]. Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland, United States

Yarrowia lipolytica is an oleaginous yeast that has been substantially engineered for production of oleochemcials and drop-in transportation fuels. It has been considered as a ‘generally recognized as safe’ (GRAS) organism in the food and nutraceutical industry. The high precursor acetyl-CoA and malonyl-CoA flux along with the versatile carbon-utilization capability makes this yeast as a superior host to upgrade low-value carbons into high-value pharmaceuticals and plant natural products (PNPs).Bacteria system in general is less efficient to express the complex gene cluster of plant natural product pathway. Unlike bacteria, yeast has developed spatially separated organelles to partition specialized metabolic functions into distinct cellular compartments. In this talk, I will present strategies to build genetic toolkits to streamline the genetic/genome modification for Y. lipolytica. I will also present strategies to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA metabolism toward engineering efficient yeast cell factories to produce complex oleochemicals, terpenes, polyketides and aromatic commodity chemicals. We identified pathway limitations and assessed genetic engineering strategies to elevate the level of acetyl-CoA, malonyl-CoA, HMG-CoA and NADPH. This work will provide a testbed for engineering Y. lipolytica and expanding nature’s biosynthetic capacity to produce complex fuels and chemicals from renewable feedstocks.

BIOT 176

Maturation of stem cell-derived skeletal myocytes promoted by micropatterning and substrate stiffness

Wendy Crone, [email protected]. University of Wisconsin, Madison, Wisconsin, United States

In this work we demonstrate the effectiveness of an engineered two-dimensional micropatterned cell culture platform for creating highly aligned myotubes from human induced pluripotent stem cell (iPSC)-derived myogenic progenitors. Myotube elongation was shown to be dependent on the micropattern feature width and spontaneous contractions were aligned with the long axis of the pattern.As a result of an optimized micropattern feature geometry on a substrate of physiologically relevant stiffness, the resulting myotubes were elongated, well-aligned, and similar to myofibers; they showed significant improvement innuclear alignment, myotube fusion, and sarcomere formation. This platform holds great potential in further understanding the process of human muscle development, as well as opportunities for in vitropharmacological studies with skeletal muscle diseases. As an example, we demonstrated that bundle-like myotubes can be produced using patient-derived iPSCs with a background of Pompe disease (glycogen storage disease type II). Culturing in this engineered platform enhanced the disease phenotype as demonstrated by the observation of abnormal lysosome accumulations.

BIOT 177

Cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases Jasmine Hershewe, [email protected], Michael C. Jewett. Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, United States

Protein glycosylation is the covalent attachment of sugar moieties to proteins. Glycosylation is ubiquitous and is key for protein stability, activity, and immunogenicity. Glycosylated proteins are important molecules in biotechnology, comprising the majority of FDA-approved biologics, and can be used as vaccines against pathogens. However, controlling glycosylation for biomanufacturing defined glycoproteins, and for understanding the roles and regulations of glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of complex, pre-built glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and contain many (>10) transmembrane helices. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained soluble yields of up to 420 ug/ml for the single-subunit OST enzyme, “Protein glycosylation B” (PglB) from Campylobacter jejuni, as well as for three additional bacterial PglB homologs. Importantly, the cell-free derived enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed, and the ability to tightly control concentrations and ratios of glycosylation components in the in vitro system allowed us to quickly optimize the system for full glycosylation of target proteins. Since the publication of this work, we have extended the approach to various OST homologs. We anticipate that this broadly applicable production method will advance glycoengineering efforts and beyond, allowing for the facile synthesis and testing of complex membrane proteins.

BIOT 178

Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform

Karen Polizzi, [email protected]. Imperial College, London, United Kingdom

Cell-free protein synthesis (CFPS) harnesses cellular machinery for the production of proteins in the absence of a living cell, providing a rapid platform for the production of proteins, prototyping genetic circuits, and the synthesis of compounds that would otherwise be toxic to a living organism. A variety of host organisms have been used for CFPS, allowing researchers to exploit the diversity of post-translational modifications and biosynthetic capabilities available in Nature. In our efforts to develop a CFPS platform derived from the methylotrophic yeast Pichia pastoris, we developed an approach that uses genetically encoded biosensors to screen different engineering Pichia pastoris strains and extract harvest times to identify the peak ribosome content, which leads to higher functionality extracts. Further optimisation of the reaction conditions using a design of experiments approach leads to a CFPS platform with yields that are increased approximately 20-fold over the starting conditions. The Pichia pastoris CFPS platform can be used to produce complex proteins such as human serum albumin and virus-like particles, suggesting it can serve as a rapid prototyping platform for the biopharmaceutical industry. Overall, this work provides a blueprint for the rapid optimisation of CFPS protocols using biosensors to assist in the design of the systems. This approach could easily be extended to the development of CFPS platforms from other species with interesting biosynthetic properties.

BIOT 179

Gaden award: Quality and quantity? Enhancing mammalian biomanufacturing performance through systems biotechnology approaches

Michael J. Betenbaugh, [email protected]. Department of Chemical and Biomolecular engineering, Johns Hopkins University, Baltimore, Maryland, United States

The traditional engineering paradigm states that you must sacrifice quality for quantity. This can be true for biomanufacturing processes in which quality can involve critical quality attributes such as the glycan structures attached to a produced glycoprotein therapeutic. As biotechnologists, we seek to break this axiom through the application of systems engineering, cell and process modeling, and synthetic biology and metabolic engineering. In our current work we are implementing these systems biotechnology tools to improve the performance of CHO cells and other production hosts. Engineering methodologies are being applied to both determine the glycosylation structures and, as needed, change these glycoforms. One approach being used is to alter the cell lines in order to both add and remove glycosylation capabilities in CHO cells. Determining which modifications are best to implement can be facilitated with the assistance of comprehensive models of glycosylation processes. Another alternative is to adjust the media and examine the role that changes to its composition including supplementation with additives has on both the product titer and glycosylation character. It is equally important to be able to evaluate the effect of these engineering modifications and thus analytical tools are used to determine the glycan patterns for glycoproteins produced in these hosts. Finally, it is also useful to dictate the final glycan structures and this is often best achieved through the use of control strategies based on our understanding of cellular glycosylation and the impact of process modifications. Such a comprehensive systems biotechnology approach will enhance our ability to generate desired profiles in terms of glycosylation and other quality attributes while minimizing the effect on the performance of CHO cells in culture, leading to enhanced qualities and yields for target biopharmaceuticals in current and future mammalian biomanufacturing processes.

BIOT 180

Quantitative mass spectrometry analysis methods for low-level CHO host cell proteins: Case study for GRP78 Kunal Bakshi, [email protected], Varnika Roy. GSK, Rockville, Maryland, United States

Host cell proteins (HCPs) are process related impurities and may impact the safety and efficacy of recombinant protein therapeutics or vaccines. Monitoring and reducing HCPs is one of the main goals of early stage process development. Availability of fit-for- purpose analytical methods to track and monitor overall and specific HCPs is key for successful HCPs reduction and control. In this case, commercially available CHO HCP ELISA kits that are commonly used during early stage development failed to detect truncated and other isoforms of 78 kDa glucose-regulated protein (GRP78). Additionally, potential association of this HCP with the antigen of interest further complicated the quantitation. Here, we describe the use of mass spectrometry based quantitative approaches to support process development, toxicology and clinical trial material testing for specific HCPs such as GRP78. This case study provides a comparison of different calibrator methods and valuable guidelines on developing fit-for- purpose analytical methods for HCP monitoring during vaccine or therapeutic biologics development.

BIOT 181

Overcoming mAb manufacturing process challenges for high concentration drug substance to facilitate subcutaneous administration

Swarnim Ranjan1, [email protected], Jessica Hung2, Kyle Skillings1, Melissa Holstein1, Sanchayita Ghose1. (1) Biologics Process Development, Bristol Myers Squibb, Devens, Massachusetts, United States (2) Biologics Process Development, Bristol-Myers Squibb, Devens, Massachusetts, United States

Ultrafiltration/diafiltration is a key step for achieving the desired final protein formulation in nearly all downstream processes. With the current industry focus on subcutaneous delivery due to ease of administration and better patient experience, there is an increasing need for higher concentration (≥100 g/L) and low volume (≤1.5 mL) formulations. However, obtaining high protein concentration using ultrafiltration poses numerous challenges, both from manufacturing process and protein stability perspectives. The objective of this work is to identify these challenges and evaluate strategies to mitigate them. The exponential increase in viscosity with increasing protein concentrations, particularly above 150 g/L causes high pressure leading to operational limitations. High viscosity also leads to low flux, resulting in long processing times during ultrafiltration and sterile filtration operations. The higher volumetric ratio of system holdup to final product pool results in undesirable yield loss and/or product dilution during product recovery. Additionally, during diafiltration and ultrafiltration, significant differences are observed in the pH and excipient concentrations between the diafiltration buffer and the formulated protein solution due to volume exclusion, charge interaction and preferential hydration effects. These effects become more pronounced with increasing protein concentration, as molecular ‘crowding’ affects protein charge due to protein-protein interactions and leads to increased volume exclusion, and can also cause protein aggregation. This work includes understanding the pH and excipient behavior during ultrafiltration/diafiltration operation and the impact of molecular properties to achieve the target excipient concentration; implementation of a plug flow strategy at low flow rate to maximize the recovery of the drug substance with minimal dilution; better understanding of the impact of viscosity on pressure drop during concentration, and evaluation of filter membranes with improved flow paths to address high pressure and low flux concerns during sterile filtration, both at lab and commercial manufacturing scales, thereby, overcoming challenges associated with manufacture of high concentration mAb product.

BIOT 182

Engineering in mammalian cells to streamline development of antibodies, antibody-like molecules and other proteins of therapeutic interest

Jennifer Maynard, [email protected], Annalee Nguyen, Ahlam Qerqez, Yutong Liu, Kevin Le. Chemical Engineering, University of Texas at Austin, Austin, Texas, United States

Engineering of antibodies and related receptors is most commonly performed using phage or yeast display, but mammalian cells are used for production. To streamline engineering of these and other membrane bound proteins, we developed a mammalian screening platform which allows for generation of large libraries and selection of variants with desirable characteristics in the manufacturing host. We have used this to engineer antibody Fab domains and showed that this platform can anticipate issues associated with glycosylation that would be missed by other screening platforms. We have also used it to identify human T cell receptors with low nanomolar affinities and increase TCR stability for expression as a soluble antibody-like molecule. Finally, we have used this platform to reduce tonic signaling issues in chimeric antigen receptors which may lead to increased persistence in vivo. Strategies to select for variants based on the desired protein activity as opposed to simple binding will be discussed.

Schematic of the episomal mammalian display system with an example Fab protein displayed on CHO cells. The displayed hu4D5 Fab was stained with ligand HER2 Fc, then anti-human Fc-Alexa Fluor 647, and anti-human IgK-FITC was added for flow cytometric detection. For flow cytometry comparison of high (Kd = 0.1 nM, hu4D5) and low (Kd = 200 nM, bD1) affinity variants, CHO cells were transfected with plasmid expressing a single variant, stained as described and analyzed by flow cytometry. Untransfected cells showed minimal staining with either reagent (data not shown).

BIOT 183

Innovation and regulatory science: Bridging processing parameters with CQAs of biotechnology products

Patrick Faustino, Jinhui Zhang, [email protected]. FDA, Sliver Spring, Maryland, United States

Regulators worldwide (FDA, EMA, ICH) have drawn attention to the importance of critical quality attributes (CQAs) for biotechnology products. To effectively assess product quality QCAs should be identified and monitored to evaluate consistent batch production and thus help to ensure optimum control of the manufacturing process of biotechnology products for regulatory acceptance. The purpose of this presentation is to provide a holistic understanding of how innovation in regulatory science can bridge processing parameters with CQAs of biotechnology products. The presentation will first highlight FDA CDER Office of Testing and Research’s vision on how innovation may help advance the regulatory science of product quality for biotechnology products; subsequently, the presentation will describe a landscape of evolving analytical platforms for biotechnology products CQAs’ identification and evaluation; in the end, several case studies will be presented to showcase the scientific efforts that OTR has taken to bridge process parameters with CQAs of biotechnology products. In summary, the presentation will highlight the innovative promise of these evolving characterization platforms and emerging technologies to support process understanding and advance the state of product quality for biotechnology products.

BIOT 184

Next generation protein a capture: Harnessing the power of nanofiber technology to intensify biomanufacturing and facilitate rapid process development

Richard Kucia-Tran, [email protected]. GSK, Stevenage, United Kingdom

Traditional packed bed chromatography has been a workhorse for the biomanufacturing industry for the past five decades, deployed in the production of everything from plasma proteins, through monoclonal antibodies to modern applications such as the manufacture of cell based vaccines. A limitation of this technology has been the inherent mass transfer constraints, inversely linking how quickly processes can be operated, and the mass throughput of the operation. As a result, it has been the long held wisdom that packed bed chromatography is a capacity and productivity bottleneck for most modern biomanufacturing facilities. Nano-fibre based chromatographic media developed by Puridify, subsequently acquired by GE Healthcare, decouples mobile phase flowrate and protein mass transfer, enabling chromatography cycles to be completed in single digit minutes, rather than hours. The work presented will highlight how this shift in processing paradigm has been utilised at GSK. Case studies will illustrate the process development work that has been performed using the PrismA Fibro for antibody capture, and to evaluate its performance, particularly at pilot scale. The data gained from these studies has been used to map out potential implementation strategies, providing either an increase in facility flexibility and therefore utilisation and/or reducing the CoGs, particularly in the context of clinical manufacturing. Furthermore, whilst the operational and financial benefits of adopting Fibro technology can be easily conceptualised, perhaps less well documented is the potential for using this technology to facilitate more rapid process development. The work presented will show how PrismA Fibro has been implemented at GSK, outside of the DSP space to which its application is most immediately apparent, in order to facilitate more rapid cell line screening and selection.

BIOT 185

Evaluation and mechanistic elucidation of recombinant hydrophobin as a crystallization inhibitor of flufenamic acid Nathanael Sallada1, [email protected], Yongjun Li2, Matthew Lamm2, Bryan Berger3. (1) Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States (2) Formulation Sciences, Merck & Co., Rahway, New Jersey, United States (3) Chemical Engineering, University of Virginia, Charlottesville, Virginia, United States

Formulation of poorly water-soluble small molecule drugs continues to be a major obstacle in bringing new drugs to market due to issues with low bioavailability. Maintaining a high degree of supersaturation in the gut is one method to increase bioavailability of poorly water-soluble drugs through addition of antinucleating agents in amorphous drug delivery systems. Hydrophobins are small, highly surface-active proteins from filamentous fungi that display resistance to degradation and potential immunological inertness, making them particularly amenable to drug formulation and delivery applications. In this work, we evaluate an engineered, recombinant hydrophobin (HFB1, class II) as a novel inhibitor of crystallization to maintain drug supersaturation of a model hydrophobic drug, flufenamic acid (FA). Model drug precipitation kinetics were tracked using a UV fiber optical method in a supersaturation- precipitation system. HFB1 was compared to several widely used polymeric crystallization inhibitors: methocel™ (A4C grade), methocel™ (K15M grade), Kollidon VA64 and HPMCAS (MF grade). Supersaturation-precipitation experiments show that HFB1 outperformed all polymers tested and has potential for use as a crystallization inhibitor in amorphous drug delivery systems. Ongoing work to be discussed in this presentation will focus on elucidation of the crystallization inhibition mechanism and design of hydrophobin variants with improved drug formulation properties.

BIOT 186

Developments in point-of-care manufacturing of human insulin using cell-free systems

Shayan Borhani1, [email protected], Govind Rao1, Douglas Frey1, Leah M. Tolosa1, James R. Swartz2, Chandrasekhar Gurramkonda1. (1) Center for Advanced Sensor Technology and Department of Chemical, Biochemical, and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland, United States (2) Chemical Engineering and Bioengineering, Stanford University, Stanford, California, United States

According to a 2014 study by the WHO, 422 million people were diabetic globally, with low to middle income communities being most effected. Additionally, in 2016 diabetes was the 7th leading cause of death globally. This increase in prevalence informs the need for changing the current manufacturing paradigm to develop new treatments and devices in an effort to make insulin more accessible. We aim to develop a manufacturing technology which allows for rapid insulin production at the point-of-care. In doing so, we utilize a variety of different cell-free platforms to express proinsulin and preproinsulin. The expressed product is then subject to an “on-column” conversion process which removes the C-peptide and signal sequence to yield a mature insulin product. This allows for more efficient production of insulin, while also reducing the burden on downstream purification steps. Additionally, when this approach is coupled with the appropriate process analytical technology, an effective and rapid validation of the final product could be achieved for real-time release.

BIOT 187

Field guide to scientific entrepreneurship

Jordi Mata-Fink, [email protected]. Entrepreneur and Inventor, Portland, Oregon, United States

Start-ups are our most efficient means of transforming innovations into products, but the process of converting a scientific advance into a valuable company is opaque and haphazard. This talk will present a systematic framework for successful innovation. We will explore how to: navigate the academic/entrepreneurial interface; define, perceive, and convey value at all stages of an idea or company; and ideate, experiment, and persuade with purpose.

Dr. Mata-Fink is a biotechnology leader, entrepreneur, author, and inventor. Based for many years at Flagship Pioneering in Cambridge, MA, his work has led to the creation of multiple valuable public and private biotechnology companies including: CYGNAL THERAPEUTICS, developing drugs that target neuronal pathways for the treatment of cancer and inflammation; RUBIUS THERAPEUTICS (NASDAQ: RUBY), developing engineered red blood cells as a safe, potent, and long-lasting cellular therapy; and EDITAS MEDICINE (NASDAQ: EDIT), developing the first CRISPR/Cas9-based genome editing therapies for human disease.

BIOT 188

Producing recombinant proteins using a novel protein expression system derived from human blood

David Burgenson1, [email protected], Gregory Szeto1, Xudong Ge2, Iordan V. Kostov3, Leah M. Tolosa4, Govind Rao5. (1) University of Maryland Baltimore County (UMBC), Baltimore, Maryland, United States (2) University of Maryland Baltimore County, Baltimore, Maryland, United States (3) UMBC, Baltimore, Maryland, United States (4) Univ of Maryland, Baltimore, Maryland, United States (5) TRC Building, Ctr for Advanced Sensor Technology, Baltimore, Maryland, United States

Cell free translation has traditionally been used to study protein expression and has been used as a basic science tool for decades. Recent advances have enabled the use of cell free expression in areas that typically employ living cells such as drug manufacturing and proteomics. Using these advances, systems have been developed that exploit the speed and simplicity of cell free protein expression, enabling the manufacturing of biologics and vaccines at the point of care as opposed to traditional centralized manufacturing. One of the earliest cell free translation systems used lymphocytes as a source of raw material for in vitro translation. However, the majority of these studies were performed nearly 50 years ago, and the general understanding of cell free protein expression and immunology has since increased dramatically. Using these advances, we have developed an in-vitro coupled transcription and translation system derived from human blood. Human blood cell free protein expression has many potential applications in personalized medicine, as well as basic science research and diagnostics. We will present data produced from the development of this system.

BIOT 189

Evaluating race specific AGE accumulation as a behavioral biomarker that can reflect optimal health in prostate cancer survivors

Courtney Thomas, [email protected]. Biological and Physical Sciences, South Carolina State University , Orangeburg, South Carolina, United States

Prostate cancer is the second leading cause of cancer-related death in men within the United States, with an estimated 31,620 deaths in 2019 alone. The racial disparity in prostate cancer is large, as African American (AA) males are 1.6 times more likely to develop and 2 times more likely to die from prostate cancer compared to their European American (EA) counterparts. While DNA and genetic mutations are often held responsible for the onset of prostate cancer, we focused on the effect of dietary and lifestyle choices on cancer progression. We found that diets high in Advanced Glycation End Products (AGEs) correlated to increased tumor growth and progression using a novel dietary prostate cancer mouse model. Overall circulating AGE levels have been observed and previously showed to be higher in AA males compared to EA males, correlating to the greater occurrence of high grade prostate cancer witnessed in AA males. In this study, two well studied AGEs, Nδ-(carboxylmethyl)-L-lysine (CML) and Nδ- (carboxylethyl)-L-lysine (CEL), were specifically analyzed for their concentration in both tumor and serum samples via GCMS. Preliminary results indicated tumors from high AGE fed mice had CML levels 65% higher than low AGE fed mice. We plan to evaluate the effect of exercise on AGE levels and tumor growth and progression as well.

BIOT 190

DNA nanostructures-templated proximity assembly and confinement of biochemical reactions

Jinglin Fu1,2, [email protected]. (1) Chemistry Department, Science Building 306A, Rutgers University-Camden, Camden, New Jersey, United States (2) Center for Computational and Integrative Biology, Rutgers University-Camden, Camden, New Jersey, United States

Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiency of multienzyme complexes depends on the spatial organization of composite components which are precisely controlled to facilitate substrate transport and regulate activities. If these cellular mechanisms can be mimicked and translated to a non-living artificial system, it can be useful in a broad range of applications that will bring significant scientific and economic impact. Self- assembled DNA nanostructures are promising to organize biomolecular components into prescribed, multi-dimensional patterns. Here, we described a robust strategy for DNA-scaffolded assembly and confinement of biochemical reactions. DNA nanostructures are exploited to organize spatial arrangements of multienzyme cascades with control over their relative distance, substrate diffusion paths, compartmentalization and functional actuation. The combination of addressable DNA assembly and multienzyme cascades promises to deliver breakthroughs toward the engineering of novel synthetic and biomimetic reactors.

BIOT 191

Enhancing protein crystallization screening results using engineered nucleation features

Kyle Nordquist1, [email protected], Tiffany Kinnibrugh1, Kevin Schaab1, Jessica L. Johnson2, Youngchang Kim2, Gyorgy Babnigg2, Andrew H. Bond1. (1) DeNovX, Chicago, Illinois, United States (2) Argonne National Lab, Argonne, Illinois, United States

Protein structure determination remains a critical facet of understanding cellular functions and developing treatments for disease, and macromolecular X-ray crystallography is still the benchmark method for determining these structures at atomic resolution. However, time- and sample-consuming bottlenecks in the processes between final protein purification and crystallization still exist. Despite advances in high throughput crystallization workflows, < 20% of crystallization screens produce positive results, and fewer crystals from these positive outcomes are of diffraction quality. DeNovX develops crystallization platforms that utilize surface energy modifications to improve crystallization outcomes while retaining diffraction quality of the crystals. Overall, we have demonstrated 1.1-8.7-fold increases in crystallization hit percentages while reducing time to crystallization by an average of 40% for a group of benchmark proteins (e.g., lysozyme, BPT, etc.) and proteins with less characterized crystallization behavior. In addition, samples prepared using engineered nucleation features generate an average of 2.5-fold more crystalline material, which is beneficial where protein supplies are limited and for emerging techniques like X-ray Free Electron Laser Serial Femtosecond Crystallography, which are acutely sample-destructive and can require hundreds or more crystals per study.

BIOT 192

Engineering of virus-derived nanoparticles with controlled architecture for diverse applications Kok Zhi Lee1, [email protected], Yu-Hsuan Lee2, Sue Loesch-Fries4, Michael Harris2, Kevin Solomon3. (1) Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States (2) Chemical Engineering, Purdue University, West Lafayette, Indiana, United States (3) Agricultural & Biological Engineering, Purdue University, West Lafayette, Indiana, United States (4) Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, United States

Nanoparticles are attractive materials for engineering applications in catalysis, sensing, electronics, photonics, and medicine as material properties are controlled by particle size and morphology. For example, increasing battery electrode surface area via nanorods increases its charge capacity, which advances essential and ubiquitous portable technologies such as pacemakers, cell phones, and other devices. However, uniform nanostructures with defined properties are challenging to produce. One strategy to improve structure uniformity and reduce synthesis costs is with metal-coating of rod- shaped biotemplates derived from plant viruses such as Tobacco Mosaic Virus (TMV) and Barley Stripe Mosaic Virus (BSMV). BSMV is particularly attractive as it is able to bind twice as much metal as TMV, increasing capacity further. However, current plant- based production platforms are non-ideal as viral architecture is coupled to viral replication and success, making it challenging to control structure for improved battery performance. Here, we develop E. coli as a production platform for BSMV virus-like particles (VLPs), independent from virus replication. Initial heterologous expression of BSMV coat proteins only produced disc-shaped structures indicative of aborted nanorod self-assembly. To successfully create BMSV-VLP nanorods, we fused the gene for BSMV coat protein to an assembly-initiating DNA sequence from TMV, demonstrating heterologous expression of BSMV-VLPs in E. coli for the first time. We further optimized the production process by improving protein expression conditions, solubility, and purification conditions. Produced BSMV VLPs range in size from 20-160 nm. Collectively, our work creates a novel platform for the synthesis of BSMV biotemplates and enables future engineering opportunities for the development of nanomaterials.

BIOT 193

Synthetic biology for high-performance protein-based materials

Fuzhong Zhang, [email protected]. Energy, Environmental Chem Engineering, Washington University, Saint Louis, Missouri, United States

Advances in synthetic biology have enabled the quantitative engineering of biological processes for the production of various metabolites. Compared to small molecules, efficient and robust production of macromolecular biomaterials is strongly lagging. Microbially produced protein-based materials (PBMs) are particularly attractive to various applications due to their tunable side-chain chemistry, high-performance, and biodegradability. However, high-strength PBMs typically have high molecular weights (HMWs) and repetitive sequences that are difficult to produce microbially due to genetic instability and metabolic burden. We have developed multiple synthetic biology approaches for the synthesis of HMW PBMs using engineered bacterial systems, including a split intein based in vitro protein ligation strategy and an in vivo seeded chain-growth protein polymerization. These biosynthetic strategies were applied to the microbial production of high-performance protein fibers, resulting in synthetic spider proteins with MW reaching 556 kDa and synthetic fibers fully replicate the mechanical performance of natural spider silk by all common metrics, i.e. tensile strength (1.03 ± 0.11 GPa), modulus (13.7 ± 3.0 GPa), extensibility (18 ± 6%), and toughness (114 ± 51 MJ/m3). We also engineered microbes to produce strong underwater adhesive that can be processed to adhesive hydrogels for various underwater repair work. These developed strategies will provide numerous novel materials with high-performance and tailored properties, enabling a broad range of new applications.

BIOT 194

Machine learning techniques for real-time estimation of critical quality attributes (CQAs) in miniature bio-therapeutic manifacturing systems

Benjamin Punshon-Smith1,2, [email protected], Govind Rao2,3, Iordan V. Kostov2,1. (1) Electrical Engineering, UMBC, Gwynn Oak, Maryland, United States (2) Center for Advanced Sensor Technology, Baltimore, Maryland, United States (3) UMBC, Baltimore, Maryland, United States

Machine learning (ML) has seen a rapid growth of interest in the field of bio-therapeutics manufacturing over the years, aiding in advancements of drug discovery, process development, and upstream optimization. These applications have benefited greatly from the wealth of real-time sensing data that companies have accrued over many generations of discovery. Most of these ML applications have been applied to the front- end or R&D phase of bio-therapeutic manufacturing with its application to drug discovery alone expected to reach a market volume of $10 B by 2024. However, the growing introduction of miniaturized bio-therapeutic platforms utilizing rapid ‘cell-free’ technologies have uncovered a growing desire for instantaneous quality assessment or ‘real-time release’. Here we propose a ML framework for the real-time estimation of critical quality attributes (CQA’s) of bio-therapeutics using in-line process analytic technologies (PAT’s) in end-to-end bio-therapeutic manufacturing. The algorithm employs a deep layer artificial neural network (ANN) with a gaussian kernel applied at the feature input to the network. A genetic algorithm (GA) is used for the optimization of the parameters for the both the kernel and ANN to maximize the performance. The framework is verified on a miniaturized cell-free bio-therapeutic manufacturing platform that produces small batch therapeutics. The platform can incorporate real-time sensing such as multi-wavelength absorbance, fluorescence, conductivity, Raman spectroscopy, size exclusion chromatography (SEC), and pressure. Furthermore, a training data set is developed by perturbing repeated purification of a target protein from a lysate-based model system. The CQA’s can be measured with techniques such as Mass Spectrometry, SDS-PAGE, Capillary Electrophoresis, functional assay, and dynamic light scattering. The real-time assessment of quality attributes such as purity, aggregation, and integrity have strong implications on both the front-end and back-end of bio-therapeutic manufacturing as we inch closer to acquiring ‘real-time release’.

BIOT 195

3D printed and polyester functional graphenic materials as osteoinductive and mechanically enhanced scaffolds for bone regeneration

Stephen Schmidt1, [email protected], Brian Holt1, Anne Arnold1, Eman Mirdamadi3, Neeha D. Arun3, Leila Daneshmandi2, Wyatt Swift-Ramirez1, Walker Vickery1, Michelle Wolf3, Cato Laurencin4, Adam W. Feinberg3,5,6, Stefanie A. Sydlik1,3. (1) Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States (2) Biomedical Engineering, University of Connecticut, Farmington, Connecticut, United States (3) Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States (4) University Connecticut Health Center, Farmington, Connecticut, United States (5) Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States (6) Mechanical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States

Traditional metal implants such as titanium, cobalt, and chromium have found wide utility in stabilizing large bone fractures, however, these come with a risk of toxicity. This risk can be mitigated with synthetic, resorbable scaffolds for bone regeneration, and have the potential to transform the clinical standard of care. We take multiple strategies to design resorbable scaffolds in functional graphenic materials (FGMs), and our recent efforts have utilized polyester-functionalized FGMs and the 3D printing of osteoinductive calcium phosphate graphene (CaPG). Polyesters including polycaprolactone (PCL), polylactic acid (PLA), and polyglycolic acid (PGA) show promise for the treatment of certain types of traumatic bone injuries due to their accepted biocompatibility and FDA approval. However, polyesters are less stiff than their metallic counterparts, limiting their application as fixation hardware to non-load bearing injury sites. To improve mechanical properties, graphene oxide (GO)-polyester composites are a promising class of biodegradable scaffolds. Initial reports of these composites are encouraging, but mechanical properties still fall short: traditional composites rely on non-covalent association between GO and the polyesters, which weakens the overall strength of the material. Herein, we present a strategy for attachment of these FDA-approved polyesters onto a derivative of GO using a robust covalent bond. While characterizing these materials, we also developed an X-ray photoelectron spectroscopy (XPS) end- group analysis method to determine polyester molecular weight. This method will aid other researchers characterizing polyesters via XPS. We are also optimizing 3D printing with CaPG, the most osteoinductive phosphate graphene FGM. CaPG intrinsically induces osteogenesis in vitro and in the presence of bone marrow stromal cells 2+ 3- (BMSCs), can induce ectopic bone formation in vivo with the release of Ca and PO4 inducerons. 3D printing porous CaPG scaffolds can be accomplished with polylactic-co- glycolic acid in a trisolvent organic ink blend, or with natural collagen via freeform reversible embedding of suspended hydrogels (FRESH). By covalently functionalizing FGMs 1) with polyesters, we create FGMs to simultaneously retain biodegradability and compatibility, but also mechanically strengthen PCL, PLA, and PGA by up to 264%, and 2) as CaPG for 3D printing, we present an effective method of obtaining porous, osteoinductive scaffolds.

BIOT 196

Clonable metal nanoparticles: Applications in electron and fluorescence microscopies

KANDA BORGOGNONI, [email protected]. Chemistry, Colorado State University, Fort Collins, Colorado, United States

Biological microscopic studies, once revolutionized by the discovery of encodable fluorophores such as Green Fluorescent Protein (GFP), engender foundational evidence for cellular processes via in situ protein labeling and tracking by fluorescence. As fluorescence microscopy is inherently hindered by the diffraction limit of light, electron microscopy unveils details of cellular ultrastructure with atomic level resolution. The primary barrier preventing biological electron microscopy from overtaking fluorescence microscopy as the main imaging modality is creating target specific contrast. While methods have been developed to generate more specific contrast agents, none have achieved the specificity of an encodable tag. Our lab initiated the development of encodable contrast agents for electron microscopy by isolating a glutathione-like metalloid reductase from a selenophilic plant capable of reducing selenium precursors into nanoparticles. After confirming the portability of the enzyme through maintenance of its activity in vitro and in vivo, modification of the wild type further enhanced its performance as a clonable selenium nanoparticle (cSeNP) tag. We then used the cSeNP tag in a proof of concept study that involved tracking filamenting protein FtsZ, our model system, in vivo using electron microscopy and elemental mapping. Once we fused the cSeNP to FtsZ, initial results generated using electron microscopy closely matched previous fluorescence microscopy studies of GFP tagged FtsZ in vivo. The next phase of this project relies on collaborating with experts in the field of 3D biological electron microscopy to generate tomograms of cSeNP decorated filaments. Ongoing work includes not only post-synthetic modification of cSeNPs to create fluorescent quantum dots (i.e. cCdSe) for applications in fluorescence microscopy, but also expansion of our clonable nanoparticle toolbox through the discovery of other metal reducing enzymes.

BIOT 197

We can do better than kcat/KM: Theory-based development of performance metrics for comparing multi-reactant enzymes

Scott Banta, [email protected]. Chemical Engineering, Columbia University, Fairfield, Connecticut, United States

Protein Engineering provides a toolbox for the creation of new biocatalysts with capabilities beyond what is found in the natural world. As new enzymes are designed, evolved and engineered, it is critical to develop figures of merit to benchmark and compare performances under different conditions. The ratio of kcat/KM is widely used in the evaluation of enzymes due to its mechanistic importance in uni-molecular (Michaelis Menten) enzymatic reactions. However, its applicability in the evaluation of multi- reactant systems is widely used but rarely justified. Therefore, we have derived new figures of merit for multi-reactant enzyme mechanisms based on the stabilization of transition states and/or the kinetic Haldane equilibrium relationships. For most bi- molecular reactions, the ratio of kcat/KiaKb (where Kia is the dissociation constant of A and Kb is the Michaelis constant of B) is best suited for comparing steady state enzyme performances. This approach can be extended to derive useful figures of merit for other more complex reaction mechanisms. The value of the new parameters for the comparison of a series of alcohol dehydrogenase mutants will be discussed.

BIOT 198

Learning the secrets of sequences with deep generative models

Debbie Marks, [email protected]. Harvard Medical School, Boston, Massachusetts, United States

What can we do with a million or a billion genomes? Understanding how variation across genomes shapes the properties of biomolecules, cells, and organisms is a foundational question in biomedicine and biotechnology. I will present examples of how generative modeling of genetic variation can give surprisingly direct answers to questions about 3D structures, dynamics and the effects of mutations. Our new work extends from the undirected models of genetic variation to deep directed models using newly developed variational autoencoders, and autoregressive models that do not depend on sequence alignments. From purely unsupervised learning, we double the improvement of prior-art for predicting the effects of mutations. I will introduce challenges for extending these methods to diverse biomedical and engineering applications, with specific examples of successful probabilistic models to generate novel functional biotherapeutics

BIOT 199

Enabling design of selective kinase inhibitors through data science

Palani Kirubakaran, Grigorii Andrianov, John Karanicolas, [email protected]. Molecular Therapeutics, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States

Protein kinases are essential to nearly every cellular signal transduction pathway. Because of this, and because of their inherent druggability, kinases have become one of the most important drug targets in the past two decades. The druggability of kinases stems from the fact that these enzymes share a strongly-conserved ATP-binding site, which is structurally very amenable to inhibition by the types of flat, aromatic scaffolds that are most accessible to synthetic chemistry. However, the human “kinome” is comprised of ~500 kinases with highly similar active sites, making it difficult to develop selective inhibitors. We have recently developed a comparative modeling approach that allows for rapid predictions of the three-dimensional structure of arbitrary (Type I) kinases. We use these models, together with large and comprehensive experimental measurements of kinase-wide selectivity for many inhibitors, as the basis for training deep learning models. These models can accurately predict the binding affinity of arbitrary inhibitor/kinase pairing, and from this data the selectivity of an arbitrary compound can be evaluated. By coupling this tool with large in silico libraries of novel chemical matter that resembles kinase inhibitors, we anticipate that potent and selective kinase inhibitors can be developed for much of the heretofore un-addressed kinome.

BIOT 200

Data-driven modeling of SHP2-regulated signaling to identify combination therapy approaches in glioblastoma

Evan Day1, [email protected], Matthew Lazzara2. (1) University of Pennsylvania, Philadelphia, Pennsylvania, United States (2) University of Virginia, Charlottesville, Virginia, United States

Targeted therapy efficacy in cancer is influenced by context-dependent, multivariate cellular responses to inhibition of the drug target. These effects often determine whether targeting a particular protein will lead to durable responses in patients, irrespective of the affinity or selectivity of the drug. SH2 domain-containing phosphatase-2 (SHP2) is a proto-oncogene in some cancers for which potent and selective allosteric inhibitors have been recently developed. While SHP2’s most well-known role is to promote activity of the extracellular regulated kinase (ERK) downstream of receptor tyrosine kinases (RTKs), SHP2 can also negatively regulate proliferation and survival signaling (e.g., via the STAT3 pathway). These divergent roles make it difficult to predict the consequences of SHP2 inhibition. For example, we recently demonstrated that in glioblastoma cells SHP2 knockdown simultaneously slowed proliferation but also increased resistance to RTK inhibitors and temozolomide chemotherapy. Our studies also revealed a role for SHP2 in regulating the expression of hypoxia-inducible factors (HIFs) in low oxygen environments, further complicating our ability to predict the effects of SHP2 antagonism in a tumor. To develop quantitative understanding of the pathways and phenotypes regulated by SHP2 in glioblastoma, we implemented a data-driven modeling approach. In cells treated with RTK inhibitors or temozolomide, we measured dynamic changes in the phosphorylation of >40 proteins over 48 hrs using Luminex bead-based immunoassays. Experiments were repeated with or without SHP2 knockdown and cultured in 21% or 1% oxygen, for a total of ~3000 distinct signaling measurements. Parallel quantitative measurements of cell cycle progression, cell death, and HIF expression were made. The matched signaling and phenotypic measurements were subjected to partial least squares regression (PLSR) analysis to identify key, phenotype- determining SHP2-regulated signaling processes across different drug treatments and levels of oxygen exposure. One therapeutically relevant prediction from our model was the observation that temozolomide resistance in response to SHP2 inhibition was mediated by a non-canonical role of the tumor suppressor PTEN, which could be overcome by combinatorial RTK inhibition.

BIOT 201 Withdrawn

BIOT 202

Neural network correlation of protein retention in chromatographic separations

Schuyler R. Reed, Abraham M. Lenhoff, [email protected]. Univ of Delaware, Newark, Delaware, United States

Prediction of protein retention times in chromatographic elution has previously been attempted using models based on protein and resin structural properties and biophysical mechanisms. QSAR models that also employ detailed 3D protein structure information but in the form of structural descriptors have also been used. In the interest of correlating retention without using full protein structural information a simpler approach was investigated here by training a neural network based on the amino acid sequence of a protein. In doing so the prediction process can be accelerated and information about elution behavior can be gained with less information about the protein structure. Retention data sets were obtained from the literature and amino acid sequences were taken from on-line databases. These sets were trained via Matlab’s neural network training tool both using full amino acid sequences and using just the amino acid composition. For each system, networks were explored with different architectures, including different layer sizes and numbers of internal neurons. Networks were found that could correlate retention times of proteins in both ion-exchange and hydrophobic interaction chromatography with R2 values exceeding 0.95, without evidence of overfitting, a potential risk because of the small size of the data set. Investigations using larger data sets would allow more extensive evaluation of the approach.

BIOT 203

Physical and chemical determinants of drug-like monoclonal antibodies

Yulei Zhang1, [email protected], Lina Wu1, Priyanka Gupta2, Emily K. Makowski3, Lilia Rabia4, Seth Ludwig4, Peter M. Tessier5. (1) Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States (2) Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut, United States (3) Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States (4) Rensselaer Polytechnic Institute, Ann Arbor, Michigan, United States (5) Chemical Engineering and Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States

The ability of antibodies to recognize their target antigens with high specificity is fundamental to their normal function. Nevertheless, therapeutic antibodies display difficult-to-predict levels of non-specific (heterotypic) and self- (homotypic) interactions that lead to various drug development challenges, including antibody aggregation, abnormally high viscosity and fast antibody clearance. We are developing bioinformatics methods for predicting the overall specificity of antibodies in terms of their relative risk for displaying high levels of non-specific and self-interactions. We find that individual and combined sets of chemical rules, comprised of maximum and minimum limits on the amounts of specific types of solvent-exposed residues in antibody binding loops, are excellent predictors of specificity for large panels of preclinical and clinical-stage antibodies. We have validated these predictions using independent sets of antibodies and mutants of antibodies with known specificity problems, and we are expanding our predictive methods using deep sequencing and machine learning. We expect that our findings can be readily used to improve the selection and engineering of antibodies with drug-like specificity.

BIOT 204

Predicting protein developability via a deep informatic/experimental hybrid

Alexander Golinski, [email protected], Katelynn Mischler, Nicole Neurock, Matthrew Fossing, Sidharth Laxminarayan, Hannah Pichman, Benjamin Hackel. Chemical Engineering, University of Minnesota, Minneapolis, Minnesota, United States

Proteins must exhibit both a designated primary function (e.g., specific binding, enzymatic activity) and developability (stability, solubility, and producibility) to become lead candidates for downstream applications. While many methods enable high- throughput discovery of primary function, unless developability is additionally screened, translational challenges often occur. Evolving developability is difficult because of the immensity of sequence space and the limited throughput of measuring recombinant yield and thermostability. We hypothesize that high-throughput developability assays will empower deep data collection and analysis to improve the design and discovery of developable proteins and combinatorial libraries. We utilized the small protein scaffold GP2 with two diversified loops as a proof of concept for predicting developability from loop sequence. 105 mutants were subjected to 3 classes of assays previously validated to be representative of protein stability and expression. A yeast-displayed stability assay differentiated mutants by the degree of proteolytic cleavage using a combination of proteinases, temperatures, and denaturants. A bacterial split-GFP assay separated mutants by expression as measured by reconstituted GFP fluorescence. A split β-lactamase assay separated mutants by solubility, stability, and expression, as the GP2-enzyme must be transported to the periplasm as a well-folded protein to remain active. The sorted populations were deep sequenced to measure clonal enrichment scores related to developability. Machine learning models were trained to predict the assay scores based upon sequence yielding an average Spearman’s correlation coefficient of 0.58 across the assays. Results confirm an increase in median stability of a disulfide stabilized loop and 2-9% increase in Spearman’s rho when utilizing an embedding and recurrent neural network over traditional modeling strategies. Also, SHuffle cells, engineered for increased expressability, were shown to increase clonal differentiation. Our model was able to reduce continued experimentation of poorly developable mutants by lowering the false positive rate of producible proteins by 5%. Continued progress will aim to predict additional developability metrics including thermostability and self-aggregation to identify a more developable subset of mutants. Long term advancements will expand the assays to assess alternative protein scaffolds, enzymes, and anti-microbial peptides.

BIOT 205

Studies on disposable screen-printed electrodes for diagnostic application of glycated hemoglobin A1c

Xiao-Chuan X. Liu, [email protected], Jessica Leng. Department of Chemistry and Biochemistry, California State Polytechnic University, Upland, California, United States

A novel amperometric biosensor prototype was fabricated using screen printing technique. The disposable single-use strips were made from conductive carbon ink and modified with fructosyl amino acid oxidase. The electrodes and conducting paths were made solely with carbon ink and characterized by conductivity and cyclic voltammetry. The biosensor showed high current output, large linearity, and effectiveness for fructosyl valine as well as human blood samples. Amperometric studies were carried out using both fructosyl valine and human blood samples. With 5 uL sample volume, the biosensor showed strong amperometric response with good linearity for a wide range (0 to 8 mM). Diabetic and healthy blood samples showed sufficient difference in their amperometric responses that correlates well with their different hemoglobin A1c levels. These results demonstrate the feasibility of using this type of inexpensive single-use biosensor strips as the basis for determining hemoglobin A1c levels for diabetic patients.

BIOT 206

Heterogeneity of neuroendocrine cells in normal prostate and in prostate cancer with neuroendocrine differentiation and localized calcitonin secretion

Ebony Thornton1, [email protected], Courtney Thomas2. (1) Chemistry, South Carolina State University, Orangeburg, South Carolina, United States (2) Biological and Physical Sciences, South Carolina State University, Orangeburg, South Carolina, United States

Prostate cancer is the second leading cause of cancer-related deaths in men. Research has shown that the overall African American (AA) male population are diagnosed with more advanced and aggressive prostate cancer than their European American (EA) counterparts. Neuroendocrine cells (NEC) are naturally found in the prostate gland as the third subset of epithelial cell type. Calcitonin (CT) peptides are markers synthesized within the prostate epithelium mainly for malignant prostates and can be found internally or on the surface of NECs. We hypothesize that location of calcitonin secretion of NECs by neuroendocrine differentiation (NED) markers will demonstrate expression from both AA and prostate tissue cells due to the NEDs signaling of localized CT. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods will designate all markers and calcitonin location in normal and prostate cancer cells.

BIOT 207

Development of a novel radiopaque alginate-based photocrosslinkable hydrogel for intravascular embolism of cerebral aneurysms

Geunho Choi1, [email protected], Jongkyeong Lim2, Joonwon Kim2, Hyung J. Cha1. (1) Chemical Engineering, Pohang University of Science and Technology, Pohang Gyeongbuk, KR-47, Korea (the Republic of) (2) Mechanical Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea (the Republic of)

Cerebral aneurysm is a cerebrovascular disorder which is characterized by a weakness in the wall of a cerebral artery or vein, causing a localized dilation or ballooning of the blood vessel. Endovascular coiling, which is non-invasive, has a short surgical time, and does not require cranial nerve manipulation, has recently attracted attention as a mean of preventing aneurysm rupture. However, incomplete occlusion by coil compaction and unstable thrombus cause recurrence of aneurysm in about 20% of cases. Alginate, a natural polysaccharide from seaweed, can have low injectable viscosity in its liquid form and high mechanical strength in its solid form. Through the development of photocrosslinkable alginate, we developed a novel biocompatible and non-toxic endovascular embolization biomaterial. We formed strand-like hydrogel fibers using catheter-based hydrogel microfibers generating devices to prevent endoleak and achieve better fills and conforms to the contour of aneurysms. In addition, the use of tantalum particles ensures radiopacity, allowing CT and MRI devices to be used. Collectively, we developed a new system for treating cerebral aneurysms which can fill the aneurysm more completely and prevent rupture.

BIOT 208

Control of population dynamics and metabolome in mixed cultures by prebiotics

Thomas J. Mansell, [email protected], Fatima Enam, Yanfen Bai. Chemical and Biomolecular Engineering, Iowa State University, Ames, Iowa, United States

The synbiotic relationship between human milk oligosaccharides (HMOs) and probiotic Bifidobacteria exemplifies prebiotic control of microbial community dynamics. Inspired by this example, we have engineered the well-known probiotic, E. coliNissle, to metabolize HMOs and used this metabolism to control population dynamics and protein expression in mixed cultures of E. coli. We accomplish this using a unique whole-cell biosensor which provides linkage-specific, quantitative detection of various HMOs. Addition of these complex substrates to synthetic microbial consortia orthogonally controls growth rate or protein expression of particular strains. We can also mimic selfish, altruistic, and social cheating ecological behaviors in these communities. In addition, we performed further metabolic engineering on our target probiotic, enabling production of short-chain fatty acids from HMOs as sole carbon sources, and ecapitulating an important function of the infant gut microbiota. Combining the two approaches, we show that by controlling population dynamics, we can ontrol the metabolome of the mixed culture. Finally, we present a high-throughput, sequencing- independent method of tracking the dynamics of an engineered probiotic in mixed culture. This work lays the groundwork for the application of directed evolution to biosynthesis of complex carbohydrates as well as the prebiotic manipulation of population dynamics in natural and engineered microbial communities.

BIOT 209

Engineering microbiomes using physical-organic chemistry

Mohit S. Verma1,2,3, [email protected]. (1) Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States (2) Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United States (3) Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana, United States

The gut microbiome is complex: billions of bacteria from hundreds of species produce thousands of metabolites. How do we engineer such complex communities? Physical- organic chemistry—i.e. the science of establishing links between the molecular structure and function of a compound in a physical system—provides one simple approach. I will share two vignettes of this physical-organic approach: the first one studies the biological activity of a short-chain fatty acid (butyrate) and the second explores the impact of carbohydrates on the microbiome. Two of the biological activities of butyrate in the colon (suppression of proliferation of colonic epithelial stem cells and inflammation) correlate with the inhibition of histone deacetylases. Structure-activity relationships were compatible with the hypothesis that butyrate acts by binding to the Zn2+ in the catalytic site of histone deacetylases. In the second example, carbohydrates provide a method for tuning the composition of the microbiome and building future synbiotics (probiotics + prebiotics). These studies offer a starting point for guiding the desired outcomes from a “healthy” gut microbiome.

BIOT 210

Accelerated virus-less generation of stable insect Sf9 cell lines for high-yield production of influenza vaccines

Christine Yee, [email protected], Prabhu Ponnandy, Andrew Zak, Fei Wen. University of Michigan, Ann Arbor, Ann Arbor, Michigan, United States Current manufacturing of vaccines in insect Sf9 cell line relies on the Baculovirus Expression Vector System, which is limited by virus-induced cell lysis. Thus, there is an interest in the field towards virus-less gene expression to simplify the costly product purification process. Current virus-less methods are based on random integration of the gene of interest into the genome, which generates a heterogeneous cell population with a wide distribution in both protein expression and growth rate. This population is unstable over time hampering the reproducibility of vaccines production. Therefore, a high-yield stable cell line is typically isolated by limiting dilution, a low-throughput process that can take up to 8 weeks. To accelerate the generation of stable cell lines, a potential solution is to use site-specific genome editing tools such as CRISPR to rapidly create more homogenous, high-yield populations. However, the lack of genomic information of the Sf9 cell line has hindered the design of key CRISPR components and limited its utilization. Taking advantage of the recently published Sf9 genome, we not only knocked out the fused lobes gene to enable expression of human-like glycoprotein, but also achieved targeted insertion of a short DNA sequence. As in other higher eukaryotes, however, a substantial proportion of CRISPR-mediated double stranded breaks resulted in non- templated repair by nonhomologous end joining (NHEJ). To increase the insertion efficiency, we aimed to knock out this competing repair pathway. We successfully employed bioinformatics tools and identified hypothetical genes responsible for NHEJ. More importantly, we confirmed their biological function, as their knockout via CRISPR resulted in stable Sf9 cell lines with improved efficiency in site-specific gene integration through homologous directed repair (HDR). These engineered Sf9 cell lines have enabled rapid generation of high-yield stable cell lines without the need for limiting dilution. This system facilitates high-throughput production of recombinant HA to develop improved vaccines for each year’s influenza season and offers an attractive platform to advance the development and manufacturing of other protein therapeutics.

BIOT 211

Withdrawn

BIOT 212

Elucidating the effects of direct physical contact between dental pulp stem cells cultivated on polybutadiene with different stiffness on proliferation and differentiation behaviors

Zijian Ma1, [email protected], Yihan Shen2, [email protected], Jessica Hofflich3, Ya-Chen Chuang3, Miriam Rafailovich3, Marcia Simon4. (1) Tianjin Nankai High School, Tianjin, Tianjin, China (2) St.Andrew's School, Middletown, Delaware, United States (3) Materials Science and Engineering, stonybrook university, Stony Brook, New York, United States (4) Stony Brook University School of Dental Medicine, Stony Brook, New York, United States Dental Pulp Stem Cells (DPSCs) have demonstrated immense potential for therapeutic purposes due to their easy accessibility as well as their capacity for self-renewal. Nonetheless, despite the increasing body of knowledge of stem cell transplantation, achieving specificity in cell responses remains challenging. In our previous study, we have shown that monodispersed Polybutadiene (PB) forms a convenient biocompatible scaffold to which cells can adhere without additional coatings. DPSCs plated on these PB substrates were able to adjust their moduli in response to the film thickness. However, a threshold at 2.3 MPa was observed after 28 days such that large amounts of biomineralized deposits would be produced if the substrate moduli were higher than this value. In this work we investigated how direct cell-cell contact of DPSCs cultured on PB substrates with different thicknesses(20nm and 200nm), representing hard and soft substrate mechanics effects, respectively, impacts differentiation pathways. The substrates were designated into 3 groups: contact group, non-contact group, and control group. Cell moduli and morphology were investigated by Atomic Force Microscopy and Confocal Laser Scanning Microscopy, respectively, after the first week of culture. Reverse Transcriptase Polymerase Chain Reaction was performed for selected gene markers: Alkaline Phosphatase(ALP), Runt-related transcription factor(Runx), and late markers, Dentin Sialophosphoprotein(DSPP) and Osteocalcin(OCN). The latter served as indicators of odontogenic and osteogenic differentiation. In addition, microarray was conducted to explore whether genetic expressions were affected by cell contacts. Finally, biomineralization images were captured on day 28 using Scanning Electron Microscopy. Our experimental results signify that DPSCs on substrates with different mechanics can alter their behaviors through direct cell-cell contact. No biomineralization was found on either hard or soft substrates when cells physically contact; however, differentiated and non-differentiated cells were capable of coexisting within a single culture when they did not contact across different substrates, where the phenotype was governed by the substrate mechanics. This study explored the influence of purely mechanical heterogeneity and the significance of cell communication, which are important for applying printed scaffolds as dentin/tooth regenerative biomaterials and promoting stem cell-based treatments.

BIOT 213

Ethanol tolerance and lipidomic analysis of protease-deficient yeast expressed with an ATP-binding cassette transporter gene of cyanobacterium

Israel Olayide, [email protected], Monica D. Rieth. Chemistry, Southern Illinois University Edwardsville, Edwardsville, Illinois, United States

Ethanol derived from biomass, a renewable energy, has the potential to be a sustainable, low emission transportation fuel. Saccharomyces cerevisiae, commonly known budding yeast, is a model organism for biological mechanisms in eukaryotic cells and a useful industrial microorganism to produce biofuel and alcoholic beverages. Genetic manipulation of yeast strains with the ability to adapt and tolerate high ethanol is an important factor to select yeast strains for efficient bioethanol fermentation. One factor that has been implicated in ethanol tolerance is the composition of the lipid membrane surrounding the yeast cells. Lipids are principal modulators of membrane fluidity and exert a crucial role in the ethanol tolerance of S. cerevisiae and other microbes. We can assess ethanol tolerance by examining the growth profile and the lipid composition of the yeast strains. In addition, membrane protein transporters for small molecules have also been suggested as a relevant factor in ethanol tolerance. In this study, we aim to produce genetically modified yeast mutants with enhanced ethanol tolerance by introducing an ATP-binding cassette transporter of a cyanobacterium Synechocystis sp. (encoded by slr0982) by way of a yeast episomal plasmid. pYES2 is a well-considered episomal yeast plasmid with a multiple cloning region that enables facile gene insertion and expression by utilizing the strong induction properties of the galactose promoter. We suggest that increased ethanol tolerance by yeast cells harboring slr0982 gene will provide a novel yeast strain for enhanced productivity in bioethanol fermentation.

BIOT 214

Enhanced therapeutic effect of therapeutic protein by co-localization of toxicity eliminating agent in nanocarrier

Seoungkyun Kim1, [email protected], Manse Kim2, Secheon Jung1, Kiyoon Kwon1, Junyong Park1, Sukhwan Kim1, Inchan Kwon1, Giyoong Tae1. (1) Gwangju Institute of Science and Technology, Gwangju, Korea (the Republic of) (2) Division of Infectious Diseases, University of North Carolina, Chapel Hill, North Carolina, United States

Therapeutic proteins are attracting much attention due to their high efficiency and specificity compared to chemical drugs. However, because the products of therapeutic proteins can cause side effects, it is necessary to reduce side effects as well as enhancing therapeutic effects. Urate oxidase (UOX) is a therapeutic protein clinically used for hyperuricemia patient. Uric acid (UA) is highly insoluble in blood and UA precipitation deposits in joint, which causes pain and swelling. Furthermore, high concentration of UA can cause kidney diseases, cardiovascular diseases, and tumor lysis syndrome (TLS). Since UOX catalyzes the conversion of uric acid (UA) to 5- hydroxyisourate, UOX is promising drug for treatment of hyperuricemia. However, UOX produces cytotoxic hydrogen peroxide (H2O2) during the oxidation of uric acid, which causes unwanted side effects. To enhance the therapeutic effect of therapeutic protein, we used UOX with catalase-mimic gold nanoparticle (AuNP) as a toxicity-eliminating agents. For co-delivery both UOX and AuNP in vivo, we employed pluronic-based nanocarrier, which showed highly biocompatibility and simple loading process. UOX and AuNP loaded in NCs showed higher UA degradation and generated lower amount of H2O2 compared with UOX alone. In addition, co-loaded UOX and AuNP showed no cytotoxicity itself and reduced cytotoxicity caused by H2O2 in vitro. Moreover, we confirmed that UOX and AuNP loaded NCs normalize hyperuricemia faster in vivo than UOX alone. Through this study, we showed that drug delivery of therapeutic protein with toxicity-eliminating agents could enhance therapeutic effect.

BIOT 215 Bioinspired-nanovesicles made from maize for therapeutic anti-colon cancer vaccine delivery

Kanokwan Sansanaphongpricha1, [email protected], Tapanee Thinbanmai1, Purichaya Sompran2, Umaporn Uawisetwathana3, Chamraj Kaewraemruaen2, Trairak Pisitkun2, Nattiya Hirankarn2, Nattika Saengkrit1. (1) National Nanotechnology Center, National Science and Technology Development Agency, Pathumthani, Thailand (2) Faculty of Medicine, Chulalongkorn University, Bangkok, Bangkok, Thailand (3) National Biotechnology Center, National Science and Technology Development Agency, Pathumthani, Thailand

Plant-derived nanovesicles have gained a tremendous attention for drug delivery due to high safety profiles, low cost of production and ability to load drugs. One of the most interesting nanoparticles derived from plants is exosomes. Exosomes are intracellular vesicles produced from golgi apparatus and endoplasmic reticulum, which entraps miRNA, proteins, and several active compounds produced from plant cells for intercellular communications. Interestingly, these extracellular vesicles carry various molecules constituents of the producing cells, which provides therapeutic effects. In addition, the substances inside the extracellular vesicles can be removed out to generate empty vesicles making them ready for drug loading. In our study, we demonstrate the use of extracellular vesicles derived from maize as a carrier for anti- colon carcinoma vaccine. We firstly isolated the extracellular vesicles from maize and subsequently removed out the endogenous miRNA, proteins or other molecules from the vesicles to obtain the empty extracellular vesicles. The nanovesicles then were modified to load with Poly I:C dsRNA, which is a strong adjuvant for immunity and ADPGK antigenic peptide for colon carcinoma. The loaded vesicles were characterized by a transmission electron microscope (TEM) and dynamic light scattering (DLS). The loading efficiency of the poly I:C and the antigenic peptide are 46% and 97% respectively, which are relatively comparable to the loading efficiency of those in a liposome control. Data from flow cytometry analysis and Real-Time qPCR suggested that the poly I:C and antigenic peptide loaded exosome-like vesicles strongly activated dendritic cell maturation and up regulated interferon- related genes, which play a crucial role in cancer immunotherapy. Moreover, the exosome-like vesicles not only protect the peptide and the dsDNA adjuvant from degradation but also enhance cellular uptake by endocytosis. Our results indicate that plant-derived extracellular nanovesicles are very promising to use as a biomolecular carrier.

BIOT 216

Incorporation of non-natural amino acids containing a tetrazine functional group into proteins for fast site-specific bioconjugation

Byungseop Yang, [email protected], Seoungkyun Kim, Inchan Kwon. School of Materials Science and Engineering(SMSE) , Gwangju Institute of Science and Technology(GIST) , Gwangju, Korea (the Republic of) Site-specific bioconjugation has been used in diverse biological applications. For site- specific bioconjugation, various bioorthogonal chemistries have been developed. Certain bioorthogonal chemistries, such as strain-promoted azide-alkyne cycloaddition, exhibited relatively short reaction rates, hindering their applications in cellulo or in vivo. Therefore, recently, inverse electron-demand Diels-Alder cycloaddition (IEDDA) attracted much attention thanks to its fast reaction rates. So far several non-natural amino acids containing tetrazine functional group were developed for site-specific bioconjugation of proteins via IEDDA. However, in vivo applications of tetrazine- containing proteins were not yet extensively studied. We investigated the site-specific incorporation of a new variant of tetrazine containing non-natural amino acid into proteins and its bioconjugation via IEDDA. The new variant of tetrazine-containing non- natural amino acid showed a much faster reaction rate than previous versions of tetrazine-containing non-natural amino acid. Using the new variant of tetrazine- containing non-natural amino acid, time-controlled in vivo imaging of protein requiring a very fast bioconjugation reaction rate is under investigation.

BIOT 217

Evaluation of pharmaceutical container for parenteral products

Youlong Ma, [email protected], Charudharshini Srinivasan, Sau Lee. US Food and Drug Administration, Silver Spring, Maryland, United States

Particulate generation in parenteral solutions can compromise quality and safety of the parenteral products. Particulates can be extrinsic (dust particles, glass fragments) or intrinsic (aggregates, corrosion of glass containers). The presence of particulates in parenteral products results in potential risk for patients and can result in costly recalls. It is of great importance to avoid particulates and ensure the safety of the vial and drugs before they are in the market. In this research, the effect of solution chemistry, storage temperature, and time on corrosion of glass vials were investigated to develop the screening technique.

Model parenteral formulations with various pHs, temperatures and storage conditions were investigated. Membrane filters with particulates and corresponding containers were characterized using orthogonal techniques. Visual inspection of stability sample at pH>10 confirmed the twinkling flakes after 6-month exposure. Microscopic techniques revealed glass flakes formation for high temperature and pH conditions, while no flakes were observed at lower temperature conditions (Figure 1).

Particulates in model formulations were found to be influenced by solution chemistry, storage time, temperature. The orthogonal techniques enabled as a reliable screening for particulates from sub-micron to visible size. Moreover, such combined techniques provided evidence for appropriate risk assessment and evaluation of pharmaceutical containers.

Figure 1. SEM images of the heel and neck regions in glass containers for high pH conditions at accelerated temperature (60°C) after approximately 6 months of exposure to model parenteral formulations.

BIOT 218

Targeted-delivery of siRNA via a polypeptide-modified liposome for the treatment of gp96 over-expressed breast cancer

Enxia Zhang1, Linying Du1, Ze Liang1, Yinan Zhao2, Wei Wang1, Pengfei Ma1, Shubiao Zhang2, Yuhong Zhen1, [email protected]. (1) College of Pharmacy, Dalian Medical University, Dalian, China (2) Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University, Dalian, China

In recent years, targeted gene therapy has led to significant breakthroughs in cancer treatment. Heat shock protein gp96 is an emerging target for tumor treatment because of its transfer ability from reticulum to tumor cell surface. CDO14 is a peptide cationic liposome developed in our laboratory with higher gene transfection efficiency and lower toxicity compared with the existing cationic liposomes. In this study, gp96-targeted liposome p37-CDO14 was constructed by modifying cationic liposome CDO14 with a gp96 inhibitor, helical polypeptide p37. p37-CDO14 could specifically bind to breast cancer with gp96-overexpression on cell membrane. Both CDO14 and p37-CDO14 showed high delivery efficiency for survivin siRNA (siSuvi) to SK-BR-3 and MCF-7 cells via obviously decreased survivin expression level and cell viability. p37-CDO14 significantly increased the accumulation of FAM-siRNA in tumor compared with CDO14. siSuvi transfected by CDO14 and p37-CDO14 could inhibit the growth of xenograft in mice and the expression of survivin in tumor tissues. The anti-tumor effect of siSuvi delivered by p37-CDO14 was much higher than that delivered by CDO14. This suggests that targeted liposome p37-CDO14 is a potential gene vector for the therapy of gp96-overexpressed breast cancer.

Fig1.Graphical abstract

BIOT 219

Galvanic exchange-induced growth of Au nanocrystals on CuS nanoplates for imaging guided photothermal ablation of tumors

Zhaojie Wang1, [email protected], Nuo Yu1, Xuan Li1, Wanjian Yu1, Shilong Han2, Xiaoling Ren1, Shiwu Yin3, Maoquan Li2, Zhigang Chen1. (1) Donghua University, Shanghai, China (2) Shanghai Tenth People’s Hospital, Shanghai, China (3) The Second People's Hospital of Hefei City in Anhui Province, Hefei, China

The conventional methods for constructing CuS-Au heterostructures in aqueous solution require the assistance of reducing agents such as NaBH4 and sodium borohydride. Herein, without the additional reducing agent, we found that CuS nanoplates can be used as efficient nanoplatform and reducing agent for constructing CuS-Au heterostructures through the galvanic exchange route. The CuS-Au heterostructures were prepared by facilely introducing CuS nanoplates into HAuCl4 solution with continuous stirring, and the size (10~45 nm) and the number (1~3) of Au nanocrystals on CuS nanoplate were controlled by adjusting the Au/Cu molar ratios and stirring time. The obtained CuS-Au heterostructures possessed strong localized plasmon resonances (LSPRs) absorption in the near infrared (NIR) region, endowing them with the rapid photothermal response and high photothermal conversion efficiency of 36.5%. Moreover, due to the high atomic number of Au element, CuS-Au heterostructures were capable of high X-ray attenuation coefficient for X-ray computed tomography (CT) imaging. When CuS-Au dispersion was injected into the tumor of mice, the tumor could be monitored by CT and thermal imaging, and then thermally eradicated under the irradiation of 1064 nm laser. Therefore, the CuS-Au heterostructures were simply synthesized through the galvanic exchange route and served as efficient nanoagent for tumor theranostics.

BIOT 220 Developing a bacterial protein-based, branched-chain amino acid biosensor for use in medical devices to predict disease progression

Lynn Wong1,3, [email protected], Hasibul Hassan2,3, Chandrasekhar Gurramkonda4, Samyukta Rao5, Govind Rao5, Leah M. Tolosa6. (1) CBEE, University of Maryland, Baltimore County, Arbutus, Maryland, United States (2) CSEE, University of Maryland, Baltimore County, Baltimore, Maryland, United States (3) CAST, University of Maryland, Baltimore County, Baltimore, Maryland, United States (4) TRC#224, University of Maryland Baltimore County, Baltimore, Maryland, United States (5) TRC Building, Ctr for Advanced Sensor Technology, Baltimore, Maryland, United States (6) Univ of Maryland, Baltimore, Maryland, United States

Altered levels of branched-chain amino acids (BCAAs) have been indicated as potential biomarkers for diseases such as type 2 diabetes, coronary heart disease, dementia and Alzheimer’s disease. Development of disease complications in patients is highly reliant on their prognosis. Thus, it is paramount to engineer sensor technologies for rapid targeted metabolite profiling, while taking into consideration economics and accessibility. Current detection and quantification methods have long turnaround times and require expensive laboratory instrumentation and skilled operators. These limitations prevent the adoption of those technologies for use in point-of-care (POC) patient diagnostics. In this study, we are developing a BCAA biosensor derived from the leucine/isoleucine/valine-binding protein (LivJ) of Escherichia coli. Cysteine mutations are strategically placed close to the binding pocket of LivJ and these sites are subsequently labeled with a polarity-sensitive dye. The mutant LivJ candidate shows an increasing fluorescence response with increasing BCAA concentration in the micromolar range, exhibiting a linear range of 0-200 uM. Immobilizing the labeled protein resulted in improved detection range and signal output of 0-1.0 mM and 20% improvement in F/F0 respectively. Studies are currently underway to develop a prototype metabolite profiling device capable of measuring at least two metabolites. This highlights the potential of LivJ as a regenerable biosensor for implementation in medical devices.

BIOT 221

Substrate synthesis and structural analysis of peptidoglycan peptidase3: Bacterial cell shape determining metallopeptidase

Jisu Park, [email protected], Kyungjin Min, Byeongmoon Kim, Hyungho Lee. Chemistry, Seoul national university, Seoul, Korea (the Republic of)

Helicobacter pylori and Campylobacter jejuni, which are known to be helically shaped Gram-negative bacteria, colonize the human mucus layer of the gastrointestinal tract and cause various gastrointestinal diseases such as chronic gastritis, peptic ulcer, gastroenteritis and gastric adenocarcinoma in humans. In the pathogenesis and gut colonization, the helical shape of H. pylori and C. jejuni is believed to be a critical factor in their motility improvement. Recently, a series of genes associated with maintaining the helical cell shape in these bacteria have been identified, most of which have been found to encode for either endo- or exoproteases that act on the peptide chains of peptidoglycan. These proteases are referred to as cell shape determining (Csd) proteins or peptidoglycan peptidase (Pgp). According to recent studies, inhibition of proteins responsible for the helical cell morphology could be useful in interference with the bacterial virulence and lifestyle, hence a very attractive therapeutic target. However, their catalytic processes and substrate recognition are largely unknown. In this study, we describe the synthesis of two substrates for Pgp3, one of the Csd proteins, and the analysis of the Pgp3-substrate complex structures of the active site of Pgp3 in high resolution.

BIOT 222

Bioresponsive fluorophore aggregation provides spectral contrast for fluorescence imaging

Kelton A. Schleyer1,2,3, [email protected], Benjamin Datko2,4, Brandon Burnside2,4, Chao Cui1,2,3, Xiaowei Ma1,2,3, John K. Grey2,4, Lina Cui1,2,3. (1) Department of Medicinal Chemistry, University of Florida, Gainesville, Florida, United States (2) Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico, United States (3) UNM Comprehensive Cancer Center, University of New Mexico, Albuquerque, New Mexico, United States (4) Center for High Technology Materials, University of New Mexico, Albuquerque, New Mexico, United States

Fluorescence lifetime and fluorescence polarization can be used to discriminate between two fluorophores emitting at the same wavelength - even between two molecules of the same species located in different chemical or physical environments. Fluorophores experience altered emission lifetime and polarization when incorporated into and liberated from macromolecules or molecular aggregates; with this in mind, we considered the possibility of creating a responsive self-aggregating fluorescent probe that would exhibit a change in these spectral properties upon exposure to a desired biochemical trigger. We developed a model compound by conjugating fluorescein isothiocyanate (FITC) to a self-assembling scaffold consisting of cysteine and cyanobenzothiazole (CBT) to create CBT-FITC. The CBT group was “caged” with a disulfide bond to make its aggregation responsive to glutathione (GSH), a biological reducing agent upregulated in cancerous tissues. We exposed the test compound CBT- FITC to GSH and examined the spectral changes resulting from its reduction-triggered aggregation in vitro. We compared CBT-FITC to a control compound (CBT-FITC-ctrl) incapable of undergoing triggered aggregation to determine the influence of aggregation on the spectral properties of the fluorophore. Steady-state absorption and emission spectra of the FITC region showed no observable changes between the two compounds. However, the aggregated CBT-FITC did exhibit other altered spectral characteristics compared to CBT-FITC-ctrl, including reduced fluorescence intensity, reduced emission polarization, and altered fluorescence lifetime in solution. These results indicate that aggregation-induced probe packing alters optical characteristics of the fluorophore, including emission lifetime and polarization, revealing a possible approach for designing responsive imaging probes via fluorescence lifetime or polarization.

BIOT 223

Tuning the efficiency of quinone methide chemistry via electronic effects

Jun Liu2,1, [email protected], Chao Cui2,1, [email protected], Ashton Sigler1, Ying Wang1, Lina Cui2,1. (1) University of New Mexico, Albuquerque, New Mexico, United States (2) Department of Medicinal Chemistry, University of Florida, Gainesville, Florida, United States

Quinone methide is a highly reactive species that can be generated from stable masked precursors. Due to their extraordinary electrophilic reactivity, quinone methide has been widely used in chemical biology as a trapping moiety. It was originally employed in the design of covalent inhibitors, then this concept has been extended to other functional molecules such as activity-based probes, and self-immobilizing fluorogenic substrates. Mostly, the quinone methide precursor adopts an orthro-difluoromethyl moiety on a phenol caged by a moiety that can be cleaved by a specific stimulus, followed by an 1,4-elimination to generate the quinone methide. Mechanisms affecting the activation reaction and the labeling step can affect the labeling efficiency. Herein, we synthesized a series of beta-galactosidase substrates with various substituents on the phenol moiety to probe the substituent effect. Fluorescent western blot experiment and flow cytometry were performed to compare the labeling efficiency of various substituents. This result can be used as a guideline for the design of activity-based probes and self-immobilizing fluorogenic substrates with high labeling efficiency.

BIOT 224

Small molecule screening for inhibitors of Escherichia coli nitric oxide defense

Wen Kang Chou, [email protected], Mark Brynildsen. Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States

The development of antibiotic resistance poses a serious threat to global health and modern medicine. Antibiotics are used not only to treat infectious diseases but also as a precaution to prevent complications after invasive medical procedures. Over time, bacteria have developed and disseminated defenses against these live-saving drugs, leading to increasing failures of antibiotic treatments, which include last resort treatments. To try to assuage this advancing health crisis, we sought to develop novel anti-infectives by targeting pathogens’ virulence, and specifically their defenses against nitrosative stress. Nitric oxide (NO) is synthesized by phagocytes, which constitute the body’s first line of defense, to eliminate invading bacteria. However, various pathogens have developed defenses against nitric oxide, allowing them to withstand immune attack, proliferate in the host and ultimately cause an illness. We aimed to identify inhibitors of bacterial NO defenses by screening for molecules that impede bacterial growth in the presence of NO, but not its absence. Of more than 8,000 compounds screened, we found 4 hits that hold promising properties. Among them, 2- mercaptobenzothiazole (2-MBT), is especially interesting because it can nearly completely thwart the aerobic NO detoxification in Escherichia coli, while not significantly undermining growth under normal growth conditions. Preliminary results suggest that 2-MBT is a direct inhibitor of the Hmp pathway, which is responsible for detoxifying NO under oxygenated conditions in E. coli. Current effort is focusing on elucidating the mechanism of action of 2-MBT, and studying the potency of its analogs to identify chemical components that are essential for its inhibitory effects.

BIOT 225

Exploiting enzymatic activity of myeloid-derived suppressor cells for payload delivery and enhanced therapeutic efficacy

Seth Burkert1,2, [email protected], Galina Shurin3, Xiaoyun He2, Alexandr Kapralov4, Michael Shurin3, Valerian Kagan4, Alexander Star2. (1) Chemistry, Lafayette College, Easton, Pennsylvania, United States (2) Upitt Chemistry Dept, Pittsburgh, Pennsylvania, United States (3) Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States (4) Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Myeloid derived suppressor cells (MDSC) have recently been identified as a target site for immunotherapy techniques due to their role in regulating antitumor immune response and tumor progression. Tumor-associated MDSC are characterized by an overexpression of oxidative machinery which is responsible for their observed suppression of innate immune responses. This enzymatic machinery has been shown to effectively degrade carbonaceous nanoparticles providing an avenue for targeted drug delivery directly to MDSC. We illustrate the use of gold-decorated nitrogen doped carbon nanotubes (Au-NCNC) for the in vivo delivery of paclitaxel to MDSC resulting in tumor growth inhibition over a 3-week period. The enzymatic degradation of carbon nanotubes and paclitaxel payload were explored illuminating a potential mechanism of MDSC to prevent effective chemotherapy treatment due to degradation of paclitaxel. Carbon nanotubes therefore represent an attractive drug delivery vehicle due to their ability to scavenge the oxidative potential of MDSC allowing for prolonged interaction of paclitaxel payload with the tumor microenvironment. Furthermore, in vivo systemic administration of paclitaxel by Au-NCNC was found to affect MDSC populations in the spleen and liver illustrating a conditioning of the immune system for proposed prevention of metastatic cancer growth. The resulting effects on tumor growth and MDSC population were not observed from systemic administration of free paclitaxel illustrating the necessity of a drug delivery vehicle with enzymatic scavenging potential for improving therapeutic efficacy. The results of this investigation illustrate how the oxidative machinery of MDSC is paramount towards their observed immune suppression and can be exploited for enhanced immunotherapy techniques.

BIOT 226

Nanopatterning protein antigens to refocus the immune response

Ana Stringari de Castro, [email protected], Ammar Arsiwala, Chad Varner, Steven Frey, Geetanjali Pendyala, Ravi Kane. Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States

The ability to design efficacious vaccines for many diseases has been hindered by existing sequence diversity in pathogen proteins and by newly-acquired mutations that enable escape from adaptive immune responses. To address these limitations, we have developed an approach for nanopatterning protein antigens to focus the immune response on conserved protein regions. This approach combines the site-specific incorporation of non-canonical amino acids into proteins with chemical modification with polyethylene glycol (PEG) or other molecules. We will first demonstrate the efficacy of the approach using green fluorescent protein (GFP) as a model antigen. We will also discuss our attempts to apply this approach to the design of improved Zika vaccines. Zika fever – caused by the Zika virus (ZIKV), a member of the Flaviviridae family – attracted global attention after its outbreak in 2015. The link between ZIKV infections and the possible development of severe central nervous system disorders was particularly troubling, making the creation of a Zika vaccine a pressing issue. While efforts to develop such vaccine are underway, current efforts do not address a major potential problem with flaviviral vaccines – antibody- dependent disease enhancement (ADE). To prevent ADE, it is imperative to elicit primarily neutralizing and ZIKV-specific antibodies. To that end, we will describe the design of a ZIKV vaccine based on domain III (DIII) of the viral envelope protein (E). Recent reports have described three different antibody epitopes on DIII - one of which is a non-neutralizing epitope due its hidden position in the context of the Zika virion. We will describe the use of nanopatterning to tune the recognition of DIII by the immune system. By chemically modifying the protein surface, we aim to direct the immune response away from epitopes that will not contribute to viral neutralization. We will also describe the multivalent presentation of the nanopatterned antigens from scaffolds in order to enhance immunogenicity. The approach described in this work should enable the elicitation of broadly protective antibodies against a wide range of pathogens and toxins.

BIOT 227

Functionalization of cotton substrates for enhanced sequestration of chemotherapeutic agents

Ophelia Wadsworth, [email protected], Javier Ortiz-Alvarado, Madison Bardot, Michael D. Schulz. Chemistry, Virginia Tech, Blacksburg, Virginia, United States

An effective treatment of hepatocellular carcinoma is chemotherapy via transarterial chemoembolization (TACE) using doxorubicin (DOX), a common chemotherapeutic agent. DOX preferentially binds to the guanine-cytosine sites in DNA and inhibits replication. TACE allows DOX to be administered directly to the tumor; however, a significant quantity still enters systemic circulation where it destroys healthy cells. To prevent toxicity to healthy cells and reduce side effects, this research seeks to develop a filter device that will be deployed via catheter downstream from the tumor to sequester residual DOX. This approach aims to limit DOX exposure to the hepatic tumor. Cellulose, the chief component of cotton fabric, has been used for drug delivery applications and, with surface modifications, for constructing biomedical materials. The strength and flexibility of cotton make it a promising material for constructing a drug- capture device. Since DOX targets DNA, we hypothesized that DNA-coated cotton fabric would be able to rapidly capture DOX from solution. Unfunctionalized cotton fabric was first assessed for DOX sequestration and then coated with functionalized silanes that served as DNA-linking moieties. 3-glycidoxylpropyltrimethoxy silane (GDPTMS), 3- isocyanotopropyltrimethoxy silane (ICPTMS), and 3-aminopropyltriethoxy silane (APTES) were applied to the cotton surface, then DNA was reacted with the surface functional groups. The presence of silanes on the surface was confirmed via X-ray Photoelectron Spectroscopy (XPS). The functionalized cotton was then immersed in DOX solution at 37 degrees C to assess capture capability over time. Ultraviolet-visible (UV-Vis) spectroscopy was used to determine the quantity of DNA appended to the surface, the quantity of DOX sequestered by each functionalized substrate, and the amount of DOX leaching that occurs.

BIOT 228

Inductive niche module: Novel angiogenesis in vitro assay system

Abdulkader Rahmo, [email protected]. SMSbiotech, San Marcos, California, United States

A unique Angiogenesis assay system (ANGIOstream) that uses a proprietary human extracellular matrix, derived from small mobile stem cells was used to capture tissue level organization of endothelial cells reminiscent to the polygonal capillary network. The mere application of an Inductive Niche Module (INM) to endothelial cells within a thin layer of Extracellular matrix initiated a process involving cell communication, differentiation, migration and collective organization, leading to a polygonal superstructure. This process initiated within 2-3 hours of INM application, was captured in detail using time laps photography in vitro. A complex multi-stage, multi-step process of organized micro vessel spatial structuring was accessible to observation without interruption. This in vitro capability would provide ample opportunities for interrogating biochemically some fundamental aspects of tissue self-organization, cell networking and spatial cell structuring.

BIOT 229

Polymerase-driven controlled synthesis of Hasselback-like DNA-inorganic hybrid superstructures

Hyejin Kim, [email protected], Jong Bum Lee. Chemical Engineering, University of Seoul, Seoul, Korea (the Republic of)

With the great advances in DNA nanotechnology, DNA has been utilized successfully as building blocks for the nanoscale to macroscale structures. However, there have been only a few attempts to expand DNA engineering to generate DNA-inorganic hybrid materials due to a lack of controllability of interactions between DNA and inorganic materials. To surpass the limits of the current synthetic techniques, we developed DNA polymerization-driven synthesis of DNA-inorganic hybrid micro/nanostructures with the precisely controlled morphologies, components, and functionalities. In particular, the strategic feeding of metal ions to the DNA polymerization process provides a driving force for generating the hybrid superstructures. As a representation, the Hasselback-like DNA-Mn hybrid structures with a large surface area were fabricated successfully in biocompatible settings by introducing manganese ions to DNA polymerization. Also, we demonstrated the use of DNA-Mn hybrids as a cell tracking agent, a contrast agent for MRI, and an electrode material for supercapacitors. Furthermore, the structural and functional features of the products can be manipulated simply by varying metal ions. Indeed, the production of various DNA-metal hybrid superstructures including DNA-Mn, DNA-Mg, and DNA-Co complexes, as well as alloy structures, are achieved. Moreover, the structural features of each hybrid are also studied concisely by first-principles calculations. Taken together, we envision that the enzyme-driven synthesis of organic- inorganic hybrid superstructures in the physiological condition is highly apt for the generation of a range of hybrid structures for interdisciplinary research and biomedical applications.

BIOT 230

Effects of partially reduced graphene oxide on the enzymatic activity of thrombin

Jonathan Lederer2, [email protected], Rebecca Isseroff1, [email protected], Abraham C. Balsam2, [email protected], Juyi Li1, Yuval Shmueli1, Miriam Rafailovich1. (1) Dept. of Materials Science and Chemical Engineering, Stony Brook University, Stony Brook, New York, United States (2) Garcia Center for Polymers at Engineered Interfaces, Stony Brook University, Stony Brook, New York, United States

Previously we had demonstrated that partially reduced graphene oxide (pRGO) enhances the enzymatic activity of microbial transglutaminase on the cross-linking of gelatin. In order to determine if this is a more general phenomenon in modification of enzymatic activity, we tested whether it has an effect on thrombin’s ability to clot fibrinogen. Thrombin, the enzyme responsible for hemostasis, converts the fibrinogen found in blood into a fibrin clot which stems blood flow and prevents blood loss. Augmenting this activity would be useful in controlling excessive external bleeding from traumatic injury.

Different concentrations of pRGO dissolved in phosphate buffered saline were added to fibrinogen solutions. Upon the addition of thrombin, rheometry was used to measure the clotting time as well as the modulus of the clot. Sharp increases in modulus, shorter clotting times, and stronger clots indicate enhancement of the thrombin, while slow increases in modulus, longer clotting time, and weaker clots signify inhibition of the enzyme. Results indicate that pRGO more than doubles the modulus and significantly reduces the clotting time. Varying the concentration of thrombin, as well as the degree of reduction of the pRGO, will be tested to understand the nature of the mechanism, while cryo-electron microscopy will be performed to determine the structures of the clots. These results suggest further future study of partially reduced graphene oxide as a modifier of enzymatic activity.

BIOT 231

Probing the interaction between protein and Holliday junction DNA at the molecular level

Dalton R. Gibbs, [email protected], Soma Dhakal. Chemistry, Virginia Commonwealth University, Richmond, Virginia, United States The Holliday junction (HJ) is a critical intermediate in homologous recombination (HR), a well-conserved DNA-repair pathway and an essential process for maintaining genetic diversity. Errors in the HR process can lead to genetic defects and certain cancers. Therefore, the knowledge of the binding interaction of Holliday junction resolvases can provide a critical insight into the origin of diseases and potential treatments. The HJ is formed in the course of strand-exchange between two homologous DNA molecules and is processed and resolved by the RuvABC resolvosome in prokaryotes. An essential step of the HJ resolution process is the binding of the junction-specific proteins and converting the junction into a more easily resolvable cruciform conformation. This step is accomplished by the binding of two RuvA tetramers in prokaryotic systems. We have previously investigated the binding interaction between RuvA and HJ using single molecule FRET, revealing molecular insights of the interaction. Currently, we have developed a force spectroscopy approach for probing the mechanical stability of the HJ- RuvA complex at the molecular level. Our preliminary results show that the protein significantly stabilize the junction, increasing its mechanical stability. These results may provide better insights of the HR process.

BIOT 232

Single molecule analysis of i-motif under topologically constrained environment

Anoja Megalathan, [email protected], Soma Dhakal. Chemistry, Virginia Commonwealth University, Richmond, Virginia, United States

Cytosine(C)-rich sequences can fold into tetraplex structures known as i-motif under acidic and near neutral pH. I-motif forming sequences are prevalent in the genome and recent studies have shown increasing evidence of their roles in regulating gene expression and replication. Since i-motif forming sequences are at complex environment inside cells, using single-molecule fluorescence resonance energy transfer (smFRET), we have recently reported the folding and conformational dynamics of human telomeric i-motif under biomimetic environment created by self-assembled DNA duplexes and nanocircles. This study revealed that the topological restrictions significantly affect i-motif folding and dynamics. Because the nanocircles we used in this study had nicks, which could lead to some flexibility within the DNA circles, we are currently investigating the behavior of i-motif using DNA nanocircles without nicks. Interestingly, our preliminary results show that the hTel sequence exhibits either an unfolded or a partially folded state on a pH-dependent manner under such topologically constrained environment. These observations reinforce that the i-motif forming sequences exhibit condition-dependent conformations/roles in various biological processes.

BIOT 233

Homodimeric variant of an aptamer generated from LIGS activates TCR-CD3ε complex lina freage1, [email protected], Deana Jamal1, Prabodhika Mallikaratchy1,2,3. (1) Chemistry, Lehman Collage, The City University of New York, Bronx, New York, United States (2) PhD Program in Chemistry and Biochemistry, Graduate Center, New York, New York, United States (3) PhD Program in Molecular, Cellular and Developmental Biology, Graduate Center, New York, New York, United States

Nucleic acid aptamers represent a broad class of nucleic acids that reveal specific binding properties towards various ligands, including low molecular-weight substrates as well as macromolecules. Through binding specific classes of molecules, aptamers can be targeted towards cells displaying such substrates of interest on their surface. We recently introduced an aptamer named ZUCH-1 against the target T-cell receptor- cluster of differentiation three epsilon (TCR-CD3ε), expressed on human T cells utilizing the method called Ligand Guided Selection (LIGS). The aptamer ZUCH-1 showed high affinity and specificity towards the desired target, T-cell receptor Complex-CD3ε. In our current work, herein we report systematic truncation followed by modification utilizing synthetic nucleic acids, notably locked nucleic acid (LNA) and a 2’OMe RNA base in anti-TCR-CD3ε to improve the aptamer’s affinity without compromising specificity. Furthermore, dimerization of the modified aptamer showed higher avidity, and the observed avidity is comparable to corresponding monoclonal antibody. Functional studies using dimeric anti-TCR-CD3ε aptamers against TCR-CD3 expressed in cultured cells demonstrated that dimeric variants can activate TCR-CD3 and this activation is comparable to its corresponding monoclonal antibody. Thus, we will introduce a first-of- a-kind aptamer-based TCR-CD3ε activator that provides a synthetic tool to investigate TCR-CD3 mediated immunological mechanisms and synthetic immunotherapeutic development.

BIOT 234

Regulatory biochemical reaction circuits for molecular sensing

Sung Won Oh, [email protected], Jinglin Fu. Rutgers University- Camden, Cherry Hill, New Jersey, United States

Smart nanodevices that integrate molecular recognition and signal production hold great promise for the point-of-care (POC) diagnostic applications. Herein, the development of a DNA-mediated proximity assembly of biochemical reactions, which was capable of sensing various bio-targets and reporting easy-to-read signals is reported. The circuit was composed of a DNA hairpin-locked catalytic cofactor with inhibited activity. Specific molecular inputs can trigger a conformational switch of the DNA locks through the mechanisms of toehold displacement and aptamer switching, exposing an active cofactor. The subsequent assembly of an enzyme/cofactor pair actuated a reaction to produce colorimetric or fluorescence signals for detecting target molecules. The developed system could be potentially applied to smart biosensing in molecular diagnostics and POC tests.

BIOT 235 Incorporation of bio-inspired polymeric coatings for enhanced Schwann cell proliferation and stem cell differentiation

Jesse Roberts1, [email protected], Shannon L. Servoss1, Jorge L. Almodovar1, Luis Pinzon Herrera1, Harris Blankenship2. (1) Chemical Engineering, University of Arkansas, Fayetteville, Arkansas, United States (2) Biology, University of Arkansas, Fayetteville, Arkansas, United States

Human embryonic stem cells (hESCs) and Schwann cells provide a compelling component for tissue engineering and regenerative medicine. hESCs and Schwann cells have potential for treating many neurodegenerative diseases and nerve trauma by bridging structural gaps that occur within the brain and the peripheral nervous system, respectively. The extracellular matrix (ECM) is a non-cellular scaffold composed of collagen, heparin, laminin, and other macromolecules to provide a structural support system for cell adhesion and communication, along with tissue development. The physical, chemical, and topological composition of the ECM plays a major role in Schwann cell proliferation and the differentiation of hESCs into functional neurons. Both hESCs and Schwann cells are extremely sensitive to properties of the matrix in that even small changes to the ECM can drastically affect cell growth. Promising studies have focused on the development of scaffolds with nano scale features that can incorporate neural differentiation enhancers. In this work, we have fabricated peptoid microsphere surfaces with multilayered films (layer-by-layer) consisting of type I collagen and heparin bi-layers. Previous studies show that the morphology of the peptoid microsphere coatings enhance cell adhesion and differentiation of hESCs to neurons and supports neurite outgrowth. The layer-by-layer coatings with collagen and heparin have shown promising results in Schwann cell adhesion and proliferation without the need for growth factors. By combining the morphology of the peptoid microspheres with the composition of the collagen/heparin bi-layer depositions, we have created a novel biopolymeric material that directly mimics the natural ECM with enhanced proliferation of Schwann cells and the differentiation of hESCs into functional neurons. This work is being extended to determine the mechanism of action, as well as incorporation of other cell proliferation and differentiation modifiers.

BIOT 236

Development of targeted chemically self-assembling nanorings (CSANs) for cell- directed immunotherapy

Ellie Hofer, [email protected], Özgün Kiliç, Jacob Petersburg, Clifford M. Csizmar, Justine Delgado, Lakmal Rozumalski, Carston R. Wagner. Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, United States

Engineering cell-cell interactions has proven to be quite valuable due to the vast number of therapeutic applications that benefit from this technology. Although genetically engineering artificial receptors onto a patient’s cells has shown some success, preparation is costly, current applications are limited, and modifications are permanent. To address some of these concerns our lab has developed a non-genetic approach to facilitate selective cell-cell interactions using various targeted protein scaffolds. To date, we have employed commonly used targeting elements such as short peptides and single chain variable fragments (scfv), and alternative binding scaffolds such as affibodies and fibronectins that are engineered to have specificity for overexpressed cancer antigens. The diversity of the CSAN construct can be expanded by fusing two scaffolds onto the same ring that target different cellular antigens, creating a bispecific nanoring.

Our lab has shown that functionalizing the CSAN construct with a cancer antigen- targeting scaffold and a T-cell targeting single chain antibody fragment (anti-CD3 scfv) forms a stochastic mixture of bispecific nanorings with the ability to traffic T-cells to the tumor. We are currently able to produce CSANs that facilitate T-cell interactions with: a) solid tumor antigens such as EpCAM, EGFR, etc. b) blood tumor antigens such as CD19, CD22, etc. c) cancer-initiating cell associated antigens such as CD133 and B7H3. Our rings have been shown to be stable in vitro and in vivo and can be used to deliver drugs or nucleic acids as well as for fluorescence and PET imaging. Finally, a distinct advantage of our construct over other bispecific immunotherapies is that we are able to disassemble our rings using the FDA approved drug trimethoprim, stopping the course of treatment.

BIOT 237

Characterization of PEF-induced cell death in annulus fibrosus tissue

Prince M. Atsu, [email protected]. Department of Cheical Engineering, Rowan University, Glassboro, New Jersey, United States Minor Outlying Islands Treatment and repair of diseased or otherwise damaged intervertebral disc will benefit hugely from IVD regeneration. There is therefore the need to develop methods to produce injectable tissue scaffold capable of enhancing IVD regeneration. Exposure of biological cells to pulsed electric fields (PEFs) and its effects has been extensively studied. The effects of PEFs on cells depends on pulse parameters such as pulse width, number of pulses and voltage. This study explores the potential of inducing cell death in the annulus fibrosus (AF) tissue while maintaining the structure and biomechanical properties using PEFs in an attempt to develop a natural scaffold. AF tissue was dissected from fresh bovine IVD and treated with PEF. The tissues were treated with 100 microsecond pulses, one set with single pulse and varying voltage from 500V to 900V and the other with multiple pulses from 10 to 100 pulses at 500V. Densities of live and dead cells in the tissue were compared after exposure using fluorescence under the confocal microscope. PEFs of 1-20 pulses caused minimal decrease in cell survival while live cells were decreased more than 50% by 50 pulses upward. Single pulses at 500V and 600V caused a decrease of less than 10% in live cells, while less than 50% decrease in cell survival was observed for higher voltages with single pulse. PEFs have a huge potential in tissue decellularization for scaffold creation for regenerative tissue engineering. Possible collaborators include but not limited to persons interested in decellularization of fibrocartilage with pulsed electric fields, annular repair of IVD and musculoskeletal pain treatment.

BIOT 238

Perovskite nickelates as bio-electronic interfaces

Fan Zuo1,2, [email protected], Hai-tian Zhang1, Feiran Li4, Henry Chan3, Qiuyu Wu5, Zhan Zhang3, Badri Narayanan3, Koushik Ramadoss1, Indranil Chakraborty6, Gobinda Saha6, Ganesh Kamath3, [email protected], Kaushik Roy6, Hua Zhou3, Alexander A. Chubykin5, Subramanian Sankaranarayanan7, Jong Choi8, Shriram Ramanathan1. (1) Materials Science, Purdue University, West Lafayette, Indiana, United States (2) Chemistry and Physics, Indiana State University, Terre Haute, Indiana, United States (3) Argonne National Laboratory, Schaumburg, Illinois, United States (4) Mechanical Engineering, Purdue, West Lafayette, Indiana, United States (5) Biological Sciences, Purdue University, West Lafayettte, Indiana, United States (6) Electrical and Computer Engineering, Purdue University, West Lafayette, Indiana, United States (7) Argonne National Lab, Naperville, Illinois, United States (8) Purdue University, West Lafayette, Indiana, United States

Functional interfaces between electronics and biological matter are essential to diverse fields including health sciences and bio-engineering. Here, we report the discovery of spontaneous (no external energy input) hydrogen transfer from biological glucose reactions into SmNiO3, an archetypal perovskite quantum material. The enzymatic oxidation of glucose is monitored down to ~ 5 × 10−16 M concentration via hydrogen transfer to the nickelate lattice. The hydrogen atoms donate electrons to the Ni d orbital and induce electron localization through strong electron correlations. By enzyme specific modification, spontaneous transfer of hydrogen from the neurotransmitter dopamine can be monitored in physiological media. We then directly interface an acute mouse brain slice onto the nickelate devices and demonstrate measurement of neurotransmitter release upon electrical stimulation of the striatum region. These results open up avenues for use of emergent physics present in quantum materials in trace detection and conveyance of bio-matter, bio-chemical sciences, and brain-machine interfaces.

BIOT 239

Fibrin-targeted fluorescent probes for the molecular imaging of thrombi

Khada Nagi1, [email protected], Sa Lay Wah1, Khanh Ha2,3, Jason McCarth2, Chase Kessinger2, Farouc Jaffer3. (1) Chemistry/Biology, SUNY Polytechnic Institute, Utica, New York, United States (2) Massonic Medical Research Institute, Boston, New York, United States (3) Mass General Hospital, Boston, Massachusetts, United States

Fluorescent imaging of fibrin could enable highly sensitive and noninvasive molecular imaging of thrombosis syndromes in vivo. The goal of this study was to develop a novel and more green synthesis of a peptide-based fibrin-targeted imaging agent that enables high-resolution near-infrared fluorescence (NIRF) imaging of deep vein thrombosis. The linear fibrin-targeted peptide was conjugated to a near-infrared fluorophore Cy7, termed FTP11-Cy7. The peptide was then subjected to intra-cyclization by formation of disulfides by air oxidation of thiols of cysteine under sonication. The NIRF peptide is based on a fibrin-specific imaging agent that has completed Phase II clinical magnetic resonance imaging trials. In vitro binding of FTP11-Cy7 to human plasma clots was assessed by using fluorescence reflectance imaging. Intravital fluorescence microscopy and noninvasive fluorescence molecular tomography-computed tomography were also performed. The fibrin-targeted NIRF agent FTP11-Cy7, synthesized using novel method, was shown to avidly and specifically bind human and murine thrombi, and enable sensitive, multimodal intravital and noninvasive NIRF molecular imaging detection of acute and subacute murine DVT.

BIOT 240

Self-assembling peptides to mitigate familial hypercholesterolemia

Victoria Harbour, [email protected], Sreya Sanyal, Zain Siddiqui, Biplab Sarkar, Ka Kyung Kim, Vivek Kumar. Biomedical Engineering, New Jersey Institute of Technology, East Brunswick, New Jersey, United States

Elevated levels of low-density lipoprotein cholesterol (LDL-C) is one of the major contributors to ischemic cardiovascular disease (CVD), the leading cause of death in the United States and globally. PCSK9, a secretory mammalian serine protease, interacts with low-density lipoprotein receptors (LDLR) on the surface of hepatocytes, leading to the receptor’s internalization and degradation. As LDLR is primarily responsible for removal of LDL-C from circulation, PCSK9 activity decreases expression of LDLR and increases serum levels of LDL-C. Accordingly, gain-of-function PCSK9 mutations lead to autosomal dominant familial hypercholesterolemia. FDA-approved anti-PCSK9 monoclonal antibodies (mAbs) have shown clinical efficacy in LDL-C reduction, lowering plasma LDL-C levels by approximately 60% without adverse events, even in patients receiving maximum dose statins. A significant drawback to anti-PCSK9 mAbs is high list price, limiting affordable access. We have engineered a self- assembling therapeutic peptide (Sle-Pep28) targeting PCSK9 to treat familial hypercholesterolemia, as an affordable alternative to mAb therapies. Sle-Pep28 is formulated as a polymeric hydrogel that gradually releases therapeutic peptides at the subcutaneous implant site. Subcutaneous delivery promotes improved pharmacodynamics over oral and intravenous administration. Promisingly, Sle-Pep28 shows specific, irreversible binding to PCSK9 in surface plasmon resonance (SPR) studies as well as significantly increased LDLR expression in hepatocytes.

BIOT 241

Proximity-induced site-specific antibody conjugation for diagnostic and therapeutic applications Chenfei Yu, [email protected]. Chemistry, Rice University, Houston, Texas, United States

Antibody conjugates utilize the selective binding ability of antibodies to deliver payloads to targets in vivo. These conjugates can be used as imaging probes for dynamic and high-resolution cell tracking or as therapeutic molecules for cancer treatment. Many conjugates are prepared using non-specific covalent interactions with cysteine and lysine residues which lead to heterogeneous products and inconsistent antibody- payload ratios. Other methods require antibody engineering, which enables site-specific antibody crosslinking but greatly reduces antibody expression yield.

To tackle these issues, we have developed a proximity-induced site-specific antibody conjugation method (pClick). This method introduces a non-canonical amino acid into an antibody affinity peptide with the help of an evolved tRNA/aaRS pair or solid phase synthesis. The ncAA allows covalent interactions with a proximal lysine residue between the antibody and affinity proteins, thus enabling the construction of antibody conjugates without the need for any antibody engineering.

To explore the applications of pClick, we first introduced between 2 and 4 radioactive tracers into Herceptin to amplify signals for high resolution tumor cell sensing and imaging. We also labeled Herceptin with auristatin and alendronate in order to make bifunctional antibody conjugates. These conjugates can be self-delivered to the bone matrix to kill tumor cells with high specificity and efficacy. pClick provides a fast and simple way to construct antibody-payload complexes and has great potential for diagnostic and therapeutic applications.

BIOT 242

Interpreting small-scale human cell culture using noninvasive sensors for dissolved CO2, dissolved O2, pH and glucose

Vida Rahmatnejad1, [email protected], Joel Tyson1, Hasibul Hassan1, Xudong Ge1, Iordan V. Kostov1, Leah M. Tolosa2, Govind Rao3. (1) UMBC, Baltimore, Maryland, United States (2) Univ of Maryland, Baltimore, Maryland, United States (3) TRC Building, Ctr for Advanced Sensor Technology, Baltimore, Maryland, United States

The concentration of nutrients influences cell growth, among which glucose is of the utmost importance as it is the main nutrient used by cells to support the rapid proliferation and effector functions. CO2 affects cell metabolism and high levels of CO2 act as toxin in cell cultures. Although the aforementioned factors play a major role in cell culture condition, tissue engineering is currently done in single-use vessels which are not equipped with systems monitoring them. As a result, cell cultures are not usually studied from an analytical point of view, and this makes these types of experiments less repeatable and reliable. The Center for Advanced Sensor Technology has developed noninvasive CO2 and glucose sensors (featuring a sampler mounted outside the vessel), pH and DO sensors (featuring a patch attached to the bottom of the vessel) to monitor cell culture environment. The low profile of the sensors makes them suitable for various kinds of bioreactors. In this study, human cell cultures were conducted in standard cell culture flasks equipped with sensors for DO, pH, DCO2, and glucose for continuous monitoring of process parameters. The analytical data were used to interpret the effect of various microenvironmental conditions on cell behavior. The results show that the use of these sensors improved understanding of the microenvironment and provided real-time information on nutrients and the metabolites for small-scale cell cultures.

BIOT 243

Investigations of AGEs (advanced glycation end products) in breast cancer

Demetria Dorsey, [email protected]. Biological and Physical Sciences, South Carolina State University, Blythewood, South Carolina, United States

Advanced Glycation End Products (AGEs) are harmful compounds, which accumulate in the body naturally and through diet. Dietary AGEs (dAGEs) have been found to be the largest contributor to AGE accumulation internally. Diets high in sugar and meats cooked in high heat, and dry environments, contain increased amounts of AGEs. Previous studies have linked AGE levels to diseases such as diabetes, and Alzheimers. Recently, AGE accumulation has been correlated to cancer onset and progression and hypothesized to increase with increased body fat. In this study, we evaluated the level of two specific AGEs, Nδ-(carboxylmethyl)-L-lysine (CML) and Nδ-(carboxylethyl)-L- lysine (CEL) to determine the quantity of these AGEs in tumor samples of obese vs non-obese patients. The levels of CML and CEL were determined using GCMS analysis.

BIOT 244

Developing a metabolic engineering toolbox for the thermotolerant yeast K. marxianus

Xuye Lang1, [email protected], Mengwan Li2, [email protected], Pamela Botero Besada-Lombana5, [email protected], Nancy A. Da Silva3, [email protected], Ian R. Wheeldon4, [email protected]. (1) Chemical Engineering, UC Riverside, Riverside, California, United States (2) Chemical and Environmental Engineering, University of California, Riverside, Riverside, California, United States (3) University of California, Irvine, California, United States (4) A242 Bourns Hall, UC, Riverside, Riverside, California, United States (5) UC Irvine, Irvine, California, United States

The nonconventional yeast Kluyveromyces marxianus is a promising microbial host for chemical biosynthesis due to its thermotolerance, fast growth kinetics, and ability to consume a wide range of carbon sources. A critical limitation thus far has been the lack of genetic engineering tools for strain manipulation. Here, we present our work in creating a metabolic engineering toolbox for K. marxianus. The toolbox includes CRISPR-Cas9 genome editing, multiplexed CRISPR interference (CRISPRi), and a standardized gene integration strategy. In addition, we have characterized a series of 25 promoters native to K. marxianus that vary in expression level. In studying these promoters, we observed significant changes in expression level as culture temperature increased from 30 to 45 °C. In the vast majority of cases expression was significantly reduced at elevated temperature. One solution to this problem was the design of a synthetic promoter that combines high expression elements from the TEF promoter and the high temperature responsiveness of the SSA3 promoter. The result was a promoter that maintains strong expression independent of temperature. Finally, for a subset of six promoters, we also evaluated protein expression and triacetic acid lactone production in xylose at different temperatures. Taken together, the genetic engineering tools presented here demonstrate the capacity to rapidly engineer K. marxianus and advance this host as a viable candidate for metabolic engineering.

BIOT 245

Engineering smart living devices toward targeted gene editing

Yu-Ju Chen, Kai-Lin Hong, Hsuan-Chen Wu, [email protected]. Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated system, microbial self-defense machinery against invasion from phages, has been repurposed into an unprecedented and robust molecular scissor for precision gene editing. While showing great promise with therapeutic potential, unfortunately, the CRISPR toolkit is, by far, lacking a proper vehicle to assist specific in vivo delivery. The pressing need to have a safe and efficient CRISPR delivery system awaits further development. To create an enhanced CRISPR delivery system, we aim to seek for solutions form Nature. Biology offers a broad spectrum of favorable features, enabling organisms to probe and respond to their microenvironments that can all be transformed into the use of synthetic biology. In this research, the probiotic E. coli strain Nissle 1917 (EcN) will be rationally programmed via synthetic biology to serve as an alternative means for effectively synthesizing, transferring and delivering CRISPR/Cas9 complexes into hosts of interest. Based on the intelligent features on the engineered EcN, we anticipate the achievement of (1) focused & enhanced delivery of CRISPR/Cas9 complexes to target cells and (2) upgraded safety without concerning random genome disruption and unregulated off-target effects.

BIOT 246

Human cerebral organoids reveal early spatiotemporal dynamics and pharmacological responses of UBE3A

Dilara S. Sen2, [email protected], Albert J. Keung1. (1) NCSU, Raleigh, North Carolina, United States (2) Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, United States

Human neurodevelopment and its associated diseases are complex and challenging to study. In particular, prenatal neurodevelopmental periods are hard to access experimentally, especially in humans. The role of UBE3A in Angelman Syndrome and Autism Spectrum Disorder is an archetype for these challenges. It is paternally imprinted in parts of the brain, exhibits complex subcellular localization, and is suspected to dynamically establish these patterns in gestation. In this work, human cerebral organoids reveal that important spatiotemporal dynamics of UBE3A occur very early in neurodevelopment. In particular, UBE3A localizes to the nucleus of neurons within a few weeks of organoid culture, with a stark transition established between EOMES and TBR1 cortical layers; these localization patterns are disrupted, and in some cases reversed, in Angelman Syndrome hiPSC-derived organoids. Organoids also exhibit early imprinting of paternal UBE3A within 6 weeks of culture, with topoisomerase inhibitors partially rescuing UBE3A levels in Angelman Syndrome organoids. One inhibitor also exhibits over two weeks of persistent rescue after just a single treatment. This work establishes human cerebral organoids as an important model for studying UBE3A biology and motivates their broader use in understanding complex neurodevelopmental disorders.

BIOT 247

Withdrawn

BIOT 248

Development of growth-driven stepwise inducible expression system in haloalkaliphilic desulfurizing bacteria Thioalkalivibrio versutus jianmin xing1,2, [email protected]. (1) Institute of Process Engineering, Chinese Academy of Sciences, Beijing, BEIJING, China (2) School of Chemical Engineering, University of Chinese Academy of Sciences, BEIJING, BEIJING, China

Highly toxic and flammable H2S gas is an environmental threat. Thioalkalivibrio versutus is an autotroph that can transform hydrogen sulfide to sulfur, in the form of extracellular globules via oxidation. Haloalkaliphilic autotrophs, like the bio-desulfurizing T. versutus, grow slowly. Weak growth makes any trial for developing potent genetic tools required for genetic engineering far from achieved. In this study, the fed-batch strategy improved T. versutus growth by 1.6 fold in maximal growth rate, 9-fold in O.D600 values and about 3-fold in biomass and protein productions. The strategy also increased the favorable desulfurization product, sulfur, by 2.7 fold in percent yield and 1.5-fold in diameter. A tight iron-inducible expression system for T. versutus was successfully developed. The system was derived from fed-batch cultivation coupled with new design, build, test and validate (DPTV) approach. The inducible system was validated by toxin expression. Fed-batch culturing coupled with DBTV approach successfully led to the first tight inducible system construction in T. versutus for improving bio-desulfurization processes by future metabolic engineering. The selected inducible system heterologously expressed the MazF toxin at high O.D600 value as a validation step. It lets the strain grow much faster than the control. The cultivation coupled with DPTV approach could be applied to other autotrophs.

BIOT 249

Hierarchically porous and water-tolerant metal-organic frameworks for enzyme encapsulation yiying sun, [email protected], Zhongyi Jiang, Jiafu Shi. Tianjin University, Tianjin, Tianjin, China Enzyme catalysis, as a green, efficient process, displays exceptional functionality, adaptivity and sustainability, which shows broad application prospects in the preparation and processing of fine chemicals such as medicine, pesticides, food and so on. Metal- organic frameworks (MOFs), a class of porous materials based on coordination between metal nodes and organic ligands, have exerted bright prospects for in situ enzyme encapsulation due to their intrinsic porous structure, designable pore size and mild preparation condition. However, MOFs for enzyme catalytic processes commonly possess weak metal-ligand coordination bond and rather small pores, which are instable in aqueous solution and present rather high diffusion resistance of reactants. Herein, we prepare a kind of hierarchically porous and water-tolerant MOFs through a facile polyphenol-treatment method for enzyme encapsulation. In brief, enzymes are firstly in situ encapsulated in zeolitic imidazolate framework-8 (ZIF-8) through co- precipitation of enzymes, zinc ions (Zn2+) and 2-imidazole molecules (2-MI). Then, tannic acid (TA, a typical polyphenol) is introduced to functionalize the surface and etch the void of ZIF-8. The hierarchically porous structure would accelerate the diffusion process of reactants, whereas the Zn-O bond in TA-Zn nanocoating would improve the structural stability against water corrosion compared to ZIF-8. Taking glucose oxidase (GOD) as a model enzyme for the catalytic conversion of β-D-glucose, the resultant GOD@ZIF-8@ZnTA exhibits the equilibrium conversion of 77.4%, which is comparable to GOD@ZIF-8 but much higher than GOD@ZIF-8@ZnTA without void etching. More importantly, the GOD@ZIF-8@ZnTA shows significantly enhanced recycling and storage stabilities compared to GOD@ZIF-8. It is expected our study provides a facile and generic method to encapsulate a broad range of enzymes in MOFs with enhanced activity and stabilities.

Schematic preparation of E@ZIF-8@ZnTA biocatalysts.

BIOT 250 Elucidating the unusual reaction mechanism of glucuronyl C5-epimerase

Deepika Vaidyanathan1, [email protected], Xia Ke2, Yanlei Yu2, Elena Paskaleva2, Robert J. Linhardt3,2, Jonathan S. Dordick1,5,4. (1) Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States (2) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States (3) Chemistry, Rensselaer Polytechnic Institute, Albany, New York, United States (4) Center for Biotechnology, Renselaer Polytechnic Inst, Troy, New York, United States (5) Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States

Chemoenzymatic synthesis of heparin represents an emerging approach to provide a substitute for animal sourced heparan sulfate and heparin. D-Glucuronyl C5-epimerase is the first enzyme that acts on the heparin precursor, catalyzing the conversion of D- glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5- epimerase has made understanding its reaction mechanism and its application in heparin and heparan sulfate biosynthesis quite difficult. The steady-state kinetic parameters, for both the forward and the pseudo-reverse reactions of C5-epimerase have been determined for the first-time using full-length polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal Michaelis-Menten behavior and the pseudo- reverse reaction displays non-saturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of additives. Surprisingly, the combination of 25 mM CaCl2 and 25 mM MgCl2 results in a forward reaction exhibiting conventional Michaelis-Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin/heparan sulfate synthesis, in the absence of 3’- phosphoadenosine 5’-phosphosulfate (PAP) also resulted in C5-epimerase exhibiting a conventional Michaelis-Menten kinetics in the forward reaction accompanied by a significant increase in Vmax. The activity and processivity of C5-epimerase as a function of polysaccharide sequence was also evaluated. The epimerase reaction is performed in deuterated environments leading to incorporation of a deuterium on the IdoA residues at the C5 position. LC-MS is used to quantify activity based on the epimerase mediated deuterium incorporation and to map the distribution of GlcNS and GlcNAc within the polysaccharide and providing information on the activity and specificity of the enzyme as a function of the sequence of the polysaccharide. The data obtained in this study provides critical information towards understanding the mechanism of C5-epimerase and improving the chemoenzymatic synthesis of bioengineered heparin specifically accounting for variability in the GlcNS and GlcNAc content.

BIOT 251

Withdrawn

BIOT 252 Impact of disulfide reduced LMW on HMW formation rate and long term stability of monocloanal antibodies

Vivekh Ehamparanathan2, [email protected], Zhijun Tan1, Angela T. Lewandowski2, cheng Du2, Letha Chemmalil2, June Kuang2. (1) Bristol-Myers Squibb, Devens, Massachusetts, United States (2) Process Development, Bristol-Myers Squibb, Hopkinton, Massachusetts, United States

High molecular weight (HMW) species and low molecular weight (LMW) species are two critical quality attributes (CQAs) for all monoclonal antibody (mAb) Drug Substance (DS) and Drug Product (DP) with well-defined specification criteria. During the long-term stability study for a mAb, we observed one DP batch to be out of specification (OOS) for HMW. Subsequent investigation found that the LMW of that DS batch was higher than normal, but within specification limits. This study was aimed at investigating the correlation of initial LMW levels in DS to the rate of HMW formation during stability studies under various stressed conditions. Interestingly, It was determined that light exposure and initial LMW level can have a significant impact on the rate of HMW formation during long-term stability. This novel study can provide a unique dimension to pursue a holistic specification strategy for both DS and DP

BIOT 253

Biophysical studies on the binding interactions of aflatoxin B1 with chicken egg albumin

Mohd A. Qureshi, Saleem Javed, [email protected]. Biochemistry, Aligarh Muslim University, Aligarh, UP, India

Aflatoxin B1, classified as group I carcinogen by international agency for research on cancer, is a secondary metabolite produced by soil fungus Aspergillus flavus and Aspergillus paraciticus. Here in this study we investigated the potential binding interaction of aflatoxin B1 with chicken egg albumin (CEA) using multiple spectroscopic methods and molecular docking. Structurally aflatoxin B1 is a coumarin fused with difuran. Fluorescence spectroscopic studies revealed quenching in the fluorescence spectra of CEA in the presence of aflatoxin B1. Hyperchromic effect in the absorption spectra of CEA was observed in the presence of increasing concentration of aflatoxin B1 using UV-absorption studies. Modified Stern Volmer equation was used to calculate the binding constant and number of binding sites of aflatoxin B1 on CEA. The binding 4 -1 constant was found to be 7.29×10 M at 293.15K between aflatoxin B1 and CEA. Fluorescence studies were also used to calculate the thermodynamics parameters like ΔG, ΔS and ΔH. The magnitude of ΔH and ΔS values were found negative suggesting van der Waals and hydrogen bonding as predominating forces in stabilizing CEA and aflatoxin B1 complex. Conformational alteration in the secondary structure of CEA upon binding of aflatoxin B1 was explored using circular dichroism spectroscopy that revealed decrease in alpha helical content in protein in the presence of mycotoxin. Molecular docking approach explicated the amino acid residue and nature of forces involved in the binding of mycotoxin to chicken egg albumin. This study will be helpful in understanding the binding pattern of the toxin to CEA and will also help in finding out the competitors having preferential binding to CEA over aflatoxin B1.

BIOT 254

Engineering the secretory pathway for increased enzyme production in Yarrowia lipolytica

Weigao Wang1, [email protected], Mark A. Blenner2. (1) Chemical and Biomolecular Engineering , Clemson University, Clemson, South Carolina, United States (2) Chemical Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

Eukaryotic cells cultures are preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and the quality control system inherent to the quality control system of the endoplasmic reticulum. A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its native protein secretion capacity and advanced secretory pathway. Conventional means of improving protein secretion includes codon optimization, amplification of the gene copy number, inducible expression, and secretory tag engineering; however, improvements to the secretory pathway are less common. In our study, secretion of the model enzyme T4 lysozyme was used to determine effective strategies for increasing enzyme secretion. By engineering a native secretion signal lip2prepro, we have successfully improved extracellular level of T4 lysozyme by 3-fold. Further improvement in T4 lysozyme secretion was achieved by co-expression of important enzymes in the secretion tag processing pathway, expansion of ER, and disruption of Endoplasmic-reticulum-associated protein degradation (ERAD). Finally, we demonstrate the generality of these approaches by expressing a wide range of heterologous proteins, including GFP, beta-galactosidase, alpha-amylase, and laccase.

BIOT 255

Post-translational modifications of interfacial mussel adhesive protein fp-5

Tae Hee Yoon, [email protected], Byeongseon Yang, Hye Ryoung Heo, Mincheol Shin, Hyung J. Cha. Chem Eng, Pohang University Sci and Tech, Pohang Gyeongbuk, Korea (the Republic of)

Marine mussel uses a network of threads called byssus to attach their body to various underwater surfaces. Until now, researchers identified numerous proteins, up to 19 types, consisting the byssus in their underwater adhesion system. Among them, foot protein-5 (fp-5), which locate on the adhesion interface, has known to have the strongest adhesion energy, and has the highest percentage of L-3,4- dihydroxyphenylalanine (DOPA) which is considered as one of the key molecules in underwater adhesion. Also, fp-5 has phosphoserine (pSer) which is another post- translational modification (PTM) expected to have various roles in underwater adhesion like calcareous substrate binding, calcium-mediated cohesion, and even phase separation. However, little has been known about the role of fp-5 in underwater adhesion because natural protein is challenging to be extracted. Actually, the amount of fp-5 is relatively low in nature and it is highly cross-linked due to its high DOPA mol%. Previously, we used in vivo residue specific incorporation technique to introduce DOPA, which can replace almost every tyrosine to DOPA (>90%). In this work, we employed chemical mutagenesis to incorporate pSer. Through combining both approaches, we prepared fp-5 molecules which have both DOPA and pSer residues and investigated expected characteristics in wet adhesion to verify potential roles of PTMs in fp-5. This study could provide insights for understanding mussel adhesion mechanism and be applied in designing bioinspired biomaterials for underwater adhesion.

BIOT 256

Engineering a cell-free system-enzyme fusion for enhanced production

Matthew Wong, [email protected], Mattheos Koffas, Georges Belfort. Howard P. Isermann Department of Chemical and Biological Engineering and the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States

Continuous research effort is being put into alternative energy production, such as biofuels, to reduce our reliance on fossil fuels. One of the stumbling blocks in this research is that production in vivo is adversely affected by alcohol toxicity and product inhibition. A solution to this problem has been previously proposed by our lab: remove the enzymes from the cells, immobilize them on resin, and run a cell-free reaction. Our previous research has yielded favorable isobutanol production compared with previous in vivo systems. An issue we encountered was that product inhibition still remained an issue, with aldehyde conversion decreasing as more isobutanol is produced. To solve this issue, we proposed genetically fusing the two ketoisovaleric acid pathway enzymes together. By carrying out this fusion, diffusional length for the intermediate can be reduced and increases the local concentration of the intermediate in the presence of the isobutanol producing enzyme. To test this theory, we designed four fusion proteins with either a rigid or flexible linker and two arrangements of our two pathway enzymes. After creating these fusion constructs and ascertaining their activity, stability measurements were conducted to test the fusions’ temperature, pH, and isobutanol stability. Using these results, optimized cell-free reactions were conducted, both in solution and immobilized, and the results compared to the previous non-fused system. Future work will examine the cofactor recycling enzyme, formate dehydrogenase, and explore if it can also be fused or co-immobilized to the resin.

BIOT 257

Reducing non-productive binding of cellulases to improve crystalline cellulose hydrolysis rates Bhargava Nemmaru, [email protected], Nicholas Ramirez, Nicholas Kravchenko, Shishir P. Chundawat. Chemical and Biochemical Engineering, Rutgers University, New Brunswick, New Jersey, United States

Deconstruction of lignocellulosic biomass has vast potential for production of biofuels and bioproducts in biorefineries. However, this potential is currently not reached due to the recalcitrance of biomass to enzymatic degradation. Various pretreatment technologies have been developed to reduce the recalcitrance of biomass. Extractive Ammonia (EA) pretreatment is one such technology which alters the crystalline structure of cellulose from its native cellulose I to cellulose III allomorphic form which is more accessible towards enzymatic degradation. However, non-productive binding of cellulases to cellulose and/or lignin is still considered to be one of the key factors reducing the overall conversion efficiency of biomass to fermentable sugars. Previous reports have suggested that the dissociation of cellulases from cellulose surface could be one of the key rate-limiting steps in enzymatic degradation of cellulose, but this hypothesis has not been properly tested yet. Here, we have systematically correlated the binding affinity and binding kinetic rates of a library of engineered endocellulase mutants to the hydrolytic activity of the endocellulase on cellulose I and cellulose III. Interestingly, we saw that an increase in overall hydrolytic activity on cellulose I and cellulose III for all engineered cellulase mutants was tightly correlated with an overall reduction in cellulase binding affinity. We also characterized the binding kinetics of mutant carbohydrate-binding modules (CBMs), appended to the full-length cellulases, to cellulose I and cellulose III, in an effort to understand whether CBM-assisted cellulase dissociation is one of the key rate-limiting step in endoglucanase catalyzed hydrolysis of cellulose to soluble sugars.

BIOT 258

Neural network based fingerprinting of monoclonal antibody aggregation using biolayer interferometry niharika budholiya, [email protected]. Chemical Department, Indian Institute of Technology, Delhi, Delhi, India

Aggregates are widely accepted to be a critical quality attribute (CQA) for biotherapeutics and believed to impact product immunogenicity. Monitoring of aggregates is typically performed using multiple orthogonal tools as any single tool is unable to offer comprehensive characterization of aggregate species over the entire size and morphology range that they can exist in. Researchers have attempted to categorize monoclonal antibody (mAb) aggregates into six classes based on their respective physicochemical properties. In this study, we have developed model based on artificial neural network (ANN) to predict the stress history of mAb contributing to the aggregate formation, based on binding sensogram profiles obtained with biolayer interferometry (BLI). It was observed that each class of mAb aggregates exhibited unique binding profiles that were characteristic fingerprint of that class. The proposed model uses principal components extracted from the mAb-Fcγ receptor binding sensogram (106 profiles from 9 stressed mAb samples) as inputs while the unique identification codes in the form of binary coded numbers are used as model outputs. The latter served as a fingerprint for each class of mAb aggregates generated by subjecting to specific stress conditions. The ANN was trained using Levenberg- Marquardt algorithm with Bayesian Regularization, using 86 sensogram profiles, in the ratio of 80:10:10 for internal training, validation and testing. The trained ANN accurately identified each single stress condition that the samples were subjected to based on their binding sensogram profiles. The model was also able to predict stress history for samples that had been subjected to more than one kind of stress with reasonable accuracy. The proposed approach therefore can be effectively employed for quality control of product quality in biopharmaceutical industry as well as for prediction of stress history of a sample.

BIOT 259

How to survive in the jungle of hydrophobicity scales: Machine learning approach to predict chimeric VLP candidate solubility

Philipp Vormittag1, [email protected], Thorsten Klamp2, Juergen Hubbuch1. (1) KIT, Karlsruhe, Germany (2) BioNTech Protein Therapeutics GmbH, Karlsruhe, Germany

In the early stage of biopharmaceutical drug development, low solubility is one of the major issues leading to the failure of drug candidates. Screening of usually more than 100 entities involves considerable experimental effort. Predictive models, such as quantitative structure-property relationship models, can contribute to reducing this effort. One example is the use of amino acid sequence-based models that are constructed using hydrophobicity scales. In recent 50 years, over 90 amino acid scales have been derived or optimized from earlier scales, which can be broadly described as hydrophobicity scales. It is a difficult task to select the best hydrophobicity scale for a new problem. Machine learning tools offer the potential to address this problem in an automated and data-dependent way. In this paper, we therefore propose a robust soft ensemble vote classifier (sEVC) that is based on amino acid sequences and experimental and theoretical hydrophobicity scales. The sEVC framework was investigated on the basis of a solubility data set of 568 chimeric Hepatitis B core antigen virus-like particles (VLPs) and 91 hydrophobicity scales. Training set size and number of included classifiers were varied to characterize model performance with regard to robustness and overfitting. Stratified sampling significantly improved model performance for small training set sizes. Monte Carlo cross-validation provided with a means to estimate model performance on external test data. During feature selection, the individual classifiers, each based on a single hydrophobicity scale, were ranked by feature importance. Patterns in amino acid hydrophobicity values in the hydrophobicity scales ranked highest and lowest granted an insight into the biological background of classification and could prove beneficial in (re-)designing candidates. The prediction accuracy on the external test set was >80% for the best models, while Matthew’s correlation coefficient was >0.6. In summary, we have developed an amino acid scale-based machine learning framework to predict biophysical properties such as solubility. This framework integrates process development acceleration and knowledge generation while maintaining statistical robustness. The use of this framework is not limited to solubility prediction of VLPs but is also applicable to other products such as monoclonal antibodies. The model could further be extended to other applications such as identification of aggregation- prone regions.

BIOT 260

Application of specifications for sterile drug products in-use stability: Industry perspective

Jason Cheung1, [email protected], Douglas Watson2, Robert Capen3, Michel Chartrain4, Monisha Dey3, Rebecca Gentile5, Sarita Mittal1, Maya Salnikova6, Andrew Semple1, David Wylie7. (1) Pharmaceutical Sciences, Merck Research Labs, Rahway, New Jersey, United States (2) Biologics & Vaccines Analytics, Merck Manufacturing Division, West Point, Pennsylvania, United States (3) Biostatistics, Merck Research Labs, Kenilworth, New Jersey, United States (4) Project Management, Merck Manufacturing Division, Kenilworth, New Jersey, United States (5) Vaccine & Biological Stability, Merck Manufacturing Division, West Point, Pennsylvania, United States (6) Regulatory Affairs, Merck Research Labs, West Point, Pennsylvania, United States (7) Analytical Research and Development, Merck Research Labs, Kenilworth, New Jersey, United States

There is relatively little in the published literature regarding industry best practices and regulatory expectations for product quality during the in-use period. There is also relatively little explicit regulatory guidance, including ICH and worldwide market-specific guidance, other than the recommendation that such stability studies must be performed. This presentation describes two strategies for controlling product quality during the in- use period, both of which are consistent with available regulatory guidance. The simplest and most straightforward approach is to set the same acceptance criteria for the in-use period as the stability specifications applied to the long-term storage condition. When this is not feasible, a different strategy can be considered whereby the in-use period is established based on alternate acceptance criteria that have been appropriately justified. For a particular program, the preferred approach is therefore based on considerations for the program, scientific rationale for specification setting, and engagement with regulators. A case study will be discussed that describes the stability studies that were performed, the data generated, and the statistical methodologies applied to support an in-use limit for a multi-use biologic drug product. For this study, the second strategy was selected due to the existence of product-specific USP and Ph Eur monographs that provided specifications for long-term storage. However, the application of either approach may be scientifically justifiable, and industry should consider both when evaluating product in-use stability limits.

BIOT 261 Colorimetric response of borohydride stabilized silver nanoparticle on interaction with organophosphates shalini shikha, [email protected]. CHEMICAL ENGINEERING, IIT DELHI, New Delhi, NEW DELHI, India

Detection of residual organophosphates in food is of paramount importance in the current scenario. Biosensors for pesticide detection may be developed by monitoring the interaction of nanoparticles with pesticides. In this report, we have studied the colorimetric response of pesticide phorate, chlorpyrifos and malathion with borohydride stabilized silver nanoparticles. The colorimetric changes are corroborated with UV- Visible spectra along with the change in particle size and zeta potential. Nanoparticle on interaction with phorate shows the visible change in color to dark green while the other two pesticides haven’t shown any visible color change. The colorimetric response of nanoparticle pesticide mixture was further studied for 12 hours of incubation and it was observed that nanoparticle-phorate mixture started decolorizing after an hour and becomes colorless after 12 hours of incubation, while other two pesticide mixtures remain same. This decolorization of phorate was correlated with the degradation of pesticides in the presence of bare silver nanoparticles. The reaction mechanism was explained by the formation of silver and sulfur atom (Ag→S) complex. The uncapped nanoparticles get stabilized by pesticides over time for chlorpyrifos and malathion while phorate undergoes degradation due to the scission of S-CH2 present in the molecule. This color change may be utilized as a colorimetric method for the detection of phorate pesticide.

BIOT 262

Highly selective in vivo editing of macrophages through systemic delivery of CRISPR-Cas9-ribonucleoprotein-nanoparticle nanoassemblies

Yi-Wei Lee1, [email protected], Vincent M. Rotello2. (1) Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts, United States (2) Univ of Massachusetts, Amherst, Massachusetts, United States

Therapeutic gene editing through delivery of CRISPR-Cas9-ribonucleoprotein (RNP) provides vast opportunities in treating genetic diseases. Several systemic administrations have been attempted through viral vector delivery in the past, but all have suffered from induced immunogenicity. CRISPR-Cas9 protein delivery has been recognized as an effectively permanent genomic DNA editing strategy due to its transient process offering improved specificity. However, one hurdle in the application of the CRISPR system for human medicine is in vivo delivery: there has been no systemic CRISPR-Cas-RNP delivery reported thus far. Herein we describe a new nanoassembled platform featuring co-engineered nanoparticles and Cas9 protein that has been developed to provide efficient Cas9-sgRNA delivery and concomitant CRISPR editing through systemic tail-vein injection into mice, achieving >8% gene editing efficiency in macrophages of the liver and spleen.

BIOT 263

Evaluation of the fatty acid composition of polysorbate 80 (PS-80)

Smruti Ragunath, [email protected], Evon bolessa, Nandu Deorkar, Celia Williams. Avantor, Bridgewater, New Jersey, United States

Polysorbates are non-ionic surfactants that are widely used in bio-pharmaceutical formulations as therapeutic agents for various diseases. The quality parameters of PS- 80, which prevents aggregation of proteins in formulations, is defined in various monographs such as EP, JP and USP. One such parameter is fatty acid composition.

Although there are established specifications for the fatty acid composition of PS-80 (EP, JP, USP) there is allowance for variation in the types and amounts. For example, there are more than twenty different fatty acids present in PS-80, some that are still not unidentified making the total specified fatty acid composition not equivalent to 100% (typically 94 – 97%). This study is focused on establishing the identity and quantity of the unknown fatty acids in PS-80.

Preliminary results indicated that the unidentified fatty acids contribute between 3-6% to the total fatty acid composition of the samples. Shorter chain fatty acids such as capric acid (<0.05 – 0.78%) and lauric acids (0.167 – 1.84%) contributed small but measurable amounts to the total composition. Longer chain fatty acids like palmitoleic acid (C16:1) and stearic acid (C18:0) contributed negligible amounts. Two of the fatty acids contributing more than 1% to the total fatty acid composition were identified as 10, 13 nonadecadienoic acid (C19:2) and eicosenoic acid (C20:1). Other long chain fatty acids identified were arachiadic (eicosanoic) acid (0.2 – 0.3%) and behenic acid (<0.057 – 0.063%).

BIOT 264

Profiling and quantitation of impurities in carbohydrate excipients used in parenteral formulations

Celia Williams, [email protected], Smruti Ragunath, [email protected], Evon bolessa, Nandu Deorkar. Avantor Performance Materials, Inc., Easton, Pennsylvania, United States

Carbohydrates such as sucrose, trehalose, mannitol, and sorbitol are used as excipients to stabilize proteins, lipids and carbohydrates in the formulation and freeze/thaw cycle of therapeutics. These excipients are typically tested, released and utilized based on compendia testing for identity, purity, elemental impurities and some related substance impurities. However, it does not include evaluation of other protein reactive impurities. For example, impurities such as b-glucans (BG) and hydroxymethylfurfural (HMF), even in minute quantities can impact protein molecule stability. This presentation will report impact of such impurities, a methodology for analysis of these types of impurities and the level of these impurities present in carbohydrate excipient products. We will present the level of HMF and BG in over 10 lots of high purity low endotoxin (HPLE) trehalose and HPLE sucrose and illustrate its impact.

BIOT 265

Specificity engineering of clinical-stage antibodies

Lina Wu1, [email protected], Alec Desai1, Yulei Zhang1, Matthew Smith1, Peter M. Tessier2. (1) Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States (2) Chemical Engineering and Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States

The ability of therapeutic antibodies to recognize their target antigens with high specificity in the complex in vivo environment is crucial to their function as safe and efficacious drugs. However, therapeutic antibodies, especially those discovered or engineered using in vitro methods such as phage or yeast surface display, often display variable levels of non-specific interactions that lead to drug development challenges such as low bioavailability and fast antibody clearance. Therefore, we have developed methods for predicting sites in antibody variable regions linked to off-target binding and for designing antibody libraries that softly randomize such sites with residues predicted to improve antibody specificity. We have evaluated this approach for improving the specificity of multiple clinical-stage antibodies that display high levels of off-target binding. To accomplish this, we have displayed our designed antibody libraries on the surface of yeast, sorted them based on their affinity for multiple types of reagents (including their target antigens and several polyspecificity reagents), and used deep sequencing methods to identify rare variants that retain high target affinity and display increased specificity. Importantly, we find that a subset of antibody sites that mediate non-specific binding do not overlap with those involved in antigen binding. We will discuss our identification of such sites and how we are exploiting them to engineer antibodies with drug-like specificity.

BIOT 266

In vitro and in silico evaluation of the effectiveness of TEMPO-oxidized nanofibrillated cellulose (TNFC) and poly(vinyl alcohol) (PVA) films for wound healing applications

Kelsey L. O'Donnell1, [email protected], Gloria Oporto2, Noelle Comolli1. (1) Chemical Engineering, VILLANOVA UNIVERSITY, Garnet Valley, Pennsylvania, United States (2) West Virginia University, Morgantown, West Virginia, United States

Different types of cellulose have proven effectiveness in wound healing applications. Bacterial cellulose is used in many instances but has challenging large scale production due to the bottom-up process and low productivity concentrations. Alternatively, TEMPO-oxidized nanofibrillated cellulose has shown potential when incorporated into scaffolds with secondary components. However, this process typically requires chemical crosslinking between components using various solvents that reverse the benefits of using a natural product. This work presents a fully aqueous, biodegradable formulation with the capabilities of releasing model molecules for therapeutic benefits in wound healing applications. The release mechanism of these molecules is analyzed using a tri- phasic release model, inclusive of initial burst release, relaxation-induced dissolution, and diffusional release, to more accurately reflect the true driving forces that control the flux of molecules through and out of the delivery platform and understand the extent of their contributions to the overall release for clinical application. BIOT 267

Molecular interactions between drug product formulation excipients and infusion containers/components

Kashmira Dilrukshan, [email protected], Pedro Smith, Andrew Ilott. Bristol-Myers Squibb, Piscataway, New Jersey, United States

At the point of intravenous dosing, most biologics are typically diluted with diluents such as saline and dextrose in infusion containers. These infusion containers are typically manufactured with a plasticized polymer. These additive polymers lend flexibility and durability to the containers. There is a potential for these plasticizers to leach into the infusion solution. The objective here was to measure and quantify the amount of plasticizers leaching from IV bags and other infusion components that are in contact with various infusion formulation compositions and to evaluate what conditions/factors influences the leaching behavior.

BIOT 268

α-Synuclein takes up stable and reversible α-helical structure in water-less environment

ANASUA MUKHOPADHYAY, [email protected]. Chemistry, Indian Institute of Technology Bombay, Mumbai, India

Intrinsically disordered proteins (IDPs) are a class of proteins having significant biological functions and they generally do not take up structure by themselves. α- Synuclein is an example of IDP which is lacks an ordered secondary and tertiary structure at neutral pH. Recently, we have reported a class of water-less protein liquids using Bovine Serum Albumin as an model system that it retains its near native protein secondary structure within the highly concentrated environment. Here we are showing a stable, spontaneous, and reversible α-helical structure formation in α-synuclein protein for the first time in a low-water (1.5 % w/w H2O) environment. Using a combination of circular dichroism and ATR-FTIR spectroscopy, we show that bioconjugation of native α-synuclein with a diamine molecule, and subsequent electrostatic coupling with the PS forms a conjugate with a retained unordered structure. Removal of water from this system provides a highly stable α-helical (≈74 %) low-water liquid system. Surprisingly, the α-helical-to-unordered state transition is completely reversible and is achieved at ≈25-30 w/w % of water in the system. Moreover, the α-helix of α-synuclein shows an extraordinary stability (>6 months) in a waterless environment.

α-synuclein takes a highly stable, reversible, and spontaneous α-helix structure under low-water conditions

BIOT 269

Protein templated gold nanoparticle formation as indicators of ioninzing radiation

Amar Thaker1, [email protected], Brent Nannenga1, Kaushal Rege2, Karthik Pushpavanam1. (1) SEMTE, Arizona State University, Tempe, Arizona, United States (2) Arizona State University, Tempe, Arizona, United States

Despite several scientific advances, determination of radiation delivered to the patient during cancer therapy remains a challenge due to the inherent limitations of existing dosimeters including fabrication and operation. Here, we describe a colorimetric nanosensor that exhibits unique changes in color as a function of therapeutically relevant radiation dose (3–15 Gy). The nanosensor is formulated using a gold salt and maltose binding protein (MBP) as a templating agent, which upon exposure to ionizing radiation is converted to gold nanoparticles. The formation of gold nanoparticles from colorless precursor salts renders a change in color that can be observed visually. The dose dependent multicolored response was quantified through a simple ultraviolet visible spectro-photometer and the peak shift associated with the different colored dispersions was used as a quantitative indicator of therapeutically relevant radiation doses. This is followed by briefly identifying the location of gold ion mineralsation in protein-gold complex through xray crystallography.

Radiation induced gold nanoparticle formation in the presence of MBP.

BIOT 270 Simple, rapidly assembled saccharide-based sensor interface that enables interrogation of antibody glycosylation and titer for improved process analytical technologies

Dana Motabar1,2, [email protected], Jinyang Li1,2, Sally Wang1,2, Chen-Yu Tsao1,2, Gregory F. Payne2, William E. Bentley3,2. (1) Fischell Department of Bioengineering, University of Maryland College Park, College Park, Maryland, United States (2) Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Maryland, United States (3) Fischell Dept of Bioengineering, University of Maryland, College Park, Maryland, United States

Recombinant monoclonal antibody (mAb) therapeutics constitute a large segment of the biologics market as they have shown success in many therapeutic areas. N-linked glycosylation on the Fc- portion of mAbs is a common post-translational modification that can have a significant impact on the mAbs therapeutic efficacy, in vivo half-life, and safety. As a result, N-linked glycan composition is considered a critical quality attribute and the glycoform profile is required for mAb characterization. Current methodologies to assess glycosylation involve complex techniques that couple extensive separation methods with mass spectrometry or nuclear magnetic resonance spectroscopy. Although these techniques provide detailed structural information about the mAb’s glycosylation state, they can be costly and require extensive time and significant expertise. For process screening and monitoring (e.g., bioreactor and downstream process vessels), such complex structural information may not be required. Instead, analytical technologies that indicate levels of product and it’s glycosylation state may be sufficient, particularly when coupled with data analytic methods that provide discrimination and model-based control schemes. To meet this measurement need, we developed an electrochemical based interface that is rapidly electroassembled onto electrodes and when linked with glycan-recognizing lectins reveals levels of mAbs with the appropriate terminal glycan moieties. Further, when coupled with a protein G:HRP reporter this interface enables process control ready electrochemical output. Further, a new thiolated protein-G construct enables total mAb titre. That is, our interface is rapidly constructed using electrode actuated redox reactions enabling hydrogel assembly from thiolated-polyethylene glycol (PEG). Then, thiolated sugar groups (e.g., galactose) and sugar-specific lectins are layered on top of the PEG hydrogel. These surfaces then provide selective capture of mAbs based on the traditional lectin-mAb binding. Our glycosensing approach is advantageous as it is simple, portable, inexpensive and can provide fast, quantifiable results for bioprocessing applications.

BIOT 271

Study of the orientation and competitive adsorption of mAbs and excipients at the air/water interface

Ankit D. Kanthe1, [email protected], Mary E. Krause2, Songyan Zheng2, Andrew J. Ilott3, Raymond S. Tu4, Charles Maldarelli4. (1) Chemical Engineering, The City University of New York, City College, New York, New York, United States (2) Drug Product Science and Technology, Bristol Myers Squibb, North Brunswick, New Jersey, United States (3) Chemistry Department, New York University, Rahway, New Jersey, United States (4) Steinman Hall, Chemical Engineering, The City College of New York , New York, New York, United States

The adsorption of biomolecules at the air-water interfaces is of crucial concern in the development of new processes for antibody-based pharmaceuticals. Loss of monoclonal antibody (mAb) at the air/water interface during drug formulations, storage, shipping and administration to the patients results into decrease in the efficacy of the drug and may promote immunogenicity. In order to solve this problem and enhance the physical stability of therapeutic mAbs the pharmaceutical industry uses a multicomponent formulation that includes surface active excipients. We have used the pendant bubble tensiometry technique to understand the nature of the adsorption of mAbs and surfactants alone followed by their competitive adsorption to the air/water interface. A combination of X-ray reflectivity (XR) and computational simulations is used to demonstrate how mAb molecules orient themselves at the air-water interface as a function of concentration. We have used experimental and theoretical modeling to interpret the electron density profiles obtained by XR that showed a flat-on orientation of mAbs at low surface concentrations and end-on conformation as surface concentration is increased. Our multidisciplinary approach has helped to provide a mechanistic molecular level detail for the first time to uncover the general understanding of the antibody interfacial behavior at the air-water interface.

BIOT 272

New diagnostic application of zinc finger proteins and graphene oxide for detecting antibiotic resistance genes

Wendy M. Cecil, [email protected], Dat T. Ha, Moon-Soo Kim. Department of Chemistry, Western Kentucky University, Bowling Green, Kentucky, United States

Zinc finger proteins (ZFPs) are the most common DNA-binding domains and multiple ZF domains can be engineered to bind to any DNA sequence of interest. In this work, a six finger ZFP was engineered to bind to 18 base pairs of DNA within the antibiotic resistance genes (ARGs) with high specificity. Target DNA within the tetM gene (tetracycline resistance gene) was recognized and DNA-dose dependence of the fluorescent signal was quantified by labeling ZFPs with quantum dots (QDs). QD- labeled ZFPs can be adsorbed onto GO via stacking interactions of aromatic and hydrophobic residues in conjunction with hydrogen bonding interaction between hydroxyl or carboxyl groups of GO and hydroxyl or amine groups of the protein. The GO surface quenches the fluorescence of the QD-labeled ZFPs when in proximity due to fluorescence resonance energy transfer (FRET). When the target DNA is added, the QD-labeled ZFPs bind to the DNA, and the DNA-protein complex is dissociated from the GO surface due to the protein conformational change, thereby restoring the fluorescence. Here, we optimized GO concentrations used in the assay and QD-labeling on ZFPs and determined the sensitivity of our system. This methodology may also be applied to any DNA of interest since ZFPs may be engineered to bind to virtually any sequence of DNA. Therefore, this work has broader applications in DNA detection and has the potential to be adapted to target other DNA of interest.

BIOT 273

Identifying triple-helix DNA motifs by CuAAC click reaction

Nanami Kikuchi, [email protected], Roee Amit. Biotechnology and Food Science, Technion Israel Institute of Technology, Haifa, Israel

Triplexes are noncanonical DNA structures, in which a single-stranded DNA or RNA binds to a double-stranded DNA molecule via Hoogsteen base-pairing. Previous studies have revealed that the binding site of well known long non-coding RNA (lncRNA) such as HOTAIR, HOTTIP, MALAT1, and NEAT1 are enriched with triplex-forming DNA motifs, and that they interact with thousands of genomic loci. Yet, the mechanism of triplex formation, how RNAs target specific triplex target sites (TTS), and how triplex formation is regulated remains unknown. To address these questions and better understand triplexes, we first explored the underlying algorithm of triplex motif. A pool of TTS and triplex-forming oligonucleotides (TFO) were incubated in vitro. Upon formation of triplex structure, TTSs and TFOs were covalently linked by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), also known as click chemistry, and analyzed using high throughput sequencing. In the same way, 5-ethynyl-2-deoxyuridine (EdU) was incorporated into U2OS cell and clicked upon triplex formation. We seek to validate triplex formation in vivo by transfecting the best candidate TFO obtained from computational analysis into the cell nucleus. Aside from biological implications, triplex based molecular biology tools and therapies are attracting broad interest among the scientific communities. In the future, we plan on using the triplex motif not only to answer the fundamental questions regarding the triplex, but to engineer single-stranded oligonucleotides that can target specific dsDNA, which could be the base for dsDNA detection, drug delivery, imaging, and gene regulation.

BIOT 274

Tunable co-gel gene delivery systems for transfection of macrophages in chronic wound environments

Anuraag Boddupalli1, [email protected], Colleen Fridley1, Tohn Borjigin1, Kristi L. Kiick2, Millicent O. Sullivan1. (1) Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Dept of Materials Sci Eng, University of Delaware, Newark, Delaware, United States

Healthcare costs associated with diabetes-related foot-wounds have increased to almost 25 billion dollars annually in the United States of America. Chronic wound environments demonstrate a prolonged inflammatory phase, with increased protease activity and complications associated with bacterial pathogens that can trigger secondary infections. There is thus a growing need to elucidate the intricacies of how immune cells migrate, proliferate and remodel the extracellular matrix (ECM), to develop effective strategies for restoring the natural wound healing response that can proceed towards formation of granulation tissue. Macrophages and their differentiated phenotypes (e.g., M1, M2) participate closely in the inflammatory and anti-inflammatory immune response in the body. There is a dearth of practical treatments, however, that upregulate the mechanisms through which M2 macrophages remodel the ECM. Using a multi-component co-gel system comprising of fibrinogen, thrombin and collagen, it is possible to deliver optimized compositions of tunable payloads that are released as cells degrade the hydrogel network. Facile gene transfection assays conducted on co- gel systems demonstrated that M2 macrophages showed increased transfectability when they were exposed to inflammatory cytokines. Further studies on characterizing this substrate-mediated transfection approach to tune the delivery of recombinant or plasmid DNA of choice will be a significant step towards assessment of macrophage- based treatments that can be effective in chronic wound environments.

BIOT 275

Treatment of plant oils with commercial and recombinant lipases towards the development of consumer products with antimicrobial activity

Jillian B. Coughlin1, [email protected], Angel Ordonez Flores1, [email protected], Taylor Rosenking2, Karin Melkonian2, Jaimelee I. Rizzo1, Eric P. Chang1. (1) Department of Chemistry and Physical Sciences, Pace University, New York, New York, United States (2) Long Island University, C.W. Post, Greenvale, New York, United States Lipases are a class of enzymes that catalyze the hydrolysis of fats. These versatile biocatalysts have been used for a variety of industrial purposes such as the manufacturing of food products, laundry detergents, and biofuels. In this study, we aim to investigate the use of both commercially available and recombinant lipases to develop topical consumer products containing derivatives of plant-based oils. Previously, we have demonstrated that specific formulations of essential oils and butters have antimicrobial activity against a variety of pathogens including Staphylococcus aureus, Escherichia coli, and, Pseudomonas aeruginosa.

Recent studies have suggested that monoacylglycerols, free fatty acids, or combinations of these types of molecules may have antimicrobial activity. To expand upon this line of research, we will use lipases to breakdown oils and butters composed primarily of triacylglycerols into mixtures of mono- and diacylglycerols, free fatty acids, and glycerol. Along with varying the source of the oil, other factors such as the type and concentration of lipase, the buffers and organic solvents, method of agitation, as well as time and temperature used will be screened. These treated oils and butters will then be infused into agar plates and their antimicrobial activity screened against the pathogens mentioned above.

Currently, we are utilizing commercially available porcine lipases for pilot studies at temperatures ranging from 25 – 50 oC. Pending successful attempts at expression and purification of a novel E. coli vector, recombinant lipases derived from Aneurinibacillus thermoaerophilus will be tested at temperatures over 50 oC. The reporter molecule 4- nitrophenyl palmitate dissolved in the plant oil demonstrates that the lipase effectively catalyzes the hydrolysis of ester bonds by the evolution of 4-nitrophenol (λmax = 405 nm at pH > 7.5) in the aqueous fraction. Conditions that produce agar plates with enhanced antimicrobial activities will be analyzed using various analytical techniques to determine their chemical composition.

BIOT 276

Increasing the stability and activity of lipase through polymer conjugation

MONICA S. RAHMAN1, [email protected], Dominik Konkolewicz1, Richard C. Page1, Julian Brown2, Reena Murphy1, Sydney Carnes1, Ben Carey Carey1. (1) Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, United States (2) Department of Chemistry, Delaware state University, Dover, DE, Dover, Delaware, United States

This research work aimed to increase the stability and activity of an industrially important enzyme, Candida antarctica lipase B (CalB). In our research work, we modified CalB with hydrophilic and hydrophobic polymers such as acrylamide (Am); dimethyl acrylamide (DMAm); block polymers of acrylamide and N-isopropyl acrylamide (NIPAm); a block polymer of acrylamide and a mixture of N-isopropyl acrylamide (NIPAM) and N- isobutoxy methyl acrylamide (NIBMA); 2-acrylamido-2-methylpropane sulfonic acid (AMPSA); a mixed polymer of DMAm and AMPSA and N, N- dimethylaminopropyl-acrylamide (DMAPA). To observe the effect of different lengths of polymers on CalB, we synthesized our polymers in three different chain lengths: DP10, DP25 and DP50 using reversible addition-fragmentation chain transfer (RAFT) polymerization method. We found a significant enhancement of activity for acrylamide (Am) and dimethyl acrylamide (DMAm) conjugates, indicating a probable noncovalent interaction of the polymers with the substrate. This activity enhancement was also found to be relatively preserved in 25% and 75% ethanol solvent system. On the contrary, hydrophobic as well as ionic polymers were not able to preserve the activity in these solvent systems. In our study, we also observed a greater stability of the conjugates in different organic solvent systems including 75% acetonitrile, 75% ethanol, 50% ethanol and 25% ethanol solvent systems. Thus, this study gives us an insight into the advances of lipase production which can improve the activity and stability of lipase in presence of denaturing organic solvents.

Increasing the stability and activity of lipase through polymer conjugation

BIOT 277

Self-assembly of an enzyme cascade with ATP channeling on DNA nanostructures Zhicheng Wang, [email protected]. Rutgers University, Philadelphia, Pennsylvania, United States

Self-assembled DNA nanostructures have been demonstrated to organize multienzyme systems with controlled geometric patterns. For example, DNA nanostructures were used to mediate the proximity assembly of enzyme cascades with enhanced and regulated activities. In this study, we assembled an enzyme pair of pyruvate kinase (PK) and hexokinase (HEK) with controlled distances on a rationally designed DNA origami. To facilitate the transport of ATP cofactors between the two enzymes, we modified the surface of a DNA origami with ATP-binding affinity to attract ATP molecules, and guide their diffusion to target enzymes. Significant activity enhancement was observed with the introduction of ATP-binding affinity channel between enzymes, as compared with enzymes without this channel. To fully understand the relationship between ATP aptamer and PK/HEK, a theoretical model also built to explain this ATP aptamer behavior.

ATP channeling on DNA origami

BIOT 278

Rational design of an amylosucrase-cyclodextrin glucanotransferase fusion to produce cyclodextrins in a unique reaction mixture Leidy R. Pico1, [email protected], Hermínsul d. Cano2, [email protected], Jorge Hernández1, [email protected]. (1) Biology, Universidad Industrial de Santander, Bucaramanga, Santander, Colombia (2) Chemistry, Universidad Industrial de Santander, Bucaramanga, Santander, Colombia

Cyclodextrins (CDs) are cyclic oligosaccharides composed of different numbers of α- (1,4)-linked glucopyranose subunits. CDs are commercially produced from starch, through the action of cyclodextrin glucanotransferase (CGTase), which generally catalyses the cyclization of starch and related α-(1,4)-glucans via intramolecular transglycosylation reactions. A recent work developed a novel enzymatic process, utilizing sucrose as raw material instead of corn starch, to reduce time and energy consumption of CDs production at industrial level. In this process, a glucosyltransferase named amylosucrase (AS) catalyses the synthesis of α-(1,4)-glucan polymers from sucrose molecules, opening the possibility of exploring other commercially available sugar-rich substrates. The aim of this project is to test the combined action of AS from Neisseria polysaccharea fused to the CGTase from Bacillus macerans, to produce CDs from cocoa mucilage, a substrate rich in sucrose and glucose. The first step involved the use of bioinformatics tools for the design and analysis of the AS-CGTase fusion protein. Wild type Protein Data Bank (PDB) structures were linked with tandem repeats of GGGS and GGGGS linker motifs, by using the Synlinker server. As a result, 3D models of AS-CGTase and CGTase-AS fusions were obtained, which were analyzed with bioinformatic tools to select the most stable ones for laboratory testing. Model selection was based on molecular dynamics simulations (GROMACS), Root Mean Square Deviation (RMSD) and Gyration Radius (RD) values as well as other structural validation parameters. The model with the highest stability was the AS-G3S-CGTase fusion, linked by GGGS motifs, with a RMSD value of 0.31 nm and a RD of 3.75 nm. Currently, synthetic genes are being constructed for the AS-CGTase and CGTase-AS fusions, for cloning into pET vectors and overexpression in Escherichia coli. A one-pot dual enzyme reaction will be implemented using the fused proteins with cocoa mucilage as a sugar-rich substrate.

BIOT 279

Novel excipient to reduce protein viscosity

Courtney ODell, [email protected], Evon bolessa, Subhabrata Das, Arvind Srivastava, Nandu Deorkar. Avantor, Somerville, New Jersey, United States

Viscosity which increases with increasing protein concentration is a major challenge in developing protein therapeutics, mainly due to protein-protein interactions. These solutions pose a challenge for both manufacturing and analysis. However, high protein concentration is desired for Subcutaneous (SC) injection development and for storage of high volume products. There are several excipients available on the market known to reduce the protein viscosity, but none are suitable for all proteins. Moreover, many of these excipients can have an adverse impact on protein stability and quality. In this discussion, we will present a novel excipient that is superior to alternatives in viscosity reduction and is also able to stabilize protein by minimizing protein aggregation formation.

BIOT 280

Gentle attachment of bioactive proteins onto graphene oxide through the Mitsunobu reaction

Wyatt Swift-Ramirez1, Stephen Schmidt1, Walker M. Vickery1, [email protected], Michelle Wolf2, [email protected], Stefanie A. Sydlik1. (1) Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States (2) Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States

Nearly six million Americans every year are affected by traumatic bone injuries. The current gold standard for treatment, bone autografts, requires surgically transferring bone from a donor site to the treatment site within the same patient. This requires two surgical sites, increasing the risk of infection as well as causing damage to the donor site. Additionally, metal implants used to provide structural support to autografts, such as screws and plates, have several drawbacks: local and systemic toxicity due to the release of metal ions, failure at the bone-metal interface, and mechanical mismatch of the bone and screw causing stress shielding of the neighboring bone, potentially leading to osteoporosis. Graphene oxide (GO) has been recently considered for use in bone implants for two main reasons, 1) the mechanical strength exceeds current standards for bone implant structural stability, and 2) GO holds inherent osteoinductivity. These properties can be used in conjunction with bioactive proteins, which are powerful in bone regeneration and unparalleled in stem cell differentiation. We present a gentle method to attach clinically relevant proteins to graphene oxide via a Mitsunobu reaction. The hydrolytically active ester bond enables controlled release of proteins from GO in situ. Successful covalent attachment of bovine serum albumin (BSA) was achieved to yield BSA-Mitsunobu graphene (BSA-MG), and controlled release of BSA was demonstrated over four weeks at targeted concentrations. The BSA released from MG was characterized to show a retention in secondary structure. This method may be used as an analogue for a protein-based osteoinductive agent such as bone morphogenetic protein 2 (BMP-2), enabling controlled release of protein-based therapeutics.

Synthesis of Bovine Serum Albumin-Mitsunobu Graphene. The tertiary alcohol of GO reacts with one of the carboxylic acid sites of BSA. GO has been simplified for clarity. BSA not to scale.

BIOT 281

Reconstitution of full-length caveolin-1 into phospholipid bicelles: Validation by analytical ultracentrifugation

Kyle T. Root, [email protected]. Lock Haven University, Lock Haven, Pennsylvania, United States

A significant hurdle in obtaining biophysical information on membrane proteins is developing a successful strategy for their reconstitution into a suitable membrane mimic. In particular, utilization of the more ‘native-like’ membrane mimics such as bicelles is generally more challenging than simple micellar solubilization. Caveolin-1, an integral membrane protein involved in membrane curvature, endocytosis, mechano- protection, and signal transduction, has been shown to be particularly recalcitrant to standard reconstitution protocols due to its highly hydrophobic characteristics. Herein we describe a robust method to incorporate recombinantly produced full-length caveolin-1 into bicelles at levels needed for biophysical experimentation. The benchmark of successful reconstitution is the obtainment of protein in a homogeneous state; therefore, we developed a validation procedure to monitor the success of the reconstitution using analytical ultracentrifugation of density-matched bicelles. Our findings indicated that our protocol produces a very homogeneous preparation of caveolin-1 associated with bicelles, and that caveolin-1 is highly α-helical (by circular dichroism spectroscopy). We believe that this methodology will serve as a general strategy to facilitate biophysical studies on membrane proteins.

BIOT 282 Changing the specificity of a guanine riboswitch to hypoxanthine

Daniel P. Morse, Adam Z. Esqueda, [email protected], Andrew S. Hong, [email protected]. Chemistry, United States Naval Academy, Annapolis, Maryland, United States

Riboswitches are RNA elements in messenger RNAs that regulate gene expression by undergoing a ligand-triggered conformational change. Riboswitches bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. We previously modified several purine riboswitches for use in vitro and, indeed, they could detect their ligands with high sensitivity and specificity. To extend the utility of our sensor design, we developed an in vitro selection strategy to isolate variants of riboswitches that have altered ligand specificity. We are attempting to find variants of our guanine sensor that can detect hypoxanthine rather than guanine. This is a challenging task because hypoxanthine is identical to guanine except for the absence of an exocyclic amino group. We partially randomized the sequence of our guanine sensor, and used hypoxanthine as the ligand during selection. Our first experiment produced only the original, wild-type guanine sensor. In hindsight, this was not surprising since the guanine riboswitch can detect hypoxanthine (albeit poorly), and it was the most abundant RNA in our initial partially-randomized RNA pool. To solve this problem we designed two new partially randomized RNA pools. In the first experiment, we increased the degree of randomization in order to decrease the abundance of the original guanine sensor. The second experiment was based on the C74U mutant of the guanine riboswitch. C74 forms a Watson-Crick base pair with the bound guanine ligand. Changing C74 to U results in a sensor that cannot bind to guanine or hypoxanthine but can bind weakly to adenine. We generated a partially randomized RNA pool in which every RNA retained a U at position 74. Since the C74U mutant cannot bind to hypoxanthine, we reasoned that we could select variants that could do so by allowing hypoxanthine to form a “wobble” pair with U74. We have begun the two new selection experiments. We will report the results of these experiments.

BIOT 283

Detection of antibiotic resistance genes via zinc finger protein and quantum dot complex with graphene oxide nanosheet

Kenneth Schlabach2, [email protected], Dat Thinh Ha2, Moon- Soo Kim1. (1) Department of Chemistry, TCCW455, Western Kentucky University, Bowling Green, Kentucky, United States (2) Department of Chemistry, Western Kentucky University, Bowling Green, Kentucky, United States

Antibiotic resistant pathogens are becoming an increasing hazard across the globe. They threaten the downfall of traditional antibiotics which tend to select for antibiotic resistance genes (ARGs). Thus, healthcare faces a critical need to discern between pathogen strains. Unfortunately current methods suffer from a variety of drawbacks such as extended wait times or highly specific working conditions. This research demonstrates a detection mechanism with both speed and specificity using novel materials and methods. The primary component of this mechanism, Zinc Finger proteins, are one of the most common DNA binding proteins in nature. Multiple Zinc Finger domains can be linked together to recognize extended DNA sequences. The target ARG used in this project was the tetracycline resistance gene (tetM). The second component is cadmium selenide quantum dot (QD), photo-fluorescent nanoparticles, covalently bonded to the ZFP. The ZFP-QD fluorescent signal was produced quantitatively dependent on target DNA concentration by mixing in solution with Graphene Oxide nanosheets (GO). Rhe ZFP- QD complex adsorbs onto the GO nanosheet to reach its lowest energy configuration. The proximity of the GO and QD will cause frequency resonance energy transfer (FRET) to occur, thus quenching any fluorescent signal caused by excitation of the Quantum Dot, When target DNA is added to the solution, the ZFP-QD complex leaves the GO nanosheet and binds to the DNA, distancing the GO and QD sufficiently to prevent FRET from occurring and restoring fluorescence as a measurable signal. While development is still in progress overall this system displays excellent potential for point of care application. Beyond this base system there is enormous potential for optimization to specific applications such as multiplexing with different QDs to detect multiple ARGs simultaneously.

BIOT 284

Fluorine-thiol displacement reaction (FTDR) for steric-free bioorthogonal labeling

Zhigang Lyu, [email protected], Zakey Y. Buuh. chemistry, temple university, Philadelphia, Pennsylvania, United States

We have developed a novel bioorthogonal reaction that can selectively displace fluorine substitutions alpha to amide bonds. This fluorine-thiol displacement reaction (FTDR) allows for fluorinated cofactors or precursors to be utilized as chemical reporters; hijacking acetyltransferase mediated acetylation both in vitro and in live cells, which cannot be achieved with azide- or alkyne- based chemical reporters. The fluorine labels can be further converted to biotin or fluorophore tags using FTDR, enabling the general detection and imaging of acetyl substrates. This strategy may lead to a steric-free labeling platform for substrate proteins, expanding our chemical toolbox for functional annotation of post-translational modifications (PTMs) in a systematic manner

BIOT 285

Effects of endogenous germination factors from various bacteria on Bacillus subtilis spore germination jingyu li, [email protected], li dong, [email protected], zequn zhang, Hongmei Wang, Fang Chen, Xiaosong Hu. China Agricultural University, Beijing, China

Spores from Bacillus species pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. It is an effective way of inducing spore germination homogenously before killing them. So the study on germination mechanism of spore is of great significance, including the screen of germinants involved in the germination of spore. In this study, we determined the effect of E.coli, B. subtilis, S. cerevisiae, L. plantarum and S. thermophilus (cell free) on B. subtilis spore germination. The bacterial supernatant from five microorganisms induced spore germination instead of the sediment. Moreover, the supernatant from E.coli, B. subtilis, and S. cerevisiae exhibited better germination effects than L. plantarum and S. thermophilus. The plate counting experiment indicated that compared with control group, the induction effects of supernatant about spore germination depended on their concentration. Furthermore, the examination of plate counting and DPA release detection confirmed that the bacterial supernatant induced the spore germination of FB85 (GRs mutant) but not the FB78 (PrkC mutant). Therefore, our data suggested that the supernatant of E.coli, B. subtilis, S. cerevisiae could include some possible agent to induce the spore germination via activate the nutrient GRs (germination receptors).

BIOT 286 Multi-target ligand-guided selection (LIGS) to generate aptamers against B-cell biomarkers

Nicole Williams1, [email protected], Sana Batool2, Hasan Ekrem Zumrut3, Mohammad Jamal2, German Sosa2, Prabodhika Mallikaratchy1,2,3. (1) PhD Program in Molecular, Cellular and Developmental Biology, Graduate Center, CUNY, New York, New York, United States (2) Chemistry, Lehman College, CUNY, Bronx, New York, United States (3) PhD Program in Chemistry and Biochemistry, Graduate Center, CUNY, New York, New York, United States

Nucleic Acid Aptamers (NAAs), are single-stranded ribo- and deoxyribo-oligonucleotide molecules, which fold into complex functional three-dimensional structures. These three-dimensional structures have the ability to bind to target molecules with high affinity and specificity. Nucleic Acid Aptamers are selected through an iterative process called Systematic Evolution of Ligands by Exponential enrichment (SELEX). Here, we implemented Ligand-Guided Selection (LIGS), a variant of SELEX, to elute aptamers against multiple biomarkers expressed in B-cells i.e., CD19 and CD20. During LIGS step, specific aptamers against CD19 and CD20 were competitively eluted using two specific monoclonal antibodies against the same target at 25°C. The eluted multiple libraries were, then sequenced using Illumina high-throughput (HT) DNA sequencing platform. The resulting sequences were analyzed using a novel bioinformatics workflow, which was designed using GALAXY, an online bioinformatics server. Two major sequence families consisting of 18 hit sequences against CD19 and 34 hit sequences against CD20 were identified based on defined enrichment values. The potential hit sequences were subsequently synthesized and screened against biomarker positive and negative cell lines to identify aptamers candidates against respective markers. Collectively, this study establishes LIGS as a state-of-the-art screening technology that can be used to generate highly specific aptamers against multiple receptor-proteins in their native state expressed on one cell population.

BIOT 287

Rapid in silico screening strategy: Development of QSAR model for antibody solubility

Xuan Han1, [email protected], Steven M. Cramer2. (1) chemical engineering, Rensselaer Polytechnic Institute, Troy, New York, United States (2) Ricketts Bldg, Rensselaer Polytechnic Inst, Troy, New York, United States

Monoclonal antibodies(mAbs) can often suffer from poor solubilities especially when high concentration formulation is needed for subcutaneous injection. In the early development stage, candidate screening based on experimental characterizations are both material and labor intensive. To facilitate the process, we developed an in silico predictive model using amino acid sequence as input to help researchers determine solubility level of molecules prior to experiments. A pool of antibodies was employed for training the model and solubility data was acquired using PEG precipitation by a high- throughput screening technique (HTS). Refined homology models of these mAbs were then built by taking protein dynamics into account. Molecular descriptors that depict surface properties of antibodies were calculated for each mAb according to their sequences as well as 3D structures. Utilizing these descriptors as input, a classification model was developed to help distinguish mAbs that showed desirable solubility from those with poor behavior. Further, we also evaluated our model using external published dataset for further validation. This work not only provides a strategy for antibody early screening based on solubility, but also sheds light into the relationship between protein surface properties and solubility where complicated protein-protein interactions are involved, providing potential guidance for designing mAbs with better manufacturability properties.

BIOT 288

Shikimate kinase: Potential target for a new antibacterial agents

Marcin Ogrodniczuk1, [email protected], Anand Motilall1, Rene Fuanta2,1. (1) Biochemistry, East Stroudsburg University, Stroudsburg , Pennsylvania, United States (2) Chemistry and Biochemistry, Auburn University, Auburn, Alabama, United States

Tuberculosis is one of the world’s leading cause of mortality from a single bacterial pathogen, with over 10 million reported cases each year. There is an alarming increase in the prevalence of drug-resistant strains, thus the need for the discovery of novel anti- tubercular agents. Mycobacterium tuberculosis shikimate kinase (MtSK) catalyzes the 5th step of the shikimate pathway, converting shikimate to shikimate-3-phosphate. The overall goal of this project is to express and characterize MtSK and screen for potential anti-tubercular agents. Transformation of XL-1 blue competent cells was performed using a pET 21b plasmid with aroK gene inserted at the multiple cloning site. Plasmids were cloned and purified and used to transform BL 21 DE3 competent cells for subsequent protein expression. With further small and large scale expression, purification and characterization, MtSK kinetic parameters would be determined prior to enzyme inhibition studies using inhibitors like avarone, a marine sponge sesquiterpene quinone and derivatives thereof.

BIOT 289

Ammonia stress triggers genome instability in CHO cells

Dylan Chitwood1, [email protected], Qinghua Wang2, Kathryn Elliott1, Aiyana Bullock3, Dwon Jordana4, Cathy Wu2, Sarah W. Harcum1, Christopher Saski5. (1) Bioengineering, Clemson University, Seneca, South Carolina, United States (2) Center for Bioinformatics & Computational Biology, University of Delaware, Newark, Delaware, United States (3) Department of Biological Sciences, Delaware State University, Dover, Delaware, United States (4) Department of Biological Sciences, Grambling State University, Grambling, Louisiana, United States (5) Department of Plant and Environmental Sciences, Clemson University, Clemson, South Carolina, United States

It is well recognized that waste product accumulation is a detriment to cell growth; however, little is known about the genotoxic properties and the influence of these compounds on global genome instability. In this study, we examined the genotypic effects of mild and high ammonia stress on Chinese hamster ovary (CHO) cells. We identified genome-wide de novo mutations and discovered variants in functional regions of genes involved in the mismatch repair (MMR) pathway, such as DNA2, BRCA1 and RAD52. Loss of function of these genes can lead to genome instability. Additionally, microsatellites were characterized against the Chinese hamster PICR assembly and discovered certain loci are not replicated faithfully the presence of elevated ammonia, which is indicative of microsatellite instability (MSI). From this characterization, microsatellite regions containing insertion and deletion (INDEL) mutations were filtered in order to identify 124 candidate loci that may be suitable biomarkers to gauge genome stability in CHO cell cultures. BIOT 290

High-throughput evaluation of antibody developability using flow cytometry

Emily K. Makowski1, [email protected], Lina Wu2, Alec Desai2, Peter M. Tessier1,2. (1) Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States (2) Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States

There are many chemical and physical requirements that must be met for antibodies to be suitable for use as therapeutics. One of the most important requirements for therapeutic antibodies is high specificity for their target antigens because off-target binding is correlated with rapid antibody clearance rates and suboptimal pharmacokinetic profiles. Therefore, we have sought to develop high-throughput methods for rapidly evaluating antibody specificity using minimal amounts of purified protein and widely accessible experimental methods. Our flow cytometry approach – which uses multiple types of polyspecificity reagents – is well correlated with other methods for evaluating antibody specificity as well as with assays used for identifying antibodies with abnormal clearance rates in vivo. We expect that these results will improve the rapid evaluation of key antibody biophysical properties linked to their developability and thereby improve the drug development process.

BIOT 291

Expanding cellular communication with electronics through the design of cellular transmitters and receivers

Juliana Pitzer1, [email protected], Eric VanArsdale1, Gregory F. Payne3, William E. Bentley2. (1) Fischell Department of Bioengineering, University of Maryland, University Park, Maryland, United States (2) Fischell Dept of Bioengineering, University of Maryland, College Park, Maryland, United States (3) Univ of Maryland Biotech Inst, College Park, Maryland, United States

Modern digital networks are enabled by the communication of transmitters and receivers about objects and events in localized environments. The information transferred is measured by the ability of the users of the network to make decisions they could not otherwise. This dynamic exchange of information, sometimes referred to as the “internet-of-things”, has revolutionized various aspects of society such as travel or financial transactions, but has largely been unable to integrate biological information. We have attempted to remedy this discrepancy by creating cellular network mimetics, such as “transmitters” and “receivers”, that are able to interconvert biological and electronic information. To send electronic information to biological networks, we designed cellular transmitters which respond to hydrogen peroxide generated by electrochemical reduction of oxygen at the surface of a gold electrode. These “transmitter” cells in turn produced AI-1, a bacterial quorum sensing molecule, to relay the information to a “receiver” population. Using this system, we were able to electrochemically regulate the gene expression of the receiver population to produce either GFP or the amino-acid tyrosine by stimulating the transmitter population with a voltage pulse. In order to electronically receive biological information, we designed a cellular population to cleave the redox silent substrate 4-aminophenyl galactopyranoside (PAPG) into the redox active molecule p-aminophenol (PAP) through expression of the β-galactosidase enzyme. We optimized this system by enhancing the cellular import of PAPG into the cytosol by the expression lacY to increase the number of PAPG importers. As a result, we were able to successfully measure the amount of AI- 1 in the media based upon the PAP current produced by our sensor cell population. These two cell-based systems represent the creation of a cellular network, which will allow for regulated information exchange to guide cellular systems.

BIOT 292

Deep sequencing methods for improving antibody discovery against complex antigens

Matthew D. Smith1, [email protected], Alec Desai1, Yulei Zhang1, Peter M. Tessier1,2,3. (1) Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States (2) Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States (3) Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States

The discovery of antibodies with extremely high specificity is important for diverse biomedical applications ranging from diagnostics to therapeutics. However, screening and evaluating antibody mutants is a slow and arduous process, especially for cases involving complex antigens that require epitope-, conformational- and/or species- specific antibody recognition. We seek to develop an integrated workflow that incorporates new approaches for antibody library design, sorting and deep sequencing methods to improve the discovery of antibodies with high conformational and sequence specificity for complex antigens (e.g., insoluble amyloid fibrils). We find that targeting specific sites in antibody binding loops for mutagenesis based on site-specific natural antibody diversity and minimizing the occurrence of specific types of high-risk residues is a powerful approach for designing antibody libraries with high specificity. Moreover, we find that sorting and sequencing antibody libraries against not only the target antigen but also against closely related non-antigens and polyspecificity reagents is critical to identify extremely rare antibody mutants that display high affinity, high conformational specificity and low levels of off-target binding. We will discuss our novel bioinformatics methods that we are developing to mine deep sequencing data and identify antibodies with high affinity, specificity and other drug-like properties.

BIOT 293

Multi-valent nano-self peptides suppress self-signaling and increase phagocytosis of antibody-opsonized targets AbdelAziz Jalil1,2, [email protected], Brandon Hayes1, David M. Chenoweth2, Dennis E. Discher3. (1) Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States (2) Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States (3) Biophysical Eng NanoBio Polymers Lab, Univ of Pennsylvania, Philadelphia, Pennsylvania, United States

Injections of foreign particles generally lead to non-specific serum adsorption with a corona rich in antibody (IgG) that binds and activates phagocytosis by macrophages. Our own cells and cancer cells inhibit such phagocytosis by displaying ‘marker of self’ CD47. While clinical combination of cancer-targeting antibodies with anti-CD47 blockade shows some efficacy against cancer, safety of these therapies remains controversial. Based on our previous study of CD47-derived polypeptides that mimic the binding and function of full length CD47, we engineer soluble nano-Self oligopeptides in multivalent form to block CD47’s receptor on macrophages, SIRPα. Bivalent nano-Self agonists prove more potent in phagocytosing antibody-targeted cells (Keff ~ 20 nM) than monovalent oligos. Bivalents also exhibit a more stable hairpin-like structure and out- compete monovalents in macrophage association, whereas scrambled oligos lack function. Soluble nano-Self oligos added to isolated macrophages suppress a passivating phosphotyrosine signal, consistent with ‘self’ signaling through SIRPα. Bivalent nano-Self oligopeptides are thus a potential alternative to anti-CD47 blockade and promoting phagocytosis of ‘self’.

BIOT 294

Developing inhibitors of UCHL5 deubiquitinase in the proteasome

Harshani Sewvandi Gurusingha Arachchige1, [email protected], Q. Ping Dou3,4, Young-Hoon Ahn2. (1) Chemistry, Wayne State University, Detroit, Michigan, United States (3) Barbara Ann Karmanos Cancer Insitute, Detroit, Michigan, United States (4) Oncology, School of Medicine, Wayne State University, Detroit, Michigan, United States

Protein degradation is crucial for maintaining the protein homeostasis in eukaryotic cells. Target proteins to be degraded are modified by poly-ubiquitination, which leads to degradation by 26S proteasome. In the proteasome, poly-ubiquitinated protein undergoes deubiquitylations before degradation at the 20S catalytic core subunits, which are mediated by deubiquitinases (DUB) associated with the proteasome. Ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5) is one of the three DUBs in the proteasome, which is located in the lid of the 19S regulatory subunits. Our preliminary data and literature showed a high level of expressions of UCHL5 in triple- negative breast cancer (TNBC) cell lines over non-TNBC, suggesting UCHL5 as a potential therapeutic target protein. However, there is no reported selective UCHL5 inhibitor. We have conducted a virtual screening of a FDA-approved drug library with UCHL5, identifying several lead compounds. Among hits, we have identified tiaprofenic acid (TA) as a potential inhibitor of UCHL5. Throughout computational docking analysis, we have designed and synthesized TA derivatives. Their inhibitory activities were evaluated by in-vitro UCHL5 enzyme assays, finding a potential inhibitor of UCHL5. BIOT 295

Expanding the promoter set for a super-critical CO2 tolerant strain of B. megaterium

Elaine M. Reece1, [email protected], Madeline J Wittwer1, Adam Freedman2,3, Janelle R. Thompson3, Kristala L. Jones Prather2, Jason T. Boock1. (1) Chemical, Paper and Biomedical Engineering, Miami University (OH), Oxford, Ohio, United States (2) MIT Department of Chemical Eng, Cambridge, Massachusetts, United States (3) Department of Civil and Environmental Engineering, MIT, Cambridge, Massachusetts, United States

In situ product extraction using the sustainable solvent supercritical carbon dioxide is a promising method to relieve end-product toxicity with potential decreased purification costs. Previously, we have engineered an environmental-isolate of Bacillus megaterium called SR7 that is tolerant of the harsh supercritical carbon dioxide conditions to produce short-chained, branched alcohols that are well extracted by the solvent. While initial attempts at engineering SR7 were successful, the limited genetic tools to further modify the organism hindered metabolic engineering progress, especially the lack of strong, constitutive promoters. To expand the synthetic toolbox for the microbe, promoter mining was undertaken from an RNAseq library for B. megaterium SR7 strain grown under a variety of growth conditions, including aerobically, anaerobically under low pressures of carbon dioxide, and under supercritical carbon dioxide. Using gene expression results, several promoters were selected that showed high-level transcription under all growth conditions for their general use in engineering this strain. Promoters were assessed by expressing green fluorescent protein (GFP) under different growth conditions and medium combinations. The promoter related to polyhydroxyalkanoate production, phaR, was found to have high, constitutive expression, comparable to the widely utilized B. megaterium xylose-inducible promoter in both strength and rapidity in minimal medium. Other promoters related to citrate synthesis and polyhydroxyalkanoate synthesis had moderate expression, permitting fine-tuned transcription. Work is underway to test these promoters under other growth conditions and within the context of biological production of alcohols. This research is broadly applicable to the growing B. megaterium engineering community, and the universality of these promoters is being explored in different strains.

BIOT 296

Molecular determinants of antibody self-association and off-target binding

Priyanka Gupta1,2, [email protected], Sandeep Kumar1, Justin M. Scheer1, Peter M. Tessier3. (1) Biotherapeutics Discovery, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut, United States (2) Rensselaer Polytechnic Institute (RPI), Troy, New York, United States (3) Chemical Engineering and Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States

Successful development of monoclonal antibodies as therapeutics requires high antibody specificity, low toxicity, low viscosity, high solubility and low levels of aggregation. Several of these properties are strongly influenced by the sequence and structural properties of antibody variable regions and especially the complementarity determining regions (CDRs). We are investigating the molecular determinants of various developability properties for multiple antibodies by systematically mutating their CDRs to alter their charge, polarity and hydrophobicity. Interestingly, we find that some mutations display favorable effects by reducing both antibody self- and non-specific interactions, while others are favorable for one property and detrimental for the other one. We are currently extending these studies to additional antibodies to evaluate their generality. We expect that these findings will improve the design and engineering of antibodies and antibody libraries with drug-like properties.

BIOT 297

Design and characterization of bispecific antibodies for in vivo imaging of pathological tau

Ghasidit Pornnoppadol1, [email protected], Alec A. Desai2, Charles G. Starr1, Peter M. Tessier1,2,3. (1) Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States (2) Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States (3) Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States

Early detection of pathological tau is vital for proper diagnosis of tauopathies including Alzheimer’s disease (AD). Noninvasive in vivo imaging requires molecular agents that can effectively cross the blood-brain barrier and specifically target pathological tau. Although tau proteins are phosphorylated at multiple sites and these chemical modifications are linked to aggregation and formation of neurofibrillary tangles, only a subset of these sites are exclusive to AD. While antibodies specific for phosphorylated tau proteins have been identified, little is known about their ability to cross the blood- brain barrier (BBB). One attractive approach for improving the delivery of biologics such as antibodies to the brain is to engineer them to cross the BBB via receptor-mediated transcytosis. Transferrin receptors are the most highly expressed receptors on the BBB that have been exploited for delivery of large molecules. Few antibodies that target these receptors and facilitate receptor-mediated transcytosis are well validated and available to the community for detailed studies of their activity in vivo using animal models. We are currently generating and testing multiple bispecific formats that combine anti-transferrin and anti-tau antibodies with the goal of optimizing their ability to retain binding to their respective targets and mediate transcytosis. We expect that these novel bispecific antibodies will be powerful agents for in vivo imaging of pathological tau.

BIOT 298 Conformational dynamics due to cofactor binding courtney A. Clark, [email protected]. Chemistry, Youngstown State University, Warren, Ohio, United States

Aldose Reductase (AR) catalyzed reduction of glucose reaction that requires NADPH as the cofactor follows a sequential ordered kinetic mechanism. The catalytically active form of the enzyme.NADPH complex which needs to be assembled first preceding to the aldehyde substrate binding. The conformational change involving a hinge-like movement of residues in flexible loop is anticipated to take place upon the binding of the diphosphate moiety of NADPH in the enzyme.NADPH complex development step. The reorientation of this loop is expected to permit the release of NADP+after the catalysis. The Translation, Libration, Screw analysis of apo AR crystal structure indicates that some of the residues in the flexible loop might move as a rigid group.

BIOT 299

Withdrawn

BIOT 300

Optimizing AEX membrane adsorbers for robust parvo virus (MVM) clearance

Sherri Dolan, [email protected]. Sartorius Stedim North America, Williston, South Carolina, United States

Abstract

The purpose of this poster is to guide users on the best practices when optimizing and sizing membrane adsorbers for virus clearance. AEX chromatography is a powerful method to clear all types of virus based on charge. Membrane chromatography devices offer many advantages over conventional chromatography resins in terms of virus binding/clearance. Compared to resins, membranes offer higher binding capacity for large molecules such as viruses, show equivalent virus clearance capabilities, have >10 fold faster flow rates, use >75% less buffer and present a flexible single-use disposable solution which does not require validation. These qualities make membrane adsorbers a perfect fit for flow-through chromatography operations where virus clearance is expected. However several considerations must be addressed when using membrane adsorbers for virus clearance: - Buffer conditions - loading capacity for virus - air removal/wetting of small scale devices - protein (or impurity) binding to membrane

This poster also presents data to support parvovirus (MVM) clearance on Sartobind membranes under challenging process conditions. MVM parvovirus is considered to be the most difficult to remove virus based on its small size and high pI. Conditions tested include: - pressure pause - high loading - non-wetted filter - non-purified virus spike - scalability from pico (0.08 ml) to nano (1 ml) devices

BIOT 301

Evaluation of pressure fluctuation in constant pressure and constant flow rate control and effects on viral clearance

Victoria Kaloudis, [email protected], Esha B. Vyas, Taka Sohka. Science and Technology, Asahi Kasei Bioprocess, Glenview, Illinois, United States

Viral filtration is an integral part of downstream processes for biopharmaceutical products in which viruses and other contaminants are effectively removed by nanofiltration. Planova filters are operated in dead-end mode by either constant pressure or constant flow rate control. In cases where strict pressure control is required, Asahi Kasei Bioprocess recommends using constant pressure control, either by house air or compressed air tanks. However, little is known about how pressure fluctuation during filtrations affects virus removal. In this study, we examined the effects of pressure fluctuation on viral clearance when using constant pressure control and constant flow rate control by peristaltic pump with one or two pump heads. A total of 16 filtrations using a protein feed of 5 g/L BSA with porcine parvovirus (PPV) spike (virus 2 2 challenge, 10.5 logs TCID50/m ) were conducted on 0.001 m Planova 20N filters according to manufacturer recommendations with pressure readings taken every 2 s. Filtrations were conducted in duplicate using constant pressure control by compressed air and using constant flow rate control by a peristaltic pump with one or two pump heads. Constant pressure runs were conducted at 8 psi (low pressure), <14.2 psi (manufacturer recommended maximum operating pressure) and 18 psi (high pressure). Target pressure for constant flow rate control with two pump heads was 6 – 8.5 psi, <14.2 psi and 16 – 18 psi, respectively. Constant flow rate control with one pump head was conducted in the range of 7 – 18 psi and <14.2 psi. The constant flow rate control runs represent exaggerated conditions with pressure fluctuation of >9 psi for constant flow rate with one pump head and relatively limited fluctuation of <4 psi for constant flow rate with two pump heads. The process volume was 150 mL, collected in two equal fractions, and PPV assay was conducted to determine viral clearance for each run. Pressure fluctuation during filtration runs did not cause marked decreases in PPV LRV under the conditions tested.

BIOT 302 Membrane adsorbers: High performance polymer brush membranes for protein separation

Mirco Sorci, [email protected], Xiaoru Li, Pranav Ramesh, Georges Belfort. Dept. of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Albany, New York, United States

The recovery of biosynthetic products, particularly biopharmaceuticals, during downstream processing is still primarily dominated by chromatographic steps. While membranes based separations would offer several advantages (e.g. higher fluxes and reduced operating times ~ higher Pe numbers; no cleaning validation required; reduced space; modular; higher flexibility; etc.), the main Achilles’ heel remains surface area, hence lower binding capacities compared with chromatographic resins. To address this limitation, we adapted the Single-Electron Transfer (SET)-Living Radical Polymerization (LRP) method to graft methacrylate monomers from commercial regenerated cellulose membranes. This polymerization method is industrially attractive, since it is robust, fast, “green” and can operate at ambient temperature in the presence of oxygen (air). Several methacrylate monomers (e.g. 2-(dimethylamino)ethyl methacrylate) were tested. Modified membranes were characterized with ATR-FTIR, SEM, AFM, Z-potential measurements; while extent of polymerization was monitored by GPC. Both static and dynamic protein (e.g. bovine serum albumin) adsorption experiments were performed to determine binding capacity. Operating conditions were also investigated to optimize membrane performance, in particular pH. In conclusion, the results demonstrate that SET-LRP grafting of polymer brushes offers the potential to create a 3D environment that can be tuned for protein adsorption, hence resulting in protein membrane adsorbers with high surface area and capacities as attractive alternative to chromatographic systems.

BIOT 303

Monitoring of antibody throughout bioprocessing by fluorescence reporter with applications for protein A column process control

Ujwal Patil1, [email protected], Mary Crum2, Binh Vu2, Atul Goyal2, Katerina Kourentzi2, Richard C. Willson2,1,3, [email protected]. (1) Biology and Biochemistry, University of Houston, Houston, Texas, United States (2) Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, United States (3) Escuela de Medicina y Ciencias de la Salud TecSalud, Monterrey, Nuevo Leon, Mexico

Measurement of product titer is pivotal in bioprocess development. A typical monoclonal antibody production process involves multiple unit operations from upstream to final product release. We have developed a target-agnostic assay to measure the concentration of IgG or Fc-fusions during the discovery, cell culture development, and in-process phases of the biopharmaceutical process train. An antibody in complex, UV- absorbing culture fluid cannot readily be detected, limiting the ability to take quick process decisions, and reducing productivity. Our fluorescence-based monitoring technology overcomes some of the current barriers by allowing homogeneous and near- instantaneous (< 30 seconds) IgG-measurements. We demonstrate application of this technology for batch and continuous IgG detection. In our approach, IgG-containing cell culture fluid is mixed with fluorescein-labeled Fc-binding ligands to produce a shift in fluorescence polarization, which can be detected in a batch or continuous-flow format. While screening various Fc-binding ligands for monitoring of human IgG we also identified ligands which signal the presence of IgG by changes in fluorescence intensity, which is readily detectable with inexpensive hardware. We observe significant shifts in both polarization and intensity in CHO cell culture fluid spiked with human IgG and can detect 0.005-0.1 g/L human IgG in protein A column breakthrough at a flow velocity of 80 cm/hr. We have successfully evaluated IgG-monitoring at a flow rate of 900 ml/hr without significant delay in fluorescence intensity signal. The fluorescence intensity information was applied to automatically switch columns when IgG-breakthrough of 0.005 g/L was reached. This technology provides a fast and reliable way to detect IgG in a flowing stream (or culture or batch samples) without elaborate sample preparation. Similar approaches can be envisioned for the detection of host cell proteins, DNA, and non-mAb products.

BIOT 304

Next generation clarification approach using chromatography of large particles

Alexei Voloshin, [email protected], Masa Nakamura. 3M Company, St. Paul, Minnesota, United States

Cell culture fluid harvesting has gone through a number of generations of approaches and technologies that implement different strategies. While several different principles have been proposed and evaluated by the industry, only two have seen deployment on a wide scale in the biopharma industry. Separation of large particles by density using a centrifuge and the cleanup of small particles through the use of depth and membrane filtration continues to be the tried-and-true approach in laboratories and manufacturing facilities worldwide. Yet, growing cell densities, increase in the difficulty and in range of cell culture biochemical and biophysical characteristics have exhausted the performance potential of both density and size strategies and of technology that implements them. As the expectations of the performance, robustness, product quality and recovery continue to increase, new separation principles are needed as is scalable technology to bring these principles into a real life process space. Here we demonstrate a new approach that uses AEX chromatography to enable cell culture fluid clarification using a chromatographic approach. Through the use of advanced non-woven materials and ligand chemistries we have enabled chromatographic separation of particles across the size range from that of mammalian cells to soluble small molecules. The result is the ability to clarify the cell culture, scale the system, and predict the quality of clarification with chromatographic precision and repeatability. This principle effectively bypasses the limitations of the present approaches and offers much higher clarification performance and robustness at the same time. We discuss the basic architecture of such chromatographic supports, scaling characteristics, performance metrics, and robustness of such an approach for deployment across the biopharma industry. Furthermore we demonstrate how this approach is implemented in a scalable single-use technology that is easily deployed at bench scale and commercial manufacturing alike. This approach enables chromatographically defined performance of the purification process from the beginning, thus simplifying the whole purification process and increasing its robustness.

BIOT 305

Integrated continuous bioprocessing using continuous countercurrent tangential chromatography (CCTC) for capture and polishing of mAbs

Oleg Shinkazh, [email protected]. Executive, Chromatan Inc, Lower Gwynedd, Pennsylvania, United States

Several recent studies have demonstrated the potential of using continuous countercurrent tangential chromatography (CCTC) for the purification of monoclonal antibody products. CCTC operates with a flowing resin slurry, exploiting traditional approaches of countercurrent staging to achieve > 10-fold increases in productivity compared to traditional batch columns in a truly steady-state low pressure (<15 psi) unit operation that can be directly integrated into a fully continuous biomanufacturing process. The featured data shows productivity and product quality outcomes from Protein A capture, as well as Anion exchange and Cation exchange polishing steps for purification of a commercial mAb. Another unique aspect of CCTC is the ability to use small particle size resins with improved binding kinetics and better system productivity but without any increase in pressure. The data also features our strategy for integrating the unit operations into a single process train incorporating an integrated in-line sampling strategy as sponsored by FDA contract ($2.5 Million award, 2017). In addition we discuss the potential of this platform to enable efficient processing of sensitive biologics because of significantly reduced residence time (<10 min from binding to elution), as well as rapid in-line buffer adjustments of eluted product.

BIOT 306

Enhanced selectivity of ion exchange resin for ADC polishing

Romas Skudas, [email protected], Annika Holzgreve. EMD Millipore, Darmstadt, Germany

Antibody drug conjugates (ADCs) have a great potential to increase the biopharmaceutical drug efficacy. Unfortunately, this great potential currently is explored at high costs due to the complexity of the manufacturing process. Especially the purification of immunoglobulins carrying different ratios of cytotoxic drugs requires the use of non-efficient chromatographic steps at narrow operation windows. The exploration of an innovative ion exchange resin enabled us to reach an efficient DAR (drug antibody ratio) species separation without applying high conductivity and/or organic solvents. Moreover, we could improve the quality of commercially available ADCs by preparative separation of each individual DAR species, regardless their manufacturing origin. The application of ion exchange chromatography showed significant economic advantages compared to hydrophobic interaction chromatography, assuring high product recovery rates without influencing ADC’s efficiency. Additionally, we were able to upscale such separation from analytical column format to preparative mode at minimal process development efforts defining a broad window of operation and reflecting the later ease of use and templatability of such method.

BIOT 307

Influence of low pH treatment on the elution behavior of monoclonal antibodies in protein A chromatography

Carolin Stange1, [email protected], Christian Dr. Frech2. (1) Institute of Biochemistry, University of Applied Sciences Mannheim, Bad Orb, Germany (2) Biotechnology, University of Applied Sciences Mannheim, Mannheim, Germany

Low pH treatment is a commonly known factor for the aggregation of antibodies and Fc- Fusion proteins. During downstream processing of these proteins, low pH conditions are used as an effective way for virus inactivation at pH 3.0-3.5, as well as for elution from a previous Protein A chromatography step. Binding to Protein A material is induced through multiple interactions between the Fc-part of the antibody with the immobilized Protein A domains and is sensitive for conformational changes. The objective of this work was to use low pH treatment to study its effect on aggregation and structural changes of monoclonal antibodies. Therefore, a Protein A purified antibody solution was acidified (pH 2.8 to 3.5) and incubated for a period of time. Afterward, the solution was neutralized and reloaded onto a Protein A chromatography column. This resulted in only a partial binding of the sample; furthermore, when analyzing the resulting elution peak by size exclusion chromatography, a double-peak elution behavior at the monomer size was observed. Both phenomena are independent of each other but occurred during the same experiment. Additionally, these effects could not be nullified through the addition of excipients to the buffer system. The results show that multiple low pH treatments can significantly change the structure and binding behavior of monoclonal antibodies and should, therefore, be kept at a minimum during purification.

BIOT 308

Membrane chromatography solutions for single-use, intensified mAb purification

Gary Skarja, [email protected]. MilliporeSigma, Burlington, Ontario, Canada

The biopharmaceutical industry is currently focusing on developing higher productivity, more flexible manufacturing processes that incorporate single-use technologies. The use of membranes for high productivity chromatographic purification processes provides compelling advantages, in comparison to tradition resin bead methods, primarily resulting from the rapid, convective protein binding mechanism that underpins membrane operating conditions. This convective binding mechanism can be exploited to achieve very short operating residence times, on the order of seconds, which enables very high productivity purification processes. However, the typical, highly permeable membrane structures that facilitate very short residence time operations exhibit modest binding capacity due to a limitation of binding site density. Advances in membrane science that have focused on resolving this fundamental trade-off have resulted in new chromatographic matrix with resin-like capacity and purification power at much faster process speed (6 seconds residence time). This webinar will focus on the development, characterization, and economic benefits of a novel Protein A affinity membrane, which paves the way for a high productivity, truly single-use chromatography platform for intensified mAb purification.

BIOT 309

Development of flow through cation exchange chromatography (FT CEX) to improve process efficiency and address on-column aggregation

Zheyuan Luo1, [email protected], Nushaba Rashid1, Gerald Terfloth1, Antonio R. Ubiera2. (1) Downstream Process Development, GlaxoSmithKline, Audubon, Pennsylvania, United States (2) UE0551, Glaxo Smith Kline, King of Prussia, Pennsylvania, United States

Cation Exchange Chromatography (CEX) is well-established and commonly used to remove aggregates in the manufacture of monoclonal antibodies. CEX can be operated in two different modes: Bind/Elute (B/E) and Flow-Through (FT). Compared to conventional B/E mode, FT CEX confer advantages in large-scale manufacturing, including substantial process efficiency gains and resulting operational cost reduction, as well as process intensification for application within a semi-continuous chromatography platform. In this work, a generalized process development approach for FT CEX step design and optimization will be presented. Firstly, a commercially-available CEX resin with low ligand density was identified and initial frontal chromatography experiments were conducted to identify flow-through operating conditions and ranges using an IgG1 protein as a model system. More specifically, univariate breakthrough studies were performed to fully characterize aggregate selectivity. Secondly, an IgG4 protein that had exhibited severe on-column aggregation behaviour when processed in B/E mode was selected and evaluated following a similar methodology. In this case, on-column aggregation was fully avoided in flow-through mode, while achieving an increase in overall efficiency and selectivity for aggregate removal. Finally, leveraging the experimental data, the value provided by in silico mechanistic modeling as a predictive tool in cation-exchange process development in flow-through mode will be discussed.

BIOT 310

Implementation of reverse-phase chromatography (RPC) to support non-platform process development analytics

Zhichao Fang, [email protected], Jeanna Allen, Nicole E. Payonk, Duncan McVey, Anurag Khetan. Biologics Development, Bristol-Myers Squibb, Pennington, New Jersey, United States

Chromatography is widely applied for protein purification. A variety of resins and columns are used by process development analytics to separate proteins based on different mechanisms, including affinity capture, ion exchange, hydrophobic interaction, and mixed mode. While these conventional schemes are sufficient to purify platform monoclonal antibodies (mAbs), more tools are required to support process development for non-platform proteins. Although reverse-phase chromatography (RPC) in general is not suitable for mAb manufacturing processes due to the addition of organic solvents, it can serve as a powerful purification approach as part of protein analytical assays. Herein, we present two case studies to demonstrate the implementation of RPC to support no-platform process development. 1. To support the clone selection for a non-mAb protein in cell line development, we performed a small scale high throughput AEX purification for 24 clones. The purified samples needed to be tested by peptide mapping for protein sequence variation and by Rapifluor-UPLC for N-glycan characterization assays. However, ELISA results indicated that the purified samples contain host cell protein (HCP) levels at approximately 10%, which caused significant interference to both peptide mapping and N-glycan analysis. These samples were further polished by RPC to reduce HCP levels. We demonstrated that protein quality and assay reliability were not impacted by the RPC polishing step. 2. A non-mAb protein which doesn’t bind to Protein A generated an analytical challenge to accurately measure the titer in clarified bulks. Cell line development needed to screen 6000 samples with titers between 0.01 and 0.5 mg/mL. To address this challenge a 3- min run time reverse-phase UPLC assay was established, which provided the accuracy, sensitivity and throughput required. In conclusion, to support process development of non-platform proteins, process development analytics should consider all separation mechanisms. The uncommon reverse-phase chromatography could serve as a powerful purification approach and analytical assay.

BIOT 311

Mechanistic understanding of aggregate reduction in a monoclonal antibody downstream process Lee Bink, [email protected]. UE0551, GlaxoSmithKline, King of Prussia, Pennsylvania, United States

High molecular weight aggregates are considered a product-related impurity due to their potential immunogenicity in patients. Aggregates are typically removed from a monoclonal antibody product stream via various modes of chromatography, including cation exchange (CEX) and hydrophobic interaction chromatography (HIC). However, formation of aggregates during downstream processing due to various stresses (shear, interfacial stress, pH, etc.) can also be regulated and/or prevented in order to minimize the total levels in bulk drug substance. Using both differential scanning fluorimetry and a measurement of aggregation kinetics, it was determined that the Affinity Chromatography step was contributing towards the denaturation of the mAb and causing a subsequent aggregation event during the low pH virus inactivation step. Implementation of a milder elution pH buffer resulted in less formation of aggregate across these unit operations. This work demonstrates how the optimization of the existing two column process can be achieved to reduce aggregation while negating the need for a third chromatography step and the necessary capital expenditure in the New Product Introduction (NPI) facility.

BIOT 312

Exploring efficiency gains with the implementation of FlowVPE into accelerated downstream process development

Thomas Lindsey, [email protected], Rashmi Bhangale, Tania Rosen, Nathan Nicholes, Leslie Wolfe, Jonathan Webb, Sigma Mostafa. Downstream Process Development, KBI Biopharma Inc, Raleigh-Durham, North Carolina, United States

FlowVPE (C Technologies) is a variable path length UV-VIS spectrophotometer that can be used to accurately measure protein concentrations across a much larger range than typical inline UV detectors on FPLC instruments. The sensitivity of the instrument is sufficient to monitor the capture and breakthrough of product using clarified harvest as the feed stream on Protein A columns. KBI Biopharma has generated data for multiple molecules demonstrating the comparability of FlowVPE UV breakthrough data to offline ProA-HPLC titer analysis for determination of Protein A resin dynamic binding capacity. Application of FlowVPE enables real-time data generation, reducing analytical support requirements for sizing ProA capture columns. This capability results in both time and cost savings in an accelerated downstream purification process development workflow.

BIOT 313

Membranes with attached photoiniferters: Uses in bioprocessing separations

Jerald K. Rasmussen, [email protected], Andrew Vail, Kristopher Richardson, Daniel O'Neal, Eli Narveson. 3M Center, 3M, Saint Paul, Minnesota, United States Controlled radical initiators, such as ATRP or RAFT initiators, have become commonplace for the synthesis of a variety of polymeric materials. The use of these initiators allows one to more easily control the molecular weight and/or the polydispersity of the resultant solution polymers. Attachment of controlled radical initiators to solid substrates, however, is quite difficult, involving multiple synthetic steps, and is often very substrate dependent. We have designed a simple method for attachment of RAFT agents or iniferters to membranes and other solid substrates by taking advantage of the hydrogen abstracting ability of Type II photoinitiators such as benzophenone. Irradiation of nylon membranes coated with a solution of benzophenone and a xanthate-functional compound results in H-abstraction from the membrane surface. The free radical is trapped by transfer of the xanthate ester, resulting in a xanthate-functional membrane. This reaction process can be extended to other types of thiocarbonylthio-containing agents, such as dithioesters, dithiourethanes, and trithiocarbonates. E-beam irradiation provides an alternative and simple mechanism for generating radicals on substrates, which can subsequently be trapped by an appropriate thiocarbonylthio-containing compound. The functionalized substrates can be coated with free radically polymerizable monomers, then irradiated with UV or LED sources, to provide polymer-grafted substrates. We have grafted a variety of ionic monomers out of aqueous solution to provide ion exchange membranes for protein purification applications. These membranes display improved protein capture performance when compared to membranes made in the absence of the controlled radical initiator. The entire process is potentially amenable to roll-to-roll manufacturing operations. Our initial results in this area will be highlighted.

BIOT 314

Development of an efficient purification process for a recombinant E. coli protein

Andrew Lees1, [email protected], Samson Grebnatsae1, [email protected], Chelsea Pratt2, William H. Rushton2. (1) FINA Biosolutions LLC, Silver Spring, Maryland, United States (2) Bio-Rad Laboratories, Hercules, California, United States

Chromatography is the heart of protein purification and the development of the chromatography process is facilitated by automated chromatography systems. Purification protocols often follow the general three step scheme of capture, intermediate and final polish. In this case study, we present the process development for a recombinant E. coli protein using new generation chromatography resins. The target protein has an acidic pI and is expressed at high levels as a soluble, properly folded intracellular protein. Thus, the lysate is full of nucleic acids and cytosolic contaminants, which are also generally acidic. Using an automated chromatography system, we screened four anion exchange resins and compared their capture capacity verses a dextran/agarose resin. A hydrophilic epoxide AEX resin with large pores provided the highest binding capacity and recovery. The capture step was then scaled up from 1 ml resin to 100 ml resin. The intermediate purification step was developed by screening HIC resins and the final polish step was developed using a second anion exchange chromatography resin. BIOT 315

Downstream process intensification with novel chromatography resins for enhanced monoclonal antibody purification

Jonathan Fura, [email protected]. Avantor, Macungie, Pennsylvania, United States

With more than a thousand antibody therapeutics currently in clinical development, the demand for improved production and purification of this class of proteins has become evident. Over the past several years, significant progress has been made to increase upstream process titers, thus creating a strain on existing downstream purification technologies. Although implementation of continuous unit operations has provided some relief, downstream processing still presents a major bottleneck in the production of therapeutic antibodies. In this presentation, we will demonstrate an integrated downstream purification process to enable significant removal of critical impurities. Through use of these affinity resins that possess high dynamic binding capacities of greater than 60 g/L, we have obtained antibodies with increased purity while reducing overall process time. Furthermore, by using select additives during process chromatography, we demonstrate this process can be fine-tuned to enhance target antibody selectivity by removing host cell protein impurities and aggregates. When used in conjunction these technologies represent a highly efficient process to obtain monoclonal antibodies for use as therapeutics.

BIOT 316

Risk-based scale-up of high-throughput chromatography systems using Bayesian statistics and mechanistic modeling

Till Briskot1,2, [email protected], Ferdinand Stueckler3, Katharina Doninger3, Tobias Hahn1, Thiemo Huuk1, Juergen Hubbuch2. (1) GoSilico GmbH, Karlsruhe, Germany (2) Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany (3) Roche Pharma Technical Development, Roche Diagnostics GmbH, Penzberg, Germany

High-throughput screening methods are used routinely to support process development in a rapid and cost-effective manner. In order to apply these methods also for process characterization, robotic scale-down models need to be established that are comparable to large-scale commercial processes. For this reason, the systematic adoption of high- throughput techniques in late-stage development has come with the need to better understand and predict scale differences. The qualification of high-throughput techniques as a scale-down model is commonly performed on an empirical base. Scale offsets are thereby mostly attributed to differences in system geometry. Recent studies have also shown that the scale offset can vary, depending on which process parameters are screened. To get deeper insights on how scale differences affect process performance, scale offsets have also been described on a mechanistic level using established chromatography models. This approach requires an extensive system characterization to effectively account for scale differences and to support scale-up. Yet a thorough system characterization can hardly be conducted in high-throughput process development. In this study we present an approach that combines Bayesian statistics and mechanistic modeling to perform a risk-based scale-up of a high-throughput chromatography system. The mechanistic model, which is calibrated using high-throughput data, will be used to predict large-scale performance based on the robotics screening readouts. Bayesian statistics is applied to account for the experimental and systematical uncertainty during calibration of the mechanistic model. In this way, the prediction uncertainty of the mechanistic model reflects the uncertainty in the scale-down model and can provide deeper insights on how scale differences affect process performance. This model-based assessment of the prediction uncertainty of high-throughput methods will therefore allow to evaluate the suitability of the scale-down model for process characterization.

BIOT 317

New tool for the next generation of antibody purification: Case study using novel protein A affinity chromatographic membrane technology

Graham Temples1, [email protected], Tom Caldwell2, Jinxiang Zhou1, Daniel Henn1, Scott M. Husson1,3, Sarah W. Harcum2. (1) Purilogics, LLC, Greenville, South Carolina, United States (2) Bioengineering, Clemson University, Clemson, South Carolina, United States (3) Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

The success of monoclonal antibody (mAb) drugs has encouraged the pharmaceutical industry to pursue high-productivity biologics manufacturing processes. The demand is driven by multiple factors, particularly growing competition and market uncertainty. In addition, there is increased demand to lower the cost of mAbs for the treatment of chronic conditions. While continuous chromatography technology has been developed to improve productivity using resin-based media, mAb purification still remains a critical bottleneck in the manufacturing process due to the slow processing speed of resins.

This presentation highlights Purilogics’ recent progress on development of novel Protein A membrane chromatographic technology (PurexaTM-A). Beyond high binding capacity and fast processing speeds, we aim to deliver a differentiated product enabling a robust process over multiple cycles. In this presentation, we will demonstrate purification performance of our prototypes using a suite of CHO-derived monoclonal antibodies. Key quality attributes such as: host cell protein, DNA, and aggregate content of the purified antibody pool will be characterized along with mAb recovery. In addition, we will demonstrate reusability of the membrane with multiple-cycle bind-and-elute studies. BIOT 318

Downstream process development strategy for reducing residual lipases in monoclonal antibody products

Michael Iammarino1, [email protected], Divya Chandra1, William McKechnie1, Sunitha Kandula1, David J. Roush2, Jennifer Pollard1. (1) Downstream Process Development & Engineering, Merck & Co., Inc., Kenilworth, New Jersey, United States (2) Biologics Process R&D, Merck & Co., Inc., Kenilworth, New Jersey, United States

Host cell proteins (HCPs) are a major constituent of the process-related impurities during biologics production. The amount of residual HCP in the final drug product is a critical quality attribute due to its potential to affect both product safety and efficacy. It is therefore a regulatory requirement to monitor the removal of HCPs during process development. The downstream process for monoclonal antibodies (mAbs) typically uses multiple chromatography and filtration steps to remove HCPs to below acceptable levels. However, for several mAbs it was found that specific HCPs known as lipases, despite being present at relatively low levels, were causing enzymatic degradation of polysorbates in the formulated drug product over time. In addition to the possible immunogenicity of these residual lipases, polysorbate degradation can potentially lead to a reduced shelf life of the drug product due to increased aggregation and particle formation. To overcome this issue, a focused effort has been made during downstream process development to track and remove these lipases, but that task is challenging as the lipases can associate or co-purify with the mAb and can be highly active even at sub-ppm levels. Process development case studies for two mAbs will be presented to demonstrate the development and analytical strategies used to address lipase clearance. Specifically, process parameters for different unit operations were investigated for their ability to remove lipases from the mAb process streams. Clearance of lipases was monitored via multiple analytical assays to enable a detailed understanding of process performance as well as deliver robust, final processes. The successes and challenges with lipase removal observed across the unit operations for the different mAbs will be highlighted along with the general strategy and approach used for tackling this downstream bioprocessing challenge.

BIOT 319

Application of an anion exchange membrane adsorber in late-stage bioprocesses

Chris Nieder1, [email protected], Tom Klimek2. (1) MilliporeSigma, Burlington, Massachusetts, United States (2) Eisai, Exton, Pennsylvania, United States

Membrane adsorbers have become a popular choice in single-use centric bioprocesses for their ease of operation and high flowrates; however, these membranes are often replaced by a packed bed chromatography operation for late-stage clinical and commercial processes. Packed bed chromatography often provides better fit for late- stage processes, as columns can be packed to scale with increased process volume and can be cycled as production needs rise. This body of work explores the potential for membrane adsorber usage in late-stage processes by pushing the limits of the membrane. High loading, up to 40 kilograms of protein per liter of membrane, challenged the membrane adsorber for HCP clearance and viral clearance. Results were used to evaluate the applicability of this membrane adsorber for a large volume, late-stage bioprocess via an economic analysis including a comparison to packed bed performance.

BIOT 320

Streptavidin-ELP-based macromolecular ligand fusions for the purification of mAbs

Kabir Dhingra1, [email protected], Akshat Mullerpatan1, Ronit Ghosh1, Pim Hermans2, Pankaj Karande1, Steven M. Cramer1. (1) Howard P. Isermann Dept. of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States (2) BAC BV, Leiden, Netherlands

The smart biopolymer elastin-like polypeptide has been successfully employed previously for the purification of mAb and non-mAb biologics using affinity precipitation. However, for each target, this involves recombinantly cloning and expressing a fusion of ELP and the respective affinity ligand. In this work, we have examined the utility of streptavidin-ELP fusions as a platform for affinity precipitation. While streptavidin usually expresses as inclusion bodies that are relatively difficult to purify, fusions of streptavidin with other proteins can enable soluble cytoplasmic expression and therefore easier purification. A tetrameric streptavidin-ELP (SELP) construct was cloned, expressed and purified from the cytoplasm of BL21(DE3) E. coli, attaining yields of > 10 mg/L of culture. Biotinylated CaptureSelect™ affinity ligands developed by ThermoFisher, were non-covalently conjugated with the tetrameric SELP, resulting in the formation of a larger macromolecular ligand-SELP complex (~190 kDa). Reversed phase UPLC, SE- UPLC and SDS PAGE analyses were employed to confirm the formation of the macro- ligand. The formed ligand complex was then evaluated for the affinity precipitation of mAbs in pure form and from a complex mixture. The results indicated 100% mAb capture in pure component as well as complete selective capture from a harvest supernatant, with greater than 80% recovery of highly pure mAb. A molar ratio of 16:1 of the ligand-SELP complex to mAb was required to achieve complete capture in from the complex feed. Importantly, no SELP was observed in the mAb eluate, indicating the preservation of the streptavidin-biotin interaction througout the process. Further, we showed that the purification efficiency of the ligand-SELP construct was maintained for multiple rounds of affinity precipitation. This proof-of-concept study demonstrates that SELP can be used in conjunction with biotinylated affinity ligands for mAb purification.

BIOT 321 Investigating effective cleaning agents and sanitization protocols for protein A chromatography through chemistry, process characterization and bacterial challenge studies

Gary Dabbagh, [email protected], Tomonori Shiotani, Yusaku Mizuguchi, Kristi Haskins, Masayoshi Nagaya. Separation Sciences Group, JSR Life Sciences, Durham, North Carolina, United States

To implement a Protein A chromatography resin in downstream processing for therapeutic monoclonal antibody, many factors are considered, such as dynamic binding capacity, impurity clearance, and pressure properties. Chemical stability and resin lifetime are important performance attributes for chromatography resin selection to characterize performance consistency and to understand the process cost over the resin’s lifetime. In this study, the chemical stability of a methacrylic resin, Amsphere A3, was evaluated against various cleaning and sanitizing reagents. Furthermore, resin lifetime was also investigated under various process buffer conditions including sodium hydroxide and peracetic acid.

BIOT 322

Process intensification feasibility studies using novel multimodal anion-exchange membrane chromatography columns

Anna Forsyth1, [email protected], Daniel Henn1, Graham Temples1, Jinxiang Zhou1, Scott M. Husson1,2. (1) Purilogics, LLC, Greenville, South Carolina, United States (2) Chemical Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

During monoclonal antibody (mAb) purification, high molecular weight species are normally removed using one or more additional chromatography steps, such as a hydrophobic interaction chromatography step. Existing multimodal ion-exchange resin- based chromatography columns combine hydrophobic interactions and columbic forces together. However, they often fail to provide satisfying capacity to remove host cell proteins (HCPs), aggregates and virus particles simultaneously, particularly in a two- chromatography-step purification scheme. Here, we present feasibility studies applying Purilogics’ multimodal anion-exchange membrane chromatography columns (PurexaTM- MQ) to remove HCP and aggregates from mAb streams purified by Protein A chromatography. The data indicates that a very high volume of antibody stream could be loaded onto PurexaTM-MQ columns to obtain purified mAbs with low aggregate and HCP levels. In addition, we conducted virus loading challenges with spiked protein and DNA in various buffer conditions. The results suggest more than a 4 log reduction of virus can be achieved. PurexaTM-MQ columns offer promising results to simplify mAb purification and achieve process compression.

BIOT 323 High-throughput non-affinity purification methodology to enable bioreactor characterization and small-scale model qualification for a recombinant enzyme

Tridevi Dahal-Busfield, [email protected], Murray Brian, Brad Whitaker, Jiuyi Lu, Jason Walther. Sanofi, Framingham, Massachusetts, United States

High-throughput, small-scale methodologies are crucial to ensure efficient, rapid and robust development of biological processes. In particular, significant advances have been made in purification process development, allowing parallel experiments to be conducted at the microliter scale, leading to dramatic minimization of resource and material requirements.

This study describes the application of such high-throughput approaches to enable the bioreactor characterization of a recombinant enzyme process. A high-throughput method for rapid and small-scale purification of bioreactor harvest was required to enable cell culture characterization and small-scale model qualification. Because no affinity chromatography solutions exist for the recombinant enzyme molecule, this effort required a novel approach. To this end, we have developed an automated high- throughput, micro-scale, two-step purification method employing resins used in the at- scale purification process that generated purified product suitable for product quality testing, requiring only milliliters of bioreactor harvest. The high-throughput scale-down Tecan method was developed and optimized to resemble the current purification process to ensure comparable product quality measured using glycosylation profiles. Similar methodologies could be applied for future programs also requiring non-affinity purification processes. In future, additional high-throughput automation techniques could be applied to completely automate this two column small-scale purification process.

BIOT 324

Evaluation of viral clearance using a high resolution CEX resin and ceramic apatite resin

William H. Rushton1, [email protected], Xuemei He1, xuemei_he@biorad.com, Irene Chen1, Akunna Iheanacho2, [email protected], Coral Fulton2, Jenifer Dean2, Louisa Vang1, [email protected]. (1) Bio-Rad Laboratories, Hercules, California, United States (2) Research & Development, Texcell - North America, Inc., Frederick, Maryland, United States

As part of the ICH Q5A guidelines, evaluating the removal of potential endogenous or adventitious viruses is done by performing viral clearance studies. These studies spike a known amount of model viruses into individual downstream process steps. A high resolution cation exchange resin and ceramic apatite resin were tested to determine their ability to remove Minute Virus of Mice (MVM) and Xenotropic Murine Leukemia Virus (XMuLV). These viruses are a standard virus panel for an early phase viral clearance studies. The operating conditions were chosen to approximate the typical running conditions used with these chromatography resins. Results for the study provide potential viral clearance values for each media under the evaluated conditions. This can assist with future development work by providing guidance as to the ability of each media to remove viruses.

BIOT 325

Using AI based descriptors to model protein chromatography

Imee Sinha, [email protected], Camille Bilodeau, Xuan Han, Shekhar Garde, Steven M. Cramer. Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States

QSAR techniques are commonly employed for the understanding of protein chromatographic behavior. These techniques utilize knowledge-based descriptors that arise from the protein’s structure and physiochemical properties. Knowledge-based descriptors rely on clever ways of quantifying the protein’s surface properties, like using software aided potential maps, measures of hydrophobicity/hydrophilicity, molecular shapes and partial charges. A new technique of descriptor calculation is the use of powerful neural networks to understand structure-based activity. A variational autoencoder network can be used to learn protein surface properties which is independent of pre-existing knowledge. A variational autoencoder is trained to efficiently reconstruct input data by passing it through an information bottleneck. The latent space of a variational autoencoder encodes information of the input in a compressed format in the form of low dimensional vectors. These vectors can be extracted and used as descriptors which accurately represent the input data. In this work, a variational autoencoder is trained to reconstruct protein data. When the autoencoder is jointly trained with a property prediction network such as retention time predictor network, the latent space of an autoencoder encodes meaningful information in the form of vectors. These latent space vectors can be exploited as meaningful descriptors which can accurately represent protein’s structural and biophysical properties. This work demonstrates the use of data driven descriptors to perform QSAR modelling and presents the opportunity to use artificial intelligence to build descriptors for property prediction.

BIOT 326

Investigation into the pH effects in multimodal chromatography using NIST mAb and its domains

Ronak B. Gudhka1, [email protected], Kabir Dhingra1, John Schiel2, Steven M. Cramer1. (1) Chemical and Biological Engineering, Rensselaer polytechnic institute, Troy, New York, United States (2) National Institute of Standards and Technology, Gaithersburg, Maryland, United States Recent work in our lab has shown the importance of certain domains of a monoclonal antibody (mAb) to be important for binding to different Multimodal (MM) chromatographic systems. Identifying the binding regions on the protein surface is of very high importance for designing efficient bioseparation processes for biologics products. Interaction of certain mAbs was governed by the (Fab)2 domain whereas other mAbs interacted via the FC region of the mAb. In this work, we extend this investigation to study the effect of pH on the binding of mAb to these MM chromatographic systems and evaluate the selectivity trends in these systems. For this study, we used NIST mAb that has been extensively studied by many research labs across the country. First, linear salt gradient chromatography experiments were performed with NIST mAb on various MM cation exchange chromatographic systems. To further investigate the regions on the protein surface that were involved in binding, the NIST mAb was enzymatically digested to get the Fab, (Fab)2 and FC domains. The retention of these domains was evaluated on MM chromatographic resins as a function of pH. There was a shift in the important domain of interaction from pH 5 to 7. While the FC domain was important for binding at pH, the Fab domain appeared to be important for binding at pH 7. By comparing these results with Electrostatic Potential (EP) and Surface Aggregation Propensity (SAP) maps under different conditions, we developed a heuristic understanding of how these shifts were a function of protein surface properties. This work has enabled us to develop a deeper understanding of how different regions on a protein surface interact with the MM chromatographic resins under various conditions and how the pH can be used to modulate the selectivity in these MM systems.

BIOT 327

Platform for purification of VHH-type antibody fragments

Tomas Bjorkman1, [email protected], Eva Heldin2, mats ander3. (1) RnD, GE Healthcare, Uppsala, XX, Sweden (2) GE Helthcare Life Sciences, Uppsala, Sweden

Protein A affinity chromatography resins have for a long time been used to purify monoclonal antibodies (mAbs) by the bioprocess industry at all scales. The binding of protein A mainly takes place in the interphase between CH2 and CH3 in the Fc region of the mAb (constant domains 2 and 3 of the Ab heavy chains However, it is known that protein A also can bind to VH3 sequences located on the variable heavy chain of the Fab region.. Thus, this binding may also be utilized for the purification of target molecules derived from the variable domain of the heavy-chain antibodies of camelids, the so called VHHs.

When developing the high capacity, highly caustic tolerant protein A resin, MabSelect™ PrismA, the VH3 interaction was re-introduced during the maturation of the affinity ligand. Here we will show examples of purification of mAb derived fragments including Fab and VHH, focusing at binding characteristics e.g. binding capacities, elution conditions and purification performance. It will be demonstrated how the VHH can be purified directly from an E. coli harvest with good yield and at high load BIOT 328

Evaluation of multimodal anion exchangers for protein recovery

Sushmita Koley1, [email protected], Mark A. Snyder2, Xuemei He2, Steven M. Cramer1. (1) Rensselaer Polytechnic Institute, Troy, New York, United States (2) Bio-Rad Laboratories, Hercules, California, United States

Multimodal chromatography has increasingly been utilized in several bioprocess based platforms due to its distinctive selectivity. Previously, our lab had reported a series of multimodal anion exchangers (MMAEX) with varying aromatic ring substitutions and its effect on protein selectivity. The nature of hydrophobicity and the ligand geometry of the bulky groups were found to play a major role in driving protein’s selectivity on the adsorbents. Probing further, we wanted to understand the recovery issues that are generally observed to be a concern when dealing with the MMAEX ligands in the industries. In regard to these, two other MMAEX ligands containing guanidine groups were evaluated. The chromatographic experiments consisted of a series of linear salt gradients that were carried out with a set of model protein library which have demonstrated to have recovery issues. The role of ligand density and ligand structure was evaluated for binding of proteins to the adsorbents and the study was compared with the parent ligand structure, Prototype 13 and a commercial adsorbent, Capto Adhere. Interesting results were observed with these MMAEX adsorbents as it was seen that while a single guanidine moiety exerted a hydrophobic type of interaction leading to recovery issues, the other structure with two guanidine moieties did not pose these problems. It was also demonstrated that a linear pH gradient could alleviate the recovery problems so observed. These results indicated that the recovery issues can be circumvented by careful modification of the ligand structure and/or meticulous use of experimental conditions to employ the multi-mode interaction of a multimodal chromatography for improvement in selectivity.

BIOT 329

Continuous ELP-based affinity precipitation for the purification of therapeutics

Manish K. Bhat1, [email protected], Akshat Mullerpatan1, Jie Chen2, Melissa Holstein2, Swarnim Ranjan2, Sanchayita Ghose2, Steven M. Cramer1. (1) Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States (2) Biologics Process Development, Bristol Myers Squibb, Devens, Massachusetts, United States

A continuous affinity precipitation process using elastin-like polypeptides (ELPs) conjugated to the Z domain of protein A was developed to purify mAbs. The setup incorporates binding of the mAb to ELP-Z, precipitation of the complex, capture and washing of the precipitates, and removal of the mAb from the ELP-Z precipitates. This system employs static mixers for efficient mixing during the binding and precipitation steps. Depth filters are used to trap ELPZ-mAb precipitates. The process was optimised for purity, recovery and robustness across multiple mAbs and a control strategy was developed to maintain process efficacy.

BIOT 330

Rethinking the Capto adhere chromatography step

Ann L. Homer1, [email protected], Brian Polilli1, Alexis Henry2. (1) BioTherapeutics Development-API Large, Janssen Pharmaceutical, Malvern, Pennsylvania, United States (2) Technical Development, Biogen, Research Triangle Park, North Carolina, United States

Capto™ Adhere Column Chromatography is the fourth column step in a purification process of a highly glycosylated protein. In early development, the functions and mechanisms of the Capto Adhere anion chromatography was an area of extensive study, capitalizing on the resins ability to separate product variants. This separation came at a cost with low step yields, no viral clearance and low process related impurity clearance. Additional product understanding indicated the product variant was active and did not need to be removed from the process. A major impediment to the process was the lack of simple yet robust techniques to quantify and qualify the target protein. Here, we describe the statistically designed experiments taken to improve the yield and process related impurity removal of the Capto Adhere chromatography step. In addition, the challenges associated with making process changes late in a development cycle for a challenging protein will be addressed.

BIOT 331

Bispecific manufacturing using controlled fab-arm-exhange (cFAE): Overview and implementation of a reduced scale model

Raphael G. Bertrand1, [email protected], Rima Naseer1, Amin Salehi1, Pedro J. Alfonso2. (1) BioTherapeutics Development, Johnson and Johnson, Malvern, Pennsylvania, United States (2) M1-2, Janssen Pharmaceuticals, Malvern, Pennsylvania, United States

Currently under legal review until 30OCT2019.

BIOT 332

Overcoming cell shear in mAb harvests using novel harvesting technology

Masa Nakamura, [email protected], Alexei Voloshin. 3M, St. Paul, Minnesota, United States

Mammalian cell culture continues to be the workhorse for expression of high value recombinant therapeutic protein modalities. As the requirements for the manufacturing process performance, product quality and safety continue to become more stringent, mammalian cell shear during clarification operation has become a critical consideration in preventing product quality degradation and in purification train design. Shearing of the cells during centrifugation and depth filtration exposes the complex therapeutic products to intracellular enzymes, such as the reductases and proteases. Additionally, cell breakage dumps difficult-to-remove proteins, such as lipases, into the process fluid. Cell shear in the primary clarification stage also produces an insoluble fragment “tail” that interferes with membranes, capture chromatography, and polishing train performance. While cell shear can be attenuated by a proper depth filter selection and running the primary clarification stage in a “true chromatography mode”, this approach is not very robust due to the non-chromatographic nature of depth filtration media and variability of cell size depending on cell line and on culturing strategy. Here we present a novel harvesting approach that utilizes chromatographically defined AEX non-woven bed that is capable of capturing cells and cell fragments using charge. Chromatographic approach to clarification enables capture of insoluble particles at very low pressures and is equally effective over very wide range of particle sizes. The result is that cell shear can be very precisely controlled and minimized to background level regardless of cell line, cell density, or cell culture conditions. Furthermore, since the clarification media is chromatographically defined, the system is highly reproducible and scalable from high-throughput screening, to manufacturing scale. This enables one to accelerate process development from discovery to commercial deployment.

BIOT 333

Recombinant proteins derived from IMAC and IEX techniques enable catalytically active palladium nanoparticles

Shadrach Ibinola, [email protected], Imann Mosleh, Hazim Aljewari, Robert Beitle. University of Arkansas, Fayetteville, Arkansas, United States

In place of chemical and physical methods, biologically guided synthesis is used increasingly as a cost-effective medium for the fabrication of nanoparticles. Palladium metal-binding sequence Pd4 (TSNAVHPTLRHL) has been demonstrated to be instrumental in the production of palladium (Pd) nanoparticles due to the histidine amino acids in the sixth and eleventh positions. By eliminating the additional cost of purification of the protein, the crude lysate of E. coli containing Pd specific protein has been proven to be a viable, cost-effective means for the synthesis of Pd nanoparticles. E. coli expression system is employed for mass production of the three copies of the palladium binding sequence fused with green fluorescence tag protein [(Pd4)3-GFP]. This protein is purified with a Fast Protein Liquid Chromatography (FPLC) apparatus coupled with Immobilized Metal Affinity Column (IMAC) and Ion Exchange Column (IEX). The catalytic properties of nanoparticles fabricated from the purified protein and the crude lysate are compared using the Suzuki-Miyaura coupling reaction. Results will be presented on the comparison of the catalytic properties of synthesized nanoparticles from crude lysate extract and purified protein. Also, the comparison of the quantity of Pd specific protein derived from the employment of IMAC and IEX techniques will be provided.

BIOT 334

Robust bioburden and endotoxin reduction using AEX non-woven chromatography

Rebecca Hochstein, [email protected], Rituparna Sengupta, Alexei Voloshin. 3M, St. Paul, Minnesota, United States

As biopharmaceutical candidate pipelines continue to grow, rapid preclinical development is key to quick and efficient identification of promising molecules and preparing them for clinical testing. While the product purity and consistency requirements at the preclinical stage are much more modest compared to those for clinical testing, the requirements for bioburden and endotoxin in the final product are often just as strict. This is due to possible interference with cell-based assays and with immunogenic response during in vivo animal studies. As the product candidates are not prepared under GMP conditions, small residual levels of endotoxin are often observed in the final drug substance at the preclinical stage.

Bioburden is typically reduced using a sterile-grade membrane filter, and endotoxin is handled using a flow-through AEX column. Both of these methods are sensitive to drug substance pool quality, flowrate, and other process variations typically observed in a research lab. Here we demonstrate how bioburden and endotoxin can be reduced using a single use, scalable, and commercially available AEX non-woven technology. This approach is capable of > 6 LRV reduction in bioburden and endotoxin reduction to water for injection (WFI) levels in a very robust manner, while maintaining 95+% recovery of the proteins from a typical formulation solution. We explore the performance of the approach as a function of isoelectric point of the target protein product, flowrate through the device, and other lab processing variations.

We believe that this approach enables rapid, robust, and scalable bioburden and endotoxin cleanup that accelerates the preclinical development of biopharmaceutical drug candidates.

BIOT 335

Process-scale ceramic hydroxyapatite columns: Packing methods, column efficiency evaluation, and post-packing handling

Louisa Vang, [email protected], Irene Chen, Xuemei He. BioRad, Hercules, California, United States

Ceramic Hydroxyapatite (CHT) has been widely used in the production of biopharmaceuticals. This mixed-mode chromatography media has unique capability of resolving biomolecules that are not separable by alternative means, owing to the synergistic interactions from the chelation of the calcium ions and the cationic exchange of the phosphoryl groups in its matrix. Multiple process and product-related impurities, such as aggregates/fragments of drug molecules, host cell proteins and nucleic acids, endotoxins, and contaminant viruses, can be effectively removed in a single chromatography operation. The rigid spherical macroporous particles of CHT offer high permeability for biomolecules and a linear pressure-flow relationship irrespective of column diameter, which allows easy production scale-up with the robustness and consistency demanded by bioprocessing. We have investigated some critical attributes in packing process-scale columns. CHT media can be packed by axial movement of column piston or constant flow of mobile phase, and no significant difference in final column quality is observed in the speed range of 100 ~ 400 cm per hour. On the basis of our results, we recommend testing column efficiency at a mobile phase linear velocity the same as column packing speed. Movement of column after packing may lead to initial minor change of plate count and/or asymmetry. However, subsequent movement does not seem to cause additional column bed settling or performance change. Practical tips for packing CHT columns will be provided in the present study.

BIOT 336

Improving the productivity of a purification process using a novel ‘in line’ product concentrator

Ziyan Deng, [email protected], Aled Charles, Richard Turner. AstraZeneca , Cambridge, United Kingdom

During the early phases of development, purification of relatively large volumes of media with low levels of recombinant protein is often required. This process is typically inefficient, because pumping large volumes through small chromatography columns is time-consuming. We have evaluated a single pass tangential flow filtration (SPTFF) disposable device that enables on line concentration of feed streams 3 – 9 fold prior to purification. This can have a significant time saving benefit to the first chromatography step and potentially an improvement in overall process productivity. This technique can be applied to all modalities such as IgGs, Fabs, DNA and viral vectors with minimal development effort once membrane compatibility has been established.

BIOT 337

Optimization of final filtration operations using single-use systems stephanie ferrante, [email protected], Andrew Koch, [email protected], Charles Raye. MilliporeSigma, Burlington, Massachusetts, United States Final sterilizing filtration is the last operation in downstream processing to assure sterility of medicinal products for human use. There is extensive regulatory guidance for final filtration encompassing both expectations for microbial retention, as well as approaches for confirming filter integrity. As single-use systems are increasingly being adopted in final filtration, there is a focus on implementing a more holistic approach to assuring sterility of the system that encompasses both filters and components.

This poster will highlight new developments in filter technology that reduce the risk of introducing contamination in pre-sterilized redundant filtration systems. They also offer opportunities for: 1. Simplified pre-use integrity testing of critical sterile filters 2. Maximized product recovery of formulated product 3. Streamlined single-use assembly design with fewer connections

These developments in assembly design reduce risk, improve operation and increase robustness of this critical filtration step. In an industry focused on driving efficiency, they offer an important advancement in process risk mitigation and yield improvements.

BIOT 338

Investigations of weak mixed-mode cation exchange chromatography for control of aggregates in bispecific antibodies

Lily Motabar1, [email protected], Matthew Aspelund1, Sender Aspelund1, Kevin Galipeau2, Alan Hunter1. (1) AstraZeneca, Gaithersburg, Maryland, United States (2) Thermo Fisher Scientific, Bedford, Massachusetts, United States

Bispecific antibodies are a class of engineered antibodies with the ability to bind to two distinct antigens. Compared to monoclonal antibodies, single chain variable fragment- IgG bispecific antibodies are prone to high levels of aggregate (10-30%), which can impact product yields and must be removed during downstream polishing steps. Mixed mode chromatography (MMC) provides a potential method of aggregate control due to its multiple modes of protein-ligand interactions, resulting in enhanced selectivity compared to traditional ion exchange and hydrophobic interaction chromatography resins. Ceramic hydroxyapatite (CHT) is an MMC resin often used as a polishing step for many high aggregate forming species; however, its scalability issues raise the need for an alternative aggregate removal step. Initial studies showed that weak mixed-mode cation exchange (CEX) resins clear aggregates with high selectivity and minimal breakthrough while operating in flow-through mode. To further investigate potential alternatives to CHT, we evaluated prototype POROS™ mixed-mode CEX resins from Thermo Fisher Scientific to study the impact of ligand density, operating pH and arginine concentration on aggregate removal ability.

BIOT 339 Chromatographic behavior of bivalent bispecific antibodies on hydrophobic interaction chromatography columns

Lucas Kimerer2, [email protected], Timothy Pabst1, Alan Hunter1, Giorgio Carta2. (1) AstraZeneca, Gaithersburg, Maryland, United States (2) University of Virginia Che, Charlottesville, Virginia, United States

The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible. Analogous behavior was observed for HIC resins with different functional ligands, matrix structures, and particle sizes. Residence time, operating temperature, and hold time were shown, to affect the elution behavior. While three peaks were obtained at short residence times and room temperature, residence times longer than about 27 min or at 45 °C resulted in a single merged peak indicating that the underlying mechanism occurs on time scales comparable to that of chromatographic separation. Holding the protein on the resins prior to elution enriched the late eluting peak indicating that multiple binding states formed on the chromatographic surface are responsible for this behavior. Tryptophan autofluorescence measurements show that stronger binding forms have increased solvent exposure indicating that surface-catalyzed conformational changes play a role. A model was developed to describe the interplay of chromatographic separation and slow conformational changes.

BIOT 340

Development of a reduced-scale model to assess sensitivities and de-risk the scale-up of continuous disc-stack centrifugation

Caroline Ahrens1, [email protected], Nicholas Levy1, Terry Benner1, Maverick Bioh1, Christopher Nieder2, John Taylor2, Mark A. Teeters1. (1) API Large Molecules, Janssen, Malvern, Pennsylvania, United States (2) MilliporeSigma, Burlington, Massachusetts, United States

Within biopharmaceutical processing, a disc-stack centrifuge followed by depth filtration is widely used in primary clarification to remove cells and cell debris prior to chromatography. The high cell densities now routine in upstream CHO cell processes have increased the risk of low yields or poor product quality (i.e. clipping or reduction) during lab, pilot and commercial harvest. Many existing reduced-scale models of disc- stack centrifuges incorporate both a shear device and benchtop centrifugation. Optimal levels of reduced-scale shear have been extensively modeled to develop processes mimicking the high levels of mechanical shear in centrifuge feed zones, the resulting cell breakage, the creation of submicron particles, and the challenges to downstream filtration. However, centrifugation process development continues to be routinely conducted at the pilot scale. Published reduced scale models often require specific equipment or expertise and do not readily generate material appropriate for subsequent depth filter sizing. Here, we introduce a simple, easy, reduced-scale shear model that is based on recirculation through a peristaltic pump. We critically evaluate the utility and consistency of metrics to characterize lab, pilot and commercial clarification including ViCELL imaging, LDH levels, turbidity, particle sizing, and downstream depth and sterile filter sizing. We use these metrics to compare our reduced-scale model to large-scale processes and recommend a decision framework to de-risk clarification during the lifetime of lab to commercial transfers.

BIOT 341

Exploring the viral clearance robustness of an AIEX membrane chromatography step for a bispecific antibody

Roger Alsop2, [email protected], Sherri Dolan2, Shi Zhuo3, Gangui Li3, Amanda Mak1. (1) SystImmune, Inc, Redmond, Washington, United States (2) Sartorius Stedim North America, Williston, South Carolina, United States (3) Baili Pharmaceuticals Co., Ltd, Chengdu, Sichuan, China

SystImmune, with Sartorius-Stedim support, has developed a robust flow through chromatography step for contaminate removal as part of SystImmune purification platform for a bispecific antibody molecule This platforms uses Anion exchange chromatography (AIEX) which is a commonly used in downstream purification processes for the manufacture of therapeutic proteins and (in this study) a bispecific antibody AIEX, when run in flow through mode, is known to capture a variety of contaminates (i.e. host cell proteins (HCP), DNA and virus) while allowing the protein of interest to flow thru. The chromatography mediated virus capture is critical for many process as it contributes to the overall viral clearance of the process which is mandated by regulatory authorities The platform utilizes Sartobind Q membrane which is a commercially available membrane based stung anion exchanger. The Sartorius membrane format offers advantages over conventional resin based chromatography: 1) a single-use format reduces validation costs, 2) up to 95% reduction in buffer consumption and 3) high flow rates results in fast process times. The original process development work was performed by SystImmune at their Redmond Washington facility and the completed process was transferred to a 300 liter Bioreactor scale at their parent organization Baili Pharmaceuticals Co., located in Chengdu, Sichuan. The viral clearance results for the confirmation run will be presented and discussed.

BIOT 342 Effect of freeze-thaw, protein concentration, and pH on antibody aggregation in bioprocess protein A chromatography

Jane Liao, [email protected], Adele Pearson, Robert Luo. Downstream Process Development, GlaxoSmithKline, Philadelphia, Pennsylvania, United States

Therapeutic proteins such as monoclonal antibodies are commonly subjected to freeze- thawing for analytical testing, shipping, and storage. Freeze-thawing stress can potentially expose protein molecules to the ice-liquid interface and induce degradation through protein aggregation. We studied the effects of freeze-thawing on aggregation of an IgG2 monoclonal antibody, specifically during Protein A purification. Several solution conditions including pH and protein concentration were found to significantly impact aggregation of the antibody during freeze-thawing. Based on these findings, the proposed mitigation strategy aims to minimize freeze-thaw related aggregation during small-scale process development, analytical testing, and manufacturing. In addition, the process parameters of the Protein A chromatography step were modified to further reduce aggregate levels.

BIOT 343

Two-stage high-throughput process development (HTPD) identifying conditions for tandem process

Tim Zhou, [email protected], Abhijeet Shirke, Dharmesh Kanani, Zhaoqing Zhang, Lu Wang. Teva Pharmaceuticals, USA, West Chester, Pennsylvania, United States

Continuous and integrated downstream processes have been the focus for manufacturing process development for biologics to reduce cost and operation complexity. In this work, we present a two-stage HTPD approach to identify and predict optimal conditions for polishing chromatography processes design, especially flowthrough mode CEX, AEX and HIC. Customized resin plates are prepared by slurry dispensing. The plate screening is performed to compare the performance of multiple chromatography resins under variant process conditions (pH, conductivity) in terms of yield and aggregate clearance. The optimized conditions of each individual resin are selected and further verified using robocolumns, where yield vs. load, as well as monomer vs. HMW breakthrough curves are plotted for frontal analysis. The study results obtained from the resin plate screening and robocolumns have good agreements; furthermore, based on the experimental data, a prediction model is established to identify the potential conditions for a tandem purification process in flowthrough mode, where two chromatography columns are operated under the same pH and conductivity. By connecting two polishing columns tandemly and operated as one unit step, much processing time and cost can be saved for better efficiency during the mAb manufacturing.

BIOT 344 Small scale modeling of disk stack centrifugation with mammalian cell culture capillary shear for a more predictive harvest process transfer

Michael R. McGarrah, [email protected], Steve Timmick, Brian Crump, Antonio R. Ubiera. Downstream Process Development, GlaxoSmithKline, Philadelphia, Pennsylvania, United States

Advances in cell culture technologies and methods in recent years have resulted in increasing solids densities at harvest. This results in a higher number of cell culture harvest processes requiring a centrifugation step at pilot and commercial scale. The traditional scaling approach used for disk stack centrifugation is to use Q/∑ theory to transfer equivalent centrifugal separation conditions between different centrifuges. However, several data sets have been generated showing that this model can be too ideal to be used in practice. Some limitations to Q/∑ theory include that it assumes unbreakable solid spheres are being separated and there is perfectly efficient separation. In practice, disk stack centrifuges vary in efficiency by design and are affected by quality of feed material. Also, for shear sensitive mammalian cell types in particular, when intact cells enter a disk stack centrifuge they are broken down by shear forces into many smaller particles. In an attempt to generate a more realistic model of expected disk stack centrifugation performance, a scale down model using capillary shear constructed of common lab parts was performed. Presented here are the results of the capillary model experiments which successfully demonstrated that sheared broth material had turbidity and particles size distribution profiles more representative of process streams generated by disk stack centrifugation compared to traditional batch centrifuged broth. Additionally, observations on the difference in shear response for PER.C6 and Chinese Hamster Ovary (CHO) mammalian cells will be discussed.

BIOT 345

Scale-up design and implementation of continuous downstream processing of viral-based products using aqueous two-phase systems

Dylan Turpeinen, [email protected], Pratik Umesh Joshi, Caryn Heldt. Chemical Engineering, Michigan Technological University, Houghton, Michigan, United States

The biomanufacturing industry desires to implement unit operations in continuous mode to simultaneously reduce overall costs and increase productivity. To benefit from the switch to continuous operation, the current downstream processing train must be updated with novel purification strategies that are high throughput, high yielding, and easily implemented. One such method is a chromatography-free separation technique using aqueous two-phase systems (ATPS). The ATPS allows for the partitioning of viral particles to a different phase than contaminant proteins directly from the harvest stage. Our previous work using a batch-mode PEG-citrate system has shown >80% recovery of porcine parvovirus (PPV) and human immunodeficiency virus-like particles (HIV VLP) with minimal contaminating proteins in the PEG-rich phase. The scaled-up continuous operation of our ATPS was achieved using an in-line helical mixer with a settler to allow for proper phase contact and separation time. Our lab has developed a bench-top scale, continuous ATPS process to purify PPV and HIV VLP from their respective crude supernatant without any loss in the recovery or purity as compared to the batch system. ATPS is a viable method for continuous vaccine manufacturing and can increase the throughput of viral-based biotherapeutics.

BIOT 346

Process development methodology to assess a new drug substance container: Freezing and thawing small scale model considerations and effect of CO2 ingress during shipping

Yanhong Feng1, [email protected], Siddharth Parimal2, Andre C. Dumetz3. (1) Biopharm, GlaxoSmithkline Pharmaceuticals Corp, Collegeville, Pennsylvania, United States (2) BPDS, GlaxoSmithKline, King of Prussia, Pennsylvania, United States (3) 709 Swedeland Rd UE0551, Glaxo Smithkline, King of Prussia, Pennsylvania, United States

Besides the required stability studies, a change in drug substance container needs a re- assessment of the freezing and thawing steps, as well as the possible impact of the shipping configuration on the product quality. This poster focuses on the process development studies necessary to de-risk a change in drug substance container. First, a small-scale model using a Blast Freezer to evaluate the effect of controlled freezing and thawing rates on product quality is presented. The experimental approach is illustrated for a monoclonal antibody. Second, the effect on the solution pH of possible CO2 ingress during shipping on dry ice is investigated to determine if a gas impermeable barrier such as a mylar bag is necessary. The results show that even though the presence of CO2 can create a pH shift, it does not occur during shipping if container closure integrity is maintained. These results illustrate a process development framework where appropriate studies performed to focus on the freezing/shipping conditions, and to guide the necessary QbD risk assessments in order to build a robust control strategy.

BIOT 347

Use of modeling to support the development of a control strategy and process characterization

Kathryn Meintel1, [email protected], Laura Wehmeyer2, Grace Tran1, Frank Yin2, George Atkins3, Christopher Pink2, Robert Gerber4, Andre C. Dumetz1. (1) BPDS, GlaxoSmithKline, King of Prussia, Pennsylvania, United States (2) PDS, GlaxoSmithKline, Collegeville, Pennsylvania, United States (3) CMC Statistics, GlaxoSmithKline, Collegeville, Pennsylvania, United States (4) PSC, GlaxoSmithKline, King of Prussia, Pennsylvania, United States Mechanistic models are available for some unit operations, however they are not yet widely used to support biopharmaceutical development. In this case study, modeling was used to support process characterization and the development of a control strategy. As a part of late phase development, following main scoping experiments, process robustness under established conditions was evaluated. Factorial Design of Experiments (DoE) was used to examine extremes of parameters on potential critical quality attributes (pCQAs). Additionally, a mechanistic model was developed for a specific part of the process. Using process knowledge and historical batch data, a process attribute risk assessment was conducted to identify potential critical process parameters (pCPPs) to investigate during process characterization mixed design DoEs. Following process characterization, both a mechanistic model and statistical polynomial model were used to define a design space in which product quality met its expected targets when the identified CPPs were varied. Based on the results of the process characterization studies, proven acceptable ranges (PARs) for critical process parameters were demonstrated to be robust within the studied design space. This case study shows how mechanistic modeling can be used complementarily to more traditional statistical modeling approaches and bring a deeper level of process understanding.

BIOT 348

Clearance of host cell protein at elevated conductivity through synthetic depth filtration media during downstream processing

Camilla Oxley2, [email protected], Vitali Stanevich2, [email protected], Mark McInnes1, Chris Nieder1, [email protected]. (1) MilliporeSigma, Burlington, Massachusetts, United States (2) Janssen, Malvern, Pennsylvania, United States

Host cell proteins (HCP) are a process-related impurity common to biological therapies that have the potential to elicit an immune response in patients and, because of this, regulatory bodies require <100ppm HCP in the drug substance. Downstream purification steps and, in particular chromatography resins, are heavily leaned on to accomplish the necessary clearance. HCP can be a complex process-related impurity in that there can be subtypes with differences in chemistry that can present a challenge to clearance using process-platform chromatography resins. Purification processes are at a point where depth filtration is commonly used for haze reduction and/or impurity clearance with product, buffer conditions commonly at a neutral pH and a conductivity of <10mS/cm. This work screened several media types e.g. cellulose diatomaceous earth, activated carbon, and synthetic media at a pH of 6.0 with varying conductivity to maximize HCP clearance and product yield. Millistak+® HC X0SP, a synthetic media depth filter, at elevated conductivity, was identified as the optimum depth filter.

BIOT 349 Process development strategies for the direct filtration of high cell density monoclonal antibody harvests

Hong Zhang, [email protected], Stijn H. Koshari, Robert Luo. Downstream Process Development, GlaxoSmithKline, King of Prussia, Pennsylvania, United States

The purpose of the harvest step in a monoclonal antibody downstream purification process is to remove biomass, particulates, and other suspended material from the cell culture broth to generate a product stream suitable for purification by liquid chromatography. Removing biomass and other particulates eases the burden on the capture column and extends its lifetime. A traditional harvest process employs a two- step approach using centrifugation combined with depth filtration. In this process, coarse solids and intact cells are first removed using a centrifuge, and the resulting centrate is further clarified by depth and membrane filtration in series. Direct filtration has the potential of greatly simplifying the overall harvest process by directly loading the harvest material on high-capacity depth filters and consequently removing the need for additional preparative steps such as centrifugation or flocculation. In this poster, we highlight process development strategies for direct filtration harvests that can increase the filter loading capacity, particularly with regard to high cell density feed streams (> 40 million cells/mL). Filter loading and facility fits were informed by metrics such as the depth filter volumetric capacity for solids and filtrate turbidity. Different types of depth filters, a wide range of fluxes and depth filter ratios, as well as different types of sterile membrane filters were explored to ensure a robust operation and scalability. The strategies discussed here can aid in transforming a trial-and-error development approach to one that is more methodological and predictive. The strategy for tech transfer including facility fit will also be discussed.

BIOT 350

Exploration of cycling and sanitization on lattice supported Protein A chromatography

Kristi Haskins, [email protected], Yusaku Mizuguchi, Tomonori Shiotani, Gary Dabbagh, Masayoshi Nagaya, Jason Chiu. Bioprocess, JSR Life Sciences, Durham, North Carolina, United States

When developing a biomanufacturing process, understanding the lifetime of a resin is important for estimating the costs associated with the chromatographic purification portion of the process and provides an understanding for how robust the purification process is. A 300 cycle purification process study which contained 0.1M sodium hydroxide cleaning in place and sanitization with peracetic acid every 100 cycles was investigated using harvested cell culture fluid (HCCF) as load material. Using the same load material, a 50 cycle study was conducted on Chromassette®, a lattice supported modular chromatography platform, packed with the same Protein A chromatography resin. After the abbreviated life cycle test on Chromassette, the sporicidal impact of PAA on B. subtilis was tested on both a column and Chromassette. Results show comparability of lifetime and cleanability between Protein A resin packed in a traditional column format and in Chromassette.

BIOT 351

Novel membrane process for highly concentrated peptides without heating and high pressure

Yuji Katayama, [email protected], Daisuke Hotta, Fumiyoshi Inoue, Mitsuru Fujita, Masato Mikawa, Noboru Kubota. Performance Materials Technology Center, Asahi Kasei Corporation, Fuji-city, Shizuoka, Japan

Concentration of pharmaceuticals is an important process toward the production of pharmaceutically active components. Conventionally, reverse osmosis (RO) membrane or distillation systems have been used for the concentration. However it is still challenging to concentrate the solution to the high concentration without deteriorating the quality. Operation of RO membrane systems need larger pump pressure than the osmotic pressure difference between the peptide solutions and permeated water. Therefore there is a concentration limit in RO membrane systems and it causes the severe membrane fouling. In addition, high pressure in the solutions also gives the negative effects on some peptide molecules. On the other hand, the distillation process requires heating, which can decompose pharmaceutically active components. Toward the process for highly concentrated peptides without heating and the high pressure, we have developed the forward osmosis (FO) membrane systems. The FO membrane systems can concentrate aqueous solutions only by taking advantage of the osmotic pressure difference between the feed solution (FS) which contain peptides and the draw solution (DS) which has higher osmotic pressure than FS. The water permeates from FS to DS with the osmotic pressure difference as the driving force, which results in the concentration of the peptide in the FS. As FO membrane system only require the osmotic pressure difference between the FS and the DS, they concentrate the peptide solution with mild conditions, which can avoid the high pump pressure and the heat, having milder fouling on the membranes. We have demonstrated our FO membrane systems can concentrate peptides and sugars to high concentration under mild operation conditions. We will also discuss the effect of concentration of various peptides with the FO membrane systems.

BIOT 352

Development of protein A membrane chromatography devices for rapid cycling

Chanis Bowse1, Gerado Cedrone1, Sean Foley1, Igor Gonzalez1, Lloyd Gottlieb1, Brad Kachuik2, Gregory Molica1, Amro Ragheb2, Rushd Khalaf1, Hua Chen2, Elena Komkova2, Graham Morse2, Kevin Rautio1, Xiaojiao Shang2, Navneet Sidhu2, Gary Skarja2, Matthew Stone1, [email protected], Paul Turiano1, [email protected], Joaquin Umana1, Melissa Vandersluis2. (1) Chromatography R&D, MilliporeSigma, Bedford, Massachusetts, United States (2) Chromatography R&D, MilliporeSigma, Burlington, Ontario, Canada

Absorptive membrane chromatography offers many advantages over traditional chromatography resins in the downstream purification of therapeutic proteins. A chromatography membrane device can be operated at much higher flow rates enabling the length of a bind/elute chromatography step to be reduced from hours to minutes. Rapid cycling of a membrane device allows the same amount of protein to be processed with a significantly reduced volume of membrane in a given time period. In addition, rapid cycling enables single-use per batch operation, which eliminates sanitization and storage requirements as the full lifetime of a membrane device is exhausted within one batch. However, membrane chromatography for the downstream purification of proteins has been largely limited to flow-through steps due to the low binding capacities of traditional filtration membranes and the large device hold-up volumes that decrease the efficiency of a bind/elute step. Herein we report the development of Protein A (PrA) membrane devices optimized to facilitate the efficient chromatographic capture of a monoclonal antibodies from clarified cell culture via a rapid cycling process. Efficient bind/elute chromatography is possible due to the novel properties of the PrA membrane and the optimized design of the devices. The PrA membranes are constructed from a hydrogel that provides significantly higher binding capacities than are possible with traditional filtration membranes. The devices were specifically designed in a flat sheet configuration to minimize the hold-up volume and facilitate efficient bind/elute chromatography processes. All the membrane device sizes utilize a flat sheet format to facilitate scaling of a bind/elute chromatography process.

BIOT 353

Multivariate low pH treatment process characterization studies for virus inactivation using an automated mixing platform

Hiren D. Ardeshna1, [email protected], Kusum Solanki2, [email protected], Lee Bink2, Gerald J. Terfloth3, Antonio R. Ubiera4. (1) Biopharm Process Development, GlaxoSmithKline Plc., King of Prussia, Pennsylvania, United States (2) UE0551, GlaxoSmithKline, King of Prussia, Pennsylvania, United States (3) UE551, GSK, King of Prussia, Pennsylvania, United States (4) UE0551, Glaxo Smith Kline, King of Prussia, Pennsylvania, United States

The Low-pH treatment for virus inactivation unit operation can exhibit a potential risk for in-process product aggregation during downstream processing for a monoclonal antibody. In this work, a full-factorial experimental design was executed using an automated and well-characterized mixing system to investigate the effect of four process parameters: low pH end point, low pH hold time, low pH titration duration, and neutralization titration duration. The instrument used is a commercially-available automated small-scale workstation, which includes a reactor, dosing units, pH and temperature probes. It enables a well-controlled and automated batch titration as compared to traditional manual pH adjustments carried out in a vessel, significantly streamlining the execution and ensuring the reproducibility of process characterization studies. The automation allows comprehensive data capture that greatly improves our ability to gain process understanding, including data analysis. Factors such as temperature, agitation, pH electrode calibration, which can cause drift in pH measurement were identified and considered for method optimization. As a result, low end pH point could be controlled within ± 0.05 of target pH, low pH hold time could be accurately defined, low pH titration duration and neutralization titration duration could also be well controlled within ± 10 % of target value. A small-scale model was qualified which can represent clinical trial and commercial manufacturing scale in terms of product quality attributes. The process characterization experimental design consisted of a two level four factor design with three center points providing the desired power to investigate any possible parameter interactions. Statistical analysis of these experiments shows that the two factors – low pH end point and low pH hold time, as well as the interaction of these factors, had a significant effect on key product quality attributes. The remaining two factors – low pH titration duration and neutralization titration duration had no significant effect on any of the product or process attributes.

BIOT 354

Comparison of protein A resins and novel protein A matrices

Talaial B. Alina1, [email protected], Paul R. Randolph2, [email protected], John Schreffler2. (1) Chemical and Biological Engineering, Drexel University, Philadelphia, Pennsylvania, United States (2) Active Pharmaceutical Ingredient - Large Molecule, Janssen Pharmaceuticals, Malvern, Pennsylvania, United States

The use of higher productivity protein A capture matrices for purification of monoclonal antibodies is becoming essential due to higher productivity bioreactor technologies. Six different Protein A resins and 3 novel protein A matrices were studied to characterize Dynamic Binding Capacity (DBC), sodium hydroxide (NaOH), resin and matrix lifetime. Additionally, batch loading, intensified batch, and intensified batch with dynamic flow loading productivities were modeled to compare costs and productivities of these intensified processes to the theoretical costs and productivities of the novel matrices. These data demonstrate novel protein A matrix productivity gains, although substantial in comparison to the current state of batch chromatography, are not as large when compared to existing protein A resins using intensified processes.

BIOT 355

Lab to manufacturing: Column packing robustness of methacrylic protein A resin

Yusaku Mizuguchi1, [email protected], Tomonori Shiotani1, Jason Chiu1, Masayoshi Nagaya1, Kaori Itaya2, Ryo Doi2, Gerald Platteau3. (1) JSR Lifesciences, LLC, Durham, North Carolina, United States (2) JSR Corporation, Tsukuba, Japan (3) JSR Micro NV, Leuven, Belgium Amsphere A3 Protein A chromatography resin is designed for advanced protein separation in downstream processing of therapeutics antibodies. The resin is characterized by high binding capacity, has an overall improved process robustness, flow characteristics, impurity removal, productivity and resin lifetime. In this study, packing of Amsphere A3 was investigated from lab- to manufacturing-scale, up to 60 cm inner diameter column. The results show that Amsphere A3 provides robustness in packing with consistent integrity and pressure properties when using various packing parameters. Furthermore, suitable unpacking protocol was successfully established for column hardware, where manual reslurry using a paddle is not possible. With this unpacking protocol, packed bed can be dispersed in an automated manner without damaging resin beads even after repeated operations.

BIOT 356

Bioprocess development: Using single-use technology to enable manufacturing scale shakedown lots

Devan Reilly, [email protected], Christopher Simons, [email protected], Wanli (Justin) Ma, Holly Ma, Adam Kristopeit. Merck & Co., Inc., West Point, Pennsylvania, United States

Single-use technologies were used to establish and demonstrate a 2000L live virus vaccine (LVV) candidate manufacturing process in an unfinished space with minimal utilities and short preparation time. The commercial process for the LVV candidate is projected to require a 2000L production reactor followed by a purification train containing filtration and chromatography steps. In the commercial production setting, this process will utilize automated large-scale chromatography skids and will require disposal of ~3000L of waste. Prior to design and construction of the commercial facility, there was a need to perform shakedown lots in order to demonstrate the process at manufacturing scale, obtain data to inform Final Manufacturing Process (FMP) development, and provide information for the commercial facility design. Through the use of single-use technologies (SUT), the aggressive timeline for the shakedown lots was able to be met.

BIOT 357

Generalized purification of viruses and virus-like particles using aqueous two- phase system

Pratik Umesh Joshi, [email protected], Dylan Turpeinen, Caryn Heldt. Chemical Engineering, Michigan Technological University, Houghton, Michigan, United States

The challenge to develop a high throughput platform method for virus-based therapeutics has been stimulating the biomanufacturing community for decades. The potential of alternate technologies that can meet the current requirements might be better than the conventional methods for larger and more complex yet fragile moieties. One of those candidates is an aqueous two-phase system (ATPS), formed by two partially miscible aqueous solutions. ATPS are known for being inexpensive, mild, environmentally-friendly, and can be easily scaled-up and processed in a continuous mode. However, the challenge of not fully understanding the partitioning mechanism had resulted in a large experimental setup to determine optimal compositions specific to different viruses. Our DOE using a tie-line framework and study of virus surface characteristics provides a method to discern the interaction between virus and phase compositions. The current experimental design in a polyethylene glycol (PEG)-citrate system at physiological pH with the porcine parvovirus (PPV, a non-enveloped virus) facilitated in achieving a high yield of 80% in the PEG-rich phase. The resulting preferential partitioning supported the hypothesis of major influence of hydrophobic and electrostatic (salting-out) interactions between virus and relative phases. The framework was then utilized to create a database for five more viruses having different characteristics of presence of envelope, pI, and size. This multivariate data is being employed to empirically predict and reduce experimental space. This work demonstrates the purification efficacy of herpes simplex virus (HSV-1), pseudorabies virus (SuHV-1), human rhinovirus (HRV-14), human immunodeficiency virus-like particle (HIV-gag VLP), MS2 bacteriophage and ways to generalize optimization of target virus in ATPS. The ATPS approach will not only help generalize virus purification but also provide a high throughput technology in virus-based therapeutics DSP.

BIOT 358

Development of a rAAV clarification platform for a viral gene therapy process

Albert S. Reger, [email protected]. MilliporeSigma, Burlington, Massachusetts, United States

Current trends in the biotechnology realm show the rapid growth of the use of viral vectors for gene therapy as one of the next generation of life changing medicines. rAAV (recombinant Adeno-associated virus) has become a popular viral vector that is able to deliver targeted genetic information to a patient and is considered non-pathogenic and safe for these patients to be treated with. Due to the life changing nature of these therapies to the patient population, the need for rapid and robust purification and manufacturing methods is a must. A key step in the purification and manufacturing of rAAV viral vectors is the clarification step. A typical rAAV manufacturing process will lyse transfected cells, releasing the rAAV viral vectors from the cell, producing a wide particle range of cellular debris that must be removed prior to moving forward in the purification process. During the early stages of developing a clarification method for a rAAV manufacturing process, it is crucial to select the correct depth filtration media that will maximize rAAV yield and provide reproducible quality filtrate. The work to be presented, focuses on the development of a clarification step by testing combinations of MilliporeSigma’s Millistak+® HC D0HC and Millistak+® HC Pro D0SP as primary depth filters and Millistak+® HC C0HC and Millistak+® HC Pro C0SP as secondary depth filters with a lysed cell culture feed containing rAAV9. A comparison of pressure profiles, volumetric throughput, turbidity of filtrate, and product yield was used to determine the optimal combination of primary and secondary depth filters. All filtration trains tested provided high quality filtrate which had high capacity on Express SHC® sterile grade filter, though total yield of product (>80%) suggests Millistak+® HC D0HC and Millistak+® HC Pro D0SP as good choices for a primary depth filter followed by Millistak+® HC C0HC as the secondary depth filter as the best option for the feed tested.

BIOT 359

Case study in raw material change control: Evaluating an alternative virus filtration post use pre-integrity testing flush solution for a monoclonal antibody manufacturing process

Morgan Ayres1, [email protected], Melani Stone1, Scott A. Tobler2. (1) Biologic Process Development and Commercialization, Merck, Harleysville, Pennsylvania, United States (2) Biologics Manufacturing Science and Commercialization, Merck and Co., Inc., Hoboken, New Jersey, United States

Evaluating the impact of vendor initiated raw material changes are a key activity for ensuring a biopharmaceutical manufacturing process maintains a state of control in response to these changes. The evaluation of an alternative virus filtration post-use flush solution (0.25 M NaOH, 1% SDS) for a monoclonal antibody (mAb) manufacturing process is used as a case study to describe this process. Triton X-100 is currently used in this manufacturing process to flush the filters after product filtration to remove any residual proteins and other foulants, and prepare the filters for a post-use filter integrity test. After 2021, triton X-100 will no longer be permitted for use in the EU due to environmental impact and an alternative post-use flush solution will have to be used. To supplement filter vendor studies supporting the use of an alternative flush solution, small-scale virus filtration runs were executed and a product specific assessment of the alternative flush solution was performed. The results of the integrity test after product filtration were compared and enhanced the understanding of the impact of the post-use flush on the integrity test outcome and demonstrated the acceptability of the alternative post-use flush solution for a range of virus filtration operating conditions. Evaluation of the post-use flush conditions at the small-scale supported the manufacturing change control and will ensure continued supply in response to raw material changes.

BIOT 360

Extension of a mechanistic model for hydrophobic interaction chromatography to account for pH changes and mixed-mode binding

Tobias Hahn1, [email protected], Theresa Laeufer1, Gang Wang2, Maria Casals Peralvarez1, Juergen Hubbuch2. (1) GoSilico GmbH, Karlsruhe, Germany (2) KIT, Karlsruhe, Germany Hydrophobic interaction chromatography (HIC) is one of the common separation modes for the purification of biomolecules. It is especially powerful as polishing step for monoclonal antibodies to remove aggregates and impurities such as host cell proteins. The separation principle is based on the reversible interaction between hydrophobic patches on the protein surface and the hydrophobic ligands of the stationary phase. Advances in fundamental understanding of HIC have been achieved by mechanistic studies and modeling work based on the Mollerup’s thermodynamic framework (2008). Mechanistic modeling provided a deeper insight into adsorption mechanism and offers time saving in process development. Strategies for model-based process development of salt- and pH-dependent IEX and mixed mode chromatography were reported by Nfor et al. (2010), and Lee et al. (2015). Modeling of pH-dependence is a necessity for practical model-based development of industrial HIC processes, as these commonly involve an alteration of the binding strength by changing the pH. In this work, we present how we extended our previously developed HIC model (Wang et al., 2016) to account for changes in the pH. Bind/elute experiments were performed by applying linear salt gradients at constant pH. The adsorption behavior of three pure proteins (lysozyme, hemoglobin, IgG) on TOYOSCREEN PPG-600M (Tosoh), was investigated for pH values near the pI and could be model successfully by introducing just a single additional model parameter. Model validation was conducted using combined linear pH and salt gradients. Furthermore, we combined our HIC model with the Steric Mass Action (SMA) isotherm to construct a mixed-mode model. A case study with the model protein glucose oxidase investigates identifiability of adsorption isotherm parameters on Capto adhere (GE Healthcare) and compared the models’ ability to match the observed peak shapes. Compared to the model by Nfor et al. which required a complex column and pore model, the newly constructed model provided equally good results with a much simpler column model and, thus, less parameters. In summary, we could successfully extend our previously developed HIC model to account for pH-changes and mixed-mode binding. The model proved to be superior to existing isotherm models and has already been used for industrial process development in service projects.

BIOT 361

Isoporous block copolymer membrane platform for downstream virus filtration of recombinant proteins

Chris Crock, [email protected], Spencer W. Robbins, [email protected], Jack Wilson, Jiawei Zhu, Rachel M. Dorin, Marty Siwak. TeraPore Technologies, Inc., South San Francisco, California, United States

Traditional filter technologies and materials are constrained by a tradeoff between selectivity and permeability. In critically important and challenging separations such as downstream virus filtration, this tradeoff translates to sacrificing either patient safety or filtration efficiency. By carefully engineering and exploiting the self-assembly behavior of block copolymers, a new type of virus filter platform has been developed to allow high liquid permeability in conjunction with virus elimination. The self-assembly of block copolymers allows them to be processed into filters with very uniform pore sizes and high pore densities, enabling very high selectivity along with high liquid permeability. In conjunction with a prefilter, these isoporous membranes are part of a powerful new technology platform for biopharmaceutical downstream virus filtration.

BIOT 362

Bench-scale evaluations of ProteinA Fibro, a single-use affinity capture platform of the next-generation manufacturing process of antibody therapeutics

Ryan Zolyomi1, Ian Scanlon4, Florence Rusly1, Kierston Shill1, Anna Graanberg2, Oliver Hardick4, Ashley Hesslein3, Hendri Tjandra1, [email protected]. (1) Biologics Development, Bayer Healthcare, Berkeley, California, United States (2) R&D, GE Healthcare, Uppsala, Sweden (3) Biologics Development, Bayer HealthCare, Berkeley, California, United States (4) GE Healthcare | Puridify, Stevenage Bioscience Catalyst, SG1 2FX, United Kingdom

With an increasing number of antibody therapeutics in development and higher cell culture titers from intensified upstream processes, manufacturing batch sizes are smaller and more diverse. As a result, single-use multi-product manufacturing facilities provide flexibility necessary to meet the changing demands. Implementing a fully single- use manufacturing platform can be challenging as bead-based ProteinA resin chromatography affinity has anchored industrial production of antibody therapeutics. Similar to ProteinA resin chromatography, a single-use alternative must consistently deliver products of high quality with low impurity content across a wide variety of antibody therapeutics. These driving forces motivated us to evaluate PrtoeinA- derivatized Fibro, a single-use chromatography technology, to advance our manufacturing platform.

Bench scale purification studies performed across a range of monoclonal antibodies demonstrate simple adaptation of the current resin ProteinA process to the ProteinA Fibro chromatography. Current process steps, buffers, and chromatography skids are directly transferable. ProteinA Fibro chromatography, however, enables rapid purification of monoclonal antibodies by offering faster mass transfer, shorter cycle times, and longer utilization within a single batch. Higher binding capacity and longer lifetimes are critical to take advantage of the productivity benefits. At bench scale, we find these two metrics are consistent across various antibodies evaluated without comprising product quality or impurity clearance. There are some nuances when developing the bench-scale Fibro chromatography due to inherent faster flow rates, higher operating pressures, broader eluate peaks, and system hold-up volumes. Characterization of these differences across multiple antibodies led to small modifications in process parameters and methods to develop a platform FibroA chromatography process at bench-scale. While experience is limited to one antibody, we experienced a similar need to make small modifications of our process as we up- scaled to pilot scale. We expect the scale-up of a Protein A Fibro chromatography process to be slightly more challenging than that of a resin process, but it is feasible as a platform process since performance is predictable.

BIOT 363

Direct observation of virus removal in PlanovaTM filter with confocal microscope

Miku Ayano, [email protected], Takumi Sato, Daisuke Nakane, Takayuki Nishizaka. Gakushuin University, Toshima-ku, Tokyo, Japan

Virus filtration has been a critical step in downstream process for the safer production of biologics products that are absolutely essential for the medical industry. Although PlanovaTM filters are widely used in this virus removal process, they are composed of the filter membrane having a complex structure, and the mechanism how viruses are trapped in membrane has not understood in detail. Therefore, we developed a new experimental system for direct visualizing fluorescently labelled biomolecules under an optical microscope equipped with a confocal optical system, and infused virus-like- particles (VLP) and protein h-IgG to the filter with a constant flow speed. As a result, we succeeded in directly visualizing at the single filer level that VLP were stably accumulated at a retention layer of the membrane and protein h-IgG passed through it.

BIOT 364

Split intein technology: Universal purification template for non-mAb proteins

Oliver Rammo, [email protected], Tatjana Best, [email protected], Vanessa Kohl. MilliporeSigma, Darmstadt, Germany

Currently, the purification of “non-standardized” or non-mAb proteins remains challenging and is associated with spending a significant effort in developing a downstream scheme for each new molecule individually. With the split intein technology we will bring a change to the market while introducing a generic downstream template to simplify this purification procedure. Due to the cleaving ability of inteins, the N- terminal intein is immobilized as the affinity ligand on the chromatographic support whereas the C-terminal intein is fused to the target as a tag. The high affinity between both inteins enables the tagged target to be purified from the lysate. The intein- mediated C-terminal cleavage reaction triggered by a change in buffer conditions, causes tagless release of the target into the eluate. The tag can be regenerated from the column and does not need to be further removed from the target such it is often the case using conventional tag purification schemes e.g. His-tag. In numerous case studies, we will present on the integrity of the C-terminal intein tag in upstream and discuss its impact to various microbial and mammalian expressed molecules (e.g. expression titer, solubility and biological activity). Further we will focus on the efficiency of the split intein downstream scheme in different process modes leading to a purity >90% of the tagless eluted target from crude feed stocks. BIOT 365

Withdrawn

BIOT 366

Characterization of ultrafiltration and diafiltration influenced by the Gibbs-Donnan equilibrium

William Cressman, [email protected], Rajiv Rao. Janssen Pharmaceutical, Malvern, Pennsylvania, United States

The demand for higher therapeutic doses paired with high-concentration drug product administration methods have driven the development of large molecule therapeutics towards increasing drug substance concentrations. Drug substance concentrations can be controlled through tangential-flow filtration (TFF), while simultaneously exchanging the active pharmaceutical ingredient (protein) into an appropriate formulation matrix. Traditionally, the TFF process uses a diafiltration buffer that is similar in excipient concentration to that of the drug substance matrix. However, increased protein concentrations may lead to association or repulsion of charged and exclusion of uncharged excipients from the diafiltration buffer, resulting in an uneven distribution of buffer excipients across the surface of the TFF membrane as supported by the Gibbs- Donnan equilibrium. The resulting drug substance has excipient concentrations that differ from the diafiltration buffer used. Changes in excipient concentration may lead to changes in pH of the final drug substance. As a component of process control strategy for a process influenced by the Gibbs- Donnan equilibrium, pH and excipient concentration of the diafiltration buffer must be offset to achieve drug substance release criteria. Manipulations to the TFF process also mitigate excipient concentration changes caused by the Gibbs-Donnan equilibrium and provide greater control over the drug substance. Studies were performed to assess various TFF process inputs, including diafiltration product concentration, diafiltration buffer excipient concentration, and diafiltration buffer pH. Data generated from the studies was used build predictive models incorporating the Gibbs-Donnan equilibrium.

BIOT 367

Accelerated tech transfer of a vaccine: Building the site while building the process

Lakshmi Madhavan, [email protected], Francis Digennaro, Alec Peterson, Nick Samuelson, Ryan Marek. Merck, West Point, Pennsylvania, United States

Rapid commercialization of vaccines plays a key role in ensuring patient access. As market demand grows, process development and commercialization groups are tasked with transferring high-quality, reliable, and productive bioprocesses on compressed timelines. A case study describing the tech transfer of the purification process for a multi-valent vaccine is presented. This endeavor required many shakedown batches to prepare for a process performance qualification (PPQ) campaign. In parallel with late- stage process development, a brand-new facility was also being built from the ground up. Several issues needed to be solved to commission the facility while still learning about the set of manufacturing processes to be commercialized there. Challenges were encountered around balancing platform capabilities with the unique needs of each drug substance component, as well as around single-use assembly limitations and unexpected scale-up issues. Some mitigating actions included deep dives into equipment design, parallel bench-scale studies, and at-scale water runs. Thanks to collaboration across many different functions, several solutions were implemented towards the goal of qualifying a highly complex set of purification processes.

BIOT 368

Withdrawn

BIOT 369

Mitigation of undetected protein aggregates affecting nanofiltration

Jason O. Moore, [email protected], Andrew W. Nields, Mamadou I. Bah, Marcia R. Kary, Susan M. Liu. Discovery, Product Development & Supply, Janssen Pharmaceuticals, Glenside, Pennsylvania, United States

Virus filtration by nanofilters is a common step during the purification of therapeutic monoclonal antibodies (mAbs). Due to the tight pore structure designed for removal of small viruses, this process is particularly sensitive to the presence of product aggregates. This study focuses on a mAb downstream process where product intermediates were found to severely plug virus filters despite having less than 1% aggregate as determined by size exclusion HPLC. Conditions of increased conductivity and pH or decreased temperature resulted in higher aggregation levels to the point where, under relatively moderate conditions, visible precipitates formed. Although these precipitates appeared to resolubilize when warmed or dialyzed against low salt conditions, product flow through a nanofilter remained a challenge, suggesting the presence of sub-visible particles. This was observed even under conditions where the protein was known not to precipitate and following additional purification steps using mixed modal anion exchange chromatography. In the study, three different techniques were applied to detect and quantify aggregates: size exclusion HPLC, dynamic light scattering, and microflow imaging. Microflow imaging was the only method determined to consistently detect sub-visible aggregates in conditions associated with plugging of virus filters. Ultimately conditions suitable for virus filtration were determined by only using buffers that minimized formation of these sub-visible particles.

BIOT 370

Progress towards improving chromosomal integration of large constructs in E. coli

Philip O'Dell, [email protected], Nicholas Kaplan, John A. Jones. Chemical, Paper, and Biomedical Engineering, Miami University, Oxford, Ohio, United States

Metabolic engineering has demonstrated the potential to produce high-value specialty chemicals with greater efficiency than traditional chemical synthesis methods. Plasmid- based expression systems enable rapid strain construction and offer good bench-scale production, but when bench-scale technology is transferred to the industrial scale, the requirement of antibiotic selection becomes prohibitively expensive and the reduced stability of plasmid-based systems is problematic for extended production phases during fed-batch fermentations. The scarless genomic integration of an optimized pathway can lead to greater industrial viability by addressing both of the aforementioned issues. Here, we present our progress towards the development of a system which can enable the scarless integration of large constructs onto the E. coli chromosome. We will present a novel method of lambda-Red mediated scarless integration which utilizes Cas9 counterselection and I-SceI mediated in vivo linearization of the donor vector. Four locations on the BL21*(DE3) chromosome were targeted for investigation into the relative expression levels of integrated pathways. We will present proof of principle data utilizing a fluorescent protein, mCherry, as well as a three-step biosynthetic pathway for a high-value small molecule.

BIOT 371

Affinity membrane for rapid, high-recovery concentration and purification of lentivirus vectors

Daniel Henn1, [email protected], Hui Ding2, Yanzhang Wei2, Jinxiang Zhou1, Scott M. Husson1,3. (1) Purilogics, LLC, Greenville, South Carolina, United States (2) Department of Biological Sciences, Clemson University, Clemson, South Carolina, United States (3) Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

Gene and cell therapies have the potential to dramatically expand the treatment possibilities for patients with conditions that have no other cures. Viral vectors, and particularly lentiviral vectors (LVs), are among the fastest growing types of gene- delivery vehicles. However, scalable production of LVs with high purity and high yield is a major challenge due to the fragility of LVs and a lack of efficient purification tools. Robust affinity chromatography products are desired to simplify the purification process and increase recovery. Here, we present a new affinity chromatographic membrane technology for the rapid purification of LVs with high binding capacity at short residence time and high recovery using mild elution conditions.

The LVs were grown from 293FT cells, and the culture supernatant with a virus titer of approximately 10^6 virus particles per mL was loaded directly onto Purilogics scaled- down syringe filter-like affinity membrane column prototypes. The affinity interaction is based on reversible binding of the LV envelope protein. LVs were eluted under mild conditions to enable high recovery of active virus. Activity was assessed by transduction efficiency of green fluorescent protein (GFP) into HEK293 cells. LV purity was determined by SDS-PAGE and ELISA assays. Additionally, we will show a side-by-side performance comparison to ion-exchange membranes in terms of binding capacity, yield, and purity.

BIOT 372

Batch, continuous or intensified batch processing: That is the question?

Sabrina Koch, [email protected], Mark Hicks, Patrick Gilbert, Hans J. Johansson. Purolite, Llantrisant, United Kingdom

Column chromatography is one of the most widely-used techniques to capture and purify monoclonal antibodies (mAbs). It continues to play a major role in the purification of mAbs, from conventional batch processing to multi-column continuous processing.

One of the main reasons for the move to intensified batch processing and continuous chromatography is the need to reduce the cost of goods (COG’s) and improve productivity. Several contributory factors either directly or indirectly affect the COG’s, from resin cost/g purified mAb, facility foot print, to buffer consumption.

In this study, we compare the latest generation protein A resin, Praesto Jetted A50, based on a uniform agarose bead against market leading batch emulsified protein A resins. The results show that significant increases in productivity and reduction of COG’s can be achieved by using purposed designed jetted resins for capture chromatography. Whether used in batch or continuous format.

BIOT 373

Development and deployment of bioreactor scale-up modeling software in Janssen BioTherapeutics Development

Christopher Canova, [email protected], Jeffrey Cohen, Kevin Clark, Eugene J. Schaefer. Janssen Pharmaceuticals, Springfield, Pennsylvania, United States

A combination of higher cellular growth processes, shortened timelines, and an ever- increasing array of biomolecules and manufacturing sites necessitated a need for increased internal bioreactor modeling capabilities in Janssen Biotherapeutics Development. In response, a first-principals based bioreactor model was created. The model probes the operating space and sensitivities for a unique facility and project combination. Data that is readily available at the laboratory scale is input into the model to make its operational predictions, which can then be transferred to the manufacturing site prior to the batch manufacture. This model was then packaged as a standalone application using Matlab and deployed for departmental use on the JnJ Appstore; This process allows for controlled deployment of model improvements and ensures that all scientists are working with the same set of assumptions and equations. A training curriculum was developed to increase departmental knowledge of bioreactor modeling and drive accountability for bioreactor scale-up to the project teams. Modeling the operational space can help to identify equipment limitations prior to manufacture and may allow for right-the-first-time processes with no need for engineering trial runs.

BIOT 374

Bioethanol production in an integrated fermentation-pervaporation system

Anjali Jain2, Ajay K. Dalai1, Satyendra Chaurasia2, [email protected]. (1) Department of Chemical and Biological Engineering,, Prof. of Chemical Engineering and Canada Research Chair in Bio-Energy and Environmentally Friendly Chemical Processing, Department of Chemical and Biological Engineering, College of Engineering, University of Saskatchewan, Saskatoon, Saskatchewan, Canada (2) Chemical Engineering, Malaviya National Institute of Technology Jaipur India, Jaipur, Rajasthan, India

Biofuels as ethanol derived from biomass through fermentation is becoming more significant as the world is facing atmospheric pollution and climate changes due to the use of petroleum fuels. Although conversion of sugar to bioethanol using yeasts does not need any water as reactant but water is required in plenty due to limit on ethanol tolerance of the yeasts. In general, 4 -8 l of waste water is generated per litre of bioethanol production. The concentration of ethanol in the fermentation broth can range from 1 to 15 wt% depending upon the source of biomass and hydrolysis process. The water content of ethanol must be reduced to less than 1.3 wt% water to produce fuel grade ethanol. Distillation is the traditional technology for the recovery of ethanol which needs a high amount of energy. As an alternative, pervaporation is claimed to be a promising purification technology for the separation of ethanol from water/ ethanol mixture and fermentation broth. Combining fermentation with pervaporation in addition to water and energy savings, will also reduce product inhibition by keeping the ethanol concentration in the broth low and simplify downstream processing as the ethanol recovered will be more concentrated. The downstream recovery of ethanol from fermentation broth costs about 30- 40% of total process cost. The MBR systems, in addition to water and energy savings, can continuously remove ethanol from the fermentation process thereby accelerating fermentation and increasing throughput. In this study, the fermentation performance of an integrated batch fermentation and pervaporation process in a polydimethylsiloxane membrane bioreactor (PVMBR) was investigated. An average of ethanol concentration in the permeate was 12.5 vol% in 8 hours. The total flux and the ethanol flux through the pervaporation unit were 257.4 gm−2 h−1 and 36.9 gm−2 h−1 , respectively. Selectivity and enrichment factor of the PDMS membrane were found to be 2.74 and 2.5, respectively. The presence of yeast and fermentation media does not have any negative impact on ethanol separation factor in PDMS PVMBR. However, they have shown the negative effect on ethanol and water flux both. MBR resulted in improved productivity and yield, as compared to the conventional fermentation system. As compared to batch fermentation, ethanol yield and specific productivity increased by 7 and 60% respectively in case of fermentation coupled with PVMBR.

BIOT 375

DOZN™2.0: Quantitative green chemistry evaluator

Ettigounder Ponnusamy, [email protected]. Corporate Responsibility, MilliporeSigma, St. Louis, Missouri, United States

MilliporeSigma created a unique web-based greener alternative scoring matrix, also known as DOZN™2.0 - A Quantitative Green Chemistry Evaluator based on the 12 principles of green chemistry. The 12 principles of green chemistry provide a framework for learning about green chemistry and designing or improving materials, products, processes and systems. DOZN™2.0 scores products based on metrics for each principle and aggregates the principle scores to derive a final aggregate score. The system calculates scores based on manufacturing inputs, Globally Harmonized System (GHS) and Safety Data Sheet (SDS) information which provide a green score for each substance and is flexible enough to encompass a diverse portfolio of products ranging from chemistry to biology based products. The DOZN™2.0 system has also been verified and validated by a third party to ensure best practices are applied. This new Greener Chemistry initiative offer customers’ an increased breadth of Greener Alternative products with confirmatory documentations to validate greener characteristics. Through MilliporeSigma’s DOZN™2.0 platform, customers can now calculate the green scores for their own processes and products. This free, web-based tool provides users with even more data so that they have more information to increase their sustainability.

BIOT 376

Structural analysis of large-scale production and purification of recombinant protein and humanized antibody

Yushu S. Ting, [email protected]. Laureate Pharma, Princeton, New Jersey, United States Recombinant proteins and therapeutic humanized antibodies are widely used in a whole range of disease fields, such as autoimmunity, cancer, inflammation and infectious diseases.

In the past, purification of protein and antibody requires specific affinity chromatographic steps. For example, large scale Protein A affinity chromatography is widely used to purify Fc specific antibody such as IgG1, IgG2(code: 5U66). Besides, other immobilized antibody column and immobilized antigen column have been used to separate recombinant protein (rIFN-α) and humanized anti-TAC monoclonal antibody. However, the antigen/antibody interaction of this affinity column for protein /antibody purification was not well understood in 1990’s.

In this presentation, protein structure analysis of rIFN-α protein/antibody binding epitope and IL-2 receptor protein /antibody binding interface are studied by x-ray crystallography using the RCSB protein database and Swiss PDF viewer Molecular Model software. 1). Study of structure analysis on interferon-alpha protein binding specificity to neutralizing monoclonal antibody (code:3UX9) was determined by the Molecular Model approach. The molecular level analysis provides structural information on the rIFN-α binding epitope, AB (24-43 AA) and Helix E (140-150 AA), against immunoglobulin Fab site. It provides the structural rationale why the target rIFN-α protein can be specifically removed from cell culture mixtures with this immobilized antibody column. 2).Also, structure analysis of antigen (IL-2 receptor) binding epitope with anti-TAC antibody IgG Fab beta barrel binding site (code: 3NFP) was determined using protein x- ray crystallography and molecular model software. The molecular level analysis provides structural rationale why the anti-TAC antibody can be isolated by its specific binding to the immobilized antigen. This resulting in elution to recover the antibody.

In summary, protein structure analysis using protein x-ray crystallography with molecular model software can be used as alternative way to study this immuno-affinity chromatography column binding mechanism for purification of recombinant protein and humanized antibody.

BIOT 377

Application of quality by design tools to guide upstream process characterization studies for a monoclonal antibody program

Balrina M. Gupta, [email protected], Linda Hoshan, Michael Nelson, Sri Madabhushi, John S. Bowers, Hong Li. Merck, Kenilworth, New Jersey, United States

Quality by design workflow and tools provide a systematic and consistent approach that helps with commercialization of products under accelerated timelines. QbD principles guide the teams to access the risks associated with product quality attributes and develop an effective control strategy for risk mitigation and management. QbD starts at a very early stage in the life cycle of a program. For the monoclonal antibody program being discussed a preliminary product quality attribute (pPQA) list was generated soon after discovery. Prior to the start of commercial process development, a commercial quality target product profile (QTPP) was updated, and a product quality attribute risk assessment performed based on its impact on safety and efficacy. Before initiating upstream process characterization (PC) studies, a parameter level risk assessment of the impact on PQA’s and process performance was done. The high and medium risk parameters from each upstream unit operation were studied as part of PC. The experimental studies were conducted in ambr 250TM which was qualified as a scale down model. The study designs and results from the executed experiments will be shown. The output of these studies will be used to develop the upstream process control strategy where the process parameters will be classified along with their acceptable ranges

BIOT 378

Mechanistic identification of CEX elution conditions for an antibody aggregate separation using Kp screening

Tobias Hahn1, [email protected], David Saleh2, Nora Geng1, Simon Kluters2, Juergen Hubbuch3. (1) GoSilico GmbH, Karlsruhe, Germany (2) Late Stage Downstream Process Development, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Baden-Württemberg, Germany (3) KIT, Karlsruhe, Germany

In 2008, Kelley et al. presented an approach to identify feasible separation conditions by measuring a partition coefficient (Kp) in the linear portion of the isotherm using high- throughput screening. The refined Kp screening approach of Genentech published by McDonald et al. in 2016 clearly showed that a smart use of automated batch binding experiments can speed up process development significantly. Using Kp screening results and empirical correlations, McDonald et al. were able to predict product yield and purity, as well as pool volume of step elutions at high load conditions. Still, the approach was not adopted industry-wide, as many experiments would be needed to create correlations for other mAb libraries. In addition, the mechanistic reasons for the approach are neglected, so that the proof of correctness cannot be provided. In this study, a rigorous mechanistic approach was applied to analyze Kp data of an industrial monoclonal antibody and a high molecular weight species. In order to obtain adsorption isotherm parameters for column modeling, a modified method for fitting batch adsorption data to mechanistic model equations is applied that relies only on the applied and measured supernatant concentrations. Apart from binding kinetics, all isotherm parameters could be identified from the Kp data which allows to simulate column runs and to obtain retention times under various conditions. For validation and to obtain the remaining kinetic parameter, three gradient elution runs in 15 mL columns were performed. All tools of model-based process development can be applied to the obtained column model, including the identification of optimal step elution conditions as originally conducted by McDonald et al. To demonstrate this, an in silico scale-up is performed that accurately describes the result of a production run in 12k fermenter scale. BIOT 379

Unexpected high levels of HHL formation observed during a tech transfer to a 2000-L single-use bioreactor: Case study

Chentian Zhang, [email protected], Eric Hodgman. Manufacturing Science and Technology, Bristol-Myers Squibb, Devens, Massachusetts, United States

An unexpected high-level of impurity formation (heavy-heavy-light chain (HHL)) was observed in a legacy monoclonal antibody cell culture fed-batch process when transferred from a 5000-L stainless steel facility to a 2000-L single-use bioreactor (SUB). Root cause analysis identified that the formation likely occurred during the production bioreactor or harvest steps. To our surprise, the lab studies mimicking the reducing environment of a single-use harvest operation did not induce HHL formation for a normal batch. Further investigation using a 5-L scale-down model of the production step identified that the root cause of HHL formation was actually the byproduct of changing sparger types and the use of a microsparger in the single-use bioreactor (Figure 1). Additional scale-down model work and material hold studies identified that the HHL formation occurs extracellularly. Overall, the evidence suggests that the microsparger itself is not inducing HHL formation; rather its presence set off a cascade of events that ultimately led to excess free thiols in the culture broth present at the end of the run. Specifically, the presence of microsparging leads to poor growth, resulting in overfeeding the culture and unconsumed free-thiol containing components that induce HHL formation. Using this knowledge we were able to replicate HHL formation using orthogonal methods and ultimately close the process characterization gap. This talk will overview the tech transfer considerations used to design the process in the initial 2000- L SUB and supporting characterization studies to characterize HHL formation. Further, this talk will also review tech transfer considerations to a secondary 2000-L SUB facility with a drilled-hold sparger that did not produce any elevated levels of HHL.

HHL formation correlated with presence of microsparging

BIOT 380

Establishment of high throughput tools for antibody crystallization process development

Lauren Rockwell1, [email protected], Emily DiMartini2, Michael Rauscher1, Sunitha Kandula1, Jennifer Pollard1. (1) Merck & Co., Summit, New Jersey, United States (2) Biomedical Engineering, Rutgers University, New Brunswick, New Jersey, United States

Historically, protein crystallization has been used for structure determination using x-ray diffraction. In addition to this traditional application, crystallization can be used in the downstream processing of monoclonal antibodies and other biological molecules. Typical therapeutic protein downstream processes can have high raw material costs, high buffer consumption, and long cycle times. Process intermediates and the drug substance are also typically held as low concentration solutions which can lead to protein instability and the need for large drug substance freezer storage space. Due to these challenges, opportunities exist for protein crystallization to be a lower cost alternative for product concentration, isolation, and impurity removal in drug substance manufacturing. Opportunities also exist within drug delivery as crystallization can be used to enable highly stable, high concentration formulations for subcutaneous delivery or to make crystalline protein suspensions with altered solubility and release properties. Due to the large size and complexity of monoclonal antibodies, crystallization conditions are often difficult to identify. Broad vapor diffusion screens used for structure determination are needed to test thousands of crystallization conditions. Conditions identified can be difficult to translate to a scalable, batch mode reaction and a wide range of process parameters need to be screened during process development. To enable rapid antibody crystallization process development at Merck, a high throughput scale down model has been established through the use of a Tecan liquid handler. This work will describe the high throughput method development, current high throughput screening results, and comparison to scale up for three monoclonal antibodies. The workflows being established for successful translation of vapor diffusion conditions to a scalable batch mode process will also be discussed.

BIOT 381

Overcoming challenges in cell culture media powder hydration with single-use mixers

Adam Sokolnicki, [email protected], Mike Furbush. MilliporeSigma, Burlington, Massachusetts, United States

Branched-chain hydrophobic amino acids support the expression of recombinant mAbs by engineered mammalian cells grown in culture, either through incorporation into the mAb or to support its synthesis. Typically present in concentrations at or below 1 g/L, advanced expression systems demand greater bcaa supplementation. Dry formulations for made-in-house medias present a challenge because these powders are notoriously difficult to wet and require specialized mixing equipment. Adapting such a process to a single-use mixer (SUM) is the focus of this presentation. Commercial single-use mixers rely primarily on a vortex, for impeller mixers, or Venturi-effect powder induction, for recirculation mixers, to hydrate powders. With bottom-mounted, offset agitators, it has been shown that vortex depth x power input-per-volume is an appropriate scaling factor for floating powder mixing at up to 3000 L. Fluid flow patterns and vortex depth are affected by liquid height inside the vessel, due to secondary flow off the vessel wall. Suboptimal vortex generation may occur at aspect ratios below 1:1 (liquid height : vessel diameter), and powder wetting is therefore unattainable. Increasing the impeller size and the agitator motor power, to boost fluid flow and therefore surface turbulence and vortex, is a successful mitigation strategy to bring wetting and dissolution into a reasonable timeframe. This strategy can ensure consistent results upon scale up.

BIOT 382

Leveraging PAT to deepen understanding of filtration-based harvest processes

Peter Liu1, [email protected], Patricia Rose1, Tyler Gable2, John Welsh1, Jennifer Pollard1. (1) BioProcess Development, Merck & Co., Inc., Kenilworth, New Jersey, United States (2) Bio and Reaction Engineering, Mettler-Toledo AutoChem, Inc., Columbia, Maryland, United States

Advances in upstream production of biologics – namely cell densities higher than 30E+6 cells/mL in fed-batch – have severely strained filtration-based harvest processes. Depth filter capacity is limited by the sheer number of solid particles to remove and protein stability is increasingly threatened by high levels of cellular impurities, especially in low viability batches. Unfortunately, due to the complex nature of depth filter media, there is little mechanistic understanding of particle removal, impurity adsorption, or cell shear. As a result, harvest development predominately relies on small-scale screening to simply rank different depth filter products by capacity or turbidity reduction. Process analytical technologies (PAT) can fill this gap and provide deeper process understanding, despite mechanistic complexities. In collaboration with Mettler-Toledo, we evaluated several PAT instruments to study the harvest process, from both a particle characterization and protein stability perspective. Particle size and count can be tracked with light scattering techniques like focused beam reflectance measurement (FBRM). This data can provide insights into both the filter and filtrate, characterizing process behavior like cake formation and particle breakthrough. The reactive environment of the batch, heightened by enzymes and other cellular impurities, can be monitored with oxidation reduction potential (ORP) probes. Even if protein damage takes time to manifest, ORP may be an early indicator of eventual product quality concerns. These PAT methods, among others that we tested, can afford scientists the opportunity to adjust process parameters in real-time and prevent failures at all scales. Once added to the harvest toolbox, PAT may provide the means to overcome high cell density depth filtration challenges.

BIOT 383

Accommodating and acclimating risk in a virtual biopharmaceutical company model

Patrick M. Hossler, [email protected], Jill Myers, Yune Kunes, Shawn Russell, Gary Hao, George Avgerinos. Biologics CMC, TG Therapeutics, Waltham, Massachusetts, United States

TG Therapeutics is a virtual biopharmaceutical company with multiple small molecule, and recombinant protein biologics in both early and late stage clinical trials. Like most small biopharmaceutical companies, balancing time, cost, personnel, and available resources to best enable process design, technology transfer, GMP manufacturing, and analytical testing is continuously challenging. This talk will highlight how TG Therapeutics accommodates and acclimates risk into process design, GMP manufacturing, process characterization, process validation, analytical testing, and addressing gaps with highlighted examples for a phase 3 monoclonal antibody. Included as part of this conversation will be specific examples of leveraging the resources we have to enable successful process development, technology transfer and scale-up to 15,000 L scale, remediating high level investigations, employing data management, as well as managing a global network of CDMO's and analytical testing laboratories. The approach and examples provided have enabled the company to persevere against significant challenges and begin to make the transition from a clinical to commercial organization. The lessons learned and perspectives of a small, virtual biopharmaceutical organization, and culture of efficiency gained as a result, will be discussed.

BIOT 384

Efficient clarification using a depth filter simplified the purification process of a therapeutic protein from the Escherichia coli system

Mark Geng, [email protected]. Bristol-Myers Squibb, Cambridge, Massachusetts, United States

Continuous centrifugation has been widely used by the biopharmaceutical industry to remove host cell protein (HCP) precipitates in the biological downstream purification processes. Although a continuous centrifugation followed by depth filtrations can achieve effective clarification at stainless steel bioprocess facilities, a single-use continuous centrifuge is not feasible at large scale. Therefore, centrifuge-free clarification using depth filters is of great interest to bioprocessing facilities that are undergoing a single-use transformation. However, depth filters’ general susceptibility to fouling presents a major challenge to maintaining a satisfactory throughput. This presentation presents an alternative clarification strategy using a single-stage depth filtration to simplify traditional clarification processes that use centrifugation and two- stage depth filters. Multiple filters were evaluated in terms of the product quality, impurity removal, and pressure. The results showed the use of a Clarisolve® 20MS media grade filter had no impact to the yield and product quality (turbidity, monomer purity and removal of HCP and a process associated impurity protein of interest). The new filter had a minimum throughput of 130 L/m2 at 20 PSI pressure limit, which provided enough capacity with less depth filter footprint. The clarification efficiency was supported by characterization of the precipitates’ particle size distribution. The process robustness was demonstrated by varying load quality and process parameters. The intensified clarification strategy using a single-stage depth filter to remove HCP precipitates greatly simplifies the process with high productivity and process robustness.

BIOT 385

Intensification of downstream purification process: Case study

Jungmin Oh, [email protected]. Avantor, Bridgewater, New Jersey, United States

Downstream purification of recombinant proteins encounters the common issue of balancing the separation of product related impurities and the number of processing steps required to achieve the desired final product purity and quality attributes. Because of the high cost of manufacturing recombinant proteins, it is crucial to develop a purification process that obtains acceptable yields while mitigating the risks of process failures and minimizing the number of process steps. Conventionally, downstream purification relies on sequential clearance of impurities using multiple chromatographic processing steps to obtain the final product. In this session, we will present a case study involving downstream processing of recombinant proteins utilizing two multimodal chromatographic processing steps, along with select additives. This process led to easier processing and removal of impurities during centrifugation and filtration and achieved recombinant protein purities greater than 95% with greater than 60% overall yield. Comparatively, traditional approaches using ion exchangers require four steps to achieve similar purity while reducing yields to approximately 40%. We also demonstrate the ability of these resins to successfully purify recombinant proteins at a manufacturing scale while maintaining product purity, quality and yield.

BIOT 386

Intensification of buffer preparation using concentrates and a pump-based dilution system

Matthew Turiano, [email protected], Adam Sokolnicki. MilliporeSigma, Burlington, Massachusetts, United States

This presentation will discuss the use of buffer concentrates and pump-based dilution to intensify buffer preparation operations. Although next generation manufacturing facilities are smaller, buffer demand remains proportional to drug product mass. The higher throughput of these facilities still requires large buffer preparation operations. However, the intensification of in-house buffer preparation to reduce tank capacity and cleanroom storage footprint as well as labor costs, may be achieved through the application of buffer concentrates plus a pump-based dilution system. Furthermore, buffer concentrates eliminate weighing of powders, environmental dust, and keep processing closed, attributes beneficial for next gen facilities. To facilitate implementing such a system, several aspects were demonstrated. On the concentrates side, the development and validation of the concentrates included generating stability data on concentrates stored in single-use assemblies, prior to dilution. Achieving pH and conductivity target specifications required accurate titration of the concentrates. On the dilution system side, pump-based dilutions provided automated buffer makeup to within 0.1 pH unit and 1 mS/cm, at greater than 20x. The overall buffer preparation time for a full suite of mAb buffers at different scales, not including QC release testing, will be compared.

BIOT 387

Leveraging process understanding for downstream process development of an antibody-based therapeutic Balakumar Thangaraj, [email protected], Jun Zhang, Ashish Sharma. Process Development, Amgen, Boston, Massachusetts, United States

As part of an orthogonal viral clearance strategy for protein therapeutics derived from animal cell lines, manufacturing processes has added virus filter (VF). These VF provide size-based exclusion of viral particles. During process development of an antibody- based therapeutic, poor hydraulic performance was observed at the VF step. The leading hypothesis focused on one of the impurities in the drug substance process leading to filter fouling. The process impurity was isolated for HD-X mass spec analysis and was determined to be partially unfolded and aggregation prone. Experiments were conducted with a new polishing resin to reduce this impurity level and assess the impact on VF permeability. The results confirmed that removal of the impurity significantly increased VF permeability. Introduction of the new resin allowed for the reduction of a number of downstream unit operations in comparison with existing process. In addition, better product quality, improved potency, increased productivity and lower COGM were observed. Results from this work will be presented and discussed with a perspective on biomanufacturing challenges for non-mAb modalities.

BIOT 388

Modeling the predictive robustness of pH titrations in downstream manufacturing processes of biopharmaceuticals

Siddharth Parimal, [email protected], Andre C. Dumetz. BPDS, GlaxoSmithKline, King of Prussia, Pennsylvania, United States pH is one of most important process parameters in protein purification processes. pH modulates biophysical interactions between target protein, impurities, buffer components, chromatography ligands, membranes, etc. throughout the series of unit operations that comprise the downstream process. The appropriate choice of pH is also often critical for in-process product stability. As such, control of pH is central to the development of robust downstream manufacturing processes of biopharmaceuticals.

In this work, theoretical models are developed to predict pH titration curves during pH adjustments in downstream processing. Contributions to buffering capacity from both buffer components and protein residues are incorporated in the model, and results are compared to experimental trends. For protein residues, the impact of utilizing sequence- based versus structure-based pKa values is also evaluated. The application of this modeling approach is demonstrated for two steps – a low pH virus inactivation step (of a mAb platform process) and a post-UFDF dilution step (before further processing to final formulation). In both cases, application of the model facilitates the identification of failure modes that can manifest because of unexpected pH variations in large scale processing. Finally, based on process knowledge gained from the models, process scale-up recommendations are made to enable and ensure robust technology transfer of unit operations where pH titrations are performed at large scale.

The approach presented here emphasizes how process development can be achieved within the quality-by-design (QbD) paradigm by the application of in silico tools during early- and late-phase process development, allowing increased process understanding and an early evaluation of process robustness to be subsequently complemented by experimental studies.

BIOT 389

Enabling acceleration: Case study in leveraging platform data sets to establish a platform raw material control strategy

Erin O. Wilson1, [email protected], Lee Bink2, Robert W. Deitcher1, Diana B. Ritz3, Mark Lankford3. (1) Manufacturing Science & Technology, GlaxoSmithKline, Collegeville, Pennsylvania, United States (2) UE0551, GlaxoSmithKline, King of Prussia, Pennsylvania, United States (3) UE0548, GlaxoSmithKline, King Of Prussia, Pennsylvania, United States

The implementation of platform manufacturing processes enables new products to be accelerated from discovery to the clinic, and ultimately to the market. GSK has defined a platform drug substance manufacturing process for new monoclonal antibody (mAb) products that enables rapid transfer from the lab to clinical manufacturing, and subsequent transfer to commercial manufacturing with minimal changes in scale and equipment. Quality by Design enables further acceleration when knowledge gained from implementation of the platform process is used to develop platform process control strategies. The development of a platform raw material risk assessment is presented as a case study in leveraging larger data sets available from the implementation of the platform processes to address the challenges of robust mAb control strategy development in an accelerated environment. The benefits include: 1) significantly reducing the resources required to complete a key component of platform mAb control strategy, 2) identification and mitigation of raw material-related risks to the platform process and 3) prioritization of raw materials in support of raw material supply chain workstreams.

BIOT 390

Role of simulation and scheduling tools in bioprocess development and manufacturing

Dimitrios Petrides, [email protected], Charles A. Siletti. Intelligen Inc, Scotch Plains, New Jersey, United States

The successful scale up and commercialization of biopharmaceuticals is a challenging task that requires collaboration of professionals from many disciplines. Process simulators can facilitate this task by assisting scientists and engineers to answer the following and other related questions: What is the impact of product titer increase on the capacity load of the downstream section, the overall throughput of a plant, and the cost of goods? What changes are required in an existing multi-product facility to accommodate the process of a new product? What is the range of variability that a process can accommodate if it operates under a tight cycle time? What is the impact of single-use systems on the demand for utilities, the environment and the cost of goods? Our experience in addressing the above questions will be presented using industrial examples in which we evaluated alternative technologies for producing therapeutic monoclonal antibodies and vaccines.

BIOT 391

Biomaterial production process intensification and analysis and optimization with process simulation tools

Dimitrios Petrides, [email protected], Charles A. Siletti. Intelligen Inc, Scotch Plains, New Jersey, United States

Analysis is a key element of process intensification and optimization. Process simulators and other computer aids facilitate this task by assisting scientists and engineers to answer the following and other related questions: What process areas are the best targets development effort in terms of return on investment? At what production scale does continuous manufacturing has advantages over batch manufacturing? What is the impact of extensive recycling on the demand for utilities, the environment and the cost of goods? Our experience in addressing the above and other related questions will be presented using an industrial example in which we evaluated alternatives for lysine production via bio-conversion.

BIOT 392

Impact of antibody disulfide bond reduction during harvest on downstream processing and product stability

Sandra Rios1, [email protected], Sketa Patel2, InKwan Han1, Hong Li1. (1) Merck, Kenilworth, New Jersey, United States (2) Celgene, Warren, New Jersey, United States

Therapeutic monoclonal antibodies (mAbs) are primarily produced in mammalian cell culture systems, which can be susceptible to disulfide bond reduction during harvest. This reduction, which is dependent on cell line and cell culture process, has been correlated with the release of reducing components from the cells and depletion of dissolved oxygen before, during and after harvest. Consequently, these factors can lead to disulfide reduction during storage at room temperature prior to initial purification via Protein A chromatography.

This work seeks to understand the impact of disulfide reduction on product quality and on the downstream unit operations. mAb disulfide bond reduction appears to be reversible as the redox potential changes during the purification, however, we observed that the levels of fragment and aggregate increase during the low pH treatment proportional to the amount of free thiol present. The increase of such product related impurities impact the process yield and in process intermediate stability. Furthermore, the drug substance produced from clarified culture with disulfide reduction clearly showed a higher susceptibility to temperature induced changes on stability, potentially indicating incomplete assembly. In addition, for several mAb cases where disulfide bond reduction has been observed, we compared it with DTT induced mAb disulfide reduction. This comparison showed a different mAb sensitivity order to reduction by DTT than the observed at harvest indicating a potential different mechanism for disulfide reduction.

BIOT 393

Real-time N-linked glycans analysis to facilitate monoclonal antibody manufacturing

Aron Gyorgypal, [email protected], Viki Chopda, Haoran zhang, Shishir P. Chundawat. Chemical and Biochemical Engineering, Rutgers University, Piscataway, New Jersey, United States

Mammalian cells such as Chinese Hamster Ovary (CHO) cells are mostly used to produce monoclonal antibodies (mAbs) for a variety of biotherapeutics on the market today. The in vivo efficacy of these biotherapeutic drugs are often modulated by their N- linked glycosylation profile which can be highly heterogenous, based on the upstream cell culture conditions, resulting in significant batch-to-batch variations. Implementation of a suitable process control strategy to manipulate mAbs glycosylation during upstream production has been a bottleneck due to the extensive time and labor required for sample preparation and glycan profile analysis. Here, we implement a fully automated and integrated on-line N-linked mAb glycans sample preparation process to monitor mAbs glycosylation profile in real time to enable advanced bioprocess control. This innovation consists of (1) an integrated sampling and derivatization set-up using a flow- injection system for sample preparation including deglycosylation, clean-up of impurities, as well as fluorescent labelling of released N-linked glycans , and (2) an on- line liquid chromatography/mass spectrometry (LC/MS) system for real-time mAbs glycan profile monitoring. We compare results from our automated sampling and derivatization system with standard methods used for glycan analysis. Our method shows promise to enable real-time process understanding, monitoring, and control for high quality monoclonal antibody manufacturing.

BIOT 394

Scale-down model qualification using ambr250 high-throughput bioreactor system for process characterization

Shaunak Uplekar1, Brandon Brino1, [email protected], Tim Broderick1, Sean McMillan1, Jameel Joyner1, Derek M. Ryan2, Gautam Nayar1, Sigma Mostafa1. (1) Process Development, KBI Biopharma, Durham, North Carolina, United States (2) Analytical Development, KBI Biopharma, Durham, North Carolina, United States

Cell culture process characterization conducted during late phase process development of biopharmaceuticals is an extremely critical but time consuming and labor-intensive stage. Traditionally, bench scale bioreactor systems have been used for scale-down model qualification (SDMQ) for this purpose. High-throughput mini bioreactor system such as ambr250 bioreactors can significantly improve the time and effort required to conduct process characterization studies. Here, we describe the approach taken to qualify ambr250 as a scale-down model of a GMP scale manufacturing cell culture process to produce a monoclonal antibody. The seed train was conducted using shake flasks and wave bioreactors up to the N-2 stage. Ambr250 system was used to establish a scale-down model for the N-1 seed stage and the production (N) stage. It was found that the cell growth, viability, nutrient/metabolite and titer profiles generated using ambr250 bioreactors were found to be comparable to the pilot and the GMP scale process. Product quality attributes such as size exclusion chromatography (SEC) profile, non- reduced and reduced capillary electrophoresis (NR/R CE) profile, charge heterogeneity using ion exchange chromatography (IEX), N-glycan and residual DNA (rDNA) were also found to be comparable. The results indicated that ambr250 bioreactor system can be successfully used for scale-down model qualification of N-1 and N stage of cell culture process and the subsequent process characterization studies.

BIOT 395

Improving mAb manufacturing productivity by optimizing buffer & media prep process flow

Pranav Vengsarkar, [email protected], Jungmin Oh, Calvin Cheah, Andrew Kalinovich, Thomas Lee, Nandu Deorkar. Research & Development, Avantor, Bridgewater, New Jersey, United States

An average biologic manufacturing process consumes hundreds of raw materials, ranging from media and supplements, to other process chemicals and single-use systems. Buffers are one of the largest constituents by volume used in the production of most modern biopharmaceutical products. The various compositions of buffers used across a biopharma manufacturing facility requires inflexible & cost-inefficient infrastructure for support and has several contamination risks. Similar problems are also present in media prep due to the inherent complexity of media compositions and the testing associated with them. Single-use technologies have become prevalent in the biopharmaceutical field due to their flexibility, reduced utility requirements and ease-of-use. Single-use powdered or liquid components for buffers offer significant advantages over their traditional counterparts due to savings in dispensing time and QC testing. Other general issues which can be addressed include GMP regulatory requirements, precise weight dispensing requirements, clumping concerns, quick purity checks and EHS concerns. Single-use raw material delivery systems in powder or liquid forms also reduce the risk of contamination by minimizing cleaning and external contact during periodic testing steps such as endotoxin testing. The modularity of these single-use systems also allows site and process specific solutions to be implemented quickly without requiring extensive capital expenditure. These single use powder and liquid formulations when combined with innovative new technologies like in-line dilution can help reduce facility footprint, labor hours and overall cost-of-goods. This presentation will give an overview of the challenges in raw material management with a risk/benefit analysis of options to overcome some of these issues.

BIOT 396

Flexible intensified manufacturing strategies to support existing and facilities of the future

Hemanth Kaligotla1, [email protected], Mandar Dixit1, Melisa Carpio2. (1) Sartorius Stedim Biotech, Pepperell, Massachusetts, United States (2) Cell Culture Technologies, Sartorius Stedim Biotech, San Jose, California, United States

As more companies move towards more diversified portfolios, drug manufacturing in biotech is evolving to require the use of different cell lines and the production of multiple types of complex proteins in the same facility. As the products vary, the manufacturing modes and volume requirements change along with the regulatory landscape. The need for scalable, flexible, and single-use manufacturing strategies is becoming increasingly important. The poster will discuss case studies on 1) demonstrating the intensification of upstream process development and scalability to commercial manufacturing, 2) demonstrating combinations of rocking motion (RM) and stirred tank bioreactors (STR) coupled with process analytical technologies (PAT) that allow for multiple seed train and manufacturing scenarios using fed-batch, intensified, or perfusion cultures, 3) demonstrating the development of counterflow single-use centrifugation on a CHO fed- batch process with ~5g/l product titer, ~8% biomass, and ~ 90% viability to double the depth filtration performance for cell culture clarification, and 4) demonstrating the flexibility that the use of novel highly productive convective capture single-batch-mode devices offer, and analyze the possible processing space concerning overall processing time, number of cycles, bed volume, and COGs. Lastly, the poster will also discuss the importance of a fully integrated single-use platform as well as the connection from upstream to downstream processing.

BIOT 397

Prediction of stabilizing formulations for single domain antibodies

Sergio Rodriguez, [email protected], Laura E. Crowell, Kerry R. Love, John C. Love. MIT, Cambridge, Massachusetts, United States Development of formulations of protein therapeutics is typically done using high- throughput screening of a range of buffering and stabilizing agents. This empirical process can take weeks, delaying the molecule’s entry to the clinic. Here, we describe the development of final formulation compositions for single domain antibodies (sdAbs) using an in silico tool that relates protein sequence, physicochemical behavior and formulation composition. We tested a constrained set of excipients based on the most common formulations found in FDA and EMA approved drugs, and determined the suitability of the formulations for a set of sdAbs by analyzing protein stability with Circular Dichroism, Intrinsic Fluorescence and Light Scattering under a range of formulation compositions. These data were used to build a tool to predict a stabilizing formulation composition, given only a protein sequence. After analyzing several sdAbs and their optimal compositions, we predict stabilizing formulation compositions for new sdAbs based only on sequence. Rapid prediction of stabilizing formulations would streamline end-to-end process development of new molecules. In the future, this tool can be extended to other classes of products, beyond single domain antibodies.

BIOT 398

Seamless scalability and challenges in the single use XDR platform

Richard Ottman1, [email protected], Zoe Klein2, Niket Bubna2, Michael Isaacs2, Gautam Nayar2, David Page2, Sigma Mostafa2. (1) Process Development, KBI Biopharma, Durham, North Carolina, United States (2) KBI Biopharma, Durham, North Carolina, United States

The ability to quickly take a process from microbioreactors to 2000L single use bioreactors has been a key success of KBI Biopharma. Operating as a CDMO requires flexibility to adapt to different cell lines and a multitude of different processes, whether developed at KBI or transferred from clients. Since 2010, KBI has successfully manufactured 140 batches with 65 different processes. We have taken a deep dive into this historical dataset to highlight the successes and challenges overcome in scaling up processes into our full scale, end-to-end single use cGMP facility. An additional goal of this data mining was to identify any cell line specific trends with incongruities between scales. These aspects will then be optimized early in development to increase efficiency during tech transfer to manufacturing. Here we demonstrate the comparability between pilot scale (200L) and manufacturing scale (2000L) batches for 3 different CHO cell lines expressing a variety of products. In developing our scale up model we have encountered and overcome several challenges. Here we highlight the challenge of overcoming elevated pCO2 levels at full scale observed with certain processes. These datasets spotlight the adaptability of KBI to successfully transfer and scale processes in single use bioreactor systems.

BIOT 399

Small-scale model for studying resin interactions with chemical sanitants Rachel Straughn1, [email protected], Rob Piper2. (1) Just Biotherapeutics, Seattle, Washington, United States (2) JustBiotherapeutics, Seattle, Washington, United States

In connected continuous antibody manufacturing, the downstream capture system can be considered part of the upstream process. Single-use components and assemblies help to extend the sterile barrier of the bioreactor, but columns and resin remain a challenge. While single-use products such as gamma-irradiated columns are available, they are costly and not conducive to low-cost antibody manufacturing. Therefore, one must rely on chemical sanitants to treat reusable materials such as resin and column housings. Just successfully implemented a sanitization procedure for Protein A chromatography capture in large-scale (500 liter) productions using a peracetic acid solution. After changing the Protein A resin used in this step, we experienced a series of contaminations at large-scale. To investigate these contaminations, we developed a small-scale model probing sanitant concentrations, hold duration, and potential resin- sanitant interactions. The data generated using this small-scale model informed concurrent large-scale column sanitization work, resulting in a sanitization procedure that successfully enabled a bioburden-free GMP continuous capture process.

BIOT 400

Impact of ammonia stress on glycosylation of the VRC01 monoclonal antibody produced in CHO cells

Claire McGraw1, [email protected], Kathryn Elliott2, Sarah W. Harcum2, Nicholas R. Sandoval1. (1) Chemical and Biomolecular Engineering, Tulane University, New Orleans, Louisiana, United States (2) Bioengineering, Clemson University, Clemson, South Carolina, United States

Chinese hamster ovary (CHO) cells are commonly used in biomanufacturing of recombinant protein therapeutics (including monoclonal antibodies) due to the human- like post translational modifications, such as glycosylation. Glycosylation of monoclonal antibodies (mAbs) is of particular interest due to the effect various glycan identities have on the effector function, structure and stability of the mAb, thus making it an important product quality attribute. To achieve a desired therapeutic effect, it could be necessary to produce an antibody with a specific glycosylation profile. In order to produce this antibody with specific glycoforms we must understand how changes to culturing conditions impact the glycosylation profile of the mAb. In this work we detect changes in glycosylation of CHO produced VRC01- a broadly neutralizing antibody currently undergoing clinical trials to treat HIV, following changes to the culturing conditions. More specifically we studied the impact of ammonia stress on both young and aged CHO cells, with different gassing and feeding protocols. We found that increased ammonia concentrations measured in the culture for all conditions tested led to a decrease in total and terminal galactosylation, as well as a decrease in sialylation of the associated glycans. Future work will look into how the conserved Asn297 Fc glycan is impacted compared to Fab glycans under various culturing conditions. BIOT 401

Overcoming cell and gene therapy manufacturing challenges by addressing open system and raw material handling

Jungmin Oh, [email protected]. Avantor, Bridgewater, New Jersey, United States

Rapid advancements in cell and gene therapy development have resulted in recent product approvals bringing these transformative medicines to market. Commercializing these products has introduced a new set of challenges unique to our industry. For example, a consistent quality product requires an overall manufacturing control strategy including control of materials, control of the process, control of any intermediates, drug substance and final drug product. The performance factors and critical quality attributes of raw materials such as endotoxin, sterility and mycoplasma can have direct impacts on the final cell and gene therapy product quality. Our presentation will provide an overview of the unique challenges facing the commercialization of these medicines – ranging from quality to logistics and contamination control. We will present case studies on achieving high quality of cell and gene therapy products while meeting regulatory compliance by utilizing closed low volume sampling systems, ready to use GMP sterile raw materials and deliver these solutions to each process step using closed loop systems. This approach will significantly reduce process risks and increase process efficiency.

BIOT 402

Pall Xpansion® bioreactor supports progenitor cell growth to >1 million cells/cm2 and proper cell differentiation

Andrew Laskowski, [email protected], Siddharth Gupta. Pall Corporation, Westborough, Massachusetts, United States

With the recent increase in early phase cell therapy clinical trials, there is a need for a manufacturing platform which can be implemented in research labs and easily scaled up to expedite process development studies, pre-clinical testing, and large-scale expansion of adherent human cells. Although various platforms such as traditional flatware and stirred tank bioreactors with microcarriers exist and have been well characterized, traditional flatware often does not allow for tight control of the cellular environment and is not scalable. Pall’s Xpansion single-use bioreactor offers a tightly- controlled, scalable manufacturing platform for cell therapy applications, allowing for expedited process development, pre-clinical testing and large scale, high-density expansion of progenitor cells while maintaining their differentiation capacity.

We have previously demonstrated efficient expansion of both epithelial cells (Vero, HEK293) and human mesenchymal stem/stromal (hMSC) in the Xpansion single-use bioreactor. Here we extend these findings by successfully expanding a proprietary progenitor cell to extremely high cell densities (> 1 million cells/cm2). The first stage of this process begins with a pluripotent stem cell line, which is expanded and differentiated into proprietary lineage-specific progenitor cells. We then demonstrate growth and differentiation of this progenitor cell in the Xpansion bioreactor using a 14- day and 21-day protocol. In summary, both protocols resulted in 1) cell expansion to > 1 million cells/cm2 and equivalent cell densities to the traditional flatware process control; 2) equivalent differentiation to a more mature cell fate as indicated by expression of key cell surface markers; and 3) self-assembly into characteristic three-dimensional structures.

BIOT 403

Substrate-functionalized single-walled carbon nanotubes as modular activity probes for optimization of lignocellulosic biomass pretreatment

Nathaniel Kallmyer, [email protected], Nigel F. Reuel. Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States

Conversion of biomass to precursor sugars and alcohols is complicated by the composite structure of lignocellulose which renders targeted cellulose inaccessible behind a matrix of hemicellulose and a coating of lignin. This structure necessitates pretreatment of biomass with chemical, physical, or biological means. Biological pretreatment with enzyme formulations or “cocktails” that degrade each component holds great promise as a sustainable method; however, current efficiencies of these formulations limit the cost-competitiveness of this strategy. While the need to improve enzyme efficiency has been recognized, methods of quantitatively measuring activity are limited by time-consuming or insensitive colorimetric and viscometric assays which require specialized equipment and often only report activity indirectly. Here, we demonstrate a library of substrate-functionalized single-walled carbon nanotubes (SWNT) as modular probes for characterization of cellulase, xylanase, and lignin- modifying enzymes. We also explore the effects of substrate substitution ratios and polymer lengths on consistency and selectivity (exo- vs. endo- preference) of cellulases using carboxymethylcellulose-functionalized SWNT. Lastly, we demonstrate the utility of such probes by screening an array of enzyme formulations with respect to cellulytic, hemicellulytic, and lignin-modifying activities with a single-wavelength fluorescence detector. We also show progress towards such screening in micro-droplet reactors. These sensors will find utility as real-time activity probes for improvement of enzyme operating and formulation conditions as well as rapid-screening tools for directed enzyme evolution.

BIOT 404

Analysis of nutrient distribution and aqueous phase from hydrothermal liquefaction (HTL) of individual food waste components for subsequent bioremediation Michael Stablein, [email protected], Aersi Aierzhati. Agricultural and Biological Engineering, University of Illinois Urbana Champaign, Urbana, Illinois, United States

Hydrothermal Liquefaction (HTL) is a thermochemical process that uses high heat and pressure to convert various biowastes into renewable biocrude oil, simultaneously both generating a metal and phosphorus rich solid phase and a nutrient rich, toxic aqueous phase. Given the promise of this technology that has achieved as much as 80% biocrude recovery, researchers have also investigated the advantages and limitations of reusing these byproducts in other applications, such as asphalt binder, adsorbents, fertilizer, and recyclable process water, etc. However, given the differences between feedstocks including manure, cornstock, algae, food waste, among others, there are considerable gaps in understanding how to best resuse the materials and the expected characteristics as a result of several reaction parameters. Thus, this work aims to elucidate the mechanisms by which some compounds are concentrated in different phases through their characterization after HTL of different types of food waste, grouped into lipid-rich, protein-rich, and carbohydrate-rich feedstocks, all collected from the campus dining hall washroom. These 8 types of food waste HTL processes were optimized for oil yield and products analyzed considering temperature and time across a range of 280-340C and 10-60min. The analysis and discussion of this work reviews considerations for subsequent anaerobic digestion and algae cultivation with respect to the nutrients and inhibitory contents of the Post Hydrothermal Liquefaction Wastewater (PHW).

BIOT 405

Recent advances in nanocarrier to transport vegetable nutrients for precision and sustainable agriculture

Luis A. Lightbourn Rojas, [email protected]. Instituto de Investigación Lightbourn, Cd. Jiménez, Chihuahua, Mexico

The increasing food demand as a result of the rising global population has prompted the large-scale use of fertilizers. However, the major problem encountered is that the applied fertilizers are often exhausted due to rapid degradation by various chemical reactions such as photolysis, hydrolysis and decomposition. On the other hand, chemicals fertilizers destroy the properties of the soil, and harmful to humans. In this regard, the amphiphile enantiomorphic colloids based on nanotechnology have the potential to surpass conventional fertilizers. The application of this nanotechnological development could increase agricultural productivity substantially, which improve the nutrient use efficiency that may reduce environmental impact and boost the plant productivity. Furthermore, controlled release and targeted delivery of nanoscale active ingredients can realize the potential of sustainable and precision agriculture.

Amphiphile enantiomorphic colloids are a most promising technology to step down the chemical release under controlled situations, reducing the current application dosage and improving efficiency of nutrients. These colloidal nanoparticles are molecular aggregates with at least one dimension between 80 and 100 nm, which can drastically modify their physico-chemical properties according to the soil conditions. This nanofertilizers can increase in nutrients use efficiency, reduces soil toxicity, minimizes the potential negative effects associated with over dosage and reduces the frequency of the application.

BIOT 406

Advanced cellulosic biofuel production using a novel pretreatment method

Shannon Hoffman1, [email protected], María S. Álvarez1, Gilad Alfassi2, Yachin Cohen2, Jose L. Avalos1,3. (1) Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States (2) Department of Chemical Engineering, Technion-Israel Institute of Technology, Haifa, Israel (3) Andlinger Center for Energy and the Environment, Princeton University, Princeton, New Jersey, United States

As global temperatures rise, biofuels are essential towards reducing greenhouse gas emissions. Corn starch ethanol is currently the most prevalent biofuel; however, to expand production sustainably, cellulosic biofuels made from agricultural waste and energy crops are needed. Furthermore, for biofuels to be used more broadly, the industry must transition from ethanol to branched-chain higher alcohols like isobutanol, which are more compatible with existing infrastructure. Despite their necessity, cellulosic biofuels have struggled to be economically solvent due to the slow rates and insufficient extent of cellulose hydrolysis achieved with current pretreatment methods. We address this challenge with a novel pretreatment technology, which significantly increases the rate and extent of hydrolysis in simultaneous saccharification and fermentation (SSF) processes. With this method, the rate of hydrolysis is increased by 20-fold compared to microcrystalline cellulose in the first hour. This technology also improves the extent of conversion, with nearly full hydrolysis achieved in just twelve hours, whereas microcrystalline cellulose does not reach full conversion within two days. Beyond demonstrating enhanced hydrolysis kinetics, we applied this technology to transition from ethanol production to isobutanol, a next-generation biofuel, in an enhanced SSF process. Cellulose pretreated using the new method produces nearly theoretical yields of ethanol within one day, and when used with a S. cerevisiae strain engineered for isobutanol production, generates isobutanol titers 6-fold higher than from microcrystalline cellulose. This new method can improve the economic viability of cellulosic biofuels through rapid and complete cellulose conversion, resulting in higher yields and shorter fermentations than existing technologies.

BIOT 407

Identifying inhibitory waste by-products in high density CHO cell culture

Bingyu Kuang1, [email protected], Seongkyu Yoon2. (1) University of Massachusett, Lowell, Lowell, Massachusetts, United States (2) University of Massachusetts Lowell, Lowell, Massachusetts, United States

Mammalian cells exhibit inefficient and poorly regulated metabolism as they tend to use as much as the available nutrients and produce waste metabolites rather than the required amount for their growth. As a result, they produce substantial waste products that create a hostile environment for the growth of cells as well as the target protein and lowers the protein quality. In order to control the metabolism and increase the productivity, it’s crucial to identify the inhibitory waste metabolites and study their pathway. The work flow for the waste inhibitor metabolomics are candidate identification, inhibitory impact verification and quantification, and control strategy development. In this study, 6 novel inhibitory metabolites were identified based on their impact on growth, productivity, and product quality. The metabolome profiles of VRC O1 (CHO K1) cell line was firstly screening in High Cell Density fed-batch cultures using high resolution global metabolomics platforms, Orbitrap LC-MS. The top 18 inhibitor candidates with high confidence in identification matching were selected by multivariate data analysis and pathway analysis. The high ranked candidates were experimentally verified by spiking them in the batch culture. Among them, 6 metabolites inhibit growth, 6 metabolites lower the antibody production, and 8 metabolites show impact on IgG glycosylation.

BIOT 408

Determination of AGEs in meat products

Breana Alston, [email protected]. South Carolina State University, Kingstree, South Carolina, United States

Diets today are normally high-heat processed foods that contain high levels of AGEs. Advanced Glycation End Products, better known as AGEs, are harmful compounds that form when proteins (as well as lipids and nucleic acids) react with sugar. AGEs can accumulate in the body via two pathways, internally through natural biochemical pathways and dietary intake. Dietary advanced glycation end products (dAGEs) are linked to the formation of several diseases associated with older age, such as diabetes, cardiovascular disease, cancer, etc. This project looks to expand on reported dAGEs found in pork chops prepared using different cooking techniques (raw, boiled, pan seared, slow cooked, and microwaved), and suggest different approaches to reduce the amount of AGEs through dietary intake. In this research, we analyzed the meat samples for quantity of Nδ-(carboxylmethyl)-L-lysine (CML) and Nδ-(carboxylethyl)-L-lysine (CEL), two well studied AGEs. Studies have shown that an increase in CML and CEL contribute to the formation and progression of tumors, and diabetes through the activation of oxidative stress. We hypothesize that the quantity of CML and CEL found in the pork chops will follow previous reports and will be reduced when cooking at lower temperatures, for shorter time periods, and in moist environments. Therefore, we expect to find the raw meat to contain the least amount of CML and CEL, followed by the boiled, slow cooked, microwaved and pan seared samples. The Gas Chromatography- Mass Spectroscopy (GCMS) was used to determine the amount of CML and CEL in the prepared pork chop samples. Results indicated a larger amount of CEL in both raw and boiled sampled (32.57, 51.56) compared to CML (1.31, 0.65). These findings were surprising since the structures of CEL and CML are similar, we believed their values would be similar in similarly prepared pork chop samples.

BIOT 409

Improved demethylation efficienty of nobiletin encapsulated in modified starch by miling process shiwei su, [email protected]. Food science, Rutgers, New brunswick, New Jersey, United States

A strain of yeast which can monodemethylate Nobiletin on the A ring was separated form Chenpi (old citrus peel). ITS results showed it is a mutant specie of Filobasidium magnum with 4 base-pairs less than the documented one.

In order to improve the demethylation efficiency, Nobiletin was encapsulated in modified starch by milling process. 5 hours milling processing produced starch-encapsulated- Nobiletin solution with particle size being around 900 nm. Lyophilized powder was applied for fermentation.

The effect on demethylation efficiency of starch-encapsulated-Nobiletin concentration was firstly tested with results showing the optimum concentration being at 15μg/ml media. The yield of the two monodemethylate Nobiletin could respectively reach 40 μg/ml and 10 μg/ml after 14 days of fermentation, which was 20 times higher as compared to that when Nobiletin was dissolved in DMSO, and 5 times higher than that of Nobiletin-starch water solution without milling process.

Encapsulation of Nobiletin in modified starch by milling process is an efficient method to boost the transformation efficiency of insoluble chemical compounds.

BIOT 410

Patterning-mechanics feedback mechanism to understand axis extension during morphogenesis Samira Anbari1, [email protected], Javier Buceta2,1. (1) Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, Pennsylvania, United States (2) Bioengineering, Lehigh University, Bethlehem, Pennsylvania, United States

During development, organs with different shapes and functionality form from a single cell. Mechanisms that drive shape, size, and morphology of tissues pose challenges for developmental biology. Within the topic of morphogenesis, tissue elongation is a necessary process in all metazoans to shape and implement their body plans. Besides recent advances, the mechanisms underlying tissue elongation are not fully understood. For example, in the particular case of the limb, it has been shown that tissue elongation cannot be explained by either a localized proliferation of cells or their oriented divisions and, surprisingly, the tissue elongates perpendicular to the direction along which cell grow. Here we propose a mechanism of tissue elongation to address this conundrum. Our model couples the mechanical properties of cells with the concept of positional information to modulate the former in a location-dependent manner. To illustrate our proposal, we use the French flag model (morphogen gradient thresholds) as well as the Turing patterning system as the mean used by cells to “know” their relative positions within a primordium. Our numerical simulations are based on the so-called vertex model that describes the energetics of tissues, and we use elasticity theory to analyze different properties of our simulations. In this context, we show that if the cell-cell adhesion is modulated as a function of the location of cells within a primordium, an auto-catalytic cell intercalation process develops, and the tissue elongates. Moreover, our results reveal that cells grow perpendicular to the elongation direction as experimentally reported. Altogether, our results shed light on the tissue elongation problem and pave the way to understand morphogenesis better.

BIOT 411

Large amplitude oscillatory shear (LAOS) flow as a metric of comparison for contemporary human blood rheological models

Matthew Armstrong, [email protected]. Chemistry and Life Science, United States Military Academy, West Point, New York, United States

Recent work modeling the rheological behavior of human blood indicates that blood has all of the hallmark features of a colloidal suspension, including shear-thinning, viscoelastic behavior, a yield stress and microstructure. After decades of modeling steady state blood data, and the development of steady state models, like the Casson, Carreau-Yasuda, Herschel-Bulkley, etc. the advancement and evolution of blood modeling to transient flow conditions now has a renewed interest. Using recently collected human blood rheological data we show and compare modeling efforts with several new models including the new modified Horner-Armstrong-Wagner-Beris (mHAWB), the viscoelastic enhanced Modified Delaware Thixotropic Model (MDTM), and more. We will compare the new approaches by ability to predict small and large amplitude oscillatory shear flow as well as uni-directional oscillatory shear flow.

This effort is followed with a discussion of novel transient flow rheological experiments applied to human blood including for model fitting purposes including step-up/step- down, and triangle ramp experiments. The family of models that can handle these transient flows involve modifications to the recently published Modified Delaware Thixotropic Model (MDTM), the mHAWB model and the Bautista-Monero-Puig Model (BMP). We fist discuss the development of the scalar, structure parameter evolution models and we compare fitting results with our newly acquired transient blood data to the models. We also highlight our novel model fitting procedure by first fitting to steady state, and while keeping the steady state parameters constant fitting the remaining model transient parameters to a series of step up/down in shear rate experiments. With the full set of parameters determined with a global, stochastic optimization algorithm the SAOS, LAOS and unidirectional oscillatory shear flow is predicted and compared to the data. Model efficacy is then compared.

BIOT 412

Auxin-like resource from the high-solid anaerobic digestion and hyperthermophilic pretreatment composting of dewatered sludge: Pathways and regulation

Yanfei Tang1, [email protected], Xiaohu Dai1,2. (1) College of Environmental Science and Engineering, Tongji University, Shanghai, China (2) Shanghai Institute of Pollution Control and Ecological Security, Shanghai, China

Efficient utilization of the auxin-like resource generated from the large amount of sewage sludge is beneficial for global carbon cycle. The semiquantitative concentration and enrichment pathways of auxin-like molecules together with the molecular structure of their attached dissolved organic matrix (DOM) were determined during the processes of hyperthermophilic pretreatment composting (HPC) and high-solid anaerobic digestion (HAD). The germination index (GI) rebound in HAD and HPC indicated the similar changes of auxin-like activity, however, the GI values of HPC 16-40 d were almost twice those during HAD 8-28 d. The enhanced fulvic acid and protein-derived (FA+PD) material (R2=0.8874) and the humic acid-like carbon (HAC) (R2=0.9052) significantly contributed to the GI rebound in digestate and compost, respectively. Indeed, auxin and auxin-like molecules mainly aromatic carboxylic acids in HAD were accumulated from the intense protein-like degradation and following active aromatic amino acid metabolism. Different from that, in HPC the evident improvement of HAC and polymerization degree of humic substances were attributed to upregulated biosynthesis pathways. FTIR spectra and MW distribution suggest the DOM of HPC concentrated more COO- and RCOOH in macromolecular or supramolecular structures, explaining the higher GI in compost though with fewer small molecular carboxylic acids. Additionally, the inevitable phytotoxicity due to excessive salinity in HAD was also caused by protein-like degradation and amino acid metabolism. Hence, auxin-like activity is essentially depended on the construction and concentration of auxin-like resources, and thereby inspiring the regulating strategies for accurate resource utilization.

BIOT 413

Molecular structure transformation of extracellular polymeric substances (EPS) and metagenomics analysis in thermal pretreatment composting with dewatered sludge: EPS reconstruction and endogenous humification

Yanfei Tang1, [email protected], Xiaohu Dai1,2, Bin Dong1. (1) College of Environmental Science and Engineering, Tongji University, Shanghai, China (2) Shanghai Institute of Pollution Control and Ecological Security, Shanghai, China

In non-lignin-dependent mode, this study proposed and then proved the hypothesis that thermal pretreatment composting (TPC) with dewatered sludge is superior for efficient humic substances (HS) formation with endogenous precursors. Extracellular polymeric substances (EPS) account for 60-80% of sludge organic matter (OM) and act as the key executive site of microbial metabolism. However, essential knowledge of the effect of thermal pretreatment on EPS transformation and functional metabolism evolution in composting is still unclear. When considering the turning point of composition and concentration of sludge OM, 110°C and 120°C thermal pretreatments (TP110 and TP120) and following 40-day composting (T1-AC and T2-AC) were conducted in comparison. TP120 and the thermal phase of T2-AC exhibited the more intense EPS deconstruction, according to the more evident enrichment of smaller molecules together with the significant humic acids (HA) release and protein-like (PN) decomposition. Subsequently, the greater enlarged EPS molecules assigned to the repolymerized HA (16-23 kDa) and PN (187-252 kDa) were found in T2-AC 16-40d, and the capacities of electron- accepting and electron-donating of T2-EPS rose again in the last 16 days finally being 3.27 and 3.47 times the levels of T1-EPS, respectively. Moreover, the initial HA exposure in T2-EPS related closely to the early higher electron exchange activity and capacity, while later HA rebound correlated significantly to enhanced Proteobacteria, Actinobacteria and pathways of amino acid synthesis, metallic complex and peptides transport. Thus, stimulating HA release from EPS in advance as redox mediates and precursors might be a novel strategy to accelerate evolutions of microbial community and functional metabolism, thereby achieving superior endogenous humification, efficient EPS reconstruction and bacterial fertilizer production.

BIOT 414

PCR-based assays for identifying transgene insertion sites in CHO genome

Jong Youn Baik, [email protected]. Biological Engineering, Inha University, Incheon, Korea (the Republic of)

Chinese hamster ovary (CHO) cell lines for biopharmaceutical production are usually established by random transgene integration, resulting in a heterogenous cell population with various transgene copy number and integration sites. In addition, cell growth, protein productivity, and quality may be affected by the transgene insertion sites in the genome. Therefore, identifying the genomic insertion site of transgenes will help better characterization of cell lines. While next-generation sequencing is routinely employed for identifying the insertion site, it requires complicated processing and analysis of sequence reads. Moreover, it is a time-consuming (4-6 weeks) and costly ($2,000-3,000 per project) technique, demanding simple, easy, and cheap methods for gene integration site analysis. There are PCR-based techniques for isolating and amplifying genomic DNA sequences neighboring the DNA junction of integrants such as Alu-PCR, A-T linker PCR and splinkerette-PCR. We explored the feasibility of these PCR-based techniques in CHO genome to identify the integration site of transgene with less resources (both time and cost). As a proof-of-concept study, we digested a plasmid vector using restriction enzymes and ligated them with splinkerette adaptors that prevent adaptor-adaptor amplification. Next, we conducted PCR and identified a band with an expected size, and verified the amplified DNA using restriction enzyme mapping. This technique was then applied to CHO genomic DNA samples to identify the integration sites of a recombinant protein gene. In conclusion, PCR-based assays would help to provide the genomic information of CHO production cells with more affordable options in the process of cell line development. BIOT 415

Back to the future: Should we innovate the past?

Olga M. Paley1, [email protected], George Parks1, Ratnesh Joshi1, Norbert Schuelke2, Raghu Shivappa3, Hang Yuan1. (1) Downstream Process Development, Takeda Pharmaceuticals, Cambridge, Massachusetts, United States (2) Takeda Pharmaceuticals International Co, Cambridge, Massachusetts, United States (3) Upstream Process Development, Takeda Pharmaceuticals, Cambridge, Massachusetts, United States

Bioprocessing technologies and best practices for therapeutic proteins continue to evolve with increasing industry maturity. The result is greater opportunities and corresponding challenges for originators considering innovative manufacturing changes for commercial products as part of ongoing life-cycle management. Various considerations such as supply robustness, project business case, regulatory strategy, and -most importantly- potential for patient benefit or risk must be weighed when assessing major process modifications. This overall cost benefit package serves as the foundation for broad organizational buy-in necessary for successful development and implementation. Takeda presents LCM case studies for two approved biologics and the technical and strategic lessons learned along the way.

BIOT 416

Synteny maps improve predictions of orthologous genes

Nicholas P. Cooley, [email protected], Erik S. Wright. Biomedical Informatics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Clusters of orthologous groups (COGs) facilitate functional and phylogenetic classification of proteins. The rapid decrease in sequencing costs and subsequent explosion of publicly available genomes has presented a need for methods of de novo COG assignment for many new genomes at once. Typically, COGs are constructed from a graph-based assembly of best hits between proteomes. These approaches begin with all-vs-all sequence comparisons to collect best hits between all proteome pairs. These best hits are then used to construct an adjacency graph, where a clustering algorithm trims clusters based on interconnectedness and strength of percent identity between given best hits.

COGs generated from best hits-based strategies tend to include many spurious sequences because the underlying hits are too permissive. In an effort to address this issue we developed a more precise method of predicting input pairs. Synteny maps provide an alternative to all-vs-all comparisons that also account for the proximity of paired genes. Gene boundaries for the paired genomes can be overlaid on the synteny map, with pairs of genes linked by shared k-mers. This process functions as a rapid substitute for best hits identification, and generates COGs with strong agreement to species trees.

Here, we present a synteny based method for predicting orthologous proteins between bacterial genomes, and construction of accurate COGs from those pairs. This work is entirely performed in the R programming language, which provides both extensibility to diverse systems and accessibility to users with minimal computational background.

BIOT 417

Picking winners: Predictive modeling for cell line selection

Jasmine Tat1, [email protected], Kim Le1, Christopher Tan1, Aaron Baskerville- Bridges2,1, Huong Le1, Kristine Daris1, Jonathan Diep1, Fides Lay1, Tony Wang1, Natalia Gomez1, Jennitte Stevens1. (1) Amgen, Thousand Oaks, California, United States (2) MIT, Cambridge, Massachusetts, United States

***THIS ABSTRACT WILL BE UPDATED, PENDING LEGAL APPROVAL***

Within the discovery-to-market development cycle for biopharmaceuticals, Cell Line Development holds a critical role in creating and selecting a cell line that becomes the Master Cell Bank for clinical and commercial biomanufacturing. Cell line generation and selection have largely been driven by resource-intensive manual cell culture processes and analytics. Analysis and decision points predominantly rely on generating large volumes of secreted protein, rate limited by cell doubling times and analytical sensitivities/development. For every project, a wealth of data is generated. However, a modular design framework has yet to leverage this data, unify processes, and transform current workflows. Through the integration of machine learning frameworks, we are shifting from an established, traditional method of manual cell culture and analysis towards a “digital cell culture” paradigm. Our strategy is two-fold: one is the implementation of nanofluidic technology to manipulate and collect data at a single-cell level, the second is utilization of machine learning to provide predictive power for cell line selection, deliver efficiency, and enable a scalable/transferable digital platform across Amgen. Integrating machine learning into our workflow will ultimately enable precise decision making, drive improved performance, better understand/control quality attributes, and dematerialize workflows.

BIOT 418

Sorting out large datasets for cell culture manufacturing process understanding

Jeremy Discenza, [email protected]. Manufacturing Technology, Bristol Myers Squibb, Chittenango, New York, United States

There no set way to assess cell culture manufacturing data, but some methods may be more efficient than others. For complex cell culture processes with hundreds of variables and interactions, we need a streamlined approach that addresses both the quality of the data and the context among variables. An overview of data analysis workflow and a case study that applies a systematic approach is presented for a manufacturing biologics process.

BIOT 419

Development of a harvest small scale model to simulate continuous centrifugation

Jianfa Ou, [email protected], Qing Wong, Michael Kagan, Dominique Monteil, Daniel Bock. Process Science, Boehringer Ingelheim, Fremont, California, United States

Disk stack centrifugation is one of the main clarification steps used in large scale mammalian cell culture processes. During process development, pilot scale disc stack centrifuge is normally used to develop and characterize the harvest process for new cell culture processes due to the lack of smaller scale model for disc stack centrifugation. In order to increase speed of development, a small scale model (SSM) of a disk stack centrifuge is of high demand to enable harvest development in an earlier stage of process development. A small scale model can also improve throughput and reduce cost. In this study, we have developed a SSM for disc stack centrifugation using a capillary shear device combined with benchtop centrifugation. Capillary tube size, length, and flow rate were optimized to generate energy dissipation rates comparable to large scale disk stack centrifuges. Additionally, shear force from multiple centrifuges at different scales were standardized and compared. Mathematical correlations were established for energy dissipation, viable cell density, cell viability, and lactate dehydrogenase (LDH) activity. Our SSM offers an approach to understand how the separation process, such as centrifuge designs, sizes, and configurations affect filtration performance and product quality. This work can speed up harvest development during process scale-up in biopharmaceutical mammalian cell culture processes.

BIOT 420

Characterization of media precipitate and determination of precipitation kinetics

Duc Hoang1, [email protected], Shaun Galbraith2, Seongkyu Yoon2, Nivedita Shipurka1. (1) Chemical Engineering, University of Massachusetts Lowell , Lowell, Massachusetts, United States (2) University of Massachusetts Lowell, Lowell, Massachusetts, United States

Process intensification of mAb production is leading to more concentrated feed media causing issues with precipitation of solids from the media solution. This results in processing problems since components in the precipitate are no longer in solution and bioavailable, this changes the media composition leading to variability in cell culture performance. The goal of this work is to characterize the feed media precipitate, and in particular to identify the components so that mitigation strategies can be developed. The precipitate was predominately found to be organic, and was analyzed with LC-MS (Liquid Chromatography – Mass Spectrometry) to identify the constituent components. Up to 16 amino acids were identified with tyrosine, histidine, arginine and phenylalanine being the most prevalent amino acids. Elemental analysis with ICP-OES (Inductively Coupled Plasma – Optical Emission Spectroscopy) and XRD (X-Ray Diffraction) suggest that metal phosphates are the predominant inorganic components. Our research revealed L-Tyrosine, L-Phenylalanine, L-Arginine and L-Histidine to be the major amino acids in solid media precipitate. Future experiments informed by this analysis can be used to determine the formation kinetics of these components, which can then be implemented in feed media design optimization and process scheduling. This work is critical for the current trends of process intensification for mAb production.

BIOT 421

Developing a genetic engineering tool box for anaerobic fungi

Ethan T. Hillman1, [email protected], Casey Hooker1, Jake Englaender2, Kevin Solomon1. (1) Agricultural and Biological Engineering, Purdue University, Lafayette, Indiana, United States (2) Purdue University, West Lafayette, Indiana, United States

Anaerobic fungi are potentially powerful platforms for biotechnology that remain unexploited due to a lack of genetic tools. These fungi have the largest repertoire of CAZymes for degradation of lignocellulose, making them particularly attractive for biofuel production, and are predicted to possess novel biosynthetic capabilities. However, a genetic toolbox that would enable further study and exploitation of these capabilities for synthetic biology remains underdeveloped. No plasmids, promoters, terminators, selection markers, reporter genes, or gene-editing systems have been characterized. Here, we present our efforts to develop this toolkit. Anaerobic fungi are naturally competent during phases of their lifecycle, which we have exploited to introduce and transiently express heterologous genes. We have leveraged existing transcriptomic resources to identify putative promoters and terminators that regulate gene expression at different that appear conserved across 6 anaerobic fungal isolates. Using our transformation protocols, we have confirmed the ability of at least one promoter candidate to promote gene expression of a flavin-based fluorescent reporter and developed a pipeline to characterize candidate gene regulatory elements. Similar experiments have also successfully introduced antibiotic selection systems, which we will extend to express alternative selection markers, fluorescent reporter genes, and CRISPR-based endonucleases. Other elements such as origins of replication and centromeric binding sequences are being developed in parallel to create a stable, plasmid-based expression system in anaerobic fungi. Ultimately, this toolbox will allow us to express genes of non-native pathways, selection markers, and engineering systems, which will unlock a variety of genome engineering possibilities for the study of fungal genetics and strain development for various synthetic biology applications.

BIOT 422 Woody fragrance in bioreactors: Insights from the development of a fungus mediated fermentation and distillation pipeline for Indian agarwood oil

Nabanita Kalita1, [email protected], Nagrani Mahajan1, [email protected], Rituparna Buragohain1, [email protected], Supriyo Sen1,2, [email protected]. (1) Ouija Biosolutions Pvt. Ltd., Guwahati, Assam, India (2) Department of Biosciences, School of Life Sciences, Assam Don Bosco University, Sonapur, Assam, India

Fragrant agarwood (Aquilaria spp.) is arguably the costliest wood in the world. The essential oil called Oudh or Agar Oil sells at above 7,000 USD for a kilogram (valued traditionally in Asian gold standard, Tola) in the global market. This aroma from Asia is increasingly becoming a component of fragrance particularly woody perfumes. Being also a superb base note and perfume retainer, its demand has consistently been high. The oil is distilled out from resin containing agarwood tissue. This tissue is formed by complex biotic and abiotic interaction between Aquilaria trees and its environment. The hard resin-impregnated wood is traditionally chipped and then soaked in open water tanks before hydro-distillation to facilitate extraction of oil. This offers an excellent opportunity for fermentation, hitherto unexplored.

India, particularly Northeast India where we are located, is one of the places of origin of agarwood aroma. In Ouija Biosolutions Pvt. Ltd. we are partnering with local agarwood perfumers to develop a novel fermentation- distillation pipeline involving a fungus- mediated fermentation bioprocess. This is a unique approach in agarwood that enhances the quality as well as productivity of agar oil. We have employed specially designed bioreactors that provide suitable conditions for microbial fermentation of resinous agarwood to improve the bioprocess leading to improved oil yield at a lesser time compared to the conventional method (Figure 1). Enzyme assays have revealed clues towards lignocellulosic substrate - enzyme action during wood –fungus interaction, which is critical for upstream process optimisation.

Our innovation is at the intersection of the traditional upstream and downstream of agar oil processing and can effectively change the way agar oil processing is carried out sustainably in the future.

Figure 1: Fermentation-distillation pipeline for a sustainable and improved method of agar oil production. Note the upstream modification of the traditional bioprocess for agar oil production.

BIOT 423

Engineering Escherichia coli for methanol-dependent growth and production

Michael Dillon1,2, [email protected], R. Kyle Bennett1,2, Gerald Har1, Maciek R. Antoniewicz3, Eleftherios T. Papoutsakis1,2, Kelley Hestmark4, Stephanie Jones4, Nyarie Dzvova4, Noah Helman4, Derek Greenfield4, Elizabeth Clarke4. (1) Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Delaware Biotechnology Institute, Newark, Delaware, United States (3) Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States (4) Industrial Microbes, Emeryville, California, United States

An abundance of methane from natural gas reserves has raised interest in engineering synthetic methylotrophs that can utilize methane or methanol, the oxidized form of methane, for growth and chemical production. Synthetic methylotrophs are non- methylotrophic platform organisms that have been engineered to utilize reduced C1 compounds. Since native methylotrophs lack many of the well-developed genetic engineering tools that platform organisms possess, the development of robust synthetic methylotrophs is of considerable interest. Our lab previously developed a synthetic Escherichia coli methylotroph that consumes 1 mol of methanol with 1 mol of glucose concurrently, making this strain “methanol-dependent.” This allows the sugar feedstock cost to be reduced by supplying a portion of the carbon substrate as methanol, which is less expensive. Using adaptive laboratory evolution and process optimization, we report improvements to growth and chemical production. One such evolved strain exhibited robust methanol-dependent growth in glucose minimal medium, whereas negligible growth was observed using the ancestral strain. Furthermore, methanol-dependent acetone and 1-butanol production was achieved via incorporation of heterologous enzymes from Clostridium. As a result of dependence, high average carbon labeling (up to 33%) in acetone and 1-butanol was observed when using a 13C-methanol tracer during fermentation. Future work will involve scale-up to a recently developed cell- recycle bioreactor system for high-titer production at bench-scale. Additionally, this work has been extended to methane utilization via incorporation of methanotrophic enzymes that allow the biological conversion of methane to methanol in recombinant E. coli. By using a 13C-methane tracer, we demonstrate incorporation of methane-derived carbon into central metabolites during gas fermentations in engineered E. coli. This work greatly expands the knowledge of the synthetic methylotrophy field by highlighting the importance of using reduced C1 compounds, alone or together with sugar-based feedstocks, for the production of liquid fuels and chemicals.

BIOT 424

Rewiring amino acid biosynthesis via regulator modulation and operon overexpression improves methanol utilization in a synthetic Escherichia coli methylotroph

Alec F. Agee, [email protected], Gerald Har, Maciek R. Antoniewicz, Eleftherios T. Papoutsakis, R. Kyle Bennett. Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware, United States

The abundance of natural gas has made reduced one carbon compounds like methane and methanol attractive feedstocks for commodity chemical and fuel production, particularly in bioprocesses where they can replace more expensive sugar substrates. While many organisms natively consume one carbon substrates, these microbes (known as methylotrophs) lack many of the well-developed genetic engineering tools that platform organisms possess, which limits their production capacity. Engineering methylotrophy in industrially popular organisms is a promising alternative – in particular, progress has been made to engineer Escherichia coli to consume methanol. However, to date, no E. coli strain has been developed that can grow on methanol as a sole carbon substrate. We have identified amino acid biosynthesis as a crucial bottleneck to sole methylotrophy using 13C labelling – while most metabolites in glycolysis and the TCA cycle show substantial methanol incorporation, labelling in amino acids is significantly lower, and many amino acids lack methanol carbon entirely. Starting with an E. coli ΔfrmA variant expressing heterologous ribulose monophosphate (RuMP) pathway enzymes, we achieved greater methanol incorporation into amino acids using a variety of metabolic engineering strategies. Improvements were made by modulating regulators of amino acid biosynthesis, overexpressing enzymes for the biosynthesis of specific amino acids, and utilizing enzyme variants that do not possess traditional allosteric inhibition. The best variants from each method were then combined, yielding additive benefits to methanol incorporation. Starting with strains generated wth these combined strategies, adaptive laboratory evolution was performed to further improve methanol incorporation. Evolved isolates demonstrate methanol utilization for the production of all proteinogenic amino acids, providing a powerful platform for engineering sole methylotrophy.

BIOT 425

Production of oxidized terpenes using P450 enzymes and their engineered enzyme fusions

Taek Soon Lee1,2, [email protected], Xi Wang1,2. (1) Joint BioEnergy Institute, Emeryville, California, United States (2) Lawrence Berkeley National Lab, Berkeley, California, United States

Terpenes are a large class of organic compounds, primarily produced by plants and constitute the main components of essential oils. Functionalization of the terpene carbon backbone using cytochrome P450 enzymes could derive more useful compounds that can be converted to higher value products. In this study, cytochrome P450 enzymes were investigated for oxidizing monoterpene (e.g. limonene, 1,8- cineole), a few oxidized terpenes were produced, including carveol, carvone, limonene 1,2-epoxide, 2-endo-hydroxy-1,8-cineole. Based on those oxidized terpenes, possible biosynthetic pathways were explored toward making biology-derived functionalized terpenes, such as monomer of the polymer.

On the other hand, terpenes are hydrophobic and volatile compounds that limit the accessibility of P450 enzymes. To optimize the production of oxidized terpenes, we developed an enzyme fusion strategy with linking terpene synthase and P450 enzyme. In an example of producing 2-endo-hydroxy-1,8-cineole, the engineered fusion showed higher efficiency from both in vitro and in vivo production results. Thus, engineering a fusion between terpene synthase and P450 presents a feasible strategy for producing oxidized terpenes, suggesting broad application during the production of terpene-based bioproducts.

BIOT 426

Gram-scale production of psilocybin in E. coli Alexandra Adams1, [email protected], Nicholas Kaplan1, Phil O'Dell2, Chantal Monnier1, Alexis Enacopol1, John A. Jones1. (1) Chemical, Paper, and Biomedical Engineering , Miami University, Oxford, Ohio, United States (2) Physics, Miami University, Oxford, Ohio, United States

Here, we will present our ongoing work on the development of an in vivo production process for the natural product, psilocybin, in the model organism Escherichia coli. Psilocybin is of particular interest to the scientific community and the general public, as it is the active component of hallucinogenic ‘magic’ mushrooms and is currently in clinical trials as a medication for treatment resistant chronic depression, post traumatic stress disorder (PTSD), and other neurological diseases. With the re-discovery of the drug’s medicinal value, efforts have increased to create a more cost-effective and reproducible method for the production of psilocybin. Our efforts in traditional genetic and fermentation conditions optimization, have yielded titers over 1 g/L in fed batch fermentation, the highest titer reported to date for psilocybin from a recombinant host. The process to achieve these titers includes screening of multiple defined and random transcriptionally-varied libraries as well as a combinatorial analysis on the effects of varying induction conditions, temperature, and media supplements on overall product titer. Our continuing work towards developing a better understanding of the production landscape of this strain as well as the development of a more efficient and stable strain to further enhance psilocybin production in E. coli will also be discussed.

BIOT 427

Genome engineering for expanded inducible gene expression in lactic acid bacteria

Samuel Rothstein1, [email protected], Thomas J. Mansell2. (1) Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States (2) Chemical and Biomolecular Engineering, Iowa State University, Ames, Iowa, United States

Lactococcus lactis plays a vital role as a starter culture in dairy fermentations, exhibits remarkable protein secretion capabilities, and is Generally Regarded As Safe (GRAS). A limitation for the use of this organism for industrial and probiotic applications is the shortage of inducible gene expression systems, an essential tool for heterologous protein expression. Using a native homologous recombination and a unique in trans antibiotic selection system, we have inserted several promoter-transcription factor pairs for orthogonal expression of target proteins. The scar-less integration of these inducible gene expression systems lends itself to iterative functionalization of different inducible gene expression systems. In particular enabled induced expression from three different orthogonal inducers. Furthermore, the methods used to introduce these gene expression systems uses a mechanism that which lends its use in lactic acid bacteria beyond L. lactis.

BIOT 428 Investigating the effects of codon optimization and ribosome binding sequences on heterologous protein expression in Synechocystis sp. PCC 6803 for improved alpha-bisabolene production

Jacob Sebesta, [email protected]. Chemical and Biological Engineering, Colorado State University, Fort Collins, Colorado, United States

Biofuels produced by cyanobacteria have the potential to reduce the cost and climate change impacts of biofuel production. Carbon fixation and conversion to fuel may completed together in the cultivation of the cyanobacteria. Organic carbon feedstocks are not required, which reduces competition for agricultural resources. Reported product titers for most molecules of interest produced using cyanobacteria lag behind what has been achieved using yeast and E. coli cultures. In part, this is due to many achievements in the control of gene expression in those organisms. We attempted to increase the product titer of the sesquiterpene, bisabolene, which may be converted to bisabolane, a possible diesel replacement in Synechocystis sp. PCC 6803 (S. 6803). One initial strain expressed the bisabolene synthase gene from the grand fir tree (Abies grandis), codon optimized by GenScript, with transcription driven by the IPTG-inducible Ptic2op promoter. This strain produced 1.1 ± 0.08 mg/L bisabolene after five days of growth in continuous light. To increase the expression of bisabolene synthase, we varied the codon usage of the bisabolene synthase gene, using commercial gene synthesis company codon optimization services as well as the academic license software, EuGene, to design four additions coding sequence. At least three ribosome binding sites (most of which were designed using the RBS Calculator) were tested for each codon usage sequence. The effect on bisabolene production of coexpression of the farnesyl pyrophosphate synthase gene from E. coli, which catalyzes the formation of the precursor for bisabolene, was also tested. Bisabolene titers ranged from undetected to 7.9 ± 0.6 mg/L. Bisabolene synthase abundance was measured by Western blot and found to be well correlated with titer. To move towards the conditions engineered strains might experience when grown in industrial settings, two strains were also tested in 12:12 light:dark cycles. Similar titers were reached after the same amount of light exposure time for both strains. One engineered strain was also tested in 1.5L photobioreactors exposed to sinusoidally oscillating light (with a 12-hour dark period) with maximum light intensity of 1,600 μmol photons m-2 s-1the bisabolene titer reached 22.2 mg/L after 36 days of growth.

BIOT 429

Adherent HEK293T cells cultured in Pall’s iCELLis® nano bioreactor with OptiPEAK HEK293T blood-free chemically defined media exhibit robust and rapid population doubling times

Andrew Laskowski1, [email protected], Todd Lundeen1, Randall Alfano2, Annie Cunningham2, Atherly Pennybaker2. (1) Pall Corporation, Westborough, Massachusetts, United States (2) InVitria, Aurora, Colorado, United States In the past five years, Pall’s iCELLis bioreactors have emerged as the leading bioreactor technology for clinical manufacturing of viral vectors and vaccines. To date, the majority of products manufactured in the iCELLis bioreactors have utilized serum- containing media formulations that were developed primarily for traditional flatware. These media formulations may not be ideal for three-dimensional culture or for mature processes in clinical manufacturing. InVitria has developed OptiPEAK HEK293T, a blood free and chemically defined media optimized for use in the iCELLis bioreactors unique dynamic environment. Chemically defined and blood-free medias provide manufacturers less regulatory barriers to overcome by mitigating the supply chain risk and variability that comes from serum. Adoption of InVitria’s media formulation in the iCELLis bioreactor can result in efficient cell attachment, cell population doubling times comparable to traditional serum-containing medias, and acceptable post seeding growth lags. In this poster, we demonstrate the successful implementation of OptiPEAK HEK293T blood-free media formulation in the iCELLis bioreactor for adherent HEK293T. We begin by demonstrating rapid and uniform cell attachment to the iCELLis fixed-bed bioreactor, followed by robust expansion kinetics found to be equivalent to those observed in serum-containing media. These results enhance the regulatory pathway for product approval by removing supply chain risk and reducing risks associated with highly variable serum composition in large scale clinical manufacturing.

BIOT 430

Integrated omics modeling of trace metal effects on lactate metabolism and product quality for improved bioprocess control

Ashli Polanco, [email protected], Ryan Graham, Hemlata Bhatia, Seongkyu Yoon. Chemical Engineering, University of Massachusetts Lowell, Lowell, Massachusetts, United States

Trace metals impact various cellular functions in Chinese hamster ovary (CHO) during mAb production including lactate metabolism, energy metabolism, specific productivity, and product quality. The impact of metals on oxidative stress, apoptosis, and mAb production are well studied; however, the mechanisms behind metal-induced changes in lactate profiles and product quality are poorly understood as the role of metal ratios and metal competition during production are difficult to characterize. Understanding the route of action of metal co-factors during production and their effects on lactate consumption and post translational modifications could improve current fed-batch processes by establishing trace metals as a process control lever. This project aims to investigate these mechanisms via an integrated “-omics” approach, where cell culture samples under various metal ratio conditions are compared based on changes in cell culture performance (i.e. lactate metabolism), mRNA expression (transcriptomics), metabolite profiling (metabolomics) as well as protein expression (proteomics) and product quality profiles. The experimental design focuses on varying the levels of three metals that have known effects on mAb glycosylation and/or lactate metabolism to different degrees – zinc, copper, and manganese. Manganese is a well-known cofactor for several enzymes involves in N-linked glycosylation, while copper and zinc can modulate glycosylation and basic charge variants, respectively. Preliminary results confirm supplementation of zinc or copper alone (up to 100uM) leads to decreased peak viable cell density and increased specific productivity across three cell lines. These two metals also demonstrate the ability to delay or inhibit lactate consumption in batch cultures, and lactate phenotypes are cell-line dependent. Glycan profiles from the DoE with Zn, Mn, and Cu will be evaluated to determine the bioprocess impact of trace metal ratios and subsequent lactate profiles on product quality attributes. Then genomic, metabolomic, and proteomic analyses of metal-spiked cultures will provide the bases for functional and pathway analysis (fluxomics). Finally, all significant findings will be integrated into a multivariate data analysis model to assess the mechanisms most impacted by metal cofactors during production and hypothesize metal ratio limits that modulate both lactate metabolism and key product quality attributes in a cell-line specific manner.

BIOT 431

Specific productivity-based clone stability study in deep-well plates

Chunyan He, [email protected], Parimala Bolisetty, Ping Xu, Sen Xu, Gabi Tremml, Anurag Khetan. Bristol-Myers Squibb, Pennington, New York, United States

Manufacturing of biological products using animal cell cultures requires a clear definition of the limit of in vitro cell age. Clone stability study is a necessary but yet time- consuming part in cell line/upstream process development. Routine stability studies compare clone performance at different population doubling levels (PDL, or generation) using a pre-defined fed-batch process. This process is often the rate-limiting step in early development. Accelerating stability study process can fasten lead clone selection and shrink FIH timeline.

In this study, we evaluated a new clone stability study method based on passaging cell specific productivity (Qp) in deep-well plates. The new stability method tracks Qp during routine cell passaging and compares Qp values at different PDLs to assess clone stability. Our results showed that clone stability ranking was comparable between the passage Qp method and the current method using a fed-batch process. Compared to our current method, the passage Qp method could reduce two weeks in stability study. Furthermore, deep-well plates can be handled by an automation system, which enables potential stability study automation in the future.

BIOT 432

Considerations during working cell bank manufacturing for long term robustness and reliability

Jie Xu, [email protected]. Merck, West Point, Pennsylvania, United States Working Cell Banks (WCBs) are a critical starting material for biologics manufacturing, yet the process by which WCBs are produced is often less characterized than the rest of the manufacturing process. The objectives of this project were to further characterize a typical WCB production process using a mammalian monoclonal antibody (mAb) cell line and identify areas of consideration during WCB process development and technology transfer/commercialization to ensure long term robustness and reliability. Case studies demonstrating enhanced WCB process understanding and characterization will be discussed, specifically: temperature sensitivity of cells during expansion stages, impact of cryoprotectant contact time during the pre-freeze stage, optimizing growth for yield enhancement, and new technology introduction including an automated cell counter to replace manual cell counting. Together, consideration of these factors during initial WCB process development will help ensure that more robust and reliable WCBs are in place for subsequent technology transfer/commercialization and mitigate potential WCB supply risks for biologics manufacturing.

BIOT 433

Search for aromagenic organisms for fermentative production of aroma in bioreactors: Early studies

Runima Das1, [email protected], Pearlin S. Naziz1, [email protected], Arup Khakhlari1, [email protected], Shiny C. Thomas1, [email protected], Supriyo Sen1,2, [email protected]. (1) Department of Biosciences, School of Life Sciences, Assam Don Bosco University, Sonapur, Assam, India (2) Ouija Biosolutions Pvt. Ltd., Guwahati, India

Aromas produced by various microorganisms are natural and have great economic potential. If this aromagenic potential can be scaled up to industrial-level production, it can be a major augmentation to the aroma diversity. The present research is therefore based on the hypothesis that aroma environments can harbour aromagenic microorganisms. We present our early findings in this paper.

We have surveyed the aromatic parts of different plants such as citrus (Citrus reticulata) leaves and rind, mint (Mentha sp.) leaves, coriander (Coriandrum sativum) leaves, guava (Psidium guajava) leaves, tuberose (Polianthes tuberosa) petals, yesterday- today-tomorrow (Brunfelsia pauciflora) petals. Similarly, agarwood (Aquilaria malaccensis) is a major aromatic plant known for its fragrant resin which is formed by a complex association of mostly microorganisms. Culturable fungi and bacteria were isolated from the samples of aromatic plant parts as well as agarwood fermentation basin by standard procedures. A total of 25 fungal isolates from different aroma associated plants and 10 fungal and 20 bacterial isolates from agarwood fermentation basins were subjected to biochemical assays such as lipase, cellulase, esterase, protease, xylanase activity (Figure 1). Their molecular identification by rDNA sequencing along with enzyme-based screening showed interesting links to aroma. Aromagenic organisms are the unexplored part of the microbiome where the enormous scope for R&D exists. We are presently investigating as to whether aromagenic organisms can stimulate fermentative production of aroma when grown in bioreactors taking agarwood fermentation as the model.

Figure 1: Search for aromagenic organisms: (a) Sample collection from aroma associated plants and (b) agarwood fermentation basin; bacterial (c) and fungal (d-f) isolates from agarwood and citrus, respectively.

BIOT 434

Utilization of the ambr®250 and a near real-time amino acid analyzer to understand the effects of common, industrially relevant mammalian cell culture stressors on process performance and product quality

Kathryn Elliott1, [email protected], Benjamin Synoground1, Christina Leuze1,2, Stephanie Klaubert3, Madison Williamson4, Christopher Saski5, Glenn A. Harris6, Sarah W. Harcum7. (1) Bioengineering, Clemson University, Clemson, South Carolina, United States (2) Molecular Biotechnology, Heidelberg University, Heidelberg, Germany (3) Chemical Engineering, Clemson University, Clemson, South Carolina, United States (4) Biochemistry and Genetics, Clemson University, Clemson, South Carolina, United States (5) Plant and Environmental Sciences, Clemson University, Clemson, South Carolina, United States (6) 908 Devices Inc., Boston, Massachusetts, United States

Despite intense efforts to improve Chinese hamster ovary (CHO) productivity, inherent cell culture stresses are unpredictable outcomes and can affect critical product quality attributes (CQAs). Common stressors in bioreactors are ammonia, lactate, and osmolarity, which are all metabolic byproducts that have been shown to adversely affect cell growth, protein productivity, and CQAs. In this study, industrially relevant levels these stressors were individually introduced to CHO cell cultures in an ambr® 250 system. Amino acid fluxes were quantified and glycosylation profiles for the monoclonal antibody, the main CQA, were characterized.

As expected, the 30 mM ammonia-stress resulted in significantly decreased growth and poorer protein quality. In contrast, the 10 mM ammonia stress did not significantly affect growth until much later in the cultures. It was observed that the ammonia-stresses (10 mM and 30 mM ammonia) caused a dose-dependent response with respect to genome instability events. Conversely, the alanine flux profiles showed significant differences between the control and 10 mM ammonia-stressed cultures. A transcriptome approach was then used to discern the underlying metabolic shift in the cells due to the 10 mM ammonia-stress. Interestingly, initial ranges of osmolarity stress did not significantly affect cell growth. However, it has been shown that lactate buildup can affect cell productivity and growth. Combined, these results could lead to more accurate process and product quality predictions.

BIOT 435

Evaluation of commercial basal and feed media for increased mAb production

Yuan Yuan, [email protected], Lisa Hodge, Amlan Das. Global R&D, Teva Pharmaceuticals, West Chester, Pennsylvania, United States

Recent trends in biopharmaceutical industry points to increased cellular productivity for mAb fed-batch process. In order to reach higher titer, the compositions in both basal and feed media need to be well balanced to meet the cell demands. We evaluated several commercially available media and feeds in a fed-batch process using high throughput system ambr15, which is an automated high throughput cell culture system that allows 24 cultivations running simultaneously at micro-bioreactor scale. Three media and three feeds were screened in a combinatorial manner in ambr15 to identify the lead basal-feed media combination. Additionally, spent media analysis was performed and key limiting components were identified for each of the media and feed pair that led to a modified medium-feed formulation. The cells in the modified medium- feed formulation demonstrated the high cell growth performance, titer and acceptable product quality attributes. BIOT 436

Evaluation of commercial media and feed for improved mAb production

Baohua Zhang, [email protected]. Teva Pharmaceuticals, West Chester, Pennsylvania, United States

Fed batch cell culture process has been a well-established process in the biopharmaceutical industry for last few decades. An emerging trend has been to develop high-titer cell culture processes to meet increasing market demands and reduce manufacturing costs. To improve the product titer and reduce the manufacturing cost, significant advances have been made in the media and feed supplements offered by commercial vendors. We evaluated several commercially available media and feeds for improved titer. Two basal media and six feed media were screened in a sequential manner in ambr15 micro-bioreactors. Spent media analysis was performed on the best media-feed pair and key limiting components were identified. Supplementation with limiting amino acids, the product titer was doubled compared with the platform process. The modified fed-batch process was further verified in bioreactors at bench scale and similar cell growth performance and product titer was demonstrated.

BIOT 437

Diving deep: Impact of raw material variability on bacterial fermentation

Nikodimos Gebreselassie1, [email protected], Ahmet Bozdag2, Heather Vu3, Qiuwei Xu3, Hari Kosanam1, Roberto Ortiz1, Jon Shanter2. (1) Vaccines Process Development and Commercialization, Merck, West Point, Pennsylvania, United States (2) Vaccines Process Development, Merck, West Point, Pennsylvania, United States (3) Analytical & Bio Chemical Tox, Merck, West Point, Pennsylvania, United States

Complex media has been a ubiquitous platform for most biotechnology fermentation processes. Components of complex media such as soy peptone, yeast extract, and tryptone are readily available and cheaper compared to defined media components. Additionally, these components provide a rich environment that promotes fast and robust growth for various organisms, enabling relatively faster process development timelines. Intended or not, these characteristics of complex medium have reduced the need for experimental studies to understand the true minimal nutrient requirements for growth of a wide variety of microorganisms. This gap in knowledge makes it challenging to identify and easily troubleshoot any media related growth defects, particularly when the microorganism’s growth is sensitive to the manufacturer lot of the undefined raw material media component. In this work, we describe growth defects encountered during the scale up of a fermentation process and the troubleshooting effort that led us to identify a raw material component as the root cause. Through follow up spiking studies of each media component, we were able to relate growth defects with the specific lot of yeast extract used in the media. Additionally, we leveraged advanced LC-MS and NMR analytical tools to better gain valuable insight into the various consumption and production profiles of more than forty nutrients throughout the course of the fermentation. Nutrient profile comparison analysis including multivariant statistical analysis revealed linkage between media components and growth performance. These findings provide an insight into the impact of raw material variability on growth performance and a path forward in using the information for media screening.

BIOT 438

Multifaceted use of ambr250 high throughput system in upstream process development

Preet Marwaha, [email protected], Michael Faiella, Bryon Martinez, Baohua Zhang, Amlan Das. Teva Pharmaceuticals, West Chester, Pennsylvania, United States

The increasing demand for biologics has led to the wide-spread adoption of high throughput systems. High throughput systems provide flexibility, efficiency, and speed to commercialization. ambr250, an automated high throughput bioreactor system allows for the independent control of 24 bioreactors. Upstream process development has traditionally been done in bench-scale bioreactors. The ability of ambr250 to control each bioreactor separately allows performing process development experimental conditions more efficiently as compared to bench-scale. We successfully demonstrated that ambr250 could be used for various upstream activities such as media-feed screening, clone selection, process development. Evaluation of commercial media and feeds was done in the ambr250, and the optimal combination was found. The ambr250 also allowed for faster clone selection. The ambr250 allowed for process development evaluation in a high throughput fashion to explore product quality.

BIOT 439

Characterization of hyperosmotic adapted CHO host cells for fed-batch and intensified perfusion process

Xiaowen Wang, [email protected], Jack Huang. Merck, Kenilworth, New Jersey, United States

Increased osmolarity of medium has immediate and prolonged impacts on cell culture performance of Chinese Hamster Ovary (CHO) cells expressing monoclonal antibodies (mAbs), including cell growth, cell-specific productivity, and product quality attributes. Cells under hyperosmotic challenge are subjected to multiple stresses, such as mitochondrial depolarization, reactive oxygen species production, and DNA damage, which can lead to cell cycle arrest and eventual cell death. However, hyperosmotic pressure correlates to high specific productivity in CHO cells, which is often observed in fed-batch culture. In addition, concentrated media have been recently implemented to maximize the efficiency and productivity of perfusion culture, which also exposes cells to these high osmolarity challenges. Therefore, cell lines that are resistant to hyperosmotic stress are needed to enable robust, productive, and flexible (intensified or non-intensified) cell culture processes. In this work, we evaluate the impact of adapting CHO cells to hyperosmotic medium. The adapted cells are characterized and compared to the parental cell lines for phenotypic changes. In addition, adapted mAb-producing cell lines are compared to parental lines for product quality attributes such as N- glycosylation and sialyation. Comparison of the cell culture performance and product quality attributes between adapted and parental cells are investigated in both fed-batch and perfusion cultures. Omics approaches are applied to the characterization of adapted cells to fully understand the cellular hyperosmotic response at the genetic and molecular levels, and to guide the design of rational cell engineering strategies to develop highly productive and hyperosmotic-resistant cell lines.

BIOT 440

Use of PAT to facilitate development and tech transfer of a cell culture process satish cheepala2, [email protected], George X. Zhou2, [email protected], Alexander Hoffman1, Katherine Lee Jennison2, Anuj Upadhyay2, Shahid Rameez2, Robert Leighty2, Megan Barron2, Shailen Singh2. (1) Merck & Co Inc, West Point, Pennsylvania, United States (2) Biologics Process Development and Commercialization, Merck Manufacturing Division, Merck & Co Inc, Kenilworth, New Jersey, United States

Development of process analytical technology (PAT) tools in parallel with cell culture development provides a mechanism to design, analyze and control biological processes through measurement of process parameters which affect critical quality attributes (CQAs). Some examples of PAT tools used in cell culture processes include capacitance probes and Raman probes, which can provide real-time monitoring of performance parameters such as metabolites, titer, and amino acid concentrations. The goals of this project are to 1) develop methods for online monitoring of metabolites and titer, using in-line Raman probe, and 2) develop in-line capacitance probe enabled real- time monitoring of VCD. Capacitance and Raman data is collected from multiple bioreactor runs and used to develop partial least square (PLS) calibration and prediction models for real time monitoring of cell culture process parameters. For example, multiple data points for VCD and metabolites derived from Raman models will enable optimization of the cell culture process to increase productivity and control the variability of the CQAs. In addition, real time monitoring of VCD using capacitance probes will help to streamline the transfer of cells to production bioreactor. In conclusion, PAT tools play a critical role in developing robust cell culture processes. Furthermore, experience gained in developing the PLS models and application at small scale will simplify the process of transferring PAT technology to manufacturing sites.

BIOT 441 Modulating cell line stability through media and process selection for a monoclonal antibody

Ashley Bui, [email protected]. Upstream Process Development, Merck, Kenilworth, New Jersey, United States

Loss of productivity during the industrial production of monoclonal antibodies is sometimes observed in recombinant Chinese Hamster Ovary cells. This work highlights a case study demonstrating upstream process development efforts to address challenges associated with cell line instability and low production yield. Two molecular mechanisms known to cause production instability are loss of recombinant gene copies after subculturing over many cell generations and epigenetic gene silencing via DNA methylation. Genetic characterization of our cell line revealed no loss of gene copies and that addition of a DNA methylation inhibitor, 5-Aza-2'-deoxycytidine (Decitabine), had a negligible effect suggesting that the change in phenotypic performance is not attributed to this typical genetic mechanism. Instead both clone stability and productivity are heavily dependent on passaging and production media, along with the feeds used as part of a fed-batch process. Evaluation of multiple commercial and in-house media and feeds led to the identification of an optimized combination, which resulted in minimal loss of stability over 60 population doublings while increasing titer in the fed- batch production cultures by 4-fold. Further development revealed that additional metal supplementation during the production step corrected the undesired metabolic profiles and phenotypes associated with cell age. Overall, we were able to develop a robust and manufacturable seed expansion and production scheme with stable and improved cell culture performance while maintaining comparable product quality attributes.

BIOT 442

Expanding the protein expression toolbox for mammalian cell line development

Noelia Blanco1, [email protected], Kristine Daris1, Eric Gislason2, Nicole Sabo1, Charilyn Tejamo1, Jennitte Stevens1, Natalia Gomez1, Huong Le3. (1) Cell Line Development, Amgen, Thousand Oaks, California, United States (2) 2Terapeutic Discovery, Amgen, Thousand Oaks, California, United States (3) Process Development, Amgen Inc., Thousand Oaks, California, United States

A CHO glutamine synthetase knockout (GSKO) host was developed in Amgen to generate high producing cell lines in shorter timelines. Although the overall performance was favorable over the current host, several steps needed further optimization. Here is described an integrated approach that includes host generation and assisted evolution, vector designing, media and bioprocess optimization that enables an efficient, fast and flexible development process. Glutamine Synthetase (GS) gene was cleaved using genomic engineering tools. Then, GSKO host was conditionally adapted to grow into cell culture media. Next, we tuned the expression of GS from our expression vectors using promoters with different strengths. As GS acts both as a selectable marker and a means to achieve gene amplification, we determined the optimal selection stringency for each promoter using various MSX concentrations. Results from these experiments determined the best combination promoter plus selection stringency. As opposed to a traditional random integration, a transposon system was used to integrate the gene of interest into actively transcribed regions of the genome, enhancing the frequency of CHO stable cell line generation with superior productivity and stability. We compared efficacy and efficiency between different transfection methods to determine the best approach to produce different Amgen modalities. Finally, through media and feed strategy optimization, we achieved a substantial increase in titer with comparable product quality. Overall, this poster highlights a holistic approach to create the novel Amgen cell line development platform that would enable Amgen to satisfy clinical supply demands with a considerable reduction in timeline and cost.

BIOT 443

Rapid production of glycoengineered proteins via CHO transient transfection

Barry F. Wolf, [email protected], Ping Huang. Abbvie, Redwood City, California, United States

Therapeutic protein N-linked and/or O-linked glycoform characteristics can impact ADCC, CDC, immunogenicity, and other important drug properties. The ability to produce proteins with a wide variety of glycosylation patterns is crucial for the testing and selection of optimal candidates to move from Discovery to Clinical development. This work demonstrates that it is possible to control a variety of glycosylation patterns, through growth medium supplementation, in a CHO transient transfection system. Through chemical supplementation, galactosylation, fucosylation, mannosylation, and arabinosylation were altered, quantified, and compared with an identical protein expressed in a stable CHO cell line and altered through similar medium supplementation. The ability to quickly and controllably express glycoengineered proteins by means of a transient CHO process can aid in speeding candidate selection and time to clinic.

BIOT 444

At-line nutrient profiling from cell media in 7 minutes with a new integrated analyzer

Jiyoung L. Anderson1, [email protected], Kathryn Elliott2, Kenion H. Blakeman1, Colin Gavin1, Sarah W. Harcum2, Glenn A. Harris1. (1) 908 Devices Inc., Boston, Massachusetts, United States (2) Bioengineering, Clemson University, Clemson, South Carolina, United States

Characterization of small molecules is essential when optimizing media formulations to improve process productivity. Simple biochemical analyzers are limited by small target lists, so LC systems are often used to perform more expansive analyses. These approaches suffer from large sample volume requirements, time consuming sample prep, and lengthy run times. Presented here is a new automated CE-MS benchtop analyzer for targeted media analysis in under 7 minutes. An analytical target list was developed spanning 33 metabolites including amino acids, biological amines, vitamins, and dipeptides. On-board, automated algorithms were used to generate quantitative calibrations and verify performance throughout data collection. The analyzer required no sample derivatization and a minimum sample volume of approximately 10 µL.

The system was first evaluated alongside the standard LC method by running a chemically-defined CHO media where the reported amino acid concentrations from both methods matched. Nutrient formulations were then analyzed with Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/F12) from five different vendors. Up to 2.5x differences between the reported formulation of the DMEM/F12 samples were observed and no formulation matched the expected component profile. Finally, the analyzer was tested doing spent media analysis of a batch fed process of CHO NIH VRC01 cell line grown under different ammonia stress concentrations on an ambr®250. All bioreactors displayed net increases in the levels of Ala, Arg, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Thr, Trp, Tyr and Val, with Gly levels showing increases in excess of 8- fold initial levels. Other media components either steadily decreased in concentration or were completely depleted by the end of culture. In the future, the rapid analysis capability of the analyzer combined with the low sample volume requirements would allow for more frequent spent media analysis to occur for real-time observation of these process changes and associated control strategies.

BIOT 445

Kinetic modeling for enhanced understanding of a fed-batch mammalian cell culture process

Steve Provencher1, [email protected], Gregory J. Nierode1, [email protected], Sarah Fadda2, Edward Close2, Diana B. Ritz1. (1) GlaxoSmithKline, Philadelphia, Pennsylvania, United States (2) Process Systems Enterprise, London, United Kingdom

Development of cell culture processes for production of biopharmaceuticals such as monoclonal antibodies (mAbs) requires extensive experimental work. For instance, media and feed composition, feeding strategy, and an array of bioreactor set-points (pH, temperature, agitation, etc.) must be optimized to maximize production of high-quality product. To reduce this experimental burden, one attractive strategy is to use mechanistic models that capture key process dynamics to streamline experiment design and accelerate acquisition of process knowledge. In this work, we developed an unstructured, segregated kinetic model of a Chinese Hamster Ovary (CHO) fed-batch mAb production process using gPROMS FormulatedProducts, a commercially-available flowsheet-based modeling platform. We fit experimental data collected using a high-throughput bioreactor platform to the model and demonstrated that the model was able to capture the dynamics of several key cell culture parameters including glucose and lactate concentration, viable cell density, and mAb titer. Global Systems Analysis (GSA) was applied to refine the model parameters based on the order of parameter sensitivity, which also provided insight into the impact of process parameters on process performance. Finally, we evaluated the utility of the model to optimize various process conditions (e.g. feeding strategy, initial seeding density) to maximize the process yield. Efforts to evaluate the model accuracy for additional CHO lineages and mAb assets are ongoing.

BIOT 446

Evaluating perfused seed trains through process modeling and cost analysis

Jeffrey Barna, [email protected], Habib Horry, Douglas Rank. MilliporeSigma, Acton, Massachusetts, United States

The bioprocessing industry is driving towards intensified processes to reduce the cost of goods and/or increase productivity. From an upstream perspective, there are two areas of focus - intensified seed and intensified production. Perfused seed is one specific upstream process that plays a role in both areas. To gain a better understanding of the benefits of incorporating perfused seed trains in mAb production, BioSolve process modeling software was used to perform a holistic cost analysis. Process models of several different perfused seed train options were generated, as well as a traditional seed train model to compare to. In this study, scenarios were run evaluating the effect of variables such as production/seed train ratio, number of production bioreactors, titer, and production duration. Results showed that under certain scenarios, perfused seed train options could deliver lower cost of goods, increase product throughput, or a combination of both.

BIOT 447

Evaluation of psilocybin production under home brewing conditions

Chantal Monnier, [email protected], Madeline McKinney, Andrew Jones. Miami University, Paulding, Ohio, United States

Promising results from clinical trials run in both the United States and abroad have increased commercial interest in the legal production of Psilocybin for future pharmaceutical applications. The production of psilocybin has been achieved through applications of metabolic engineering and fermentation optimization, using a strain of Escherichia coli that has been engineered for maximum performance. Due to the success of psilocybin production in a controlled bioreactor environment, this sparks a curiosity on whether psilocybin can be produced in an unregulated setting (i.e. home brewing). This study will evaluate the potential to home brew psilocybin using an engineered microbe that has been optimized for use in a bioreactor setting. To accomplish this task, we will attempt to produce psilocybin using only equipment and chemicals that are available to the general public at reasonable cost and under sterility conditions consistent with typical home brew applications. We will use High Performance Liquid Chromatography (HPLC) to analyze the psilocybin titer and to compare home-brewing and bioreactor production conditions. All work with scheduled drug compounds is performed in an approved laboratory and all required safety and waste disposal regulations were followed.

BIOT 448

Metabolic engineering for the improved methylation of natural products

Alexis Enacopol, [email protected], Chantal Monnier, J. Andrew Jones. Miami University, Lemont, Illinois, United States

Escherichia coli has shown to be a suitable and sustainable host of production of a variety of methylated natural products including psilocybin, vanillin, and certain anthocyanins. For these pathways to work efficiently, the E. coli host must provide enhanced production and regeneration rates for the releveant precursor and co-factor metabolites needed for these pathways. Here, we focus on optimizing the efficiency of the methylation reaction catalyzed by the S-adenosylmethionine (SAM) - dependent psilocybin methyltransferase (psiM). Efforts to improve methylation performance through appilication of transcriptional optimization, targeted gene deletions, and substrate supplementation techniques will be presented. We focus our efforts on SAM regeneration while also considering the complexities of the methionine pathway regulation on overall cellular health.

BIOT 449

Modified feed formulation strategy for increased mAb productivity

Mathangi Madhavan, [email protected], Charles Hall, [email protected], Lisa Hodge, Kirby Neilon, Amlan Das. Global R&D, Teva Pharmaceutical, West Chester, Pennsylvania, United States

CHO cells have been the industry workhorse for producing therapeutic proteins for the last several decades. There has been a lot of focus on cell growth and antibody productivity to obtain high titers to meet clinical/commercial demands. Improvements in media and feed formulations have been one of the major contributors for obtaining high titer fed batch processes. We evaluated different feeding schemes on cell growth and cell productivity in the ambr15 system. The ambr15 is a high throughput, automated bioreactor system for 24 parallel cultivations at the 10 –15 mL microbioreactor scale. Lowering the feed led to increased cell growth and spent media analysis led to the identification of limiting media components. Supplementation of the limiting components, while lowering the feed, improved the productivity. Furthermore, we were able to successfully implement this process at bench scale.

BIOT 450 Impact of trace metal variability on CHO cell culture processes and application of mitigating control strategies

Mike Weiss, [email protected], Nicole Larmore, Kevin Clark, Jesse Ruben, Steven Savage, Jeffrey Simons. BioTD, Janssen, Malvern, Pennsylvania, United States

Variability in trace metal levels within CHO cell culture processes is known to impact CHO cell metabolism, with potential effects on product quality and process performance. Sources of this variability may include raw materials used in the bioreactor media and potential leaching from process equipment. The ranges in trace metal levels were quantified across multiple lots of a chemically defined media, and potential introduction of trace metals from equipment contact was quantified under worst-case process conditions for metals leaching. The observed ranges for each metal were compared to theoretical process values (i.e., media formulation targets), and experimentally evaluated for their impact on multiple CHO cell culture processes. Control strategies were developed to limit trace metal variability to a range demonstrated to be acceptable to the CHO cell culture processes.

BIOT 451

Development of modular Cas9 and lambda-red based system for chromosomal integration of large DNA constructs in Escherichia coli

Nicholas A. Kaplan, [email protected], Phil O'Dell, J. Andrew Jones. Department of Chemical, Paper, and Bioengineering, Miami University, Fort Wayne, Indiana, United States

As proof-of-principle metabolic engineering projects transition out of the laboratory for scale-up and commercialization, genomic integration of DNA constructs is realized as a necessity for strain stabilization and economic feasibility. However, techniques for integration of large biosynthetic pathways in E. coli are laborious and often scar- forming. Here we present our progress towards the development of a modular, lambda Red and Cas9 based system for site-specific and scarless chromosomal integration of large fragments in Escherichia coli. Protocol design is highlighted to demonstrate simplicity and modularity of this system. Efforts include integration of entire biosynthetic pathways, characterization of promoter at different genomic loci, and investigation of effects of fragment size on integration efficiency. A timescale for the complete process of integration is shown to illustrate the benefit of this platform for future industrial and academic efforts in the field of metabolic engineering. Finally, an economic benefits analysis is included to highlight the importance of chromosomal integration in commercial applications.

BIOT 452

Withdrawn BIOT 453

Model simulations to facilitate the scale-up of a CHO cell culture bioreactor process

Laura Prosperi, [email protected], Jeffrey Cohen, Christopher Canova, Mark A. Teeters. Janssen, Malvern, Pennsylvania, United States

A bioreactor model describing mixing and gas transfer is applied to facilitate the scale- up of a CHO cell culture process from a 2,000L to a 15,000L bioreactor scale. Multiple simulations of the 15,000L bioreactor were run to identify ranges of mixing and gassing parameters (e.g., agitation rate, air sparge cap) that provide outputs that are comparable to those observed at the 2,000L scale. Outputs include CO2 profile, interfacial cell loss, Kolmogorov eddy scale, and blend time, among others. In addition, model simulations were applied in a sensitivity analysis across ranges of input parameters or routine process variations in viable cell density. The model simulations are an efficient and cost-effective method to identify and reduce bioreactor process scale-up risks.

BIOT 454

Identifying glucose feeding profiles to control growth rate in CHO cells using precision pumping in the ambr®250 bioreactor system

Stephanie Klaubert1, [email protected], Kathryn Elliott2, Madison Williamson3, Bradley Skelton2, Cameron Schnabel2, Benjamin Synoground2, Sarah W. Harcum2. (1) Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States (2) Bioengineering, Clemson University, Clemson, South Carolina, United States (3) Clemson University, Clemson, South Carolina, United States

Fed-batch bioreactors are the current industry standard for production using CHO cells due to ease of scale-up and compatibility with large-scale manufacturing. In fed-batch microbial cultures, nutrients are continuously or semi-continuously fed, while the effluent may be removed and if so, discontinuously. Typically, fed-batch culture conditions are used to adjust metabolic rates and overcome substrate inhibition by controlled substrate feeding. Currently, most CHO cell culture fed-batch feeding strategies utilize daily bolus feeding. This discontinuous feeding approach often relies on off-line daily measurement of a key substrate (e.g., glucose) that targets to maintain this metabolite at or above a desired concentration. With daily bolus feeding, cells are typically not limited in growth and, therefore, exhibit an unrestricted exponential growth rate. By establishing an exponential feeding profile, a desired growth rate could be achieved by maintaining CHO cells in a state of substrate limitation according to a predetermined desired growth rate, much like already commonly practiced for microbial fermentations.

Preliminary data shows IgG production increases as cell growth begins to slow. A lower controlled growth rate may allow for redirection of cellular resources to protein production and reduce the burden of oxygen transport at high cell densities. Thus, it was hypothesized that slowing the growth rate earlier in the fed-batch phase for CHO cells would result in increased IgG production by allowing cells to redirect cellular resources to IgG production for a longer duration and ultimately result in higher IgG concentrations with improved protein quality. Pumping systems within the ambr®250 were used to control glucose feeding to achieve desired submaximal exponential cell growth rates. The results of this study show the impact of controlled glucose feeding on the growth profiles of CHO cells in fed-batch cultures and protein productivity.

BIOT 455

Clone ranking methodology for expedited process development using parallel workflows

Dane Grismer, Hongxia Chen, Rich Harper, David Chang, Yanjing Li, Niket Bubna, [email protected], Althaf Hussain, Gautam Nayar, Sigma Mostafa. KBI Biopharma, Durham, North Carolina, United States

Despite demonstration of clonal origin of cell lines, the mutations rates in CHO cells with increasing age in culture result in genetic diversity. Investigation of generational stability to fulfill regulatory expectations adds significant timelines to cell line development, which may bottleneck expedited workflows. We have established a comprehensive methodology to minimize relative instability of top clones identified for process development and establishment of a robust manufacturing process. The procedure for selecting a top clone includes tracking maintenance of rank order from static to shaken stages during cell line development, using standardized methods for cloning, development of culture medium sensitive for appropriate selection pressure, mapping flow cytometry-based diversity in subpopulations, and implementation of cell culture fed batch conditions. The fed-batch bioreactor study performed using a platform cell culture process is used to assess manufacturability and product quality. Clone performance is tested in a seed train followed by production in ambr15 or ambr250 bioreactors. A clone ranking system using weighted rank orders has been developed and successfully utilized for multiple cell lines. Outputs from this study are measured in a quantitative and qualitative approach and will be described. This integrated strategy for clone selection provides the basis for determining the top candidate clone while allowing for flexibility with different cell line types, molecule types and product filing status.

BIOT 456

CelliconTM perfusion solution

Allyson Fournier1, [email protected], Alison M. Dupont2. (1) R&D, MilliporeSigma, Bedford, Massachusetts, United States (2) EMD Millipore, Bedford, Massachusetts, United States Heightened cost pressures are challenging biopharmaceutical facilities to operate with increased speed and flexibility. Process intensification has become a strategic focus aimed at efficiently utilizing existing facility assets to deliver increased productivity. A key example of this is utilizing perfusion in place of traditional batch or fed-batch processes in upstream. Adoption of perfusion in N-1 bioreactors yields a reduction in process time and/or increase in manufacturing capacity without increasing volume capacity. A key technology within the perfusion application is the mechanism for removing fluid from the bioreactor while maintaining the cells in the system. Historically, the technologies available for retaining cells in the bioreactor have had limitations either mechanically, or with separation performance aspects such as throughput, efficiency, product retention, or process scalability. Due to recent advances in the cell retention technologies, in parallel to advancements in cell line and media development, perfusion processes are becoming a more viable alternative to expand performance beyond what traditional batch/fed-batch platforms can attain. To address this market need, MilliporeSigma is developing a family of pre-sterilized, high-throughput perfusion filtration solutions/systems with ease-of-use in mind and scalable from bench to production.

BIOT 457

Using RNA-seq to characterize underlying gene expression changes in observed alanine metabolic shifts of ammonia-stressed Chinese hamster ovary (CHO) cell cultures

Benjamin Synoground1, [email protected], Kathryn Elliott1, Christina Leuze2, Tom Caldwell1, Cameron Schnabel1, Sarah W. Harcum1. (1) Department of Bioengineering, Clemson University, Clemson, South Carolina, United States (2) Deparment of Molecular Biotechnology, Heidelberg University, Heidelberg, Germany

Recombinant cell cultures used in the production of biopharmaceuticals naturally produce and accumulate ammonia (NH3) as a metabolic waste byproduct, largely as a result of glutamine metabolism. At the conclusion of a typical 14-day Chinese hamster ovary (CHO) cell culture, ammonia levels have the potential to reach concentrations as high as 10-20 mM. This accumulation of ammonia in the surrounding media is toxic and inhibitory to mammalian cellular metabolism and has been shown to decrease cell growth, protein productivity, and protein quality. Preliminary metabolite data from parallel fed-batch CHO cells cultured in ambr®250 bioreactors, treated with either 10 mM ammonia or untreated, identified distinctive alanine metabolic profiles due to the treatment. To further examine the changes in the alanine profiles under ammonia- stressed conditions, samples from 0, 3, 6, and 9 days were sequenced via RNA-Seq. The subsequent read data was processed through a bioinformatics pipeline to determine differentially expressed genes and enriched gene sets to characterize transcriptome changes related to alanine metabolism. Since ammonia is also well- known to affect critical quality attributes, glycosylation transcripts, as well as other major differentially expressed genes and pathways were identified. BIOT 458

Characterization of single-use bioreactor 5:1 turndown ratio hydrodynamics

Evan Schlaich, [email protected], Colleen Clark, Sen Xu, Gabi Tremml, Anurag Khetan. Biologics Development, Bristol-Myers Squibb Company, Pennington, New Jersey, United States

Single-use bioreactors (SUBs, or disposable bioreactors) are widely used for clinical and commercial production of biologics. Reduced cross-contamination, floor space, down-time, and design rigidity commonly seen with traditional stainless steel reactors are some of the advantages to disposable bioreactors. Despite these benefits, minimal results have been reported utilizing the turndown ratio; a marketed advantage of the Xcellerex XDRTM system where the operating range can be expanded to a lower working volume. Here we present the successful application of the 5:1 turndown ratio and demonstrate the ‘short fill’ capability of the XDRs with the potential to replace rocker bioreactors.

Initially, we evaluated an Xcellerex 200 L bioreactor at 40 L working volume using scaled process parameters (gassing, agitation, and control schemes) to that at an initial working volume of 130 L. We observed minimal cell growth with these conditions. Under the low aspect ratio (H/T=0.25), we hypothesize that radial mixing remained inadequate over our standard agitation operating range (P/V at 12 to 20 W/m3). As a result, an increase in bubble rupture events occurred at the free-surface and mixing heterogeneity became detrimental. Computational fluid dynamic modeling revealed areas of fluid stagnation. In order to increase mixing uniformity and bubble-throw across the tank diameter, higher agitation speed was favored but found to cause significant vortexing. In this high mixing regime, surface turbulence was also linked to a decreased growth rate - likely due to bubble entrainment and the introduction of sparged gas directly into the vortex. To overcome fluid discontinuity yet minimize vortex formation and air entrainment, power-to-unit-volume was increased beyond standard operational levels (P/V at 107 to 209 W/m3), agitation was switched to up-pumping, and overlay-based oxygen control was implemented. Under such scheme, cell growth in the XDR was comparable to that in rocker bioreactors for multiple antibody producing-cell lines. Furthermore, cells taken directly from the turndown expansion and placed into production had identical growth and production characteristics to traditionally expanded cultures. Taken together, we demonstrated that a single XDR vessel can be run at a 5:1 working volume first as a seed tank and then operated as a production bioreactor, with no need for additional system modifications for simplified N-1 seed and production operations.

BIOT 459

Going all in: New technology evaluation for legacy cell culture processes for 3 live virus vaccines Lisa Toback, [email protected], Christopher McColgan, Kristin Valente. Global Vaccine Technical Operations, Merck , West Point , Pennsylvania, United States

For licensed manufacturing processes, the opportunity to evaluate new platform technologies is rare due to hurdles with product comparability and potential regulatory impact. When presented with this unique opportunity, there are a few approaches one can take including: a slow build of independently optimizing each unit operation for the novel technology as an iterative process, or conversely utilizing a “one-size fits all” approach that leverages the current manufacturing processes with scaling based on standard scaling factors (the “all in” approach). In this example, the approach to utilize the manufacturing processes with standard scaling (surface area) was selected. With any approach there are pros and cons. Benefits of this approach included: (1) optimizing speed so proof-of-concept evaluation for three antigens was completed within 3 months from equipment receipt, (2) enabling direct comparison to current manufacturing process, and (3) limiting the required process development . During the evaluation unexpected challenges were encountered such as: steep learning curve- interfacing and overcoming challenges in the novel technology, uncovering of uncontrolled variables, and limited applicability direct comparison to current processing outputs. This poster discusses the decision process for evaluation of a novel technology and demonstrates successful, accelerated evaluation of process modernization by the “all in” approach for three legacy vaccine products.

BIOT 460

Withdrawn

BIOT 461

Electrochemically-tunable, simultaneous CRISPR inhibition and activation through OxyRS promoter in Escherichia coli

Sally Wang1, [email protected], Narendranath Bhokisham2, William E. Bentley1. (1) Fischell Dept of Bioengineering, University of Maryland, College Park, Maryland, United States (2) IBBR-Plant Scis Bldg, Rm 6142, University of Maryland, College Park, Maryland, United States

CRISPR-Cas9 technology is a powerful genome-editing tool that originated from the bacterial immune system. Harnessing the ability to target any specific sequence, CRISPR-based genetic regulation techniques such as CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) have been developed subsequently. We have previously reported an electrochemically-tunable CRISRPa system through the OxyRS promoter in the presence of electrode-generated hydrogen peroxide. The OxyRS promoter, or the intergenic region of genes oxyR and oxyS, serves as the promoter for both coding regions. oxyR encodes a peroxide-sensing activator, which upon induction of hydrogen peroxide activates the expression of a regulon associated with oxidative stress. Specifically, this includes oxyS, which encodes a small, regulatory RNA that can further activate or repress the expression of other genes associated with eliminating oxidative stress. In this study, we intend to improve our electrochemically-inducible CRISPR system by using CRISPRi to inhibit oxyS expression in the genome to enhance OxyRS promoter activity. First, we will demonstrate that genomic oxyS expression can be successfully repressed with CRISPRi by targeting its coding region and furthermore result in a positive feedback loop on the OxyRS promoter. With this positive feedback loop, we anticipate showing an increase in the gRNA expression for CRISPRa since it is placed under the OxyRS promoter. We will also explore the possibility of getting improved CRISPRa response with the increased gRNA level.

BIOT 462

Cell culture process optimization for reducing protein aggregation

Shaunak Uplekar1, [email protected], Camila Rodrigues1, Brandon Brino1, Douglas Silva2, Gautam Nayar1, Sigma Mostafa1. (1) Process Development, KBI Biopharma, Durham, North Carolina, United States (2) Analytical Development, KBI Biopharma, Durham, North Carolina, United States

Aggregation of protein produced in cell culture processes is a critical attribute that affects its product quality. Protein aggregation is generally considered as a detrimental quality attribute essentially due to its potential to cause immunogenic responses in patients in addition to its negative impact on drug product formulation. Thus, most of the drug products have very low specifications for the aggregate levels. Aggregate levels can be reduced through downstream processing by achieving their clearance. However, this approach is typically associated with decreased product yield. Here, we present a case that has very high levels of protein aggregation in cell culture environment. Seed train and cell culture production was conducted using a Chinese hamster ovary (CHO) cell line. Cell culture studies were conducted using high throughput mini bioreactor systems such as ambr15 and ambr250. The samples were tested for aggregate levels using size exclusion chromatography (SEC). A design of experiment (DOE) based approach was used to screen different process parameters such as dissolved oxygen, pH, temperature and ionic strength of basal medium. In addition, a one-factor-at-a-time (OFAT) strategy was used to screen several additives such as chemical chaperones, metal ions, surfactants and amino acids. DOE approach identified process parameters such as pH that proved to be significant in the statistical model. Also, the OFAT strategy identified several additives that significantly reduced the aggregate levels. Finally, a combination of all the factors that provided significant improvement individually, were combined which provided further improvement in reducing the aggregate levels. This indicated a possible synergistic effect of the factors chosen for reducing the aggregate levels.

BIOT 463 Algorithm development and application for glucose feeding of CHO cell culture for process development

Steven Savage, [email protected], Stefanie Berges, James Price, Amalie Levy, Abbey Weith. Janssen Research & Development, Malvern, Pennsylvania, United States

Increasing demand for biotherapeutics has compressed industry timelines for the development of clinical manufacturing processes for new molecular entities (NMEs). This poses challenges to early process development teams that have limited information regarding newly generated cell lines. To meet this challenge, we have developed a statistically robust glucose algorithm capable of predicting daily glucose feed set points in order to maintain residual culture glucose at a desired level. The algorithm allows early process development teams to focus development efforts on process factors other than glucose feeding (e.g., complex nutrient feeding). The algorithm was developed by collecting process glucose data from 162 small-scale runs across six cell lines with varying phenotypes. The algorithm utilizes residual glucose values to the calculate the current glucose consumption rate along with historical cell density changes to predict the glucose consumption rate over the next 24-hour period. The predicted glucose consumption rate is used to calculate a glucose feed set point in order to maintain culture glucose at a desired level. Compared to process analytical technology, this approach offers a simple way to leverage historical glucose data to provide a generalized approach for maintaining glucose levels during early development. This algorithm has been applied to more than six early development cell lines and demonstrated consistent glucose control within desired residual glucose set point range. We believe this approach provides early development programs with an easily implementable method to control glucose without any preexisting knowledge of a cell line’s glucose consumption demands.

BIOT 464

Evaluation of retrovirus-like particle expression in fed-batch and perfusion as a function of cell density

Arshan Nazempour, [email protected], Douglas Rank, Michael Phillips, Audrey Chang, Herb Lutz. MilliporeSigma, Bedford, Massachusetts, United States

Viral safety is a key concern in the growing field of biologics produced by mammalian cells as viral contamination could have serious clinical consequences. Viral contamination can arise from different sources including but not limited to cell bank with latent or persistent virus infection, viruses expressed endogenously by the cells, raw materials, contaminated reagent from an operation unit such as affinity chromatography and handling. To ensure the safety of human therapeutics, it is not only important to understand the sources of viral contamination but also required by regulatory agencies to ensure prevention, detection and removal of such contaminants. Retrovirus-like particles (RVLPs) are expressed endogenously by Chinese hamster ovary cells and are considered as one of the sources for viral contamination. As so, it is integral to biologic manufacturing to investigate the life cycle and to quantify the level of RVLPs throughout bioprocessing unit operations. Considering today’s industrial trend toward continuous bioprocessing, there is a need to carry out studies into the life cycle of RVLPs in newly developed solutions/unit operations. One such area of interest is perfusion bioreactor processes. Compared to traditional fed-batch approaches, perfusion mode provides a favorable environment to the cells by continuous metabolite removal and nutrient addition. Rapid removal of toxic byproducts along with continuous supplement of fresh medium enable achievement of high cell density within perfusion bioreactors. However, the impact of increased cell density on RVLP expression has not been studied. In this poster, we present our investigation on evaluating the level of RVLPs measured by RT-qPCR as a function of cell density in fed-batch and perfusion bioreactors. To the best of our knowledge, our study is the first direct comparison of retrovirus content between the two different modes of bioreactor operation. This has implications for differences in the risk from therapeutics produced by each mode.

BIOT 465

Preferential expansion of vascular endothelial cells versus smooth muscle cells using chitosan surfaces

Ryan C. Mangulabnan, [email protected], Howard W. Matthew. Chemical Engineering & Materials Science, Wayne State University, Detroit, Michigan, United States

Vascularization of engineered tissues has proven to be challenging. In vitro assembly of arterioles or venules requires seeding with endothelial cells (EC) and smooth muscle cells (SMC). However, contact inhibition of EC often results in rapid overgrowth by SMC. Likewise, faster SMC growth rates necessitate expensive antibody-based methods to purify EC from mixed suspensions. Thus, simple techniques for preferentially expanding EC would reduce the cost of achieving high-purity EC cultures from mixed primary suspensions. Since chitosan films were previously shown to support EC growth while performing poorly as a SMC culture substrate, we studied the composition of mixed EC/SMC populations on chitosan films as a method for enriching the culture with EC. EC and SMC mixed suspensions were obtained by internal collagenase digestion of ovine aortas. Mixed suspensions were seeded onto tissue culture plates in M199 w/ 20% FBS and medium was changed every 3 days. After 14 days, cells were subcultured onto either plastic or chitosan-coated dishes. Chitosan dishes were coated by covering with 1% chitosan in 1% acetic acid and air dried for 24 hrs. Dried films were rinsed with 1 M NaOH and neutralized with PBS before cell seeding. Cultures were stained at intervals with DAPI, DiI-AcLDL, and FITC-anti-SM a- actin to quantify ECs and SMCs. Cultures initially contained 14.7% SMC. After 40 days on chitosan, SMC content was reduced to 0% and 98% of cells were positive for the endothelial marker DiI-AcLDL. On plastic, the SMC were reduced to only 6%. Results suggest that preferential expansion of EC vs. SMC is enhanced on chitosan films. Ongoing studies are examining the role of EC purity and EC-SMC signaling in these phenomena

BIOT 466

Development of a yeast-based system to elucidate phytosteroid biosynthesis

Yanran Li, [email protected], Shanhui Xu. UC Riverside, Riverside, California, United States

Plant natural products are important sources of pharmaceuticals, with more than 10% of the WHO listed essential medicines of flowering plant origin. However, the biosynthetic pathways of most phytochemicals are not fully elucidated - enzymes catalyzing certain steps in the proposed pathway are unknown. Without the comprehensive understanding of the biosynthesis of plant NPs, it is challenging to produce these expensive molecules and difficult to advance the discovery of novel plant NPs from the rapidly growing transcriptome data. Baker’s yeast Saccharomyces cerevisiae has been demonstrated to be a promising biotechnological production platform for plant natural products. Thus, S. cerevisiae has been considered to be a promising heterologous host to simultaneously reconstitute and elucidate plant natural product biosynthesis. Here, through a combination of metabolic engineering, strain evolution, and pathway reconstruction, a yeast-based system was developed to reconstitute and thereby elucidate the biosynthesis of phytosteroid and derivatives.

BIOT 467

Accelerating cell line development with rapid single cell and population level discovery on the Berkeley Lights Beacon

Christopher Tan, [email protected], Kim Le, Ewelina Zasadzinska, Jasmine Tat, Jonathan Diep. Amgen, Thousand Oaks, California, United States

The Berkeley Light’s Beacon is a nanofluidic cell culturing device that allows simultaneous culturing of thousands clonally isolated cells on a single chip. We have previously reported the utility of this technology to Cell Line Development through its ability to generate biologics manufacturing quality CHO cell lines with improved assurances and documentation of clonal derivation. Here, we report that the Beacon can be employed to isolate cell lines with higher specific productivity, shorter cell line development timelines, and improved prediction of population dynamics. The differentiating application of the technology is its ability to perform light and fluorescent-based live cell imaging assays at multiple time points to garner phenotypical information about each clonally isolated population prior to export and expansion for additional screening or genetic characterization. Berkeley Light’s commercially available developed Spotlight® assay utilizes a fluorescently conjugated anti-Fc probe to detect secreted protein in individual pens. The assay allows quantification of the steady-state secreted protein level in each pen at a specific time point. It is not clear whether measured secreted protein produced by a few cells in a nanofluidic environment at an early timepoint is predictive of future productivity at large-scale productions with many orders of magnitude more cells, in a different production environment, and over several days. Using a retrospective multi-regression approach, we find Spotlight® scores are highly predictive of cell specific productivity. With the ability to predict specific productivity at an early stage with a few cells, we also demonstrate how single cell subcloning processes can be initiated earlier in the typical cell line development campaign. This has enabled the generation of highly productive subclones in under two months. Combined, a cell line development platform integrated with the Beacon technology platform enables production of stable clonally-derived cell banks with higher productivity, shorter development timelines, and improved phenotypic characterization.

BIOT 468

Engineering complementary pairs of binding proteins via directed in vitro selection

Ki-Joo Sung1, [email protected], Eric Miller1, Patthara Kongsuphol2, Subha Baniya3, Hui Qi Aw-Yong2, Vivian Tay2, Yuxuan Tan2, Farah Kabir1, Karl Pang-Yeo2, Isabel Kaspriskie1, Hadley Sikes1,2. (1) Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States (2) Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore, Singapore (3) Biochemistry, Wellesley College, Wellesley, Massachusetts, United States

Sandwich immunoassays are commonly used for in vitro diagnostics of infectious diseases. In this format, one binding protein is immobilized to some surface and captures the target biomarker, while a second binding protein also binds to the biomarker to produce a signal for detection. This format requires the simultaneous binding of affinity reagents to non-overlapping target epitopes. Typically, affinity pairs are identified via post facto functional analysis of clones that were not selected for complementarity. Furthermore, antibodies are commonly used as the affinity reagents in diagnostic immunoassays; however, due to the limitations of antibodies for in vitro applications—including variable stability and issues with cross-reactivity—alternate binding proteins have been investigated to replace antibodies in in vitro diagnostics.

In order to directly engineer pairs of binding proteins against our target biomarkers, we developed the Rapid Affinity Pair Identification via Directed Selection (RAPIDS) process. This method uses in vitro libraries with directed evolution in order to enable the efficient identification of affinity reagents that function together as complementary pairs. For this study, we used the reduced-charged Sso7d protein (rcSso7d) as an alternative binding protein due to its inherent thermal stability, ease of genetic modification, and low-cost production in bacteria.

We used RAPIDS to develop highly-specific affinity pairs against biomarkers of Zika virus, and sepsis. We also incorporated negative selections to ensure specificity and low cross-reactivity of the identified clones. Without additional trial-and-error screening, these affinity pairs exhibited utility in multiple assay formats including bead-based assays, well-plate ELISAs, and paper-based assays. The RAPIDS process applies selective pressure to hundreds of thousands of potential affinity pairs to efficiently identify complementary pairs that bind to separate epitopes without binding to one another or non-targets. With the RAPIDS method, we demonstrate efficiency, versatility, and increased control over the development of functional affinity pairs for use in diagnostic assays.

BIOT 469

Engineering and mechanistic analysis of carbohydrate binding domain assisted transglycosylation reactions for a novel protein design of multidomain transglycosidases

Chandra Kanth Bandi1, [email protected], Tucker Bergin2, Madhura Kasture1, Antonio Goncalves1, Sai Venkatesh Pingali3, Jinshan Gao4, Heather Mayes2, Shishir P. Chundawat1. (1) Chemical and Biochemical engineering, Rutgers University, Piscataway, New Jersey, United States (2) Chemical Engineering, University of Michigan, Ann Arbor, Ann Arbor, Michigan, United States (3) Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States (4) Department of Chemistry & Biochemistry, Montclair State University, Montclair, New Jersey, United States

Glycans are been actively studied in recent years owing to their growing recognition and importance in regulating diverse biological processes. In nature, glycans are synthesized using membrane associated glycosyl transferases which are challenging to study, engineer or express for large scale synthesis. Alternatively, glycosyl hydrolases (GHs) can be reverse engineered and modulated to synthesize sugar polymers using their inherent transglycosylation property. Most GHs or glycosidases catalyze the hydrolysis of glycosidic bonds driven by catalytic domains (CDs) that are appended to autonomously folded non-catalytic protein domains called carbohydrate binding domains (CBMs). CBMs are thought to mostly improve hydrolysis activity through substrate localization in the proximity of CDs because of their ability to recognize and bind to carbohydrates. However, their impact on transglycosylation reactions is still poorly understood. Here, we study the influence of CBMs on transglycosylation activity of a novel chimeric transglycosidase enzyme design for synthesis of b-glucans. Surprisingly, we observed that some unique CBM designs help recover transglycosylation and partial hydrolytic activity of nucleophilic amino acid residue mutants of GHs. The molecular mechanism responsible for these unique multidomain chimeric transglycosidases was explored systematically using rational protein engineering and biochemical assays. Structural analysis using small angle X-ray scattering (SAXS) and molecular dynamics simulations reveal the presence of inter- domain flexibility, protein-protein domain interactions, and an unusual SNi-like front- facing mechanism for transglycosylation reaction using activated donor sugars. Furthermore, the CBM domain design was integrated to other GH family enzyme to show how this chimeric transglycosidases design strategy could be extended to other carbohydrate-active enzymes (CAZymes) for synthesis of glucans. Our results arguably corroborate an evolutionary relation of some engineered transglycosidases to native glycosyl transferase designs based on the similarities of the unusual SNi type nucleophilic substitution mechanisms employed by multidomain CAZymes towards formation of glycosidic bonds using activated donor sugar groups.

BIOT 470

Engineering Antimicrobial Proteins: Co-evolutionary models aid molecular discovery

Benjamin Hackel, [email protected], Seth Ritter, Tsvetelina Baryakova. Chemical Engineering and Materials Science, University of Minnesota - Twin Cities, Minneapolis, Minnesota, United States

Antibiotic-resistant infections are a pressing challenge in healthcare and agriculture. Antimicrobial proteins (AMPs) present a compelling opportunity for efficient discovery of a suite of potent, selective therapeutics with diverse modes of action. This seminar will discuss the development and use of protein engineering platforms to enhance stability, selectivity, and potency in AMPs via a hybrid of bioinformatics-guided library design and high-throughput discovery assays. Co-evolutionary sequence models enhanced library strategies to engineer enhanced stability and activity in endolysins against Clostridium perfringens and vancomycin-resistant Enterococcus. A high-throughput sequence depletion assay elucidated the sequence-function relationship in the oncocin AMP against E. coli. In addition to identifying enhanced AMPs, these methods could be broadly applied to protein engineering.

BIOT 471

Light-responsive monobodies for dynamic control of customizable protein binding

Cesar Carrasco-Lopez2, [email protected], Jared E. Toettcher1, Jose L. Avalos2. (1) Molecular Biology, Princeton University , Princeton, New Jersey, United States (2) Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States

Customizable, high affinity protein-protein interactions, such as those mediated by antibodies and antibody-like molecules, are invaluable to basic and applied research and have become pillars for modern therapeutics. The ability to reversibly control the binding activity of these proteins to their targets on demand would significantly expand their application in biotechnology, medicine, and research. Here we present, as proof- of-principle, a monobody whose binding to an SH2-domain target can be controlled with light. Constructed by inserting a light-responsive protein into a structurally conserved loop of the monobody fold, this light-dependent protein binder we call OptoMonobody (OptoMB) displays as much as a 300-fold increase in its binding affinity when conditions are changed from blue light to darkness, with complete reversibility. Our OptoMB shows light-dependent binding affinity to SH2 domain both in vitro and in mammalian cells. To demonstrate that our anti-SH2-OptoMB retains high selectivity for its target, we used it to purify SH2-tagged proteins directly from crude E. coli extract, in what we call light- controlled affinity chromatography (LCAC), achieving as much as 99.8% purity in one step of purification and more than 40% yield. This OptoMB belongs to a new class of light-sensitive protein binders we call OptoBinders (OptoBNDRs) which, by virtue of their ability to be designed to bind any protein of interest, have the potential to find new powerful applications as light-switchable binders of untagged proteins with high affinity and selectivity, and with the temporal and spatial precision afforded by light.

BIOT 472

Functional molecular imaging using calcium activated MRI reporter proteins

Harun Ozbakir, [email protected], Arnab Mukherjee. Chemical Engineering, University of California, Santa Barbara, Santa Barbara, California, United States

Optical imaging of intracellular calcium using genetically encoded calcium indicators is a mainstay of neuroscience research. Recent advances in multiphoton microscopy have extended the use of fluorescent calcium indicators to deeper layers in the animal brain. However, fluorescence techniques provide a limited field of view, which hinders the ability to study brain-wide patterns of neuronal activation. In contrast to optical imaging, MRI is ideally qualified for tomographic imaging of the entire mammalian brain with a spatial resolution approaching ~100 μm although MRI techniques suffer from a lack of compatible genetic reporters that can specifically detect molecular markers of neuronal activation such as calcium. In order to bridge this critical gap, we have purposed calprotectin as the first genetically encoded MRI sensor for calcium. This protein is a calcium-activated Mn(II)-sequestering protein that is usually secreted by neutrophils to combat pathogens. Importantly, it is only in the presence of calcium that it switches to a Mn(II) bound state, which produces a different paramagnetic relaxation effect in comparison to unbound state. For this purpose, we expressed this protein in E. coli, purified using affinity chromatography, and measured MRI signal strength (T1 relaxation) in vitro in the absence and presence of saturating amounts of calcium, where we observed up to 20% change. Additionally, we measured the dynamic range of the sensor and showed that it can detect calcium with a mid-point detection sensitivity of ~20 μM. We have also engineered stable Chinese hamster ovary (CHO) cell lines expressing calprotectin and observed up to ~ 40% increase in MRI signal strength upon introduction of saturating amounts of calcium to cells. We are currently working on optimization of this protein’s performance before performing a proof-of-concept assessment in living animals. The most recent in vitro, cellular, and in vivo results will be presented.

BIOT 473

Supercharged protein cargo-templated assembly of Archaeoglobus fulgidus ferritin cages

Joshua A. Bulos, [email protected], Zhiheng Wang, Maegan A. DeLessio, Rui Guo, Jeffery G. Saven, Ivan J. Dmochowski. University of Pennsylvania, Philadelphia, Pennsylvania, United States

Extensive work has been conducted in the field of encapsulation of cargo by a supramolecular protein complex. Many examples exist of loading a variety of cargo molecules, from enzymes to inorganic nanoparticles, using protein cages such as viral capsids, carboxysomes and ferritins. These have been used for a variety of applications, ranging from nanoparticle synthesis to developing novel methods for drug delivery. Recent studies have shown templated self-assembly of thermophilic ferritin (AfFtn) from Archaeoglobus fulgidus around a supercharged protein, GFP(+36), as well as around enzyme-GFP(+36) fusions. Here, the charge requirements of protein cargo to template self-assembly of AfFtn using GFPs is investigated with a spectrum of different surface charges (GFP(+28), GFP(+14), GFP(+9), GFP(-30)). Similarly, the surface of human carbonic anhydrase II (hCAII), a zinc metalloenzyme that hydrates CO2 into - HCO3 , was redesigned using computational protein design for encapsulation within AfFtn without the need to fuse it to GFP(+36). The designed hCAII variants retain catalytic function. The AfFtn-hCAII complexes are highly active and can potentially be used for CO2 fixation in extreme temperature conditions.

BIOT 474

IPROv2: Iterative protein redesign and optimization suite of programs with improved modularity and flexibility

Soodabeh Ghaffari, [email protected], Veda Sheersh Boorla, [email protected], Ratul Chowdhury, Costas D. Maranas. Chemical Engineering, Pennsylvania State University, Sate College, Pennsylvania, United States

IPRO is an iterative protein redesign and optimization tool that uses molecular mechanics calculations and Mixed Integer Linear Programming (MILP) to tailor an existing protein for targeted binding properties. Briefly, IPRO comprises of a backbone perturbation followed by an amino acid and a rotamer selection step by solving an optimization problem using MILP, target molecule redocking, complex energy minimization, computing interaction energy metrics in assessing whether or not to retain the design. IPRO has been successfully used for several applications ranging from redesign of pores for targeted solute separation, improved enzymes and high-affinity antibody variable fragments where covalent and non-covalent interactions are scored using CHARMM potential functions.

The current implementation of IPRO has a default script ‘Start_Experiment.py’ which user can use to not only restrict substituent amino acid choices for non-conserved binding pocket residues, but also enforce catalytic distance (angle, and dihedral) constraints by just answering a series of Yes or No questions. However, the force-field calculations use the same energy terms as listed in CHARMM irrespective of the fold- class of the protein at hand. Herein, we introduce a more flexible and modular version of IPROv2 written in a convenient Python interface. IPROv2 supports the use of four different force-fields CHARMM, AMBER, GROMOS and Rosetta. Additionally, it supports the use of Rosetta’s Monte Carlo Simulated Annealing based algorithm for rotamer optimization besides IPRO’s MILP. So, user can now design their own protocols by combining and customizing different available algorithms with varied force- fields. Also, in IPROv2, the dependency on GAMS (a licensed software which can formulate an optimization problem as a mathematical model) has been removed by directly generating the optimization model in MPS (Mathematical Programming System) file format which can be input to CPLEX solver to solve the MILP problem.

IPROv2 will be used to systematically evaluate these different force-fields and algorithms based on their ability to recapitulate structural details for designed proteins. These evaluations will be performed separately for different fold-classes of SCOPe database using experimental structures of designed proteins. The results of these evaluations will guide user to create fold-specific protocols and possibly increase the chance of success in protein design.

BIOT 475

Lasso peptides as hyperstable antimicrobials

A James Link, [email protected]. Princeton University, Princeton, New Jersey, United States

Lasso peptides are miniature proteins (14-30 aa) that are defined by a unique macrocyclic, threaded structure. This structure endows lasso peptides with protease resistance as well as thermostability and resistance to denaturation by organic solvents. We have developed genome mining methods to predict novel lasso peptides from genome sequences and synthetic biology tools for the high level heterologous production of these peptides. This talk will focus on the unique biophysical properties of lasso peptides as well as how these biophysical properties translate to antimicrobial activity.

BIOT 476

From sequence to process: In silico DSP development based on quantitative structure-property relationships

David Saleh2,1, [email protected], Gang Wang2, Federico Rischawy2, Simon Kluters2, Joey Studts2, Juergen Hubbuch1. (1) Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Baden-Württemberg, Germany (2) Late Stage Downstream Process Development, Boehringer Ingelheim, Biberach, Germany

During the last years, in silico process optimization and characterization have become a key technology in early and late stage development and the focus is turning more and more to mechanistic models for chromatography processes. Nevertheless, the complex and time-consuming model calibration inhibits the implementation of mechanistic modeling workflows in current development strategies. Quantitative structure-property relationship (QSPR) models have the potential to mitigate the risks related to parameter estimation by inverse curve fitting to experimental data. Here, we will present our multidimensional modeling pipeline, leading to the digital twin of a cation exchange (CEX) chromatography process from the primary sequence of the monoclonal antibody (mAb). Starting with the primary sequence, homology models were built for 21 IgG1, IgG4, and complex antibody-formats. Protein surface analysis resulted in >1000 structural descriptors per molecule, including shape-, patch-, and region-specific descriptors. Multiple high-throughput and lab-scale experiments were conducted for all mAbs. The comprehensive dataset enabled the calibration of a transport-dispersive model and the steric-mass action isotherm. QSPR models correlated structural properties to mechanistic chromatography model parameters. The established models were then used for the prediction of isotherm parameters and entire chromatograms of mAbs not involved in the training data set. Further, the correlations found between certain descriptors and isotherm parameters increased the understanding of the binding- mechanism in CEX chromatography. Feature selection revealed which subdomains of the mAb contribute to the adsorption process. Our work demonstrates a bottom-up approach for building mechanistic chromatography models for therapeutic antibodies.

BIOT 477

Modeling host-cell protein retention to expedite ion-exchange resin screening for flow-through polishing

Chase Herman2, [email protected], Xuankuo Xu1, Steven J. Traylor1, Sanchayita Ghose1, Abraham M. Lenhoff2. (1) Biologics Process Development, Bristol-Myers Squibb, Devens, Massachusetts, United States (2) Univ of Delaware, Newark, Delaware, United States

The effectiveness of flow-through anion-exchange polishing steps requires retention of a variety of impurities that differ in their strength of adsorption, and the relatively weak adsorption of some host-cell protein (HCP) impurities may lead to inadequate clearance. Such effects may be amplified by intrinsic retention differences among different resins. Models that can capture this sensitivity would be useful in directing experimental design efforts. To that end, the present study seeks approximate models to predict HCP adsorption equilibria from protein sequences and to integrate them into column models for flow-through chromatography. In the absence of HCP structural information, coarse-grained calculations of molecular-level interactions are performed using a colloidal representation of the protein and the resin, making use of HCP sequence and other structural information. Adsorption energetics are estimated from numerical solutions to continuum electrostatics equations. The resulting retention predictions are compared to those from colloidal models, and their accuracy is assessed by comparison to column experimental data.

BIOT 478

High resolution membrane chromatography: Modeling and simulation of mAb charge variant separation

Jan Hedrich1, Manuel Phillipe Díaz Carreño1, Alina Wulff1, Romas Skudas2, Christian Dr. Frech1, [email protected]. (1) Biotechnology, University of Applied Sciences Mannheim, Mannheim, Germany (2) Merck KGaA, Darmstadt, BW, Germany

Due to the increased performance of novel upstream processes, continuous downstream processes, which allow higher throughput, are gaining more importance in industry, since they increase yield and decrease process time manifolds. Hence, further efficient technologies are being developed recently. Among new emerging technologies in downstream processing, membrane chromatography holds excellent potential for the purification of proteins. Their high level of downstream productivity and versatility is achieved by combining the functionality of resin-based columns with the high flow rates of filter membranes. The major applications of membrane chromatography are the purification of large biomolecules like viruses, VLPs and protein complexes, and the removal of contaminants like DNA, HCPs etc. in flow-through mode. In this study, a prototype ion-exchange membrane was used for high resolution separation of protein variants in a bind and elute mode. As a midsize model protein, a monoclonal antibody with three charge variants was used at low and high protein loadings. To elucidate the elution behavior of the three monoclonal antibody charge variants, mechanistic modeling was applied using linear pH and salt gradient elution experiments. Utilizing the model parameters in in-silico simulations, chromatograms are predicted and the separation has been optimized. The performance and binding capacity of the membrane is compared to a commercially available resin-based column. BIOT 479

Modeling of industrial ion-exchange chromatography using a colloidal multi- component adsorption isotherm

Till Briskot1,2, [email protected], Felix Wittkopp4, Ferdinand Stueckler3, Tobias Hahn1, Thiemo Huuk1, Juergen Hubbuch2. (1) GoSilico GmbH, Karlsruhe, Germany (2) Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany (3) Roche Pharma Technical Development, Roche Diagnostics GmbH, Penzberg, Germany (4) Roche Pharma Research and Early Development, Roche Innovation Center Munich, Roche Diagnostics GmbH, Penzberg, Germany

Model-based process development of ion-exchange chromatography has become increasingly important in recent years. Efficient modeling of these processes requires knowledge of the full adsorption isotherm that describes how resin properties, buffer conditions, and other process parameters affect the adsorption equilibrium. Commonly, the mathematical description of the isotherm is based on a stoichiometric representation of the adsorption process according to the law of mass action. Due to the abstract nature of stoichiometric models, model extensions are usually performed on an empirical base and are often associated with a significant increase in model complexity. They are therefore only valid within a narrow operating range and can be difficult to calibrate in the case of complex industrial processes. In the presented work we apply an alternative, non-stoichiometric protein adsorption model based on a colloidal representation of proteins in an industrial setting. We could previously demonstrate that this type of model is valid over a wide pH range and that it can predict the behavior of charge variants based on differences in the primary protein structure. The presented work further describes non-linear protein adsorption in practical multi-component systems. The benefit compared to conventional stoichiometric models and the industrial applicability of the model are demonstrated by describing the separation of an industrial monoclonal antibody from multiple product related impurities.

BIOT 480

Protein conformational changes during chromatographic separation

Giorgio Carta, [email protected]. Chemical Engineering, University of Virginia, Charlotttesville, Virginia, United States

Proteins can exhibit conformational changes on the surface of resins used for chromatographic separations resulting in complex elution behaviors both for ion exchange chromatography and for hydrophobic interactions chromatography. Both unfolding with loss of secondary structure and reversible conformational changes have been reported. In either case, recovery and separation efficiency are impacted. Additionally, these phenomena complicate the development of mechanistic models since the classical, thermodynamics-based approaches for the description of protein surface interactions do not take such phenomena into account. In this presentation, we review recent efforts to understand and model chromatographic elution of bivalent bispecific antibodies whose multi-domain characteristics result in particularly complex elution behaviors.

BIOT 481

Identifying preferred binding regions in multimodal chromatography using covalent labeling and liquid chromatography/mass spectrometry

Ronak B. Gudhka1,2, [email protected], Kabir Dhingra1,2, Dimitri Zagorevski2, Steven M. Cramer1,2. (1) Chemical and Biological Engineering, Rensselaer polytechnic institute, Troy, New York, United States (2) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States

Recent biophysical investigations into protein-ligand interactions carried out in our lab have helped us to develop a deeper molecular level understanding of the binding of biological products to Multimodal (MM) chromatographic systems. These biophysical investigations employed Nuclear Magnetic Resonance (NMR) spectroscopy to get the residue specific binding information for various proteins. However, NMR spectroscopy requires the use of 15N-labeled and assigned proteins that can be challenging for large proteins like monoclonal antibodies (mAbs) or viral capsid proteins, which are generally greater than 50kDa. Hence, there is a need to employ a different technique that can be applied to a broader range of proteins as well as binding conditions. To address this, we used a combination of covalent labeling and Liquid Chromatography/Mass Spectrometry (LC/MS) to identify the regions on the protein surface that are involved in binding to the chromatographic resin. In this study, we evaluated the binding region on small model protein Ubiquitin (Ubq) using Diethyl pyrocarbonate (DEPC) as a covalent label for binding to various MM chromatographic resin systems. DEPC is a versatile covalent label that can readily react with several nucleophilic amino acid residues on the protein surface. First, a DOE study was designed to identify the optimum DEPC:Protein ratio as well as the reaction time for covalent labeling of Ubq in solution. These optimized conditions were then used for the on-resin labeling experiments of Ubq. By a comparative study of the in-solution and the on-resin labeling reaction, the residues involved in binding with the chromatographic resin were identified. The binding region identified using this technique was confirmed with the results obtained from NMR spectroscopy. This combined covalent labeling and LC/MS approach allowed us to develop a molecular level understanding of binding of Ubq to different MM chromatographic systems. This work, which requires very small amounts of samples, has potential applications for identifying the residues on surfaces of complex biological products like mAbs, bispecific antibodies, viral capsid proteins, etc. for binding to various chromatographic systems. This rich binding region information would guide us to design efficient separation processes a priori for new biological products that are associated with product-related variants.

BIOT 482

Mechanistic modeling of ligand density variations in anion exchange chromatography

Gabriela Sanchez Reyes1, [email protected], Heiner Graalfs2, Christian Dr. Frech1. (1) Biotechnology, University of Applied Sciences Mannheim, Mannheim, Germany (2) Merck KGaA, Darmstadt, Germany

Ion exchange chromatography (IEC) is a widely used unit operation in the purification of therapeutic proteins. However, the performance of an ion-exchange process depends on a complex interrelationship between several parameters, such as protein properties, mobile phase conditions, and chromatographic resin characteristics. Consequently, batch variations of IEC resins play a significant role in the robustness of these downstream processing steps. Ligand density is known to be one of the main lot-to-lot variations, affecting protein adsorption and separation performance. The use of a model-based approach can be an effective tool for comprehending the impact of parameter variations (e.g. ligand density) and their influence on the process. The objective of this work was to apply mechanistic modeling to gain a deeper understanding of the influence of ligand density variations in anion exchange chromatography. To achieve this, 13 prototype resins having the same support as the strong anion exchange resin Fractogel® EMD TMAE (M), yet only differing in ligand density, were analyzed. Linear salt gradient elution (LGE) experiments were performed to observe the elution behavior of BSA and a monoclonal antibody. An extended stoichiometric displacement model for ion-exchange chromatography, describing the dependence of ligand density variations on protein retention, was successfully applied. Additionally, a lumped rate model was used to perform in silico simulations, which proved the reliability of the model.

BIOT 483

Impact of multimodal ligand density on free energy of adsorption of proteins to ligand-immobilized SAM surfaces: Molecular dynamics study

Mayank Vats, [email protected], Shekhar Garde, Steven M. Cramer. Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States

Multimodal chromatography presents unique selectivity in bio-separations, but a fundamental understanding of differences in multimodal (MM) ligands’ behavior still eludes us. Recent simulations work in our lab on MM-ligand immobilized self assembled monolayer (SAM) surfaces has demonstrated that ligand flexibility and density differences can result in surfaces with relatively large patches of hydrophobicity and charge. These self-associated ligand patterns have also been found to be persistent in chromatographically relevant higher salt concentrations and could govern protein- surface interactions. Here we develop that premise using enhanced sampling in molecular dynamics simulations of these systems. First, INDirect Umbrella Sampling (INDUS) was used to study the hydrophobic nature of surfaces where immobilized MM- ligands were found to aggregate and create hydrophobic and charged patches, and then compared with low ligand density surfaces without ligand self-association. To further understand the importance of ligand clustering in protein adsorption, free energies of binding of model proteins to a series of SAM surfaces with varying MM- ligand densities were calculated from potential of mean force (PMF) measurements performed using umbrella sampling. The possibility that water-mediated interactions during binding and unbinding at the protein-SAM interaction interface show hysteresis- like behavior was also investigated. Correlations between the calculated free energies and corresponding patch distributions for those ligand surfaces, inform us about the role of ligand density driven self-association in adsorption. Our study presents an understanding of hydrophobic behavior in multimodal systems at different ligand densities and connects multimodal resin surface properties with density dependent behavior during protein adsorption, which in turn has implications for surface property driven multimodal resin design and selection.

BIOT 484

Systems design and synthetic construction of influenza virus for vaccine production

Thu Phan1, [email protected], Christopher Stach1, Ryan Langlois2, Wei-Shou Hu1. (1) Chemical Engineering and Materials Science, University of Minnesota, St Paul, Minnesota, United States (2) Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, United States

Traditional productions of flu vaccine rely on the rescue of influenza viruses through transient transfection of multiple plasmids encoding the virus genome. The viruses generated are then used to infect embryonated eggs or cultured cells in the manufacturing process. Because of the high mutation frequency of the replication of the virus’s RNA genome, a limited number of infection cycles is used to amplify the viruses. The manufacturing process thus relies heavily on the virus's innate replication. Not only does this reliance make the vaccine less effective but it also leads to long response time to a pandemic situation. We took a synthetic approach by dismantling virus replication regulation and controlling the kinetics of the expression of various viral components instead. To characterize virus replication kinetics, we performed a transcriptomic analysis of the three types of viral RNA components in a single-cycle infection. We developed a novel informatics pipeline using total RNA-Sequencing that allows for the quantification of all viral RNA species: the temporary template cRNAs, genomic vRNAs and mRNAs. The kinetics of the expression of all RNA components for different viral genes served as a guide in our design of the expression system for virus production. We constructed an expression platform that allows for the generation of virus components in mammalian cells, which can be employed for virus production without the use of seed virus. This synthetic platform not only allows for more efficient, high- fidelity generation of flu virus but also provides opportunities to optimize the kinetics of virus gene expression for higher virus titer.

BIOT 485

Utilizing native regulation of heterologous secretory pathway genes in yeast engineered for expression of human proteins

Neil Dalvie1, [email protected], Yuchen Yang1, Joseph Brady2, Kerry R. Love1, John C. Love3. (1) Chemical engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States (2) Massachusetts Institute of Technology, Cambridge, Massachusetts, United States (3) Building 76 Room 261, Massachusetts Inst of Technology, Cambridge, Massachusetts, United States

Mammalian enzymes and chaperones are often needed to express human proteins with complex folding and glycosylation requirements. In alternative hosts, however, exogenous secretory pathway proteins can be inactive or toxic when overexpressed or localized to the wrong cellular compartment. Engineered promoters have been traditionally used to rationally tune the expression of heterologous genes, but the toolbox of expression cassettes is limited for non-model organisms. Native promoters are an elegant alternative to engineered promoters, encompassing native induction and trans regulation when driving expression of a heterologous gene. Recently, simplified gene editing tools and the accessibility of genomic and transcriptomic sequencing have expanded the toolbox of expression cassettes to include the entire host genome. Here, we use native promoters in Komagataella phaffii to achieve both rational tuning and inducible expression of both endogenous and exogenous secretory pathway genes to achieve uniform engineered glycosylation and protein folding while avoiding toxicity. Native promoters and regulation provide a nearly unlimited toolbox of available gene expression cassettes in non-model organisms, exemplifying the advantages and flexibility of efficient, markerless gene editing tools.

BIOT 486

FLAMES: Transcriptional biosensor to evaluate cellular flavin regulation

Nolan T. Anderson1, [email protected], Rachel Taylor1, Zoe Imansjah1, Arnab Mukherjee1,2,3. (1) Chemical Engineering, University of California, Santa Barbara, Goleta, California, United States (2) Chemistry, University of California, Santa Barbara, Santa Barbara, California, United States (3) Center for Bioengineering, University of California, Santa Barbara, Santa Barbara, California, United States

Introduction. Riboflavin is a key metabolite that provides enzymatic cofactors for oxidative metabolism in cells. Unlike most bacteria and fungi, animals do not synthesize flavin. Thus, microbially-sourced flavin is used as a dietary supplement in agricultural feed, food, and medicine. These efforts would benefit greatly from high throughput techniques to rapidly detect intracellular flavin to enable large-scale strain engineering and candidate drug screening. However, current techniques for measuring flavin largely rely on chromatography, which is neither scalable nor usable in live cells. To address this challenge, we report development of a flavin-actuated metabolic sensor (FLAMES) based on a flavin responsive riboswitch (known as ribB) recently discovered in E. coli. Approach and results. RibB natively regulates flavin homeostasis in E. coli by turning off flavin synthesis when flavin content in cells is sufficient, binding to cellular flavins and arresting expression of downstream flavin synthesis genes. To build FLAMES, we first combined ribB with the constitutive PLtetO-1 promoter and a strong ribosome binding site (Bba_B0030). Next, we inserted an mCherry reporter downstream of this regulatory sequence (Fig. 1A) and incorporated these parts in the pSC101 plasmid. The resulting sensor operates as a flavin deficiency detector by activating mCherry expression in low flavin concentrations. Using cell-free expression, we first quantified dose-response by measuring change in fluorescence as a function of flavin concentration and estimated a dissociation constant of 0.62 ± 0.30 µM (N=3) (Fig. 1B) . We validated performance of the biosensor in cells, observing a 48.5 ± 7.0% decrease (N=3) in mCherry fluorescence when exogenous flavin is increased from 0 to 50 µM (Fig. 1C). Here, we present these results and our recent efforts using directed evolution to maximize the dynamic range of the sensor.

BIOT 487

Data mining for mechanistic models using enhanced symbolic regression

Nicole Beauregard1, Mark Marten2, Steven Harris3, Ranjan Srivastava1, [email protected]. (1) Univ of Connecticut, Storrs Mansfield, Connecticut, United States (2) Engineering Bldg 314, UMBC, Chem., Biochem. Environ. Engr., Baltimore, Maryland, United States (3) University of Manitoba, Winnipeg, Manitoba, Canada

Biological systems can often be represented as complex mechanistic networks illustrating the intricate relationships among biological species. Large quantities of data are often generated from experiments investigating the existence or function of different pathways or mechanisms. The ability to predict biological networks from experimental data would be invaluable for biotech or pharmaceutical industries as a means for rapid identification of network bottlenecks, determination of potential drug targets, or optimization of bioprocesses. Even with advances in machine learning and computing power, the ability to deconvolute networks to identify the mechanistic network from data is still a challenge today.

This work examines the use of enhanced symbolic regression via genetic programming as a technique for inferring biochemical networks from data. Biochemical networks can be decomposed into systems of differential equations using assumptions from mass action kinetics. Genetic programming, a type of evolutionary algorithms, uses recombination and mutation principles to evolve equations over generations. This algorithm is notorious for its computational intensity and long simulation times. We implemented heuristics to this genetic program based on domain knowledge of our system to constrain both the complexity of the equations generated and the parameter search space. To assess the feasibility of the enhanced algorithm, we produced a synthetic dataset using a known system of equations describing intracellular viral kinetics. The known system consisted of three ordinary differential equations and six parameters. After 100 generations, we were able to evolve an ensemble of models that bound the dataset. To validate the model, we perturbed the initial conditions and still saw good performance from the ensemble of models. The enhanced symbolic regression technique shows good promise as a technique to infer information about biochemical networks from data. However, it is clear that to handle the challenges posed by larger system, heuristics grounded in domain specific knowledge will be needed to reduce the search space to a manageable size.

BIOT 488

Using a multi-omic approach to understand and model eukaryotic signal transduction

Mark Marten1, [email protected], Cynthia Chelius1, Liliane Ribeiro2, Jyothi Kumar3, Samantha Reese3, Nicole Beauregard4, Stephen Lincoln4, Walker Huso1, Ranjan Srivastava5, Steven Harris6. (1) Engineering Bldg 314, UMBC, Chem., Biochem. Environ. Engr., Baltimore, Maryland, United States (2) University of São Paulo, São Paulo, Brazil (3) University of Nebraska-Lincoln, Lincoln, Nebraska, United States (4) University of Connecticut, Storrs, Connecticut, United States (5) Univ of Connecticut, Storrs Mansfield, Connecticut, United States (6) University of Manitoba, Winnipeg, Manitoba, Canada Eukaryotic signaling networks are composed of interconnected systems of regulatory proteins and are involved in controlling nearly all biological processes. To study these networks, our collaborative team is working toward combining phosphoproteomic and transcriptomic data with genetic-algorithm programming to build dynamic mathematical models.

As a model system, we are studying the fungal Cell Wall Integrity Signaling (CWIS) pathway, which is activated upon wall stress leading to expression of several wall-repair genes. To assess system-wide dynamic response to stress, we perturbed A. nidulans cultures with CWIS activating agent micafungin, collected 13 phosphoproteomic and transcriptomic samples over 10 and 120 min respectively and found over 1800 genes and 430 phosphorylation sites show significant dynamic change. Omics-based hypotheses were confirmed with experiments, and our collective data indicate previously unknown connections with (i) actin regulation, (ii) endocytosis, and (iii) septum formation as critical cellular processes responding to activation of CWIS.

While the CWIS pathway induces expression of some wall repair genes, it is unclear how numerous others are regulated. To address this question we used a strain lacking the last kinase in the CWIS pathway, MpkA, rendering the pathway inactive. To begin, we compared MpkA+/- strains, at a single time point, in the absence of wall stress. Over 2500 genes and 200 phosphorylation sites showed differential expression and occupancy respectively. Omics-based hypotheses were confirmed with experiments, and our collective data indicate previously unknown connections with wall maintenance, branching regulation and iron metabolism. Seeking alternate regulatory pathways, we carried out dynamic experiments with the MpkA- strain taking phosphoproteomic and transcriptomic samples over 10 and 120 min respectively. We will report on our findings from these experiments and how we are using these data, along with a genetic algorithm programming approach, to develop models to describe signal transduction and resultant changes in gene expression.

BIOT 489

Studying the dynamics of gene expression patterns in fed-batch processes to gain a better understanding of process

Arpan Bandyoapadhyay, [email protected], Alexei Podtelezhnikov, Nicholas Murgolo, Jonathan Cacciatore, Vijay Janakiraman. Merck & Co, Kenilworth, New Jersey, United States

Chinese hamster ovary (CHO) cells are the industrial workhorses for recombinant protein production. Despite their importance, very little is known about the molecular and gene regulatory mechanisms that control cellular phenotypes during the biomanufacturing process. While the cells grow and produce the protein in the bioreactor, it is expected that the expression pattern of genes will change and that such changes may give indications of the cellular state in terms of viability, growth, and productivity. Further, perturbations in the process can also lead to changes in expression patterns. In one of our recent studies, we analyzed the dynamics of gene expression changes during a fed-batch process. The changes in gene expression profiles were examined across key process steps such as a temperature-shift and feed additions. Further, we examined the changes in gene expression patterns in response to variations in the levels of a trace metal. Pooling the transcriptome data, we generated clusters of genes having similar gene expression patterns generating an expression baseline for future comparisons and provide a more nuanced understanding of the changes in cellular physiology during a fed-batch process. Building such datasets will help us link various phenotypic traits to relevant biomarkers and better equip us to respond effectively to future manufacturing challenges.

BIOT 490

Towards antimicrobial, anti-resistance chemotherapeutic strategies using large- scale chemical-genetics

Eachan O. Johnson1, [email protected], Emma Office1, Sabine Ehrt2, Eric Rubin3, Christopher Sassetti4, Dirk Schnappinger2, Deborah T. Hung1. (1) Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, United States (2) Weill Cornell Medical College, New York, New York, United States (3) Harvard T.H. Chan School of Public Health, Boston, Massachusetts, United States (4) University of Massachusetts Medical School, Worcester, Massachusetts, United States

With the rise in antibiotic resistance, new drugs are desperately needed against bacterial infections, especially tuberculosis (TB), a complex disease caused by Mycobacterium tuberculosis (Mtb) which kills 1.5 million people per year. Combining chemistry and genetics, we developed a new strategy called PROSPECT for rapidly identifying many new small molecule inhibitors of Mtb and for elucidating their mechanisms of action (MOA), by performing large-scale chemical screening on pooled genetic libraries containing 250-400 barcoded strains hypomorphic for individual essential genes. We created barcoded hypomorphic strains for 474 of the ~625 essential genes in Mtb and developed a multiplexed, whole-cell assay to measure strain abundance. Applying the approach with activity-enriched and unbiased libraries of >100,000 compounds, we characterized >25 million potential chemical-genetic interactions.

Using machine learning, we identified >40 novel compounds against known MOAs, including new classes of inhibitors of DNA gyrase, mycolic acid biosynthesis, and folate biosynthesis. We also identified a pair of chemically distinct new inhibitors, BRD-9327 and BRD-8000, of a novel target, EfpA which are synergistic and display distinct interactions with EfpA. High-level resistance to one compound increases the sensitivity to and reduces the emergence of resistance to the others even when we applied antimicrobial monotherapy sequentially. Thus, the combination of BRD-9327 and BRD- 8000 represents a proof-of-concept for the novel strategy of applying chemical-genetics to design of antimicrobial combination chemotherapy in which mutual collateral sensitivity is engineered and exploited.

BIOT 491

Metabolomics process modeling: Systems biology approach to understand variability in commercial biologics cell culture processes

Amanda Lewis1, [email protected], Sophia Liu1, Serhiy Hnatyshyn2, Jan-Lucas Ott1, Eric Garr1, Bethanne M. Warrack1, Michael D. Reily3. (1) Bristol-Myers Squibb, Devens, Massachusetts, United States (2) PCO, Bristol Myers-Squibb Company, Princeton Junction, New Jersey, United States (3) Mail Stop L14-05, Bristol-Myers Squibb, Princeton, New Jersey, United States

The biopharmaceutical industry strives to develop and operate efficient, robust, reproducible commercial biologics processes. A major challenge of industrial biologics processes is optimization of cell culture conditions to increase productivity while maintaining consistent product quality. The cell culture operations, which involve the use of live cell hosts, have historically introduced significant variability to the overall process. Technological improvements which include the implementation of advanced cell line engineering, chemically defined media, quality by design (QbD) development approaches, and in-line and at-line monitoring, have significantly reduced process variability. Nonetheless, performance variability remains a challenge for many commercial programs. This variability in turn can impact both product yields and product quality. Even small performance differences can become significant in low-yield processes with large campaign sizes, or processes manufactured at multiple sites. The ability to understand and eliminate sources of variability is greatly enhanced by augmenting the quality and quantity of data available from commercial campaigns.

Metabolomics Process Monitoring (MPM) is a data-driven approach to understand sources of manufacturing variability on a cellular level. Here we present a case study of MPM implementation in a legacy commercial biologics program. First, we describe how the MPM workflow was successfully integrated into a commercial manufacturing process. Second, we discuss novel data normalization techniques developed to enable long term trending. Third, we describe the selection of an orthogonal projections to latent structures (OPLS) model to link systems biology and process data. Finally, we share key mechanistic insights obtained from the case study, and provide a vision for how MPM can enhance commercial biologics capabilities going forward.

BIOT 492

Alternative carbon sources enable flexible, methanol-free Pichia pastoris perfusion processes with increased volumetric productivity

Andrew Biedermann, [email protected], Neil Dalvie, Kerry R. Love, John C. Love. Chemical Engineering, MIT, Cambridge, Massachusetts, United States

Strategies for rapid, low-cost biologic production could shorten process development timelines and improve global access to biologic drugs. Pichia pastoris is a promising production host, due to its fast growth rates to high cell density, and high productivity of heterologous proteins with low host cell protein impurities. Most Pichia pastoris processes use the methanol inducible AOX1 promoter, due to its high expression and tight, inducible regulation, which enable rapid outgrowth and more consistent product quality. While these characteristics have resulted in widespread use of this promoter system, using methanol as a carbon source and inducer results in additional metabolic stress and process safety concerns. In this work, we demonstrate improved process productivity with reduced heat generation and oxygen consumption, using genetic engineering and alternative carbon sources to obtain flexible, methanol free process operation. We conducted a broad screen of 30 alternative carbon sources, characterizing growth and productivity of a rotavirus vaccine subunit product, P[8]. RNA- seq was used to better understand how the choice of production carbon source influences gene regulation and specific productivity. The results of these analyses identified a new production carbon source which enables improved volumetric productivity for several protein products, relative to our original methanol-based perfusion process.

BIOT 493

Augmenting dissolved oxygen control in intensified cell culture processes

Jonathan Wang1, [email protected], Kai Hoeffner2, [email protected], May Chin2, Jiuyi Lu1, Jason Walther3, Armin Opitz2, Franqui Jimenez2, Jean McLarty1. (1) Cell Culture Development, Biologics Development, Sanofi, Framingham, Massachusetts, United States (2) Manufacturing Science and Technology, Sanofi, Framingham, Massachusetts, United States (3) Purification Development, Sanofi, Framingham, Massachusetts, United States

Sanofi’s continuous biomanufacturing platform affords numerous advantages over both legacy perfusion processes and traditional fed-batch systems. Process intensification allows for an order of magnitude higher cell density resulting in increased volumetric productivity. Intensified processes rely heavily on automation with fewer manual operator inputs in order to ensure robust operations.

Dissolved oxygen probes in intensified high density cell culture processes are plagued by gas bubble adherence related noise. Excessive noise may lead to suboptimal oxygen supply resulting in hypoxic cell culture conditions that impact growth and product quality. A root cause analysis was performed to identify factors such as raw materials, equipment, and automation strategies that might influence ability to control dissolved oxygen. We propose a strategy to address common problems observed in dissolved oxygen control and present the proof of concept obtained in an at-scale production vessel.

BIOT 494

Continuous monoclonal antibody (mAb) biomanufacturing and integrated process analytics for mAb N-linked glycans analysis

Viki Chopda1, [email protected], Aron Gyorgypal2, Rana Said1, Ari Alter1, Shishir P. Chundawat1, Haoran zhang1. (1) Chemical and Biochemical Engineering, Rutgers- The State University of New Jersey, New Brunswick, New Jersey, United States (2) Chemical and Biochemical Engineering, Rutgers University, Flemington, New Jersey, United States

The transition from batch to continuous manufacturing is imminent for the production of biotherapeutics like mAb due to the distinct advantages offered by the latter such as increased productivity, lowered costs, and improved product quality. Continuous biomanufacturing also offers increased flexibility and robustness in process operation over traditional batch processes. However, the development and optimization of continuous manufacturing of biotherapeutics requires a detailed understanding of the bioprocess fundamentals that are often possible only through a thorough analysis of batch and fed-batch operations. Furthermore, one of the challenges associated with continuous mAbs production is the necessity to closely monitor drug product critical quality attributes (e.g., mAb glycosylation, aggregation) to implement real-time process control and ultimately meet FDA’s regulatory requirements. Here, we aim to establish a bench-scale upstream mAb production platform integrated with advanced process analytics to facilitate critical modeling driven analysis and derisking of continuous biomanufacturing processes. We show results from our ongoing efforts to establish this test-bed platform for a model mAb (Trastuzumab) production using batch and fed-batch cultivation of a recombinant mammalian cell line. We evaluate the impact of several upstream process parameters (e.g., media composition) on critical quality attributes like mAb glycosylation. Next, we highlight an integrated sampling system developed to facilitate on-line liquid chromatography/mass spectrometry-based analytics for real-time monitoring of mAb N-linked glycosylation profile to modulate the upstream bioreactor cell culture process. Finally, we discuss efforts for integration of the continuous upstream bioprocess platform with a validated upstream process model to achieve real time process monitoring, optimization, and control for mAb production. BIOT 495

Manufacturing of a trivalent subunit vaccine in a single campaign

Laura Crowell, [email protected], Neil Dalvie, Joseph Brady, Mary K. Tracey, Kerry R. Love, John C. Love. Koch Institute at MIT, Cambridge, Massachusetts, United States

Vaccines, along with most other protein biologics, are currently manufactured in large- scale, centralized facilities. Such manufacturing paradigms lack the flexibility and agility required to quickly address global epidemics and have led to stagnant coverage and missed immunization targets due to failures in campaigns or supply chains. Multivalent vaccines are particularly challenging since each subunit is manufactured individually, requiring multiple campaigns to produce a single batch of drug product. New manufacturing methods, and associated process development strategies, are needed to improve the flexibility and dependability of vaccine manufacturing at low costs. We have previously demonstrated a small-scale, integrated and automated manufacturing platform capable of rapidly producing a variety of recombinant proteins with phase- appropriate quality for early development. Our platform comprises three modules for fermentation via perfusion, straight-through chromatographic purification, and formulation, combined with a holistic strategy for process design. Here, we demonstrate the application of our platform to manufacture a trivalent rotavirus vaccine in a single campaign through co-expression and co-purification. We first developed and deployed processes for the production of each individual subunit antigen, while maintaining similarity in process operating conditions. Our initial processes showed a 40% reduction in the number of steps as compared to the original manufacturing method for these molecules. While process-related variants were removed, product-related variants including N-terminal truncation, glycosylation, and aggregation persisted. In order to maintain process conditions compatible with all three subunits, we addressed product- related variants at the molecular level by strategically altering specific amino acids. We then created a single Pichia pastoris strain that expressed all three engineered subunits. Finally, a single manufacturing campaign was executed on our system. Successful co-expression and co-purification of all three subunits was confirmed by RPLC, SEC and LC-MS. Process-related impurities were reduced below typical regulatory guidelines. We believe that this work demonstrates the feasibility of manufacturing a multivalent vaccine in a single campaign. We hope that such process intensification will enable more flexible and agile manufacturing of vaccines to meet global needs.

BIOT 496

Implementation of continuous processing for the downstream purification of vaccines

Tiago Matos1, [email protected], Ayobami Adegbite1, David Hoying3, Marc Wenger2. (1) Vaccines Process Development, Merck, West Point, Pennsylvania, United States (2) WP42A 20, Merck Research Laboratories, West Point, Pennsylvania, United States (3) Penn State, State College, Pennsylvania, United States

The increasing worldwide demand for vaccines along with intensifying economic pressure on health care systems underlines the need for further improvement of vaccine manufacturing. In addition, regulatory authorities are encouraging investment in continuous manufacturing (CM) processes to ensure robust production, avoid shortages, and ultimately lower the cost of medications for patients. As of January 2019, the FDA has approved five products from manufacturers using CM. All were, however, small molecules. Our goal is to develop continuous downstream processing for viral vaccines, using strategies such as simulated moving bed chromatography (SMB), countercurrent chromatography, single-pass tangential flow filtration (SP-TFF), and inline filtration and diafiltration (IF/DF). Recently, these techniques have been successfully implemented for smaller biopharmaceutical molecules to address product instability, improve process efficiency, and reduce facility footprint and capital cost. However, the large size of viruses, virus-like particles, and other biological macromolecules has complicated the application of these methods for vaccines. Membrane chromatography is often presented as an optimal approach for viral vaccine processing due to its large pore sizes and convective mass transport properties. We therefore explored continuous processing with membrane chromatography for a viral vaccine candidate by implementing simulated moving bed (SMB) chromatography in a 4-column mode. SMB improves the efficiency of the chromatography step by enabling higher loads and multiple cycles of column use. The separation performance of SMB was compared to a conventional batch chromatographic capture step used at full manufacturing scale. The product step yield while operating in SMB was comparable to batch mode. Furthermore, the total amount of membrane area required for SMB was a tenth of that needed for batch operation, while keeping total processing times similar. Since SMB uses smaller column sizes, it can take advantage of the high flow rates achievable for membrane chromatography that are not typically possible at larger column scales due to the flow rate limitations of many chromatography skids. To achieve a fully continuous purification train, we subsequently integrated these continuous chromatography platforms with SP-TFF and IL/DF, taking advantage of larger pore membrane options available to viruses.

BIOT 497

Improving plant cadence and utilization through cell culture seed train and production process intensification

Niket Bubna, [email protected], Jake Kim, James Hamlin, Gautam Nayar, Sigma Mostafa. KBI Biopharma, Durham, North Carolina, United States

Increased pace of biologics development in order to reach the clinic faster has led to tremendous pressure on first in human (FIH) drug substance manufacturing plants. Even though innovations in single-use cell culture and purification technologies have aided faster clinical entry, our current efforts are directed to efficiency gains through improved plant cadence and utilization. As a contract development and manufacturing company using disposable technologies, we have investigated techniques to reduce overall cell culture process duration to enable introduction of more products in the plant. The main goal for an inoculum train is to adapt cells out of thaw and generate enough viable cells to initiate the production-stage. Traditional approaches such as 100-200 million cells/mL vials or bags of frozen cells can be used for directly inoculating seed bioreactor bags, but due to long-term cell bank stability concerns these may not be readily implemented for all FIH products. We have assessed and implemented use of 30-50 million cells/mL vials and use of multiple cell bank vials to initiate the seed train. High volume culture vessels (1.5-2.5 L working volume) have also been successfully used to limit the number of subcultures required. These approaches have led to over 30% reduction in inoculum train duration for the platform process and have been implemented even for expedited process development projects. Apart from maintaining a strict seed train schedule, production perfusion cell culture (ATF) can also been used for process intensification. Use of perfusion production processes can lead to 20-30% higher volumetric productivity. Results from two cell lines comparing perfusion performance with fed-batch will be presented. These efforts toward intensifying seed train and production stages can lead to improved plant cadence by adding more manufacturing runs or reducing the number of runs required per year for each molecule.

BIOT 498

Withdrawn

BIOT 499

Integrated approach to semi-continuous drug substance manufacturing

Mehdi Ghodbane, [email protected], Ashley Reeder, Nidhi Thite, Sobhana Sripada, John Starcevich, Patrick Thompson, Gregory Laslo, Rick Atwell, Brad Kauffman, Patrick Pohlhaus, Jessica R. Molek, Antonio R. Ubiera. GlaxoSmithKline, King of Prussia, Pennsylvania, United States

In recent years the biopharmaceutical industry has been exploring the utilization of continuous drug substance manufacturing to reduce facility footprint, labor requirements, and operating costs while maintaining target product output. The vision within GlaxoSmithKline (GSK) for a semi-continuous manufacturing platform is to maximize equipment utilization and reduce the capital investment required for future facilities, resulting in a flexible and small manufacturing platform that can support an expanding portfolio of assets with varying BDS manufacturing demands. To explore the technical feasibility of this manufacturing paradigm, a laboratory scale development facility has been designed and built to serve as a ‘sandbox’ for technology development. Within the laboratory, technology assessments, process development, and process derisking can occur such that technology transfer and future manufacturing operations risks can be mitigated. This presentation will focus on the integrated approach GSK has taken focusing on three main aspects; equipment design, analytical strategy, and data management. First, the process flow, facility layout, automation control strategy, and key philosophies that drove the design of the system will be discussed. Furthermore, the combination of inline, online, and at-line analytical methods implemented to monitor critical quality attributes throughout the process will be outlined. Finally, the framework for the data management which allows for automated real time process monitoring, historization, data aggregation, and post-campaign visualization will be discussed. A facility designed with these capabilities can enable automated process operation, improve process monitoring, and drastically reduce documentation burdens. Together, an integrated semi-continuous platform designed with these capabilities promises to reduce labor costs and improve process control, in addition to the benefits that can be realized from continuous unit operations alone.

BIOT 500

Process characterization strategy for continuous manufacturing

Erin Franco1, [email protected], Jack Huang2, Nelson Chavez1, Madiha Khurshid1. (1) Amgen, Inc, Thousand Oaks, California, United States (2) Merck, Kenilworth, New Jersey, United States

Continuous Manufacturing (CM) offers many benefits over traditional fed batch bioreactor processes. The continuous perfusion of CM allows for increased culture productivity and an extended harvest period. However, CM cell culture runs can be significantly longer than traditional processes. The extended length of the process can prove challenging for completing Process Characterization (PC) studies efficiently. Understanding the steady-state of the process allowed us to greatly reduce the time needed to evaluate each condition. By implementing a creative PC design strategy that tests multiple conditions within the steady-state portion of a single bioreactor run, we were able to reduce the PC study timeline by >60%.

BIOT 501

Navigating the unique manufacturing and supply chain challenges in cell and gene therapy and what we can learn from our past experiences

Rolf A. Ramelmeier, [email protected]. Sangamo Therapeutics, Brisbane, California, United States

Gene and cell therapies present a new wave of modalities to treat and potentially cure disease. They are considerably more complex compared to therapeutic proteins and small molecules. While we can leverage much of our knowledge and experience in that space, cell and gene therapies will require a significant investment in technical and scientific understanding as well as a different approach for manufacturing and supply chain to meet the needs of the patient. This presentation will address the unique challenges to process and analytical development, which is still in the early stages of its evolution and frankly is like the wild west right now. This talk will also address why manufacturing and supply chain need a lot of attention, from starting materials, vector banking, vector and cell production, and frozen vials to chain of custody. Given the huge growth in gene and cell therapy clinical trials and need for clinical material, considerable pressure has been put on both CDMOs and innovator companies to garner the right manufacturing capacity. These complex therapies demand more oversight and control via the technical experts which may not reside at the CDMOs, which further challenges our ability to reliably manufacture these compounds. Sangamo, an early pioneer in the gene and cell therapy space, is addressing many of these challenges. This will be highlighted as well in the presentation.

BIOT 502

Intensification of the baculovirus/Sf9 production platform by developing a media formulation and feed addition strategy

Krishanu Mathur, [email protected], Anastatia Neuman, Charles Ly, Peter Slade. Voyager Therapeutics, Cambridge, Massachusetts, United States

Explosive growth in the field of gene therapy has enabled numerous therapeutic candidates to accelerate into the clinic and advance towards commercialization. This rapidly increasing clinical demand necessitates manufacturing processes with ever- increasing productivity that also consistently meet a high standard of product quality. The current Baculovirus/Sf9 platform, despite its many advantages over mammalian systems for rAAV production, is used in batch mode with little intensification being used since its introduction over 20 years ago. Commercially available media solutions cannot support high cell density infections; which causes steep drop in AAV productivity. This productivity drop is attributed to nutrient deficiency in cell culture at high densities. However, a lack in understanding of the Sf9 cell metabolism has inhibited the implementation of strategies which can sequentially promote Sf9 cell growth, baculovirus infection, and AAV production. This presentation highlights Voyager Therapeutics’ efforts at intensifying the Baculovirus/Sf9 platform. Spent media analysis combined with empirical DOE and mixing design studies has led to identification of key nutrients involved in different phases of Sf9 cell growth and AAV production. These learnings have enabled development of a robust basal media formulation and feed concentrate that can be used together for a fed-batch manufacturing process. This presentation also discusses proof of concept work to show compatibility of the Baculovirus/Sf9 platform with an ATF perfusion system. High cell density infections can be sustained for longer durations, resulting in a significant increase in overall productivity. Work is continuing to further intensify the process as well as understand the effects of an intensified process on AAV product quality. BIOT 503

Fundamental approach to understanding selectivity trends for bispecific antibody purification by multimodal chromatography

Siddharth S. Parasnavis1, [email protected], Matthew Aspelund2, Wai Chung2, Steven M. Cramer1. (1) Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States (2) Purification Process Sciences, AstraZeneca, Gaithersburg, Maryland, United States

Monovalent bispecific antibodies (bsAbs), a class of biological product that enables the binding of multiple targets while retaining the conventional monoclonal antibody (mAb) structure, are becoming of increasing importance in a wide range of clinical applications. During cell culture, incorrect pairing of the heavy and light chains of the bsAb lead to the formation of various ‘mis-paired species’ that have very similar surface properties as the target bsAb and pose significant purification challenges. While multimodal (MM) chromatography is often used as a polishing step after Protein A capture, there is limited fundamental understanding of the resin and protein surface properties that drive the separation of the bsAb product from the mis-paired variants. In this work, the retention behavior of the bsAb, the parental mAbs, and two key mis-paired variants (coeluting with the bsAb in the Protein A step) were studied on a set of homologous MM cation exchange (CEX) resins under various conditions to identify selectivity trends. To understand how the constituent domains contributed to the observed behavior on the multimodal systems, chromatographic evaluation was carried out on Fab, Fab2 and Fc fragments obtained from these species by enzymatic digestion. The observed behavior from these experiments were then tied to surface property maps such as electrostatic potential (EP) and surface aggregation propensity (SAP) maps as well as novel surface property clusters to identify key surface patches that are important binding sites for MM systems. Experimental techniques were then used to confirm the surface patches on the parental mAbs that are important for binding on mixed-mode systems found using in silico surface property mapping tools. The techniques presented in this work have important implications for the development of a workflow for the purification of bsAbs, for the design of bsAbs for biomanufacturability as well as for understanding MM ligand design and applications.

BIOT 504

Achieving a potent AAV using the insect cell/baculovirus systems

Shamik Sharma, [email protected], Jeffrey Slack, Krishanu Mathur, Andrade Hendricks, Harvir Grewal, Chris Brown, Peter Slade. Voyager Therapeutics, Cambridge, Massachusetts, United States

The baculovirus/insect cell system has proven to be a robust production method for the manufacture of adeno-associated virus (AAV) for gene therapy. It is an attractive method when compared to the triple transfection mammalian process. The baculovirus/insect cell system does not require the production of DNA plasmids, produces a high percentage of full capsids, has similar over all productivities, and is easily scalable. Although the current baculovirus process is adequate for drug manufacturing, it has seen little optimization since it was first introduced to produce recombinant proteins over 20 years ago. Recently there has also been evidence of decreased vector potency some AAV serotypes. Our work has focused on redeveloping the baculovirus/insect cell system to create a highly productive process, producing potent AAV. This has been accomplished by first engineering the baculovirus genome to predictably control AAV viral protein (VP) ratios, resulting in a baculovirus expression system that has the potential to dial-in AAV potency based different protein ratios. We have also focused on increasing overall productivity by addressing bioreactor parameters and media for Sf9 host cell growth, baculovirus infection and AAV production. It is our goal that this work will result in a state-of-the-art process for AAV production on par with current IgG manufacturing in CHO cells.

BIOT 505

Scaled-down, high-throughput optimization of transfection for lentivirus production for use as gene therapy vectors

Sneha Gopal1, [email protected], Ronit Ghosh1, Adam Osborne2, Lindsay Hock2, Marshall Poirier2, Tanzim Uddin2, Anudeep Ramchetty2, Jill Zemianek2, Kun Fang2, Gretchen Gee2, Sadettin Ozturk2, David McNally2, Steven M. Cramer3, Jonathan S. Dordick4. (1) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States (2) MassBiologics, Mattapan, Massachusetts, United States (3) Ricketts Bldg, Rensselaer Polytechnic Inst, Troy, New York, United States (4) Center for Biotechnology, Renselaer Polytechnic Inst, Troy, New York, United States

Lentiviral vectors (LVVs) are emerging as a promising tool for gene and cell therapy. Due to the ability of LVVs to deliver genetic payloads to both dividing and non-dividing cells, they possess significant potential for introducing therapeutic genes to cells of interest. LVVs are typically produced by transfecting envelope, packaging and transfer plasmids into Human Embryonic Kidney 293 (HEK293) or other suitable cell lines. Nevertheless, several challenges remain in the production process including low titers and a lack of suitable scale-up production. To address these concerns, we developed a high-throughput microwell platform for upstream transfection optimization leading to higher yields of LVVs. Several commercially available cell media were evaluated for their ability to support high cell densities. BalanCD – a commercially available HEK293 suspension cell medium was able to support both high cell densities and generate high transfection efficiencies, and was, therefore, selected for further screens. To optimize transfection, screens were performed to establish optimal amount of DNA/cell, viable cell density at time of transfection, optimal ratios among packaging, envelope and transfer plasmids, and the ideal transfection reagent to DNA ratio. Comparisons of optimal conditions across scales were then made for both batch and perfusion scale-up modes. BIOT 506

Withdrawn

BIOT 507

Understanding scalability of Mustang® Q devices in lentiviral vector purification using DOE approach

Aydin Kavara1, [email protected], Kurt Boenning1, Julio Huato1, Todd Sanderson1, Cristina Peixoto2, Tiago Faria2, Rene Gantier1, Mark Schofield1. (1) Bioprocess R&D, Pall Corporation, Westborough, Massachusetts, United States (2) iBET, Oeiras, Portugal

Lentiviral vectors (LVVs) are increasingly used in treatment of acquired and inherited genetic diseases. Production and purification of the LVVs remains challenging due to low titers from upstream processes and the low stability of the virus. A typical LVV purification strategy might include an AEX step positioned after clarification and before tangential flow filtration and final filtration. Thus, anion-exchange (AEX) chromatography has a key role in purification of LVVs. As the AEX step is positioned directly after clarification, relatively large volumes have to be processed. This makes membrane chromatography an ideal choice as it can be operated at and above 10 membrane volumes per minute. Despite widespread application of AEX membrane chromatography, there has been little published effort to understand the scalability of membrane devices when transitioning from filter plate experiments to small and then larger capsules. Here we implemented a Design of Experiment (DOE) approach to optimize the bind and elute conditions of LVVs in Mustang Q filter plate experiments and then transferred these conditions to 0.18 mL and 1 mL Mustang XT Acrodisc® Units. Gradient and step elution experiments were carried out to understand the role of salt and additives in desorption of LVV from the membrane surface. In addition, we compared the performance of Mustang Q devices to other AEX sorbents. Under optimized conditions, the observed capacity of Mustang Q capsules was 2.4 x 1011 to 1.5 x 1012 transfecting particles/mL of membrane. In summary, a scalable LVV purification protocol was developed for facile transition from Mustang Q filter plate to larger XT capsules.

BIOT 508

Ultrafiltration behavior of adeno-associated viral vectors: Understanding the implications of choice of process parameters and device format on commercial manufacturing

Abhiram Arunkumar, [email protected], Nripen Singh. Technical Operations, Voyager Therapeutics, Cambridge, Massachusetts, United States Recombinant adeno-associated viral vectors (rAAV) are increasingly used as the vector of choice in gene therapy. The purification process of rAAVs involves cell lysis, clarification of the lysed cells to separate the supernatant containing the rAAV, benzonase® treatment, followed by several purification steps. The final step is an ultrafiltration/diafiltration step to concentrate the viral vector and place it in the formulation buffer matrix. While the ultrafiltration behavior of proteins and monoclonal antibodies are very well understood, rAAV transport in ultrafiltration membrane modules are not. This work provides data to demonstrate the impact of membrane molecular weight cut-off, transmembrane pressure, crossflow-rate and the choice of constant TMP-mode or constant permeate flux-mode on the process performance and product quality (recovery of rAAV and aggregation). Several process runs using pressure-dependent ultrafiltration and pressure-independent ultrafiltration are compared to identify the most robust operating condition. Finally, the choice of reusable ultrafiltration modules is compared against single-use ultrafiltration modules keeping product quality, cost of goods and process performance as performance indicators for commercial rAAV manufacturing.

BIOT 509

High-resolution structure determination by microcrystal electron diffraction (MicroED)

Brent Nannenga, [email protected]. Arizona State University, Tempe, Arizona, United States

Microcrystal electron diffraction, or MicroED, is a method that is capable of determining high-resolution structures from very small and thin 3D crystals using a transmission electron microscope. For MicroED, the crystals used are several orders of magnitude smaller than what is required for single crystal X-ray diffraction experiments, making this technique valuable for targets where crystal growth and optimization are difficult. MicroED has been successfully used on microcrystalline samples including proteins, peptides and small organic molecules, in many cases to very high resolutions. In this presentation, the MicroED workflow will be described and recent applications of the method will be highlighted.

BIOT 510

Rational design of a genetically encoded Xe-129 NMR contrast agent for Zn2+ detection

Zhuangyu Zhao, [email protected], Serge Zemerov, Ivan J. Dmochowski. Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States Xenon-129 magnetic resonance imaging (MRI) has greater sensitivity and selectivity over traditional H-1 MRI methods, due to xenon’s hyperpolarizability, which provides ~105 signal enhancement compared to standard collection of room-temperature Xe-129 nuclear spins, low intrinsic Xe-129 NMR background signals and large chemical shift range. Hyperpolarized Xe-129, coupled with chemical exchange saturation transfer, also known as hyper-CEST, has previously enabled picomolar detection sensitivity using small-molecule-based and genetically encoded contrast agents. However, design of biocompatible Xe-129 MRI contrast agents that respond to biologically important substances remains challenging. To this end, we have developed a genetically encoded Xe-129 NMR biosensor for Zn2+ detection, which holds promise for tracking Zn2+ in pancreatic β-cells, prostate tissues and Aβ amyloid plaques, etc. To achieve Zn2+ sensing, we engineered maltose binding protein (MBP), a known Xe binding protein, into a Zn2+-responsive smart sensor by employing computational methods to identify a mutation site. A complex salt bridge in MBP was found to be required for generating hyper-CEST signal, which was therefore re-designed into a Zn2+ binding site. Zn2+- dependent hyper-CEST signal was observed at +50 ppm, referenced to free Xe in aqueous solution, and this signal can be readily detected using only 1 µM contrast agent which gave rise to 0.17 ± 0.02 contrast. The contrast agent showed no response to biologically abundant Mg2+, Ca2+ and Fe3+ and a weak response to Cu2+, demonstrating good ion selectivity. These results highlight the huge potential for utilizing MBP as a versatile protein system to design novel Xe-129 MRI contrast agents.

BIOT 511

Use of surface plasmon resonance to characterize A2AR and Gαs association

Kirsten S. Koretz1, [email protected], Claire McGraw2, Anne S. Robinson3. (1) Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States (2) Chemical and Biomolecular Engineering, Tulane University, New Orleans, Louisiana, United States

G protein-coupled receptors (GPCRs) are expressed widely throughout the human body. They are the largest family of receptor proteins and all are composed of characteristic seven transmembrane alpha helices. Upon activation by ligand binding, the receptor shifts conformations, initiating an intracellular signaling response. The first step in this signaling cascade consists of the dissociation of a G protein from the GPCR as the receptor structure changes into the active conformation. The G protein proceeds to activate and amplify further downstream signaling via enzymes and second messengers (e.g. cAMP). In this work, the GPCR of interest is the adenosine A2A receptor (A2AR), the most well-characterized of the four adenosine receptors. In an effort to better understand the structure-function relationship of the receptor, A2AR has been crystallized bound to a variety of ligands, as well as a mini-G protein. The crystal structures elucidate the structure of the receptor in different active and inactive states, but all the structures truncate nearly 25% of the receptor at the C-terminal end. This truncation aids in crystallization of the receptor, as the C-terminus is long and unstructured; however, removal of such a large percentage of A2AR could lead to a structural snapshot that is quite distinct from the authentic native structure. Previous work from our laboratory observed ablation of the downstream signaling response in both yeast and mammalian cells upon truncation of the C-terminus, suggesting a role for the C-terminus in either G-protein binding or conformational changes. Here, we successfully characterized the interaction between A2AR and Gαs by measuring association and dissociation rates via surface plasmon resonance (SPR). We also compared the data to that of A2AR truncated at amino acid 316 (A2AΔ316R), one of the truncations used in receptor crystal structures. Comparison of Bmax, KD, kon, and koff indicates that while both receptors associated with Gαs, A2AΔ316R had a decreased Bmax and kon, as well as an increased koff, which produced an increased KD. These results suggest that the decrease in cAMP production upon truncation of the A2AR C-terminus seen previously may be related to a reduction in formation of GPCR-G protein complexes in addition to a faster complex turnover.

BIOT 512

Two-step inactivation model of α-amylase: How do non-aqueous solvents influence the enzyme activity?

Arshad Khan, [email protected]. Chemistry, Penn State, State College, Pennsylvania, United States

α-Amylase is an enzyme present in our saliva and pancreatic secretions and is responsible for the break-down of starch into sugar molecules. It undergoes reversible inactivation by first dissociation of calcium ions followed by irreversible inactivation of apoenzyme by heat. Since non-aqueous solvent suppresses the ionic dissociation step, the enzyme achieves a greater stability in non-aqueous solvents. The effect of heat and non-aqueous solvent will be discussed on the basis of the two-step model.

BIOT 513

Tissue-specific dystrophin isoforms have different stabilities and functional roles

Vaibhav Upadhyay1, [email protected], Sudipta Panja1, Swati Bandi1, Krishna Mallela2. (1) Dept of Pharmaceutical Sciences, University of Colrado Denver, Aurora, Colorado, United States (2) Pharm Sci box C238-P15, Univ of Colorado Denver, Aurora, Colorado, United States

Mutations in Duchenne muscular dystrophy (DMD) gene that affect the expression of dystrophin protein lead to a number of disorders collectively called dystrophinopathies characterized by muscle degeneration and loss. Dystrophin is a large protein (427 KDa) made up of N-terminal calponin homology (CH) domains, 24 spectrin repeats, cystein rich (CR) domain and C-terminal (CT) domain. Numerous isoforms of dystrophin are known to exist due to alternative splicing and usage of alternate promoters. These variations occur in the functionally important N-terminal and the C-terminal domains. The N-terminal domain mediates interaction with actin, whereas the C-terminal domain interacts with cytosolic proteins like dystrobrevin and syntrophin. The impact of these isoform variations on the structure and function of dystrophin is unexplored. Dystrophin isoforms have differing tissue specificity and thus the differences in their structure- function become important in understanding tissue specific symptoms of dystrophinopathies. We probed the impact of these variations on structure-function relationship of dystrophin. We found that minor N-terminal variations in the CH domain of dystrophin isoforms modulate their thermodynamic stability and actin-binding function, thus leading to specificity in dystrophin-actin interactions in various tissues. We also characterized the yet unexplored CT domain of dystrophin, and the variations in the CT domain of dystrophin lead to differences in the stability and binding specificity towards its binding partners. These results show that sequence variations in tissue specific isoforms do govern thermodynamic stabilities, thus affecting tissue-specific dystrophin expression, and dystrophin interaction networks. These differences should be considered before designing effective therapeutic strategies for dystrophinopathies that specifically affect different tissues with a myriad of symptoms.

BIOT 514

Structural studies of an apoptotic signaling receptor complex, proNGF-p75NTR- Sortilin, of neuron using cryo-EM

Purbasha Nandi, [email protected], Po-Lin Chiu. School of molecular Sciences , Arizona state university , Tempe, Arizona, United States

One of the prominent causes of neurodegenerative diseases is the dysregulation of downstream signaling of the neurotrophin receptor p75NTR in complexation with various neurotrophin ligands like Nerve Growth factor (NGF), Brain-Derived neurotrophic factor (BDNF) or Trk’s(Tyrosine Kinase receptors). In healthy neurons, p75NTR upon binding to ligands like mature NGF and TrkA assists the proliferation of neuronal cells leading to cell survival. On the contrary in aging cells or diseased conditions, the levels of the precursor of NGF (proNGF) is dramatically high, and its interaction with p75NTR aided by another transmembrane protein Sortilin, has neurotoxic effects, leading to neuronal atrophy or fatal cell apoptosis. Also, intramembrane proteolysis of p75NTR generates a substrate for γ-secretase, which is crucial in regulating increased levels of amyloid-beta (Aß) peptides in Alzheimer’s disease. To elucidate the governing principles of this unfavorable signaling, we seek to identify the molecular mechanisms underlying the preferential affinity of pro-NGF over NGF for p75NTR, activating the apoptotic pathway in presence of coreceptor Sortilin. Due to the small, flexible and highly disordered structure of the pro-domain of NGF, traditional X-Ray Crystallography or NMR prove cumbersome. Hence, single-particle cryo-electron microscopy is a promising tool to characterize the structural and mechanistic details of protein-protein interactions at near-atomic resolution. Identification of the critical amino acid residues can provide useful insight into potential drug designing for related neurological disorders. Initially, each of these recombinant membrane proteins is being purified individually by using the Sf9 insect cell system for overexpression, followed by the analysis of sample purity using size exclusion chromatography, Western Immunoblotting, and negative staining TEM.

BIOT 515

Understanding reaction mechanisms of monoterpene synthases using atomistic simulations

Hoshin Kim1, [email protected], Narayanan Srividya2, Bojana Ginovska3, Bernd M. Lange2, Simone Raugei4. (1) Physical & Computational Science Directorate, Pacific Northwest National Laboratory, Richland, Washington, United States (2) Inst of Biological Chemistry, Washington State University, Pullman, Washington, United States (3) K1-83, PNNL, Richland, Washington, United States (4) Chemical Materials Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States

Monoterpene synthases (MTSs) catalyze the production of a diverse array of terpenoid products, which are widely used as key ingredients for many industrial applications including pharmaceuticals and biofuels. MTSs control challenging reactions — such as hydrogen atom, hydride, and proton transfers — in a selective and stereospecific manner. However, despite their importance and versatility, a general understanding of how these enzymes achieve high specificities and efficiencies remain elusive. In this study, we performed molecular dynamics (MD) simulations of three MTSs, (-)-limonene synthase, bornyl diphosphate synthase, and 1,8-cineole synthase, aimed at understanding how these enzymes control selectivity by exploiting steric confinement to promote specific configurations of catalytic intermediates. Mechanistic hypotheses based on MD simulations were validated against experimental mutagenesis studies. The potential impact of key residues on product specificity will be discussed.

BIOT 516

Decoding the role of amino acids attributes for the prediction of protein thermostability: Machine learning and statistical approach

Vinay Kumar Duggineni, [email protected], Shashi Kumar, Parag Arvind Deshpande. Quantum and Molecular Engineering Laboratory, Chemical Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India

The engineering of thermostable enzymes is particularlly relevant for enzyme engineering. Industries seek to use environmentally friendly enzymes in their processes. Thermostability of proteins in general and especially thermophilic proteins has been a subject of a wide variety of studies based on theoretical and experimental investigations. Enzymes typically function at temperatures below 60o C and denature if exposed to higher temperatures. In contrast, a small portion of enzymes can withstand higher temperatures as a result of various structural adaptations. Understanding how proteins adapt to functions at high temperatures is important for deciphering the protein attributes that dictate protein stability. The present work focuses on employing various supervised and unsupervised machine learning algorithms as well as attribute weighting approaches to find protein attributes that contribute to enzyme thermostability. Specifically, we compared two groups of enzymes: mesophilic and thermophilic enzymes. Further, a combination of attribute weighting with supervised and unsupervised clustering algorithms was used for prediction and modeling of protein thermostability from amino acid composition properties. This study demonstrates the feasibility of predicting thermostability irrespective of sequence similarity and is expected to serve as a basis for engineering thermostable enzymes.

BIOT 517

Purification of Cas9/sgRNA complexes from residual sgRNA by ultrafiltration

Ivan Manzano1, [email protected], Grace Vezeau2, Neil Taylor1, Howard Salis1,2, Andrew L. Zydney1. (1) Chemical Engineering, Penn State University, University Park, Pennsylvania, United States (2) Agricultural and Biological Engineering, Penn State University, University Park, Pennsylvania, United States

CRISPR technologies have created exciting opportunities for gene editing and novel biotherapeutics, using an engineered single guide RNA (sgRNA) that binds to specific DNA sites enabling modification / regulation of targeted genes. The development of CRISPR-based therapeutics requires robust methods for downstream processing, including the purification ribonucleoprotein complex from residual free RNA. We present the first study on the potential use of ultrafiltration membranes to separate Cas9/sgRNA complex from free unbound single guide RNA. The Cas9 protein was produced in E. coli NiCo21 (DE3) cells transformed with pET- 28a-Cas9-Cys plasmid containing the S. pyogenes Cas9 with a 6xHis tag. Cells were grown in a 5L bioreactor, lysed, and clarified, with the lysate purified by Nickel Affinity Chromatography using a 5 mL HisPure Ni-NTA column. A 48 kDa sgRNA was synthesized from a DNA template consisting of the T7 promoter sequence, a guanosine nucleoside to promote efficient initiation of transcription, and the appropriate DNA sequence. The DNA was prepared from a gBlock fragment and amplified by PCR. T7 RNA polymerase was used for in vitro RNA synthesis using the T7 HiScribe Kit. Synthesized RNA was purified using phenol:chloroform extraction and then coupled to the Cas9 protein. We performed stirred cell filtration experiments with the individual Cas9 and sgRNA to identify appropriate conditions for separation. Key variables were the membrane molecular weight cutoff (50 – 300 kDa), the membrane chemistries (PES and RC), and the filtration velocity. We separate the Cas9/sgRNA complex from free sgRNA via diafiltration, with samples analyzed by gel electrophoresis using SYBR Gold and SYPRO Ruby dyes for visualization of the RNA and Cas9, respectively. Preliminary data showed effective removal of the sgRNA using a Biomax 100 kD membrane at filtrate flux around 30 µm/s (100 LMH). These results demonstrate the potential of using ultrafiltration for the purification of ribonucleoproteins from free unbound RNA, providing a low-cost scalable system suitable for large-scale production of these new biotherapeutics. BIOT 518

Development of downstream purification process for lentiviral gene therapy vector

Ronit Ghosh, [email protected], Sushmita Koley, Sneha Gopal, Jonathan S. Dordick, Steven M. Cramer. Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States

Lentiviral vectors have been increasingly employed as gene delivery vehicles for the treatment of a variety of genetic and acquired human diseases. As gene therapy treatments progress from pre-clinical to clinical phases, there is a growing demand for high purity and concentrated lentiviral vectors. However, the current manufacturing process presents major challenges such as low virus production titers and viral vector stability leading to low downstream recoveries. This work presents a workflow for robust, fast and scalable process development for LVV purification. Viral vector stability was first evaluated in different mobile phase conditions spanning a range of pH, ionic strength, and buffer species to identify the optimal conditions for the screening of chromatographic media. The stability results suggested that the vector was stable in a pH range of 6.5-7.5 and the stability was observed to decrease above or below this range. The effect of salt concentration on vector stability was also investigated and it was observed to reduce at higher ionic strength likely due to increased osmotic pressure. The results of this study helped to devise a robust purification strategy with the use of chromatographic resins, membranes and/or monoliths which could provide stable lentiviral vector. Process development stage included high-throughput screening of resins in a bind-elute mode using 96-well slurry plate format to minimize time and material requirements. Following this, the top candidates were evaluated in miniature column setting for obtaining resolution/selectivity under different gradient and loading conditions and ranked based on capacity, yield and impurity clearance criteria. A lab- scale column was performed to ensure the validity of these findings along with tracking the levels of process-related impurities like HCP and hcDNA. Finally, the employed screening strategies and resulting purification schemes aided to guide the development of a GMP process for the manufacturing of clinical-grade lentiviral vectors for gene therapy applications.

BIOT 519

Evaluation of non-immunoaffinity chromatographic methods for adeno- associated virus capture in Sf9-baculovirus system

Matthew Luther, [email protected], Aditya Ansodaria, Abhiram Arunkumar, Nripen Singh. Technical Operations, Voyager Therapeutics, Cambridge, Massachusetts, United States

As recombinant Adeno-Associated Virus (rAAV) based therapies continue to show increasing clinical progress, the business and scientific case for robust and efficient manufacturing processes grows proportionally. As such, traditional rAAV downstream processes should be evaluated for robustness and efficiency at each unit operation of the manufacturing process. In this study, multimodal and ion-exchange resins were evaluated as alternative chromatographic capture options for purification of rAAV produced in an Sf9- Baculovirus system. rAAV recovery and the clearance of process related impurities were evaluated along with resin reusability. The result of this evaluation provided valuable insights in developing an rAAV purification process that ensures production of pure and high-quality drug products.

BIOT 520

Robust clarification for adeno-associated virus vectors via depth filtration

Thomas Parker1, [email protected], Elina Gousseinov1, Youness Cherradi2, Claire Scanlan1. (1) Technology Management, MilliporeSigma, Burlington, Massachusetts, United States (2) Technology Management, Merck Chemicals N.V./S.A., Overijse, Belgium

The bioprocessing industry widely embraces depth filtration for cell culture clarification of recombinant proteins produced in mammalian cell lines; however, Adeno-Associated Virus (AAV) processes present a unique harvest challenge due to a potential lysis step and the surface characteristics of the target product. While efficient at removing cell debris and other insoluble impurities, many depth filters are optimized for adsorption of soluble impurities for monoclonal antibody process streams, where target products are relatively high concentration and have a slight net positive surface charge. Since AAV have slightly acidic isoelectric points, these viruses may interact with positively charged adsorptive depth filters at typical harvest pH ranges. Process development, therefore, requires thorough screening and investigation of depth filters prior to selection for AAV clarification. In light of these concerns, MilliporeSigma reviewed internal data to understand trends of depth filter performance for clarification of AAV from SF9 and HEK293 cell lines; additionally, a controlled screening experiment confirmed findings from data mining activities. This body of work presents the results from these two investigations, which suggest Millistak+® depth filters to be a robust solution for clarification of multiple AAV serotypes from SF9 and HEK293 cell cultures. Additionally, the study demonstrates scalability from bench scale to process scale, giving confidence for implementation in cGMP. Robust performance, ease of use in manufacturing, and scalability make depth filtration an ideal selection for clarification of AAV.

BIOT 521

Use of mechanistic modeling in process development for lentiviral vector purification Sushmita Koley, [email protected], Scott Altern, Ronit Ghosh, Steven M. Cramer. Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, United States

With the advent of gene therapy and its potential to provide a cure for a variety of diseases, there has been an increasing interest in lentivirus as a therapeutic vector as well as for transfection of cell therapy agents. However, there is an urgent need to improve the yield of downstream processes for producing lentiviral vectors. The objective of this work is to generate mechanistic models that bridge the gap between high throughput screening and process scale chromatography to reduce time and experimental efforts for process generation with gene therapy agents. This work includes development of a modeling strategy for predicting the elution behavior of lentivirus and removal of product and process-related impurities such as HCP and hcDNA using various ion exchange platforms. The paper presents an efficient workflow to facilitate process development using a combined high throughput screening and modeling approach and demonstrates its utility using a number of formats including resins, membranes and monoliths.

BIOT 522

Membrane-assisted precipitation and redissolution of virus-like particles: Approaches for an integrated capture step

Nils Hillebrandt1, [email protected], Philipp Vormittag1, Thorsten Klamp2, Juergen Hubbuch1. (1) KIT, Karlsruhe, Germany (2) BioNTech Protein Therapeutics GmbH, Mainz, Germany

Virus-like particles (VLPs) are an emerging modality among biopharmaceuticals applied in the field of cancer and infectious diseases. VLPs are produced by heterologous expression in various hosts, such as E. coli. Intracellular expression prescribes lysis of the cell as initial purification step, leading to a broad contaminant spectrum of cell debris, proteins and nucleic acids. Capture of VLPs from clarified lysate therefore has to reduce protein and nucleic acid contaminants and ensure high recovery of VLPs. The comparably large size of VLPs is a drawback in chromatographic separation; however, it qualifies VLPs for membrane separation and precipitation. Capturing VLPs by precipitation and subsequent redissolution reduces host-related contaminants and provides an alternative to chromatographic separations. Membrane filtration processes are easily scalable and allow for control of shear stress. Thus, utilizing cross-flow filtration for precipitation-based purification of VLPs merges key advantages of both unit operations. This integrated approach facilitates scalable separation, wash and redissolution of precipitate. In this work, an integrated membrane-based precipitation and redissolution capture step was developed for Hepatitis B core Antigen VLPs. Using a commercial 0.2 µm membrane, precipitated VLPs were retained while smaller contaminants were washed out by diafiltration. Subsequent depletion of precipitant lead to redissolution of VLPs. Soluble VLPs were able to pass through the membrane into the permeate stream where in-process monitoring of 280 nm UV-absorbance and conductivity allowed for automated peak fractionation. Protein contaminant removal was highly efficient resulting in >95% protein purity whereas nucleic acid depletion was incomplete. This was addressed by developing two alternative process variants, which incorporated a preliminary Benzonase treatment or an integrated chromatographic flow-through step, respectively. Both variants further reduced nucleic acid content by >90% and below a concentration of 5 ng/µL, maintaining high protein purity and yield. In summary, the developed capture step lays the groundwork for a scalable, automated and fully integrated VLP purification process.

BIOT 523

Streamlining polyvalent vaccine purification: Case study for a custom, dual- purpose, affinity chromatography resin

Kelley Kearns, [email protected], Warren Kett, Karol Lacki. Avitide Inc., West Lebanon, New Hampshire, United States

The complexity of vaccine purification is underscored by the diversity of antigen types (e.g., live-attenuated viruses, virus-like particles, subunits/recombinants/polysaccharides/conjugates, toxoids) as well as the frequent need to purify multiple antigens for use in a single vaccine formulation, i.e., polyvalent vaccines. The use of affinity chromatography, which provides high recovery with unmatched purity in a single step based on highly specific interactions with the target molecule, can greatly simplify the purification process.

This case study demonstrates the invention of an affinity chromatography resin to selectively capture and elute multiple protein targets that are used in a proprietary vaccine formulation using a fusion of two target-specific ligands. Development of the affinity resin was challenged by the large size of the vaccine constructs and their high propensity toward aggregation. The performance of the resin was optimized using a high-throughput screening methodology, testing variations in ligand density, ligand fusion linker, base bead matrix, size, activation level, and conjugation chemistry, average pore size, and the bead-to-ligand spacer molecule. The final custom resin is capable of purifying the two different antigens, each from a separate feedstream, at greater than 80% yield and achieving > 99% purity in a single step. This is another example of a ligand discovery and affinity resin optimization process designed for bioprocessing that has been successfully applied to various other macromolecules, including bispecific antibodies, therapeutic proteins, and gene therapies.

BIOT 524

Two-step purification process for H1N1 virus using ion exchange chromatography resins Wei Zhang1, Duy Tien Ta1, Kai Ling Chu1, Mark A. Snyder2, mark_snyder@biorad.com, William H. Rushton2, [email protected]. (1) Downstream Processing Group (DSP), Bioprocessing Technology Institute (BTI), Singapore, Other, Singapore (2) Bio-Rad Laboratories, Hercules, California, United States

Yearly outbreak of influenza causes millions of cases of infections worldwide, with about ten percent being fatal. The pandemic risk of the disease and the high mutation rate of the viruses have urged scientists to continuously and rapidly develop new kinds of vaccines with higher qualities. In this work, we demonstrate the development of vaccine production process against the Influenza A H1N1 subtype, using recently developed ion exchange resins. The aim is to develop a low cost process while maintaining high quality and safety of the product.

H1N1 virus was propagated in serum free medium using Vero cells as a host and released into the culture supernatant. The supernatant was separated by centrifugation, heat-treated for virus inactivation, followed by Benzonase treatment and concentration by TFF. H1N1 virus concentrate was then captured on a wide pore anion exchanger and subsequently polished by wide pore cation-exchanger. High purity and good overall yields were achieved with this purification. Digital-droplet PCR (ddPCR) with high sensitivity was used as the main quantitative assay throughout the whole process. All purification steps used here are scalable and can be adapted for large-scale production, making the process is an efficient alternative for Influenza A H1N1 vaccine production.

BIOT 525

Early culture pH controls antibody glycosylation using traditional and PAT methods

Madiha Khurshid, [email protected], Prince Bhebe, Li Zhang, Tony Wang, Erin Franco, Nelson Chavez, Eleanor Le, Richard Wu, Jack Huang. Amgen, Inc, Thousand Oaks, California, United States

Control of critical quality attributes (CQAs) is essential for the manufacture of high- quality biologics. Control over glycosylation of antibodies within specified ranges can be challenging but is crucial for product efficacy, immunogenicity and pharmacokinetics. Particularly for biosimilar products, the glycosylation ranges for demonstrating similarity to the innovator product can be very small. Changes in bioreactor process parameters at the manufacturing scale could impact glycosylation outside the specified ranges. Several bench-scale experiments were conducted to understand the relationship between TAF and early culture pH. It was demonstrated that the TAF of the product harvested was affected by pH early in the culture. Given this information, pH control was improved using traditional univariate setpoint control in the early phase of bioreactor culture. This resulted in lots with acceptable TAF. More sophisticated bench-scale studies were carried out to understand the effect of initial pH in combination with other levers with the aim of constructing a predictive model. The pH was intentionally manipulated in early culture to create a defined disturbance in TAF. A Process Analytical Technology (PAT) strategy was used to monitor and control TAF at large-scale.

BIOT 526

Early activation and late resolution of the unfolded protein response in fed-batch culture associated with high productivity CHO cells

Dyllan Rives1, [email protected], Tara Richbourg2, Sarah W. Harcum3, Mark A. Blenner4. (1) Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States (2) Genetics and Biochemistry, Clemson University, Clemson, South Carolina, United States (3) Bioengineering, Clemson University, Clemson, South Carolina, United States (4) Chemical Biomolecular Engineering, Clemson University, Clemson, South Carolina, United States

Chinese hamster ovary (CHO) cells are common protein production platforms due to efficient post-translational modification machinery and endoplasmic reticulum (ER) quality control; however, secretion levels needed for industrial cell lines can lead to an imbalance in ER homeostasis. Accumulation of improperly folded proteins is a particularly challenging bottleneck in cell line engineering and initiates the unfolded protein response (UPR). This research aims to demonstrate higher specific productivity results in unavoidable ER stress by measuring the UPR in cell lines engineered for high-levels of recombinant protein production. In fed-batch culture, we achieve high and low specific productivity with two CHO cell lines, one producing immunoglobulin G (IgG) and one producing erythropoietin (EPO-Fc), respectively. Western blot and quantitative polymerase chain reaction (qPCR) were used to investigate the UPR time course, correlated to product titer. In comparison to the parental cell line controls, both CHO cell lines exhibited early activation of the IRE1 and PERK pathways. By Day 3 of fed-batch culture, spliced XBP-1 mRNA expression increased up to 64 fold, and both ATF4 and CHOP mRNA expression increased up to 8 fold. Transcriptomic analysis was conducted to monitor UPR targets and identify additional signaling transduction pathways activated during fed-batch culture. To determine if highly productive lines have been selected for the ability to cope better with ER stress, the UPR was monitored in fed-batch cultures of CHO cell pools with reduced IgG production via shRNA interference. Lastly, we explore the impacts of combining chemicals known to enhance productivity or reduce ER stress on cell productivity, titer and the UPR.

BIOT 527

Maintaining productivity over extended durations for perfusion processes

Amanda Underhill2, [email protected], Supal Vyas2, Nikita Chauhan2, Paul Gramlich2, Nitya Jacob1. (1) Amgen, Thousand Oaks, California, United States (2) Pivotal Drug Substance, Amgen, Cambridge, Massachusetts, United States Manufacturing process platforms in the biopharmaceutical industry are evolving from low biomass, large volume fed-batch processes to include continuous, sustained high biomass, small volume processes. This transition provides the opportunity for advancing innovative solutions to new process development challenges. During the development of one such intensified process, several different cell culture process modes were evaluated: steady state perfusion, non-steady state intensified batch and fed-batch. Productivity remained constant through the duration of the culture for the non-steady state intensified batch and fed-batch process modes but decreased in the steady state perfusion mode. Interestingly, the overall productivity was found to correlate negatively with culture viabilities. Investigation of this decrease in productivity revealed a correlation to cell age and subsequently to a decline in copy number. Implementation of selective medium for perfusion effectively reversed the decline in productivity, maintaining it without impact to product quality. As platforms in the biopharmaceutical industry continue to evolve, these solutions can be leveraged in the development of a robust perfusion process.

BIOT 528

Defeating product quality challenges for biosimilars through cell culture process development

Yogender Kumar Gowtham1, [email protected], Richard Ottman1, Srivatsan Gopalakrishnan1, Niket Bubna1, Gautam Nayar1, Sigma Mostafa2. (1) Upstream PD, KBI Biopharma, Cary, North Carolina, United States (2) KBI Biopharma, Durham, North Carolina, United States

Biosimilars are a growing interest in the biopharma industry as they provide affordable alternatives, shorter regulatory path and due to the near-term patent expiry on several blockbuster biologics. However, biosimilar process development is challenging as it requires comparable product quality and efficacy to the innovator, which is established by matching physiochemical properties. We have established a toolbox to regulate product quality for biosimilars through upstream process development. Two case studies demonstrating use of the toolbox will be presented. In the first case study, differences in charge variant distribution were observed due to cell line expression systems (CHO vs NS0). Media and feed screening experiments and process parameter optimization were conducted to achieve a 3-fold increase in acidic charge variants to match with the innovator product quality profile. In the second case study, lower ADCC activity was observed due to lower levels of high mannose species. A screening study was used to investigate the impact of several factors including feed supplement, osmolality, and pH to modulate N-glycan profiles, particularly mannose species. Results from the study showcasing at least 2-fold increase in mannose levels and consequent improvements in ADCC levels will be discussed.

BIOT 529 More efficient site-specific integration in CHO cells enabled by a CRISPR- mediated genome editing reporter system

Nathaniel K. Hamaker1,2, [email protected], Kelvin H. Lee1,2. (1) Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Delaware Biotechnology Institute, Newark, Delaware, United States

Chinese hamster ovary (CHO) cells continue to be the biomanufacturing platform of choice for the production of therapeutic glycoproteins, including monoclonal antibodies. The traditional cell line development workflow involves random integration of the transgene encoding the recombinant protein of interest. Random integration inherently leads to high levels of clonal heterogeneity and necessitates the tedious screening of multiple clones to isolate those with suitable performance characteristics. In contrast, site-specific integration (SSI) results in transgene knock-in events at user-defined genomic target sites, including prevalidated hot spots capable of supporting highly active and stable expression. SSI has therefore emerged as a powerful cell line engineering technique by enabling the accelerated development of production cell lines with more predictable performance and product quality. Despite these benefits, attempts to perform CRISPR-mediated SSI in CHO cells are typically met with remarkably low efficiencies, hindering efforts to isolate clones in which precise, rather than random, integration has occurred. Numerous methods have been proposed to remedy similar shortcomings observed in other mammalian cell types, yet none have been systematically evaluated in CHO. To overcome this obstacle, we developed the SSIGNAL (Site-specific Integration and GeNome ALteration) reporter system to facilitate rapid screening of CRISPR-mediated genome editing strategies. The system uses fluorescent signals to indicate genotype states, enabling data acquisition via standard flow cytometry instrumentation without the need for single-cell cloning or deep sequencing. The system was applied in CHO cells to test several current approaches for targeted integration to determine the most effective double-strand break repair strategy with the goal of improving efficiency. Ultimately, the development of robust SSI protocols may be implemented to improve long-term stability of transgenes for better cell culture process control.

BIOT 530

New strategy for identification of DNA zip codes via targeted rewiring of endogenous loci in mammalian cells

Meng Zhang1, [email protected], Pankaj Chaturvedi2, Andrew Belmont2, Huimin Zhao1. (1) Chemical and Biomolecular Engineering, University of Illinois at Urbana- Champaign, Urbana, Illinois, United States (2) Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States

Genetic materials inside the nucleus are not randomly distributed but spatially organized. For example, certain genomic loci are preferentially located at the nuclear periphery, while others are more likely to be found at nuclear interior. However, the underlying mechanisms that regulate such spatial distribution of the genome remain largely unknown. Originally discovered in Saccharomyces cerevisiae, “DNA zip codes” are short DNA sequences that encode spatial information targeting towards defined compartments in the nucleus. Similarly, in higher eukaryotes such as mammalian cells, DNA sequences around 5-10kb in size have been identified for spatial targeting from ~200kb bacterial artificial chromosomes (BACs) harboring human genomic sequences. However, previous strategies a have two major drawbacks: 1) the screening relies heavily on random integration of candidate DNA fragments, which suffers from no control over the copy number, no prior knowledge about the landing sites, and potential bias towards open chromatin regions; 2) the construction of BAC libraries by traditional recombineering method is labor-intensive and time-consuming. To overcome these bottlenecks, here we present a new strategy that couples the specificity of CRISPR/Cas9 system together with the large-cargo capability of serine recombinases to achieve targeted rewiring of preselected endogenous loci in mammalian cells. Moreover, direct cloning techniques are applied to quickly generate BAC library (20~100kb) with desired deletions in order to accelerate the library construction for downstream screening. Using this strategy, the targeting function of each modified BAC can be evaluated at the same, defined locus, which excludes undesired chromosomal context effects associated with random integration. Therefore, our strategy enables the dissection to be carried out in a more consistent, reliable and efficient manner. Collectively, we anticipate this strategy to expand the current paradigm of large-scale genome engineering in mammalian systems, leading to novel knowledge about gene regulation on an epigenetic level.

Schematic of the overall workflow

BIOT 531

Development and characterization of a RNA-sensing spatiotemporal gene regulation program for mammalian systems

Victoria M. Hunt, [email protected], Kelvin H. Lee, Wilfred Chen. Chemical Engineering, University of Delaware, Newark, Delaware, United States

To effectively probe gene function and reprogram cell regulatory networks, it is critical to have technology platforms that provide precise and accurate targeting of genes. The use of CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) for targeted silencing or upregulation of transcription, respectively, is currently one of the most utilized technologies for predictable control over gene expression. The CRISPR system allows for sequence-specific targeting of genes but inherently lacks the ability to incorporate useful endogenous signals for spatiotemporal control of gene expression. Here we present the design and further characterization of a class of riboregulators through the incorporation of toehold riboswitches into sgRNA scaffolds. This artificial circuit is able to detect the presence of specific RNA and switch on transcriptional level gene regulation through RNA-RNA strand displacement reactions, which are governed by predictable Watson-Crick base pairing. These synthetic constructs can be programmed to process specific information within the cell including changes in native metabolism and stress responses. We demonstrate the programmability and adaptability of these engineered systems to control gene expression in mammalian systems with minimal infidelity. This system can be engineered to regulate a reporter protein through both repression and activation, with the activation phenotype greater than 3-fold as compared to the control. We show the robustness of our system can be modulated by altering the toehold length of the riboswitches. We also demonstrate the ability to create a cascade-type system to control protein degradation. We envision these synthetic riboregulators can be applied in a variety of contexts within mammalian systems such as improving mammalian cell productivity and mAb product quality. This technology has the potential to function as an RNA-based master regulator for autonomous cellular control to direct specific phenotype in CHO cell cultures.

BIOT 532

Effects of media supplementation on CHO-produced antibody glycosylation: Dynamic changes in glycoform profiles throughout batch culture

Evan Wells1, [email protected], Kevin McFarland2, Madison Greer1, Liqing Song1, Michael J. Betenbaugh2, Anne S. Robinson1. (1) Chemical Engineering, Carnegie-Mellon University, Pittsburgh, Pennsylvania, United States (2) Department of Chemical and Biomolecular engineering, Johns Hopkins University, Baltimore, Maryland, United States

Monoclonal antibodies (mAbs) are glycoproteins that have become leading therapeutics for treating numerous cancers and chronic and inflammatory illnesses. These proteins are produced mainly in Chinese hamster ovary (CHO) cells for several advantageous reasons including scalable culture, high productivity, and suitable glycosylation characteristics. The traditional focus for improving mAb production has centered on raising antibody yields of individual batches by either increasing culture length, manipulating process conditions, or selecting higher productivity clones for commercial production. Despite the stunning progress and improvements to antibody yield in the last twenty years, a continuing challenge is to identify process conditions that balance maximum titer with optimal critical quality attributes (CQAs). Glycosylation is a CQA that impacts antibody stability, serum half-life, and effector functions (ADCC & CDC). The pathway for antibody glycosylation is non-template- directed and heterogeneous process, with nearly a dozen enzymes across both the ER and the Golgi apparatus participating in protein modification. Many improvements to instrumentation and methods have enabled faster and higher resolution glycosylation analysis, but mechanistic insights about glycan variations between cell lines, media formulations, and process conditions still remain scarce. Here, we utilize a design of experiments (DoE) approach to determine how different media supplementation dynamically impact glycosylation throughout a batch process of antibody-producing CHO cells. Glycan profiles will be compared with metabolic information throughout the culture’s duration to determine how nutrient availability contributed to overall glycosylation, and factorial analysis will be shared to assess effects and statistical impact of each supplement on titer, [metabolites], and glycan profile.

BIOT 533

Genetically engineered immunity for cancer: Development and global regulatory approvals of chimeric antigen receptor (CAR) T cells and what’s coming next

Bruce Levine, [email protected]. UPenn Medicine, Philadelphia, Pennsylvania, United States

Since the 1990’s, we have conducted clinical trials of gene modified T cells. Gene editing has created T cell resistant to HIV infection. Chimeric antigen receptor (CAR) T cells targeting CD19 on B cells leukemias and lymphomas have induced durable complete responses in patients who are relapsed or refractory to all other available treatments. This synthetic biology technology has now undergone global multi-center clinical trials and recently received FDA, EMEA, Canada, Switzerland, Japan, and Australia approvals (KymriahTM, Novartis) in relapsed/refractory acute lymphoid leukemia in children and young adults as well as in diffuse large B cell lymphoma. Translation of these technologies from research bench to clinical application requires integrated scientific, engineering, clinical, and regulatory expertise. New designs for genetically engineered T cells include switches and potency enhancements that will be required for targeting solid tumors. The road forward for wide patient access to these uniquely personal cellular therapies depends not only on scientific progress in targeting, gene modification and cellular manipulation, but also on meeting automation, engineering, clinical site onboarding, and health policy challenges.

BIOT 534

Turning gene therapy into medicine: Approval of LUXTURNA™ (voretigene neparvovec-rzyl)

Kathleen Reape, [email protected]. Spark, Philadelphia, Pennsylvania, United States

This presentation will describe some of the clinical, regulatory, and CMC challenges encountered in the development program for voretigene neparvovec-rzyl for use in patients with vision loss due to biallelic RPE-65associated inherited retinal dystrophy. From a translational standpoint, a large animal model (Briard dogs) provided robust nonclinical proof of concept. However, developing a clinical program to support the regulatory approval of a gene therapy for a rare inherited retinal disease for which there were no other treatments available at the time was far from straightforward. For this program, a novel primary endpoint was developed (the multi-luminance mobility test or MLMT), that was relevant to the clinical deficits specific to this condition. Other clinical, regulatory and CMC challenges had to be overcome. Gene therapy trials require long- term follow-up of clinical trial participants, and this is currently ongoing. There are unique challenges associated with the development of gene therapies for rare diseases where no approved product currently exists. It is critically important to engage early and often with patients, families, advocacy groups, researchers, investigators and regulators, to ensure that the clinical development and regulatory strategies support the demonstration of efficacy and safety in individuals with rare diseases.

BIOT 535

Systematic engineering of virus-like particles to identify optimal characteristics for nanoparticle delivery

Bon Ikwuagwu1, [email protected], Emily Hartman2, Danielle Ercek1. (1) Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, United States (2) Chemistry, UC Berkeley, Berkeley, California, United States

Self-assembling protein scaffolds known as virus-like particles (VLPs) are promising delivery vehicles for diagnostic and therapeutic applications. VLPs are easily produced in a bacterial expression system, are biocompatible, and have defined chemical handles that can attach targeting ligands. However, there are some key challenges to be solved. In particular, VLPs are highly stable and must come apart upon entry to the target cell. Moreover, it is difficult to control their size relative to other delivery platforms. Here, we engineered VLPs based on the MS2 bacteriophage by combining comprehensive codon mutagenesis, one-pot selections, and high-throughput sequencing to create protein fitness maps in a strategy we termed Systematic Mutagenesis and Particle Selection (SyMAPS). Using SyMAPS, we identified several mutations that confer a desired acid instability, as well as a single point mutation that uniformly alters the size and geometry of the VLP. We were intrigued by the mutation that alters the size of the VLP because size is a known characteristic that affects accumulation in tumors. Therefore we applied similar mutations to MS2 homologs to understand if the mutation’s effects were generalizable, and went on to identify alternative mutations that would confer the same size shift. Here, we present the detailed results of this suite of studies, and discuss the role of our engineered VLPs both for theranostic applications and as tools for investigating the role of particle size on tumor penetration.

BIOT 536

Solving macromolecular drug delivery with a proteomic barcoding platform

Nicole McNeer1, [email protected], Sara Wiener2, Pascal Egloff3,5, Markus Seeger3, Paolo Cifani4, Alex Kentsis4,1. (1) Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York, United States (2) Albert Einstein College of Medicine, New York, New York, United States (3) Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland (4) Molecular Pharmacology, Sloan Kettering Institute, New York, New York, United States (5) Linkster Therapeutics AG, Zurich, Switzerland

Targeted agents such as antisense oligonucleotides, nanobodies, peptidomimetics and other engineered macromolecules offer compelling means for therapy of conventionally “undruggable” interactions in human disease. However, the efficacy of these bulky agents is severely limited by barriers to tissue and intracellular delivery. Based on recent methods by Egloff et al, in Nature Methods 2019, we developed a platform technology to design, screen, and profile cell penetrating peptides (CPPs) tagged with unique mass spectrometry-assignable peptide barcodes (“flycodes”), for combined delivery and tracking of macromolecules. We assembled libraries of CPPs, based on putative penetration domains of human pathogens, and new computational algorithms for generation of physicochemically related sequences. To profile such libraries, we developed a method to tag CPPs with short peptide barcodes designed for detection by high-resolution mass spectrometry, achieving sub-attomolar (hundreds of molecules per cell) limits of quantitation in subcellular compartments. Proof-of-concept studies of novel CPPs demonstrate improved intracellular and nuclear delivery in diverse cell types, as validated by live-cell confocal microscopy of fluorophore labeled molecules. Using this approach, we developed improved cell-penetrant peptidomimetics to block activity of oncogenic transcription factor MYB in human leukemias, and electroporation-free delivery of Cas9 nuclease complex for therapeutic gene editing of sickle cell disease cells. In all, this approach enables high-throughput screens of macromolecular therapeutics with efficient and specific delivery and penetration properties in cells, tissues and living organisms.

BIOT 537

Investigating the impact of surface chemistry on the uptake of ultrasmall iron oxide nanoparticles by cancer cells

Akshay Narkhede, [email protected], Jennifer A. Sherwood, Angelo Antone, Kasie Coogan, Yuping Bao, Shreyas Rao. Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, Alabama, United States

Glioblastoma multiforme (GBM) and breast cancer brain metastasis represent the most aggressive brain-related malignancies with a survival period of only 4 - 15 months. This is, in part, due to the ineffectiveness of current diagnostic as well as therapeutic techniques in specifically labeling and targeting cancer cells. Herein, we have investigated the utility of ultrasmall iron oxide nanoparticles (USIONPs) (<4 nm) to label U87, glioblastoma cell line (primary brain cancer) and MDA-MB-231Br, brain metastasizing variant of MDA-MB-231 triple-negative breast cancer cells in vitro. Specifically, we investigated the impact of quinic acid and tannic acid surface coatings on the uptake of USIONPs by cancer cells respectively. We, qualitatively and quantitatively, observed a higher cellular uptake of quinic acid- coated USIONPs by both cancer cell lines compared to the tannic acid-coated counterpart at 4, 24 and 72 hr time points. In addition, Prussian blue staining at early timepoint (4hr) revealed the trafficking of quinic acid-coated USIONPs at the cell membrane. Further, the blocking of cancer cells with free quinic acid resulted in a significant reduction in the uptake of quinic acid-coated USIONPs, indicating their targeting capability through interactions with certain cancer cell surface receptors. Quinic acid and its derivative are known to interact with P-selectin membrane receptors, which are typically overexpressed in cancer cells, tumor microenvironment, and metastatic niche. To this end, we qualitatively confirmed the expression of P-selectin in U87 and MDA-MB-231Br and demonstrated that blocking P-selectin receptors significantly reduced the uptake of quinic acid-coated USIONPs by MDA-MB-231Br cells. Overall, our work establishes the potential of quinic acid-coated USIONPs to be utilized as a platform for tumor-targeted drug delivery and in imaging and detection of primary and metastatic tumors and provides a strong proof-of-concept for future in vivo studies.

BIOT 538

Nanoparticle-loaded exosomes for targeted breast cancer therapy

Mengmeng Zhai, [email protected]. chemistry and biochemistry , university of oklahoma, Norman, Oklahoma, United States

Exosomes as cell-original nanovesicles are ideal carrier for targeted delivery of therapeutic cargos such as gene, drugs and nanoparticles. However, the lack of effective loading methods and low efficiency of the cargos have impeded their therapeutics applications. As a new type of gene, drug and nanoparticles delivery vehicles, exosomes have several advantages, such as natural protection of encapsulated cargos, low immunogenicity and cytotoxicity, nanoscale size, high cellular uptake, and modifiable surface proteins. In this work, we integrated phage display and nanotechnology to develop a novel strategy for loading nanoparticles into exosomes for breast cancer therapy. We constructed a novel nanocomplex, where the exosome internalizing peptide modified nanoparticles were loaded into the tumor-homing exosomes. The nanocomplex was used as a novel “drug” to achieve enhanced cancer killing efficacy. This effective loading methods significantly improved the cancer cell killing efficiency in vitro and in vivo.

BIOT 539

Computational model of in situ forming implantable drug delivery devices

Tamara Kinzer-Ursem, [email protected]. MJIS 3084, Purdue University, West Lafayette, Indiana, United States

Implantable, bioresorbable drug delivery systems offer a unique alternative to current drug administration techniques; allowing for patient-tailored drug dosage while also increasing patient compliance. In situ forming implants (ISFIs) undergo phase inversion solidification and then slow degradation when introduced into the aqueous environment of the body, resulting in controlled release of the encapsulated drug. In this work, we present a comprehensive mathematical model of polymer implant degradation and drug release. We implement a multi-scale modeling approach to investigate both the precipitation of polymer into a solid implant in the first few days, and the degradation and erosion of the ISFI over the next few weeks; thereby allowing for the accurate prediction of the drug release profile from ISFIs of varying molecular weight. Initial drug burst dynamics were modeled by applying the analytical solution of diffusion as a weighting function for the solidification of the polymer. Finite difference methods were used to solve the degradation and erosion of the polymer due to the hydrolysis of ester bonds, which produce carboxylic acid terminate oligomers. These acidic oligomers along with acidic drug further catalyze the degradation of the polymer, therefore it becomes necessary to evaluate the acid dissociation on a much smaller timescale. Comparing to experimental data for varying PLGA copolymer molecular weight (Fig. 1), the computational model was able to accurately predict the drug release with a 6.7% error, and a negligible 2.0 % in predicting the immediate drug burst. The model thus accurately predicts drug release of various molecular weight polymers and provides mechanistic explanation for non-intuitive increases in drug release seen in some polymer mixtures. In the future this model can be used to accelerate the ISFI design process, and to further elucidate important physical aspects of the system that are not intuitive and might otherwise elude discovery.

BIOT 540

Liposomal azithromycin reduces post-ischemic cardiac inflammation

Ahmed Al-Darraji2, David Henson1, Vincent Venditto1, [email protected], Ahmed Abdel-Latif2. (1) Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky, United States (2) University of Kentucky, Lexington, Kentucky, United States

Ischemic heart disease is the leading cause of morbidity and mortality in the US and is often caused by myocardial infarction (MI). The inflammatory response associated with MI leads to an increase in circulating bone marrow cells, particularly monocytes and macrophages. This response is crucial for removing dead tissue and initiating the healing process. However, exacerbated inflammation following MI can be detrimental and is associated with infarct expansion, poor cardiac remodeling and adverse clinical outcomes. Currently, there are no therapies to treat the inflammation that occurs after MI. Modulating inflammation represents a paradigm shift in the treatment of MI. Therapeutic transition of macrophages to a reparative phenotype, known as alternative macrophage polarization, represents a promising strategy in reducing scarring and heart failure after MI. To address this, we have prepared a formulation containing azithromycin (AZM), a clinically approved antibiotic that has been shown to induce alternative macrophage polarization. The lipid composition of the formulation enables 30 mol% AZM to be packaged in the lipid bilayer and a resulting formulation diameter of 203 ± 1 nm. Intravenous administration of liposomal azithromycin (L-AZM) to mice after MI indicates rapid uptake by macrophages and monocytes with significant accumulation in the cardiac lesion after injury. Notably, less than 1% of liposomes are present in cardiomyocytes. Using in vitro and in vivo assays, L-AZM is found to reduce the proinflammatory cytokine milieu (e.g. TNFα and IL-1β), while increasing the anti- inflammatory cytokine profiles (e.g. IL-10, TGF-β). Finally, L-AZM is shown to preserve cardiac function, reduce lesion volume post MI, and improve overall survival. These data indicate that L-AZM is a promising candidate for continued pre-clinical evaluation and translation to treat patients experiencing a myocardial infarction.

BIOT 541

On-demand delivery of therapeutic proteins using light responsive hydrogels

Paige LeValley1, [email protected], April M. Kloxin2. (1) Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Chemical Biomolecular Engineering, University of Delaware, Newark, Delaware, United States

Hydrogels that respond to both stimuli applied externally and within the body have found widespread use for the on-demand and controlled delivery of biologics, ranging from growth factors to antibodies. Systems that provide on-demand control over the release of a therapeutic payload are of interest in creating treatment with a high level of control over dose and timing of administration. In particular, light responsive hydrogels are advantageous for creating platforms with on-demand protein release. One commonly used photolabile chemistry within the biomaterials community is nitrobenzyl (NB) based linkers, which respond to cytocompatible doses of long wavelength UV light without observed detriment to cell populations. However, the widespread use of NB-linkers for drug delivery is hindered by its slow degradation at higher wavelengths of light that can better penetrate skin. To combat this, coumarin-based linkers have been exploited as they have been shown to degrade in response to blue light, which can penetrate skin more effectively than ultraviolet wavelengths. Here, NB and Cmr linked hydrogels are compared for the release of model bioactive proteins in response to network degradation by irradiation with either long wavelength UV or short wavelength blue light. The degradation of NB and Cmr linked hydrogels was characterized using in situ rheology, where NB hydrogels degraded with long wavelength UV light at a similar rate to Cmr hydrogels degraded with 400 – 500 nm light. Additionally, the degradation of bulk hydrogels through a pig skin layer, a good mimic for human skin, was examined to test the validity of using light based systems as subcutaneous depots for protein delivery. Excitingly, degradation of both NB and Cmr hydrogels was observed over minutes to an hour at a light intensity of 10 mW cm-2, indicating that light based degradation occurs on time scales used for approved light-based therapies. Finally, the release of a model bioactive monoclonal antibody from photodegradable hydrogel networks was demonstrated. The concentration of the released antibody was determined using reverse phase chromatography and its bioactivity assessed using a toxin-based cell assay. Overall, light responsive hydrogel-based depots can be designed for the encapsulation and on-demand release of bioactive proteins, as well as combined with other degradation motifs (e.g., reducing or aqueous environment cleavable linkers), toward personalized therapeutic regimens.

BIOT 542 Bioinspired nanoscale drug delivery platforms from stimuli-responsive polymers and liposomes

Sonbinh T. Nguyen, [email protected]. Dept. of Chemistry, Northwestern University, Evanston, Illinois, United States

This presentation describe efforts from our group to create a platform of polymer-caged nanoparticles for the targeted delivery of anticancer drugs. In our approach, narrowly dispersed cholesterol-functionalized poly(acrylic acid) chains were grafted to drug- encapsulated liposomes via a simple drop-in procedure. The resulting polymer-grafted liposomes (PGLs) possess surface-active carboxylate groups that can be crosslinked with telechelic linkers to afford highly stable polymer-caged nanobins (PCNs). The surface-grafted polymers in PGLs and the polymer cage in PCN both greatly enhanced the stability of the drug-loaded liposome core in blood serum. Most importantly, their pH-responsive characteristics can be used to trigger the release of the encapsulated drug payloads under specific acidic conditions. The PGL and PCN concepts can be extended to include lipoplexes and spherical nucleic acids (SNAs) with gene- knockdown applications. In addition, they can be applied to the selective, targeted delivery of multiple drugs, or a combination of drugs and imaging agents, to specific cancerous sites.

BIOT 543

Roles of endocytosis and FcRn-mediated recycling in IgG transport across an in vitro blood-brain barrier

John S. Ruano-Salguero, [email protected], Kelvin H. Lee. Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware, United States

Immunoglobulin G (IgG)-based therapies for numerous neurodegenerative indications are being evaluated in clinical trials as first-in-class disease-modifying agents. Although promising, poor brain delivery of IgG (~0.01% of the administered dose) – attributed to the restrictive phenotype of brain endothelial cells (BECs) that form the blood-brain barrier – necessitates dosing regimens that have elicited severe adverse effects and raised questions regarding treatment cost and accessibility. Successful strategies to improve IgG brain delivery, such as antibody engineering, must first identify the processes regulating IgG transport across BECs. To overcome the experimental complications of in vivo models/methods that have contributed to conflicting hypotheses, mainly concerning the neonatal Fc receptor (FcRn) that regulates IgG recycling and transport in peripheral endothelial cells, IgG transport and trafficking was systematically evaluated using an in vitro model of brain endothelium derived from human pluripotent stem cells. Complementary super-resolution and live-cell fluorescence microscopy were used to correlate intracellular distributions and transport rates for IgGs exhibiting disparate FcRn engagement. Relationships between processing and transport were further explored using modified IgG and other macromolecules, which revealed the impact of endocytoic pathways on transport rates. Collectively, our findings shed light on the factors influencing the rate of brain entry for conventional therapeutic IgG and also provide possible routes to engineer improved variants with enhanced brain delivery.

BIOT 544

Amphotericin-B loaded compressed nanofibrous oral tablets as a potential drug delivery vehicle for Leishmaniasis

Mrunalini K. Gaydhane, [email protected], Anindita Laha, Chandra S. Sharma, Saptarshi Majumdar. Chemical Engineering, IIT Hyderabad, Medak, Telangana, India

Amphotericin B is a drug of choice for life threatening disease Leishmaniasis called “kala azar” and severe mycotic infections. Every year Leishmaniasis affects lives of 12 million people whilst fungal infections affect 1.2 billion worldwide. The Amphotericin B molecules are amphipathic in nature and comes with the lipid formulations to be only administered parenterally. Low bioavailability (<0.9%) and high toxicity inside the oral track due to its macromolecular and self-aggregative nature limits oral administration of Amphotericin B. The current research unveils an efficient route to address the toxicity, stability and release kinetic of Amphotericin B, in a controlled manner at realistic pH condition inside the Gastro-intestinal tract. The Gelatin nanofibers have chosen as an excipient for the drug molecule which are cross-linked with a chemical cross-linker, glutaraldehyde for only 8 minutes to improve its water resistance and thermo-mechanical properties. The as spun drug loaded gelatin nanofibers are then compressed to form oral tablets and the release kinetic are studied. The compressed nanofibrous oral tablets (CNOT) containing 20 mg drug / 500 mg weight, has shown the zero order release profile for 10 consecutive days. The antifungal assay against Candida albican species showed resistance towards the fungi till seventh day. The cytotoxicity assay was investigated to find out if there was any toxic effect of glutaraldehyde and amphotericin B onto L929 mice fibroblast cells, which inferred 100% cell viability for 1st, 3rd and 5th day culture. Thus, CNOT offers prolong release of Amphotericin B, which will surely enhance patient’s compliance. It protects the drug from premature degradation in high acidic pH condition in stomach. Also, it reduces the infusion related toxicities of lipid based amphotericin B in much more cost efficient manner.

BIOT 545

Enhancing tissue repair via peptide-mediated gene and drug delivery

Millicent O. Sullivan, [email protected]. University of Delaware, Newark, Delaware, United States

We have developed new strategies to employ extracellular matrix (ECM)-inspired peptides as drivers of gene and drug delivery during bone and skin repair. This talk will specifically highlight the use of collagen mimetic peptides (CMPs) as tethers that enable integration of DNA polyplexes and antimicrobial agent-loaded liposomes within collagen/fibrin hydrogels (“co-gels”) for applications in wound healing. Chronic wounds are a growing unsolved health problem with a prevalence rate of 1-2% of the general population in developed countries. Two key issues hindering repair include limitations in growth factor activity and accessibility, and the prevalence of infection and biofilm formation in the chronic wound environment. However, while a variety of ECM, growth factor, and antimicrobial treatments have received wide attention, clinical efficacy has been limited. Matrix-mediated delivery offers a lower cost pathway for growth factor delivery that addresses the confounding issues with recombinant protein therapies through its capacity to induce sustained, localized production of bioactive growth factor by endogenous cells; moreover, matrix represents an ideal depot to enable sustained delivery of antimicrobials. In our study, we demonstrate spatio-temporally-controlled PDGF gene transfer by conjugating CMPs to PDGF-encoding polyplexes, such that the polyplexes were stably tethered into the co-gel via CMP strand invasion into the collagen triple helix. In vitro results confirmed that the polyplexes were highly efficient in transfecting fibroblasts to induce PDGF expression and subsequent cell proliferation and migration. Simultaneously, we demonstrate that CMPs also provide useful tethers for incorporation of sustained liposomal depots for antimicrobials that enable improved resistance to infection over prolonged time periods. In vivo excisional wound healing studies in mice established faster wound closure using both CMP deliveyr approaches: CMP polyplex-loaded co-gels induced faster wound closure with minimal scar formation as well as improved collagen production, myofibroblast activity, and collagen orientation. Meanwhile, CMP-tethered antimicrobial liposomes offered increased resistance to bacterial infection at the surface of the wounds, resulting in improved healing. These results are indicative of the potential of matrix-modified biomaterial scaffolds in effective treatment of chronic non-healing wounds.

BIOT 546

Peptide nanocluster vaccine formulation impacts immune response

Alexandra Tsoras, [email protected], Julie Champion. Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States

Peptide subunit vaccines are desirable because they increase control over the induced immune response and safety of a vaccine by reducing the risk of off-target responses. The immunogenicity of most peptides, however, is often insufficient to produce robust and lasting immunity. Many materials and delivery vehicles have been developed to incorporate target antigens to improve the induced immune response while maintaining specificity and control. We have developed peptide nanoclusters (PNCs) as a subunit peptide vaccine formulation that has shown potential to increase immunogenicity of peptide subunit vaccines. PNCs are comprised of crosslinked peptide antigen, and have been successfully synthesized from several therapeutically relevant peptides as small as 8 amino acids in length. However, synthesis of the PNCs requires either adding residues to the peptide epitope sequence or engaging amino acids within the antigen epitope in conjugation chemistry to form a stable material with sufficient antigen incorporation. It is important to understand if and how these modifications and the stability of the crosslinking itself affect immune responses to inform the formulation of peptide vaccines for induction of the desired immune response with a wide variety of peptide antigens. To understand the effects of peptide sequence and crosslinking, several types of PNCs were synthesized by desolvation from the model peptide epitope SIINFEKL, which is derived from the immunogenic protein ovalbumin. In some variations, SIINFEKL was altered to include extra residues on each end strategically chosen to enable more conjugation chemistry options, as well as to increase the size of the peptide beyond the minimal presented epitope. Additionally, both reversible and irreversible crosslinkers were used to form the PNCs. These variations were evaluated for immune cell responses in vitro. Dendritic cell antigen presentation and antigen-specific T cell activation depended on the type of peptide formulation. Further, dendritic cell presentation did not always correlate with strong T cell activation. These results, as well as those of in vivo immunization with the PNC variants will be discussed. The knowledge gained in this work will be applied to disease-specific PNC synthesis to maximize immunogenicity and, ultimately, efficacy of peptide subunit vaccines.

BIOT 547

Nanocarrier design using inverse Flash NanoPrecipitation: Ensuring therapeutic stability and achieving controlled release of peptides and proteins

Chester Markwalter1, [email protected], Robert Pagels2,1, Betul Uralcan1, Akiva Gordon1, Istvan Pelczer1, Pablo G. Debenedetti1, Robert K. Prudhomme1. (1) Princeton University, Princeton, New Jersey, United States (2) Optimeos Life Sciences, Princeton, New Jersey, United States

The encapsulation of biologics (peptides, proteins, nucleic acids) into nanocarriers to achieve novel therapeutic behavior has been envisioned for decades, but clinical translation has been hampered by complex production strategies. The methods that have been developed, such as liposomes or double emulsion techniques, are limited by low loading (the fraction of the total formulation mass composed of the biologic). To address this unmet need, we have developed a self-assembly process to form polymeric nanocarriers comprising a biologic in a hydrophilic core, encapsulated by a poly(lactic acid) shell, and stabilized by a poly(ethylene glycol) brush. Called “inverse Flash NanoPrecipitation”, the technique uses scalable processing steps to achieve biologic loadings that are 5-15x higher than values achieved for liposomes or the double emulsion process (9-27% versus <2%).

While improved loading and scalable processing are both important metrics for formulations of biotherapeutics, it is also crucial that the biologic remains stable to processing and that suitable release profiles are achieved using the nanocarrier. We describe a detailed assessment of chemical and physical stability of several water- soluble compounds and model biologics in the process. We have characterized degradation and refolding behavior for therapeutics ranging from antibiotics to β- galactosidase using assorted techniques: HPLC, NMR, circular dichroism, SEC, and/or activity measurements. While robust proteins like lysozyme and horseradish peroxidase recovered full activity, β-galactosidase lost nearly all initial activity. We therefore detail an approach that seeks to stabilize sensitive proteins to the processing steps using depletion-driven attractions to reversibly cluster the biologic. In the second part of the talk, we highlight release profiles achieved as a function of nanocarrier composition. We describe the mechanism of release, the associated design principles, and discuss potential uses of the technology.

BIOT 548

Cytosolic delivery of therapeutically-relevant proteins by site-specific incorporation of endosomolytic peptides for the treatment of breast cancer cells

Rachel M. Lieser1, [email protected], Daniel Yur1, Wilfred Chen2, Millicent O. Sullivan3. (1) Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Chemical Engineering, University of Delaware, Newark, Delaware, United States (3) University of Delaware, Newark, Delaware, United States

Efficient intracellular delivery of proteins remains a significant challenge despite its immense therapeutic potential for enzyme therapies and genome-editing. Proteins often require active strategies for intracellular delivery due to their membrane impermeability. In addition, accessing the cytosol or subcellular compartments requires internalized proteins to escape the endosome to evade lysosome degradation or exocytosis. Past efforts to deliver proteins to intracellular targets have often relied on cell-penetrating peptides and lipid nanoparticles, which have shown promise, but often suffer from low potency, limited in vivo activity, and a lack of generalization. Endosomolytic peptides, which are able to fuse with the endosomal membrane following a pH-triggered conformational change, have shown promise in nanoparticle systems, but only a handful of these peptides have been tested for cytosolic protein delivery. Further studies are necessary to understand the full potential of these peptides for protein delivery and determine how to use these peptides more generally for delivering proteins that vary in size and composition. Our previous work site-specifically conjugated epidermal growth factor (EGFR)-targeting peptides to proteins to explore the effect of ligand valency and spacing on protein internalization in EGFR-overexpressing breast cancer cells. High EGFR ligand valency and clustering was associated with robust internalization as compared to unmodified protein, however, the internalized protein remained entrapped in the endosome leading to its eventual degradation. In this study, EGFR ligands and multiple endosomolytic peptides were site-specifically conjugated to the protein to enhance endosomal escape. Two endosomolytic peptides with differing mechanisms of endosomal disruption, e.g., the pore-forming and the carpet-like mechanisms, were explored for their ability to deliver a model maltose-binding protein to the cytosol. Both peptides decreased lysosomal protein degradation and enhanced endosomal disruption, although the carpet-like sequence was more effective. Furthermore, this approach was used to deliver p19 siRNA binding protein, for siRNA mediated gene silencing, and caspase, for enhanced cell apoptosis, demonstrating the modularity of the approach. These results, combined with the growing body of literature, may provide insight for future delivery systems so that intracellular protein therapies become more clinically relevant.

BIOT 549

Modelling sustained release through non-destructive computed tomography xutao shi1, [email protected], Stijn Koshari3, Abraham M. Lenhoff2, Norman J. Wagner3. (1) Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Univ of Delaware, Newark, Delaware, United States (3) Dept of Chemical Engineering, University of Delaware, Newark, Delaware, United States

Sustained-release systems improve the bioavailability of biopharmaceutical products and eliminate the necessity for a frequent administration schedule of the drug. A better understanding of such delivery systems, and therefore a more efficient system design, can be achieved by using mathematical models capable of predicting the drug release profiles a priori. However, these models are often formulated phenomenologically, and morphological details such as the early-stage pore formation of the sustained-release system are often neglected or assumed without proper experimental characterization. In this work, we utilized non-destructive X-ray computed tomography (CT) to quantitatively characterize the internal structure of a PLGA-based sustained release system. We modified an existing mechanistic sustained-release model and incorporated the structural characteristics obtained from segmentation of the three-dimensional CT reconstructions. The model predictions were compared to the release profiles obtained from experimental studies. The results demonstrate the importance of structural porosity in sustained release and the potential of a CT-aided mechanistic model to predict the drug release profiles of a PLGA-based sustained-release system.

BIOT 550

Pretargeted delivery of PI3K/mTOR small-molecule inhibitor-loaded nanoparticles for treatment of non-Hodgkin lymphoma

Kin Man Au1, [email protected], Andrew Wang1, Steven Park2. (1) Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Carrboro, North Carolina, United States (2) Levine Cancer Institute, Atrium Health, Charlotte, North Carolina, United States

Overactivation of the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling has been identified in a number of hematological malignancies, including non-Hodgkin lymphoma. BEZ235 is an effective dual PI3K/mTOR inhibitor, but it was withdrawn from early-phase clinical trials owing to its poor solubility and on- target/off-tumor toxicity. Here, we present the development of a novel nanoparticle (NP)-based pretargeted system for the therapeutic delivery of BEZ235 to CD20- and HAL-DR-expressing lymphoma cells for combination targeted therapy. The pretargeted system is composed of dibenzocyclooctyne-functionalized anti-CD20 and anti-Lym1 antibodies (Abs) as the tumor-targeting components and azide-functionalized BEZ235- encapsulated NPs as the effector drug carrier. The pretargeting Abs were tailored to undergo rapid bioorthogonal ligation with the effector NPs at the target site, enhancing the therapeutic efficiency through increased amount of BEZ235 retained inside the tumor. Using four established lymphoma cell lines with different CD20 and HLA-DR antigen densities as examples, we demonstrate that the dual Ab pretargeted strategy effectively raises the number of NPs retained on the target tumor cells. This improves the in vitro antitumor activity of BEZ235 through the inhibition of the PI3K/mTOR pathway. Comprehensive in vivo studies using Namalwa and Raji mouse models confirm that the dual Ab pretargeted system effectively inhibits tumors growth, improves survival, and reduces the systemic side effects of BEZ235. Our data demonstrate that a novel NP-enabled pretargeted system consolidates the use of kinase inhibition with Ab- directed targeted therapy and improves the therapeutic window of small molecule inhibitors.

BIOT 551

EASY Process: ProteinA free, disposable and continuous monoclonal antibodies purification process

Benoit Mothes, [email protected]. Purification Process, Sanofi, Vitry sur Seine, France

Process simplification underlying EASY Process has one main goal : bring the monoclonal antibody production process to higher productivity while keeping purification simple and independent from complex technologies.

The main objective of such development was to make this new process the most versatile as possible, purifying in the same way, and without heavy modifications, all kind of modalities proposed to DSP development teams (mabs, Fabs, multispecific…). At least for first consequence, the reduction of development duration from weeks to hours. Then to open the possibility to purify in a continuous manner, using disposable technologies, thus increasing the productivity and reducing the batch preparation times. Finally to incorporate this process in a compact and mobile unit concept - plug & play - adapted to all types of facilities. In other words, key drivers leading to design EASY Process is the elimination of chromatographic steps (ie no more columns, resins, expensive ProteinA, buffers, huge chromatographic systems) moving to a very simplified process : -No more preparation of dozen of buffers, columns packing, equilibration, regeneration and sanitization of equipments -No more storage and control of media with disposable technologies -Process cycle time shortened to complete the entire purification in less than 5 hours taking into advantage a novel state-of-the-art disposable technologies (ie membrane adsorbers and filters) . -Continuous processing with small and mobile purification units in order to allow maximum process closure, automatisation to avoid potential contamination and human- based errors

Finally, a huge reduction of CoGs associated to monoclonal antibodies purification and a first disposable mobile purification unit created.

BIOT 552

Evolution of the recovery process train to overcome challenges in intensified biologics processes

Joseph J. Perry, [email protected], James Angelo, Juan Wang, Chao Huang, Melissa Holstein, Xuankuo Xu, Sanchayita Ghose. Bristol-Myers Squibb, Devens, Massachusetts, United States

Interest in intensified processing strategies for mAb biologics has grown industry-wide due to the promise of significant cost savings resulting from efficient consumable utilization, increased productivity, and more efficient changeover. However, the implementation of these strategies is accompanied by new challenges. As upstream process development drives higher cell densities and more productive cultures, impurities such as host-cell protein, enzymes, and residual DNA are observed at higher levels, presenting a greater challenge to downstream purification. While one challenge is related to absolute levels of impurity clearance throughout the purification process, this should not be the only consideration. For example, residual DNA is not considered to be problematic in relation to downstream clearance, but the influence of high DNA concentrations on stability of clarified bulk matrix and capture resin lifetime is elevated when multicolumn continuous capture (MCC) is implemented, especially in cases where enhanced resin utilization is prioritized over productivity.

This presentation demonstrates the application of an Emphaze AEX Hybrid Purifier as the tertiary stage in a harvest train to overcome challenges such as matrix and product stability of in-process pools, fouling of guard filters and resins, and impurity clearance through an intensified downstream purification process, specifically of product sourced from an upstream high density fed-batch cell culture processes.

BIOT 553

Nanoparticle assisted protein crystal nucleation: Towards crystallization in downstream protein purification

Caroline McCue, [email protected], Henri-Louis Girard, Kripa Varanasi. Mechanical Engineering, MIT, Cambridge, Massachusetts, United States

Slow protein crystal nucleation is a major barrier to using crystallization as a separation and purification strategy in protein manufacturing. This work investigates the use of functionalized nanoparticles in solution to act as templates for initiating crystal nucleation. We used lysozyme, a protein with well-characterized crystallization conditions, to evaluate the nucleation rates and induction times of crystals grown on the functionalized particles. The nucleation rates and induction times were measured using an emulsion based setup and an image recognition algorithm to count the number of crystals that formed in drops over time. On a microfluidic chip, a supersaturated solution of lysozyme was mixed with a stream containing precipitants and functionalized nanoparticles. The functional groups used include bioconjugates, which bind covalently with biomolecules, and are commonly used in diagnostic imaging and targeted drug delivery. Surface adsorption measurements on larger flat functionalized surfaces were used to further examine the mechanism by which the functional groups enhance crystal nucleation. This work suggests that functionalized nanoparticles enhance crystal nucleation, and could help enable crystallization to be used as a purification step in protein manufacturing by reducing the time required to nucleate protein crystals.

BIOT 554

3D print your own chromatography column: Novel materials for the direct printing of ion exchange monoliths

Ursula Simon, [email protected], Struan Hattersley, Sulaiman Sarwar, Simone Dimartino. Institute for Bioengineering, The School of Engineering, The University of Edinburgh, Edinburgh, United Kingdom

3D printing allows the fabrication of complex structures down to micron scale according to digital models. This capability enables to create ordered highly reproducible porous structures for chromatography in contrast to traditional random stationary phases. Despite the increasing interest in ordered stationary phases, the application of 3D printing in the field of separation sciences remains limited due to the lack of materials which are suitable for chromatography as well as compatible with current 3D printing methods.

In this work, we present novel methacrylate-based materials which allow direct printing of fully functional ion exchange monoliths. This was achieved by incorporating monomers containing quaternary amine and sulphopropyl moieties in the material formulation prior to 3D printing. The smallest printable feature size, was limited to 300 µm. To increase the printed monolith’s surface area, porogenic agents were employed in the printing formulation and successfully washed out after the printing process. The novel developed materials were analysed in terms of their porous structure, permeability, chemical resistance, mechanical stability as well as their binding capacities for model proteins.

For protein separation experiments ordered structures such as the Schoen Gyroid and Schwarz Diamond were explored as novel stationary phase geometries with bed porosities of 50 % and pores at submicron scale. The 3D printed monoliths showed good reproducibility between different printing batches in terms of protein binding as well as porous structure, comparable to commercial material.

BIOT 555

Process intensification of mAb purification for efficient impurity removal hirohisa takeuchi1, [email protected], Son Jeonghyun2, kyo Izumida2, Masato Suenaga2, Olga Paley1, Norbert Schuelke1. (1) Takeda Pharmaceuticals International Co., Cambridge, Massachusetts, United States (2) Takeda Pharmaceutical Company Limited, Hikari, Japan

The present work is a case study in applying process intensification principles to purification development of a commercially suitable manufacturing process for a monoclonal antibody at Takeda. A previously developed process for the antibody faced significant challenges such as insufficient impurity removal efficiency and low throughput. Hence, the existing process was intensified to remove impurities more efficiently and establish an economically and environmentally friendly process by increasing the load capacities of each step and reducing the associated number of cycles and buffer consumption demands. In addition, by combining the flow through mode of hydrophobic interaction chromatography with a reduced bed height, resin consumption and operation time were reduced, while impurities were nonetheless removed more efficiently than the existing process. The present result from this study will be used as a basis for a future assessment for a semi continuous interconnected process.

BIOT 556

Integration of normal flow filtration into downstream continuous bioprocessing Jessica R. Molek, [email protected], Mehdi Ghodbane, John Starcevich, Patrick Thompson, Antonio R. Ubiera. Biopharmaceutical Process Development, GSK, King of Prussia, Pennsylvania, United States

Normal flow filtration (NFF) is ubiquitous throughout the purification process for biopharmaceuticals. Common applications of NFF within the downstream platform include removal of cell and cell debris, of precipitated particulates, of microbial contaminants, or of virus and virus-like particles. In NFF the pressure-driven feed stream flows perpendicular to the membrane, where process- and product-related impurities are retained primarily due to size exclusion while allowing the product to pass through unretained. While NFF is relatively straight forward to introduce into a batch process, implementation within an integrated processing environment is not immediately obvious due to the semi-continuous nature of the operation. This work explores the use of a duty/standby strategy to operationalize and allow NFF unit operations to fit within the integrated semi-continuous downstream processing framework. Using this approach, failure modes and thus links between quality attributes and process parameters must be reconsidered to develop robust control strategies that are informed by process analytical technologies (PAT). Processes for each NFF unit operation are defined based on small scale characterization of the system, as well as by the requirements and limitations of the manufacturing environment, such as overall process run time, desired filter change out rate, and capability of available PAT and automation systems. A comprehensive analysis of a proposed duty/standby-based strategy for normal flow filtration within an integrated continuous process, relative to traditional batch processing, will be discussed, highlighting the benefits, efficiency, and level of robustness that can be achieved with this approach.

BIOT 557

Harvest chromatographic clarification enables simplification and compression in vaccine production

Lei-yun Boone2, [email protected], Chris C. Koehler1, [email protected]. (1) 3M, Havertown, Pennsylvania, United States (2) GlaxoSmithKline Vaccines, Rockville, Maryland, United States

Vaccine production and purification relies on the complex interplay between multiple unit operations and is not standardized: production strategies vary from vaccine to vaccine. The adoption of new technologies and advances in manufacturing capabilities can help foster accelerated vaccine discovery, development and deployment. Implementation of multifunctional single-use technology during vaccine purification provides an avenue to simultaneously reduce process complexity and compress the production process. When 3M™ Emphaze™ AEX Hybrid Purifier, a single-use anion exchange chromatography product, is incorporated in the purification scheme of recombinant protein-based vaccines it provides the potential to simplify the production process.

In this investigation, the production of a vaccine target was considered where primary clarification is currently accomplished using two-stage depth filtration. This strategy serves to remove large debris, however, soluble impurities such as Host Cell Proteins (HCPs) are not effectively cleared. As a result, HCP removal is accomplished downstream in the incumbent process using a series of chromatographic separations., Emphaze AEX Hybrid Purifier provided a higher reduction of soluble impurities compared to clarification using depth filtration alone. The appreciable reduction of the challenging HCP with Emphaze AEX Hybrid Purifier warranted exploration of potential downstream modifications, which can facilitate process compression and simplification. In addition, Emphaze AEX Hybrid Purifier is amenable to process condition manipulation which can improve product recovery.

BIOT 558

Evolution of intensified downstream processes

Judy Chou, Ashley Hesslein, [email protected]. Bayer HealthCare, Berkeley, California, United States

Bayer will discuss their work in creating intensified downstream processes.

BIOT 559

Next generation bioprocess development

Gregg Nyberg, [email protected]. Process Research & Development, Merck & Co, Inc., Southborough, Massachusetts, United States

Rapidly delivering diverse biotherapeutics to patients requires efficiently developing innovative, robust manufacturing processes and technologies. Successful bioprocess development organizations must simultaneously optimize for speed to clinic, speed to launch, robust supply and low cost. Flexible manufacturing platforms that are robust across diverse modalities are foundational. State of the art platforms are highly productive and deployable in reconfigurable facilities that facilitate rapid, cost effective capacity scaling. Such platforms increasingly rely upon intensified and continuous manufacturing technologies. This talk will review enabling technologies and strategies for evolving legacy manufacturing platforms.

BIOT 560

Oxygen mass transfer coefficient with sparged bubble size analysis in stir tank bioreactors

Yusuke Tomioka, Takao Ito, [email protected]. MilliporeSigma, Tokorozawa, Japan Stir tank bioreactors are widely used in mammalian cell culture process for various scales of biomanufacturing. One of the successful bioreactor scale-up is achieved from the understanding of an operation parameter for efficient mixing and gas transfer etc. Volumetric oxygen mass transfer coefficient (kLa) is widely used to determine gas transfer capacity of bioreactor operation, that is refracted on the bioreactor design, such as impeller, vessel height and diameter, gas sparger shape and capacity. A lot of engineering approach has been discussed to determine kLa value, but the size of the bubble and distributions has not been verified with the current model. Bioreactors have multiple type of sparger to supply oxygen to fit various culture condition, the generated bubbles also have different diameters and size distribution, that affects the specific interfacial area. The correlation of engineering parameters from modeling are not well characterized yet. In this study, the determination approach of mass transfer coefficient with bubble diameter and size distribution analysis was evaluated by using MobiusR single use bioreactor. We verified the performance of the bioreactor mass transfer coefficient with general mechanical models and analyze the correlation between mass transfer coefficient and sparged bubble specifications, also discuss about the unique sparger design and characterization.

BIOT 561

Implementation of the Beacon nanofluidic platform into a manufacturing cell line development process

Jonathan Diep, [email protected], Kim Le, Christopher Tan, Huong Le, Jasmine Tat, Ewelina Zasadzinska, Jennitte Stevens. Amgen, Thousand Oaks, California, United States

The manufacture of therapeutic biologics relies on understanding and harnessing mammalian cells to secrete desired proteins. Generating a highly productive cell line however typically involves long timelines and resource intensive, due to the need to screen large numbers of candidates in protein production studies. As a result, miniaturization and automation strategies are often employed to allow for reductions in resources and higher throughput. However, current automation approaches rely on the use of standard cell culture vessels and bulky liquid handling equipment. The Beacon platform from Berkeley Lights offers to eliminate these bottlenecks through growing cells on custom nanofluidic chips. Berkeley Lights’ OptoElectro Positioning (OEP) technology projects light patterns to activate photoconductors that gently repel cells to manipulate single cells on nanofluidic culturing chips. Integrate fluorescence imaging capabilities allow for analytics of cells in real time. Together, using this fully integrated technology platform, common cell culture tasks can be programmed through software, allowing maintenance and analysis of thousands of cell lines in parallel on a single chip.

We describe the ability to perform key cell line development work on the Beacon platform. We demonstrate that Chinese hamster ovary (CHO) cell lines can be isolated, cultured, screened, and exported at high efficiency. We then compare this process head to head with a FACS-enabled microtiter plate-based workflow across four active biologics programs. Resulting subclones from both Beacon and FACS processes were evaluated in small scale production experiments and bench-scale controlled bioreactors comparing growth, productivity, and product quality. We demonstrate the ability to generate a diverse and comparable set of candidate clonal cell lines with reduced resources. Additionally, we performed validation experiments to demonstrate that the Beacon process provides high assurances of clonal derivation and zero cross- contamination events. Together, the Beacon enables high quality production cell lines with improved data packages, detailed tracking, high assurance of clonality, reduced resources, and decreased cycle times.

BIOT 562

Seed train intensification by implementing WAVE perfusion bioreactors

Linda Hoshan, [email protected], Sara Rodriguez, Ashley Bui, Jack Huang. Biologics Upstream Process Development, Merck, Kenilworth, New Jersey, United States

Both WAVE bioreactors and N-1 perfusion bioreactors have been widely applied as separate steps in seed train expansion for mammalian cell culture. Although N-1 perfusion bioreactor is an effective tool for high seed density process, there are needs and interests to streamline the passaging scheme and replace this complicated and labor-intensive unit operation. Hence, an optimized, highly-efficient and simple seed train process is highly desirable to improve this platform. In this case, leveraging WAVE perfusion bioreactors at bench top scale (50L) for seed train expansion can reduce the number of required expansion steps and can potentially be used for direct inoculation of production bioreactors at the manufacturing scale. In this work, we first optimized the cell-specific perfusion rate and operation parameters for both the N-2 and N-1 WAVE perfusion bioreactors using one CHO cell line expressing a recombinant monoclonal antibody. The optimized perfusion rate schedule is able to maintain similar daily cell growth rate at high cell density and is successfully applied to three CHO cell lines, with similar cell culture performance. Exponential growth was observed throughout the seed expansion reaching > 15 × 106 vc/mL and > 80 × 106 vc/mL for the N-2 and N-1 perfusion bioreactors, respectively. This process was successfully scaled up from a 10-L WAVE perfusion bag to a 50-L WAVE perfusion scale. Cell cycle analysis also confirms that cell cycle phase profiles are comparable throughout the WAVE perfusion bioreactor step. Importantly, inoculation of cells from different stages of WAVE perfusion bioreactor demonstrated to have similar cell culture performance and product quality attributes, comparing to the platform N-1 perfusion bioreactor process. Using three different CHO cell lines, we showed that WAVE perfusion seed cultures can improve process efficiency by optimizing the number of seed expansion bioreactor steps with simplified operations. BIOT 563

High-throughput holographic imaging technologies for rapid monitoring of cell density, viability, and cell health in bioreactors

Melissa Hill2, [email protected], Kevin Huff2, Gregory Laslo2, Martin Pisaneschi1, Geoffrey Esteban1, Sayantan Bose2. (1) Iprasense, Clapiers, France (2) GSK, Philadelphia, Pennsylvania, United States

Viable cell count (VCC) and viability are key cell culture process parameters during biopharmaceutical manufacturing. As such, accurate measurements of these attributes are critical both in process development and GMP manufacturing. Existing technologies to measure cell count and viability in bioreactors are not only expensive but also pose certain restrictions in terms of time required for measurements, equipment-to-equipment variability, and high sample volume requirements. This work aims to describe the use of a novel, label-free holographic imaging technology (NORMA) to enable rapid and accurate measurements of not only VCC and viability, but also cell health based on changes in refractive index of the cells. Holographic cell imaging is an inexpensive solution over current technologies for measuring cell density and viability from bioreactors. The rapid and high-throughput readout coupled with a dilution-free, low sample size makes it suitable for measurements in bioreactors across scales including microscale bioreactors. The robustness of this novel technology was tested across multiple cell lines, media types, and process conditions. Both VCC and viability were reliably measured up to 40 million cells/mL without dilution. Refractive index of the cells was observed to change with phases of cell growth which consistently correlated with cell health in the reactor. The high-throughput version of the NORMA technology (NORMA 4S) was successfully integrated with the advanced microscale bioreactor (ambr15) system for timely, automated sampling and rapid measurement of VCC and viability using just 20 µL of cell suspension enabling more frequent measurements than conventional technologies.

BIOT 564

Fabrication and characterization of a 3D-printed plate insert for dynamic co- culture applications

Sharif M. Rahman1, Rachael N. Coates1, Conner P. Allison2, David Quiring1, Jordan R. Remont2, Erika M. Lewis2, Elizabeth C. Martin2, Adam T. Melvin1, [email protected]. (1) Chemical Engineering, Louisiana State University, Baton Rouge, Louisiana, United States (2) Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, Louisiana, United States

Cellular communication has been studied extensively by scientists, clinicians and engineers, as it is a critical component in development and disease. In order to communicate, cells emit extracellular signaling molecules (e.g., growth factors) which regulates if a cell will proliferate, migrate, or undergo apoptosis. The most common method is the Transwell assay which has several weaknesses: (i) an inability to directly visualize the cells, (ii) the invasion of the cells in the top chamber into the polycarbonate membrane, (iii) very low cellular yields for post-experimental analysis. To address this, we developed a novel co-culture approach that utilizes a 3D-printed plate insert to dynamically co-culture two different cell types in the same 10 cm petri dish by creating two distinct culture chambers above and below an agarose hydrogel. The agarose hydrogel physically separates, yet chemically connects, the two culture regions and is coated with poly-D-lysine to allow for the adhesion, spreading, and growth of adherent cell lines in the top layer. The approach design is user-friendly, affordable, and allows for dynamic and tunable co-culture conditions while maintaining similar growth patterns as compared to off-chip controls in tissue culture plastic (TCP). Experimental and COMSOL simulations show the rapid diffusion of biomolecules and oxygen across the hydrogel reaching a steady state within ~12-18 hours. The plate insert approach allowed for the direct visualization of cell lines in both the top and bottom chambers facilitating the study of independent cell growth and morphology. The plate insert was used to study the interactions between two different breast cancer cell lines, triple negative (MDA-MB-231) and ER+ (MCF7) co-cultured with adipose-derived stem cells (ASCs). Recent studies have shown that ASCs play a critical role in promoting metastasis and drug resistance in breast cancer patients. Using our novel co-culture approach, we identified enhanced cellular growth and overexpression of EMT- associated genes in the MDA-MB-231 cell line when they were cultured in the presence of ASCs. We also observed increased drug resistance in the MCF7 cell line in the presence of ASCs, evident through increased proliferation. Our approach allowed for the independent harvesting of both cell lines in numbers sufficient for downstream PCR and Western Blot analysis to identify changes in the genotypic and phenotypic signature of cancer cells.

BIOT 565

High-throughput toxicological screening on 3D hiPSC cultures and derived progeny

Andre L. Rodrigues1,2, [email protected], Sneha Gopal3, Vibha Narayanan3, Tiago G. Fernandes1,2, M. Margarida Diogo1,2, Joaquim M. Cabral1,2, Jonathan S. Dordick3. (1) Department of Bioengineering , Institute for Bioengineering and Biosciences - Insituto Superior Tecnico, University of Lisbon, Troy, New York, United States (2) The Discovery Center for Regenerative and Precision Medicine, Instituto Superior Tecnico, University of Lisbon, Lisbon, Lisbon, Portugal (3) Department of Chemical and Biological Engineering and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Instute, Troy, New York, United States

Pluripotent stem (PS) cells, both embryonic and induced pluripotent stem cells (iPSC), have the capacity for prolonged self-renewal and differentiation into numerous distinct cell lineages. Human PS cells (hPSC), therefore, may be a reliable source of donor cells for transplantation therapies and for drug screening. These applications require tight control over the in vitro culture microenvironment to obtain the desired hPSC differentiated derivatives with a high efficiency and functional quality. Microscale high- throughput culture systems are useful tools for mimicking and controlling hPSC’s in vivo microenvironment unveiling the complex interactions between cell-cell and cell- soluble/immobilized factors. In this study, we establish a robust and reproducible 3D microarray platform for expansion and differentiation of hiPSC into neural and cardiac progenitors, ultimately enabling the analytical quantification of the effects of growth factors and small molecules that impact cell viability and phenotypic characteristics. This platform also enables to perform high-throughput studies on the toxicity of small molecules towards hPSC-differentiated derivatives and evaluate different cellular sensitivities to pharmaceuticals.

BIOT 566

Systematic evaluation of high-throughput scale-down models for single-use bioreactors (SUB) using vvm as the criterion

Rubin Jiang, [email protected]. Merck , Kenilworth, New Jersey, United States

Developing representative bioreactor scale-down models is a critical part in process development and characterization. Here we present a systematic analysis of high- throughput scale-down models (ambr®250, 250 mL) developed for 500 L or 2,000 L single-use bioreactors (SUB) using engineering approaches. A main scaling criterion, gas throughput (vvm), was studied for scale-down model development after analyzing bioreactor hydrodynamic environment and gas transfer characteristics. Two different processes with distinguished peak cell densities (12 -14 vs. 20 - 25 × 106 cells/mL) for a monoclonal antibody (MAb-A) were evaluated. We demonstrated that scaling-down processes using similar vvm as the criterion was feasible in reproducing the large-scale gas transfer characteristics for both cell densities, though more challenging in high- density conditions. Furthermore, a range of pCO2 levels could be generated in the ambr®250 bioreactors through vvm modulations for evaluating the impact of pCO2. The same approach was applied to two other MAbs with comparable results across scales. Practical aspects of ambr®250 operations, especially the impact of background air sparge rate and antifoam addition on dissolved oxygen (DO) control and gas transfer, were also discussed. Understanding and managing the gaps between small- and large- scale bioreactors is expected to streamline scale-up effort, and equally importantly scale-down activities for process characterization.

BIOT 567

Development of a Platform media and buffer control strategy leveraging scale- down media mixing models and at-scale data lakes

Christopher VanLang, [email protected], Amanda Laidler, Liz Valentine, George Boras, Amy Nehring, Alyssa Fidanza, Elena Banegas Nunez, Ben Dionne. Process Development, Amgen, West Greenwich, Rhode Island, United States A heavy use of a platform media and buffer preparation process allows for the agile development of bioprocesses over a diversity of biologics processes. We have built upon that platform by carefully interrogating and standardizing the individual process steps. With small-scale mixing models, we have built a mechanistic understanding of how various process parameters impact media preparation. Combining these physical insights with a wealth of at-scale data, we can design a flexible media and buffer preparation platform that can better support complex manufacturing processes in an efficient manner.

To enhance our process understanding of the major contributors to performance, we developed mixing models at the 7-L scale using a power per unit volume geometric scale-up method to reflects media preparation up to the 20,000 L scale. Given the role of dissolved carbon dioxide in bicarbonate buffered solutions, it was expected that carbon dioxide entrainment would impact pH. However, process parameters that impact dissolved carbon dioxide levels have been unknown. We present a few case studies that tested hypotheses of media preparation consistency using this mixing model. In one scenario, the order of addition influenced the media pH; changes to the order of addition resulted in a 25% recovery in cell culture productivity. In another scenario, mix time was identified as a major contributor to pH. Increasing the mix time increased the pH by 0.015 units which improved the consistency of at-scale batching. These small scale mixing models help to better translate the role of mixing dynamics on process consistency.

These small-scale findings can be combined with real-time process monitoring from data lakes to define a proactive control strategy. Building statistical models to relate process inputs with process outputs, we can leverage plant data to identify potential causes of process variability. Along with the small scale data, representative plant experience can be fed back into the media and buffer platform and aid early stage development. For instance, we have used plant information to compare the process capability of shared media across various scales and mixing geometries to better define the expected process performance. We outline our strategy to define and harmonize the media and buffer preparation process and showcase best practices using small scale models to build process understanding.

BIOT 568

KLa characterization of O2 and CO2 allows for improved prediction of process variables at pilot scale

Xiaoming Liu1, [email protected], Jeffrey C. Swanberg1, Chao Ma1, Jongchan Lee2. (1) Process Development, Bristol-Myers Squibb, Maynard, Massachusetts, United States (2) Mailstop L-2, Bristol-Myers Squibb, East Syracuse, New York, United States

Mass transfer, including oxygen transfer and carbon dioxide stripping, is critical to cell culture performance in a bioreactor system. As a process scales up from bench scale, it is crucial to make sure oxygen demand is met and carbon dioxide accumulation remains below levels that reduce process productivity or product quality. In this study, complete profiles of oxygen transfer coefficient, kLa O2, and carbon dioxide stripping coefficient, kLa CO2, were established in 500L and 5L bioreactors. A range of scale dependent parameters were studied and different sparger types were compared. Sparging flow rate was found to be the dominant factor for both oxygen transfer and carbon dioxide stripping in both scales, while headspace was not a significant contributor. Van’t Riet equations were established for mass transfer coefficients and applied to real process data. Maximum oxygen demand in pilot scale bioreactor was predicted using kLa O2 and small scale process data. The average error of prediction was 6.3% for six different cell culture processes. Both kLa O2 and kLa CO2 data were used to explain CO2 stripping limitations at the pilot scale. An online pCO2 control strategy was then applied to small scale processes to successfully reproduce pilot scale pCO2 profile.

BIOT 569

Scalability of impurity precipitation via a heterogeneous mixing application of the population balance

Mohamed Agoub1, [email protected], Steven J. Traylor2, Jing Guo3, Weixin Jin1, Helen Hua1, Xuankuo Xu2, Russell Diemer3, Abraham M. Lenhoff3, Sanchayita Ghose1. (1) Bristol-Myers-Squibb, Westford, Massachusetts, United States (2) Biologics Process Development, Bristol-Myers Squibb, Hopkinton, Massachusetts, United States (3) Univ of Delaware, Newark, Delaware, United States

The isoelectric precipitation of impurities during viral inactivation neutralization has proven to be difficult to adequately characterize across scales. Namely, efforts have been made to connect critical mixing factors, such as Reynolds number or specific energy, to the observed particle size distributions in order to develop a scalable model of impurity precipitation. However, experimental results have suggested that these factors alone are insufficient in describing the precipitating system across scales. The objective of this work is to further investigate variables contributing to the mechanism, facilitated by simulation of the mixing vessel using computational flow dynamics, and quantifying particle formations via a sectional population balance model. Experiments were conducted for one scaled-up system and two scaled-down systems of different geometries to quantify the trajectory of particle size distributions via flow microscopy. CFD analysis of each system displayed the prominence of heterogeneous mixing conditions in lab-scale experiments, lending credence to the possibility that non-uniform shear rates throughout the vessel impacts the shape of the particle distribution. This heterogeneity can be represented via a two-compartment mixing model, illustrated as individual regions of high- and low-energy agitation exchanging material. A parameter set can be generated to describe these compartments, and subsequently applied to the population balance equations to predict the particle distribution.

BIOT 570 Development of a robust control strategy to reach target excipient concentrations during drug substance buffer exchange by UFDF

Grace Tran1, [email protected], Andreas Sophocleous1, Ryan Muthard1, Andre C. Dumetz2. (1) GlaxoSmithKline, King of Prussia, Pennsylvania, United States (2) 709 Swedeland Rd UE0551, Glaxo Smithkline, King of Prussia, Pennsylvania, United States

Buffer and excipient composition of a drug substance are, in regulatory submissions, registered details. As a part of late phase product development work packages, the formulation robustness is typically supported by stability studies looking at the effect of buffer and excipient concentrations. Excluded volume and Gibbs-Donnan effects have long been known to cause an off-set in the formulation of protein solutions when utilizing UFDF. These phenomena can result in a deviation from the target concentration when using diafiltration buffer at final formulation concentrations. The recent emphasis on the development of buffer and excipient methods has shown that those off-sets on the concentration of formulation components can be significant especially for formulation at high protein concentrations. In a few extreme cases, the concentration of formulation components can potentially be outside the range characterized by supporting stability studies. Here the principles behind excluded volume and Gibbs-Donnan effects are reviewed focusing on the process parameters that affect their magnitude. Different experimental approaches to reach the target composition during UFDF are presented. Both cases of platform (mAb) and non-platform (recombinant) protein molecules are covered. The results are then presented in the perspective of Quality by Design for the development of a control strategy for a late phase asset.

BIOT 571

Streamlining process characterization efforts using the high-throughput ambr® crossflow system for ultrafiltration and diafiltration processing of monoclonal antibodies

Lara Fernandez-Cerezo, [email protected], Steven W. Benner, Jennifer Pollard. Downstream Process Development & Engineering, Merck & Co., Inc (USA), Kenilworth, New Jersey, United States

Final development stages for biopharmaceuticals often involve process characterization (PC) studies to gain process knowledge and understanding in preparation for process validation. One common approach to conduct PC activities is by using Design of Experiments (DoE), which can provide data for the product quality attributes, operating ranges and system parameters. Qualified scale-down (SD) systems are typically used to conduct these studies. For example, for an ultrafiltration/diafiltration (UF/DF) application, a traditional SD system is fitted with: a 88 cm2 or 0.11 m2 Pellicon flat-sheet tangential flow filtration (TFF) cassette; a diaphragm crossflow pump; a retentate valve to control mean transmembrane pressure (ΔΡTMP); and a feed/retentate recirculating tank connected to a diafiltration buffer tank. This system, however, requires hundreds of milliliters of material per run and can only conduct one experiment at a time. This poses a challenge in resources, in terms of material, time and personnel, as there could be 20+ experiments required for a typical DoE UF study. One solution to circumvent this is, the use of high-throughput screening systems which enable parallel experimentation by only using a fraction of the material requirements. Recently, Sartorius Stedim Biotech has commercialized the ambr® crossflow system to meet this need by only using 10 cm2 proprietary cartridge and 5 mL channel hold-up volume. In this presentation, the performance of this system will be compared with a traditional pilot-scale TFF system for a monoclonal antibody process during a PC study. 2 Studies across a range of ΔΡTMP (12 to 18 psi); QF (3 to 9 L/min/m ); and diafiltration concentration (35 to 55 mg/mL) were conducted in both scales. The main comparative metrics used will be: the permeate flux as a measure of the membrane performance; and the aggregate content as a measure of the product quality. Recovery and turbidity values will also be shown.

BIOT 572

Pilot-scale bioreactor production of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures

Kantharakorn MACHAROEN1, [email protected], Min Du1, Seongwon Jung1, Karen McDonald1,2, Somen Nandi1,2. (1) Chemical Engineering, University of California Davis, Davis, California, United States (2) Global HealthShare Initiative®, University of California Davis, Davis, California, United States

Plant-based biopharmaceuticals have become more attractive due to several advantages of using whole plants (molecular farming) or plant cells as a production platform. For example, plants do not produce prions, and they are unable to propagate human pathogens like mammalian cells. Plants also provide proper protein folding and complex protein post-translational modifications. Unlike whole plants, plant cell cultures can be grown in low cost chemically-defined media in a contained sterile environment such as a bioreactor, which makes the system controllable, reproducible, and compatible with regulatory guidelines such as current good manufacturing practice (cGMP). However, there are limited studies on scale-up to pilot-scale bioreactors, especially in conventional stainless-steel stirred tank bioreactors (SSTBR), to produce recombinant proteins in plant cell suspension cultures. In this study, we successfully scaled-up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticides exposure, from metabolically regulated transgenic rice cell suspension cultures in a 40-L pilot-scale SSTBR. Employing cyclical operation together with the efficient simplified-process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab-scale bioreactor studies, we found consistent maximum total functional rrBChE production level of 46-58 µg/g fresh weight in four cycles over 80 days of operation. Here we demonstrate the potentially cost-effective and scalable platform for rrBChE production as a result of the simplified bioreactor operation and the low cost of plant cell culture media (~40 times less expensive than CHO cell culture media).

BIOT 573

Industrial biotechnology award: Development and scale-up of the commercial fed batch refolding process for Lumoxiti, an anti-CD22 two chain immunotoxin

Alan Hunter, [email protected]. AstraZeneca, Gaithersburg, Maryland, United States

Lumoxiti (moxetumomab pasudotox) is an orphan drug and the first approved recombinant immunotoxin relying on an antibody fragment for selective targeting of cancer cells. It is composed of the VH and VL portions of an anti-CD22 antibody genetically fused to a truncated form of Pseudomonas exotoxin (PE38) by a peptide linker. Immunotoxins are similar to the Antibody Drug Conjugate (ADC) concept where a toxic payload (in this case PE38) is directed against a target of interest (CD22) using an antibody or antibody fragment. The Lumoxiti CMC process development team, responsible for development of a commercializable manufacturing technology, overcame numerous obstacles and industrialized a truly innovative approach to protein refolding to bring this promising therapy to patients. Industrial protein refolding presents a number of unique challenges. In particular, low refold titers and high levels of misfolded protein and aggregates commonly encountered in refold reaction mixtures often prove exceedingly difficult to purify, leading to low yield and high cost of goods. In this work, we present the development, scale-up, and validation of a commercial immunotoxin refolding and purification process. Limitations of the existing clinical process are explained. The development strategy is summarized and data is presented describing improvements made to the process. Results from the 950L commercial scale refolding process are provided, demonstrating a greater than fivefold improvement in volumetric productivity compared to the clinical process. Lastly, several novel challenges that arose during late stage development are discussed including identification and control of low level expression of endogenous DE3 bacteriophage with a recA deficient E.coli host strain, as well as validation strategy for a process where the two polypeptide chains comprising Lumoxiti are expressed and recovered in separate fermentation unit operations.

BIOT 574

Production of antibody-drug conjugates through maleimide-diene coupling to noncanonical amino acids

Ronald J. Christie1, [email protected], Andre St. Amant2, Javier Read de Alaniz2, Changshou Gao1. (1) Antibody Discovery and Protein Engineering, AstraZeneca R&D, Gaithersburg, Maryland, United States (2) Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, California, United States Rapid generation of antibody-drug conjugates (ADCs) is limited by chemistries available for drug attachment. Here, we present diene-containing noncanonical amino acids (ncAAs) capable of undergoing fast and selective Diels-Alder reactions with maleimide following incorporation into antibodies. Diels-Alder bioconjugation (DAB) provides practical and performance-enhancing benefits related to production and the resulting physical properties of bioconjugate therapeutics such as ADCs. Manufacture of ADCs using DAB can be achieved in a one-step process that doesn’t require addition of catalysts, pH adjustment, or chemical activation of antibodies. Since DAB was designed from the perspective of ADC payloads, it utilizes clinically validated drugs and thus eliminates the need for concurrent development of new compounds. DAB brings unique advantages related to conjugate stability and antibody design options that are not realized with current cysteine-based approaches that use the same maleimide payloads. For example, DAB solves position-dependent stability issues that plague thiosuccinimide-linked ADCs due to the retro-Michael reaction at physiological conditions. Also, drug can be attached via DAB at antibody positions where cysteine introduction interferes with natural protein disulfides. While DAB has been successfully applied towards development of ADCs, the technology is poised to enable further applications that expand the scope of protein-based therapeutics such as polymer-, peptide-, nanoparticle-, and nucleic acid-bioconjugates.

BIOT 575

Activity-based discovery and optimization of agonist antibodies

John S. Schardt, [email protected], Hark S. Jhajj, Ryen O'Meara, Peter M. Tessier. Chemical Engineering and Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States

Agonist antibodies that activate intracellular signaling via binding to extracellular receptors have emerged as a new paradigm in disease treatment (e.g., in cancer and neurodegenerative diseases). Despite this promise, the discovery of extremely rare antibodies capable of initiating receptor-mediated cellular activation remains a major biotechnological bottleneck. To address this issue, we have developed a high- throughput, direct function-based screening method to generate antibody variants with high agonist activity against the T-cell receptor Ox40. Our approach uses mammalian cells engineered to report intracellular, Ox40-mediated signaling via the NF-kB pathway in a manner that can be robustly detected using flow cytometry. By combining this cellular reporter system with the display of libraries of antibodies on the mammalian cell surface, we are able to directly screen for agonist activity in a high-throughput manner. We will discuss our validation of this approach using clinical-stage agonist and antagonist antibodies as well as our isolation and characterization of novel antibody variants that activate the Ox40 receptor. We expect that this type of activity-based screening method – which can be readily adapted to target other cellular receptors – will be valuable for improving the rapid and reliable isolation of agonist antibodies for diverse biological and therapeutic applications.

BIOT 576

Site-specific conjugation of antibodies to a protein carrier for intracellular antibody delivery

Anshul Dhankher, [email protected], William T. Studstill, Manuel E. Hernandez, Julie Champion. Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States

The application of antibodies as therapeutics is restricted to a small number of extracellular targets since antibodies cannot cross the cell membrane. To target intracellular proteins, we developed a self-assembling protein carrier (Hex carrier) that binds the conserved Fc region of antibodies and delivers them intracellularly. The carrier is comprised of a self-assembling hexamer barrel with an Fc binding domain (SPAB) fused to each monomer of the barrel. Characterization of serum stability of the Hex carrier fully loaded with Immunoglobulin G (IgG) showed that antibodies loaded on the carrier were exchanging with the excess IgG present in simulated serum conditions. In order to use Hex for in vivo drug delivery applications, antibodies needed to be conjugated to Hex to prevent exchange with serum IgG. The SPAB domain contains two phenylalanine residues directly facing the Fc region in the binding interface between the two proteins. To create a covalent linkage between the Fc region and SPAB domain, a non-canonical amino acid analog of phenylalanine, para-azido-phenylalanine (pAzF), was incorporated into the Hex carrier. Upon exposure to UV light, pAzF forms a reactive group that inserts into nearby C-H and N-H bonds of the protein backbone. Incorporation of pAzF was confirmed by ESI mass spectrometry and the Hex proteins containing pAzF (pHex) retained their binding function to the Fc region of antibodies. Upon crosslinking of IgG to pHex, antibody exchange with serum IgG was significantly reduced compared to the original Hex proteins and a non-crosslinked pHex control. Finally, the crosslinked pHex carriers showed significantly higher delivery of fluorescently labeled IgG inside of cells compared to Hex without pAzF. Ongoing work on the pHex proteins is examining the biodistribution and pharmacokinetics of pHex carriers in a mouse model and future work will test the delivery of a functional antibody to a relevant disease target.

BIOT 577

Development of key control points to manufacture antibody drug conjugates with high payloads

Daniel Custar1, [email protected], Engin Ayturk2, Thomas R. Wagler1, Michael Kaufman1. (1) CMC, Mersana Therapeutics, Cambride, Massachusetts, United States (2) Mersana Therapeutics, Shrewsbury, Massachusetts, United States

XMT-1536 is an antibody drug conjugate (ADC) in a Phase 1 clinical trial that targets cancer cells containing NaPi2b antigens, typically expressed in 90% of ovarian and non- small cell lung carcinomas. This ADC is comprised of a highly water soluble and biodegradable polymeric linker platform, Dolaflexin, in which 3-4 auristatin F- hydroxypropylamide (AF-HPA) payload molecules are bound to Dolaflexin and 3-4 Dolaflexins are conjugated to the antibody, leading to a high payload ADC with a wide therapeutic window. The hydrophilicity of the Dolaflexin polymer allows for the high drug-to-antibody ratio (DAR), while maintaining antibody pharmacokinetics and drug-like properties of the ADC. The Dolaflexin platform has inherent heterogeneity that can be managed and controlled to provide reproducible critical quality attributes, which have led to good batch-to-batch consistency. Similarly, the bioconjugation of Dolaflexin to the antibody has well-defined controls in place, as a result of carefully designed screening conditions and well-executed DoE studies, to produce ADCs with consistent key quality attributes. The process development and control points of bioconjugation of Dolaflexin to antibodies to generate a robust manufacturing platform with high DAR ADCs will be discussed in greater detail.

BIOT 578

Engineering immunogens to elicit broadly neutralizing antibodies

Steven Frey1, [email protected], Chad Varner1, Ammar Arsiwala1, Tania Rosen1, Michael Currier2, Martin Moore2, Ravindra S. Kane1. (1) School of Chemical & Biomolecular Engineering, Georgia Tech, Atlanta, Georgia, United States (2) School of Medicine, Emory University, Atlanta, Georgia, United States

A challenge in the development of an effective vaccine is the creation of an immunogen that elicits a broad immune response. This challenge stems from the variability of viral proteins across strains, such that it may be beneficial to direct the immune response to conserved antigenic sites. Respiratory syncytial virus (RSV), for which there is currently no licensed vaccine, displays a fusion (F) protein that contains both variable and conserved regions. This protein has an antigenic site called site Ø, which has been shown to elicit potent, neutralizing antibodies and has therefore been considered important in the formulation of an RSV vaccine. However, this site is also the least conserved on the F protein across RSV subtypes. Therefore, we hypothesized that directing the immune response away from site Ø and refocusing it towards more conserved parts of the RSV F protein might serve to better elicit broadly neutralizing antibodies. Thus far, we have created an RSV F protein with inserted glycans that shield site Ø to demonstrate that RSV F-based vaccines with a shielded site Ø can still be broadly protective. In future studies, we will test our hypothesis that site Ø-specific antibodies are less likely to be broadly neutralizing than antibodies directed towards the other, more conserved sites of RSV F. The RSV F protein is just one example of an immunogen we are engineering to elicit a broadly protective immune response. These engineered immunogens may ultimately help to inform the design of broadly protective vaccines.

BIOT 579

Tuning macromolecular fate through site-selective chemical conjugation of a novel half-life extender

Aimee Usera1, [email protected], Claire Adcock1, Frederic zecri1, Jun yuan1, soohee park1, Alexandra Bruce2. (1) Global Discovery Chemistry, Novartis Institute for Biomedical Research, Cambridge, Massachusetts, United States (2) Aro Biotherapeutics Company, Philadelphia, Pennsylvania, United States

Peptides and proteins offer a natural entity to tackle high value medicinal targets. However, native biologics oftentimes have a short half-life which makes translation to patients extremely difficult, if not next to impossible. We have addressed this longstanding challenge by developing novel site-selective protein chemistry methods to install a proprietary half-life extension technology. Discovery of a histidine-directed modification enables N-terminal site-selective labeling of proteins and peptides. Transglutaminase mediated chemoenzymatic labeling of lysines allows for site-specific conjugation to peptides, proteins, and antibodies. To address the specific challenge of rapid clearance, we identified a novel albumin binding fatty acid with ~ 50 fold greater affinity than native myristic acid. Further SAR through biacore binding resulted in an optimized lipid extending the half-life of proteins and peptides to that of albumin. In applying these technologies to the extremely short acting serelaxin peptide, we show that this long acting conjugate analog extends the exposure and shows dose responsive target engagement towards a goal for improved treatment for heart failure. Utilizing our newly developed toolbox of technologies, we have prolonged the pharmacokinetic and pharmacodynamic profile of a diverse set of macromolecules and provided a potential path forward toward developable biotherapeutics of the future.

BIOT 580

Kinetic analysis of cellular internalization and expulsion of unstructured D- chirality cell penetrating peptides Manibarathi Vaithiyanathan1, Hannah C. Hymel1, Nora Safa1, Olivia M. Sanchez1, Jacob H. Pettigrew1, Cole S. Kirkpatrick1, Ted J. Gauthier2, Adam T. Melvin1, [email protected]. (1) Chemical Engineering, Louisiana State University, Baton Rouge, Louisiana, United States (2) LSU AgCenter Biotechnology Lab, Louisiana State University, Baton Rouge, Louisiana, United States

Cell penetrating peptides (CPPs) offer great potential as delivery agents for otherwise cell impermeable therapeutic molecules in addition to allowing for the introduction of novel biosensors to track intracellular processes. However, the majority of identified CPPs are unstructured which makes them susceptibility to proteolytic degradation and limits their overall utility. To address this, researchers have explored how altering the sequence identity or structure of the CPPs enhances both uptake efficiency and intracellular stability. Prior work from our lab found that utilizing a β-hairpin secondary structure for the CPP enhanced intracellular stability; however, it diminished overall uptake efficiency when compared to an unstructured, scrambled L-chirality sequences. Thus, a tradeoff was identified between uptake efficiency and intracellular peptide stability. The focus of this work is to expand upon these findings by incorporating D- chirality amino acids in unstructured CPPs to identify peptide sequences with superior uptake kinetics and long-term stability. Intracellular proteases and peptidases cannot recognize D-chirality peptides which allows them to exhibited extended lifetime in the cell. A small library of D-chirality CPPs were synthesized and their performance was evaluated using a series of time, concentration, temperature and energy dependent studies. A three-fold increase in cellular uptake was observed with the D-chirality peptides over their L-counterparts, with a slight alteration in the mode of entry. While majority of uptake occurred by direct penetration, CPP internalization was partially controlled by endocytosis followed by endosomal escape and cytosolic access. The inversion of chirality from L- to D- provided increased protease resistance where the D- CPPs exhibited a half-life of ~600 min - a 20-fold increase in comparison to a half-life of ~ 30 min for its L-counterpart. Interestingly, it was observed that a percentage of the internalized D-CPPs were expelled from the cells in a time-dependent manner until the cell reached an effective steady state. Mechanistic studies identified that cells exported the D-CPPs via the exocytosis pathway and that it occurred in an energy-dependent manner. These findings highlight the potential and utility of D-chirality CPPs, but also identify an expulsion method suggesting a new trade-off exists between uptake kinetics, intracellular stability, and intracellular retention.

BIOT 581

Multivalent polymer−peptide conjugates: General platform for inhibiting amyloid beta peptide aggregation

Xing Jiang2, Abby J. Halmes2,1, [email protected], Giuseppe Licari2, John W. Smith3, Yang Song2,1, Edwin G. Moore1, Qian Chen4, Emad Tajkhorshid2,1,5, Chad Rienstra1,5, Jeffrey S. Moore2,1,4. (1) Chemistry, University of Illinois at Urbana- Champaign, Urbana, Illinois, United States (2) Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States (3) Materials Science and Engineering, University of Illinois at Urbana- Champaign, Urbana, Illinois, United States (4) Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States (5) Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States

The aggregation of amyloid beta (Aβ) into fibrils has been implicated in the progression of Alzheimer’s Disease (AD). While many inhibitors of this aggregation have been developed, few have taken advantage of multivalency effects. We demonstrate a family of multivalent polymer-peptide conjugates (mPPCs) which inhibit Aβ fibril formation. mPPCs consist of a polymer/peptoid ligand appended to a poly(N-(2- hydroxypropyl)methacrylamide) backbone. In this study we investigate the effects of polymer molecular weight, peptide/peptoid ligand, and ligand loading on inhibition efficiency to inform mPPC design. While previous work in our group has demonstrated these effects in limited systems, expanding the library of mPPCs tested has highlighted the general effectiveness of mPPCs as Aβ inhibitors. ThT fluorescence experiments showed complete inhibition of fibril formation by most mPPCs, and dose dependence studies demonstrated the importance of ligand charge in inhibition. DLS experiments along with molecular dynamics simulations suggest that mPPCs form globular structures due to ligand-ligand interactions. These interactions influence the proximity of the ligands and their accessibility to Aβ. Ultimately this library of compounds is valuable as a platform for furthering our understanding of the mechanism of Aβ aggregation.

BIOT 582

Current and future challenges in monoclonal antibody purification

Hanne Bak, [email protected]. PMPD, Regeneron Pharmaceuticals, New York, New York, United States

33 years after the first approval of a monoclonal antibody for therapeutic use, the biopharmaceutical industry has close to 100 therapeutic monoclonal antibodies approved or in review in the EU or the US, many of which use highly similar purification platforms. Meanwhile, we have seen a rise in new powerful therapeutic modalities that offer possibility of permanent cures for serious diseases and reduced dosing frequency. As such, a relevant question would be: “Is there really more we need to do with monoclonal antibody purification?”. The short answer is yes. This keynote presentation will discuss current challenges as well as future trends in the purification of therapeutic monoclonal antibodies for human use.

BIOT 583 Protein A fiber chromatography from research and early development to scale-up and manufacturing of monoclonal antibodies

Anna Graanberg1, [email protected], Martin Hall1, Lars Rosengren1, Anders Nilsson1, Ian Scanlon2, Matthew Townsend2, Oliver Hardick2. (1) R&D, GE Healthcare, Uppsala, Sweden (2) R&D, GE Healthcare, Stevenage, United Kingdom

A new Protein A fiber chromatography format has been developed as a complement to traditional column chromatography. The Protein A fiber matrix has an open pore structure where mass transfer is governed by convective flow which allows high mAb binding capacities at very short residence times. This results in cycle times of minutes instead of hours as compared to traditional column chromatography. The short cycle times allows for purification of many samples in short time which makes the pre-packed, small-scale Protein A fiber unit a versatile tool during e.g. clone selection and up-stream cell-culture media development. In the downstream process the time savings in process development will be significant where screening of different buffer conditions and lifetime studies including hundreds of cycles can be performed in less than 24 hours. In larger scale, the Protein A fiber operated in a rapid cycling manner allows full utilization of the Protein A lifetime during one batch. The pre-packed and pre-sanitized single- batch disposable format eliminates column packing and reduce the risk of bioburden incidents. Examples will be given from small scale, where automation and a combination of capture and desalting steps will increase the throughput in up-stream process development as well as wash and elution buffer optimization in downstream process development. However, the focus will be on larger scales where multiple manifolded fiber units of different sizes, and number of cycles during operation will match the mass of mAb from the bioreactor. The compatibility of fiber units with current chromatography systems regarding system flow rates, back pressure limitations and dead volumes will also be demonstrated. Productivity, throughput, buffer consumption and purification performance will be compared to traditional and rapid cycling Protein A column chromatography.

Protein A fiber unit (0.6 L) operated on a chromatography system with a disposable flow path. Cycle time <6 minutes, maximum pressure 3.6 bar. MV=Matrix volume. The insert shows the open structure of the fiber matrix.

BIOT 584

Improvement in protein A resin lifetime by implementing poly- diallyldimethylammonium chloride (pDADMAC) flocculation coupled with gradient depth filtration

Philip Szymanski, [email protected], Akshat Gupta, Dana Kinzlmaier. MilliporeSigma, Burlington, Massachusetts, United States

The economics of the protein A capture step in mAb purification rely on effective resin utilization over multiple cycles. To this end, pDADMAC-based flocculation and subsequent filtration with gradient depth filters was evaluated as a method of cell culture clarification to reduce the impurity burden on protein A resin. The effects on column performance, measured using yield, binding capacity, backpressure, and impurity clearance, were investigated over 160 cycles. Results were compared with a lifecycle assessment of protein A capture of the same feed stream clarified using traditional depth filters without flocculation. Improvements in maintenance of dynamic binding capacity, column backpressure evolution over number of cycles, and host cell protein (HCP) levels in protein A eluate were observed. Process consistency in terms of product concentration in the eluate and eluate volume was also maintained over the 160 cycles. Cost of goods analysis was performed to quantify the economic benefits of the enhanced clarification strategy on the holistic clarification and capture process. Additional wash optimization to further improve the HCP clearance is currently being investigated.

BIOT 585

ProteinA Fibro chromatography as a platform affinity-capture of the next- generation manufacturing process of antibody therapeutics

Ryan Zolyomi1, [email protected], Kierston Shill1, Ian Scanlon2, Florence Rusly1, Anna Graanberg3, Oliver Hardick2, Hendri Tjandra1, Ashley Hesslein1. (1) Biologics Development, Bayer HealthCare, Berkeley, California, United States (2) Puridify, GE Healthcare, Stevenage Bioscience Catalyst, SG1 2FX, United Kingdom (3) R&D, GE Healthcare, Uppsala, Sweden

With an increasing number of antibody therapeutics and higher cell culture titers from intensified upstream processes, manufacturing batch sizes are smaller and more diverse. Consequently, single-use multi-product manufacturing facilities are utilized as they provide the flexibility necessary to meet these changing demands. These driving forces motivated us to evaluate ProteinA-derivatized Fibro, a single-use chromatography technology, in meeting the goals of our future manufacturing platform.

Implementing a fully single-use manufacturing platform can be challenging as bead- based ProteinA resin chromatography has anchored industrial production of antibodies. ProteinA resins have an excellent record of clearing impurities while consistently delivering high quality products. Typically, large columns are used during clinical manufacturing to reduce processing times and are thus rarely exhausted of their entire lifetime capacity. This results in under-utilization, high initial costs and storage concerns. Fibro ProteinA enables rapid purification of monoclonal antibodies by offering faster mass transfer, high capacity, and fuller utilization within a single production batch. It offers the potential of delivering the same product quality as that of ProteinA resin with a potential cost- and time saving during clinical productions. Bench-scale Fibro studies performed across a range of monoclonal antibodies indicate its feasibility as a platform approach. To assess its scalability, pilot-scale case studies were conducted. They demonstrated its single-use facility fit, productivity benefits, and product-quality comparability to those of resin-based processes. Additionally, these pilot-scale studies addressed concerns over their adoption due to the disadvantages of membrane-like technologies, namely higher buffer consumption, larger elution pool volumes, and shorter device lifetimes. As Fibro units were scaled up, the buffer consumption, elution volume and lifetimes were found to be comparable to those of resin-based affinity chromatography.

We evaluated the feasibility of implementing FibroA chromatography into a future mAb manufacturing platform. While there remain uncertainties surrounding long-term process economics at a manufacturing scale, this technology allows for productivity increases, cost-savings, and flexibility. We envision its potential for a paradigm shift in manufacturing antibodies in the future.

BIOT 586

Impact of mobile phase modulators on capacity and impurity clearance in protein A chromatography: Insights from mechanistic models

Vijesh Kumar1, [email protected], Xuankuo Xu2, Steven Traylor2, Sanchayita Ghose2, Abraham M. Lenhoff3. (1) Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Biologics Process Development, Bristol-Myers Squibb, Devens, Massachusetts, United States (3) Univ of Delaware, Newark, Delaware, United States

Protein A affinity chromatography is the dominant capture step in monoclonal antibody manufacture. Since it is the first unit operation in a succession of chromatography steps, process understanding using a mechanistic model can be efficacious. Although mechanistic models based on Langmuir isotherms are widely used, the physical significance of the model parameters is not always considered in detail. In this work, we have calibrated such a model using complete breakthrough curves in the presence of various mobile-phase modulators at different pH values. Both modified Langmuir and colloidal-based models accommodating pH effects were incorporated in the model. The isotherm models were coupled with the general rate model, including pore and surface diffusion effects. The dynamic binding capacity was shown to be impacted by the load concentration (0.5-10 mg/mL), kosmotropic salts (sulfates, phosphates, and citrates) and organic phase modulators (ethanol, isopropanol). The maximum binding capacity within the Langmuir isotherm and the protein-protein repulsion within the colloidal isotherm led to significant differences in the protein-surface binding affinity extracted from the models under these conditions. The parameters obtained from breakthrough curves were used to model the effects on product recovery of wash and elution buffers for mobile phase modulators and pH. The pH buffer titration on the Protein A column is one of the challenges in modeling elution peak shape and thus pool volume. This was overcome by modeling the column with a buffer titration curve run without protein load. The impact of the removal of host-cell protein and protein aggregate with mobile phase modulators and its pH was found to be important. Overall, this work demonstrates that mechanistic models can provide broad insight into protein binding behavior inside the column and hence utilize the knowledge to develop an optimal process.

BIOT 587

Mechanistic understanding of protein A resin lifetime in continuous multi-column mode

Jing Guo, [email protected], Jiayue Yang, Dharmesh Kanani, Lu Wang, Mi Jin. Teva pharmaceuticals, West Chester, Pennsylvania, United States Operating Protein A chromatography in continuous multi-column mode significantly improves resin capacity utilization and productivity compared with that in batch mode. However, the Protein A resin lifetime performance in continuous multi-column mode is still not well understood. As two or more columns always stay connected in the load zone in continuous mode, it is possible that the resin lifetime behaves differently than that of a single column in batch mode. We performed a resin lifetime study with a novel Protein A resin, running in a continuous 3-column mode, for 150 cycles. The resin binding capacity and yield results over cycles were compared with those from a single column running in batch mode for 150 cycles. Chromatography general rate model coupled with Langmuir adsorption isotherm was used to fit the breakthrough curves to investigate changes in resin properties, including maximum adsorption capacity and protein diffusivity coefficients, to understand the underlying mechanisms of resin fouling and ligand degradation. This knowledge facilitates predicting product loss of continuous multi-column Protein A chromatography after a certain number of cycles, and in developing corresponding strategies such as adjustable product loading and effective resin cleaning methods.

BIOT 588

Influence of excipients during antibody purification on Protein A chromatography

Carolin Stange1, [email protected], Supriyadi Hafiz2, Tanja Henzler2, Christian Dr. Frech3. (1) Institute of Biochemistry, University of Applied Sciences Mannheim, Bad Orb, Germany (2) Merck KgaA, Darmstadt, Germany (3) Biotechnology, University of Applied Sciences Mannheim, Mannheim, Germany

The production of antibodies and Fc-fusion proteins involves several downstream processing unit operations. The widely used purification template with Protein A chromatography, virus inactivation at low pH, and subsequent ion exchange chromatography steps is mostly able to remove impurities like aggregates, host-cell proteins, and viruses, which could affect the safety and efficacy of the product. The low-pH elution during Protein A chromatography, as well as during virus inactivation may induce aggregation. Preventing protein aggregation during these unit operations instead of removing the multimeric forms during subsequent polishing steps would be a more efficient strategy. Excipients have shown that they can minimize aggregation levels in the final product formulation. The objective of this work was to apply excipients, like polyols, in buffer systems, to study their impact on several aspects during Protein A chromatography and virus inactivation. Therefore, a pH gradient system on three different Protein A materials was applied. The results include their effect regarding elution patterns, stabilization of the monomer antibody, host-cell protein removal, and virus inactivation rates, as well as the effect on binding capacities in following cation exchange chromatography. The results show that the addition of excipients can have a beneficial effect for the purification during Protein A chromatography and virus inactivation, without harming the subsequent chromatographic steps. BIOT 589

Amino acid metabolism in CHO cells

Maciek R. Antoniewicz1, [email protected], Michael J. Betenbaugh2. (1) University of Delaware, Newark, Delaware, United States (2) Department of Chemical and Biomolecular engineering, Johns Hopkins University, Baltimore, Maryland, United States

CHO cells require complex media for cell growth and protein production. The major components of industrial media are amino acids, however, relatively little is known about the metabolism of amino acids in CHO cell cultures. Here, we applied advanced 13C-flux analysis tools to elucidate the metabolic flow of the amino acids in a fed-batch CHO culture that overproduced IgG. Carbon flows were tracked throughout the growth phase and changes in metabolism were quantified when cells transitioned from growth phase to stationary phase. In addition, we quantified how changes in amino acids profiles in the medium translated to changes in cell growth and protein production. To trace each amino acid individually, custom media formulations were used, where each medium formulation was depleted of a specific amino acid. A labeled 13C variant of the depleted amino acid was then added to the medium at the desired concentration. CHO cells were then grown in fed-batch culture. As the cells metabolized the labeled amino acids, this resulted in a redistribution of 13C-atoms which we quantified using GC-MS for both extracellular metabolites (including lactate, amino acids and the IgG product) and intracellular metabolites (including free intracellular metabolites, cell proteins, lipids and carbohydrates). This allowed us to calculate the fraction of each amino acid that was used for cell growth, protein production, lactate formation and energy generation. We also investigated the effects of labeling in both the batch and fed-batch stationary phase. Finally, we investigated the effects of varying amino acid concentrations. Each amino acid was added to the medium at a lower or higher concentration compared to the base medium and changes in metabolic fluxes were compared in order to determine the precise impacts of amino acid concentration changes on the metabolic flux profiles. Taking all of this data together, we are now building a predictive kinetic model that relates how the metabolism of CHO cells can be predicted from amino acid profiles. In future work, model predictions will be experimentally validated as a means of optimizing the amino acid composition of industrial culture media.

BIOT 590

Integration of a mechanistic kinetic model in metabolic characterization of cell lines

Meghan McCann1, [email protected], Conor O'Brien1, Irfan Hodzic3, Sofie O'Brien2, Jeanette Caronia2, Samira Azarin1, Frank Swartzwelder3, Wei-Shou Hu1. (1) Chemical Engineering and Materials Science, University of Minnesota, Twin Cities, Saint Paul, Minnesota, United States (2) Biomedical Engineering, University of Minnesota, Twin Cities, Minneapolis, Minnesota, United States (3) Upstream R&D, MilliporeSigma, St. Louis, Missouri, United States

Consumption of nutrients and production of metabolites lead to environmental changes over the course of cell-culture processes. Cell metabolism both drives and is affected by these changes that are often accompanied with decreased productivity and product quality. The general metabolic characteristics of cells lines derived from the same host cell are generally similar, however, differences in the metabolic profiles in culture are commonly seen. We hypothesize that the difference in cell line metabolic performance is the result of variability in the composition of metabolic machinery and the topology of regulatory network, and that the effect of those variations can be captured and predicted by a mechanistic metabolic model adapted for a particular cell line. In this work, we systematically characterize the transcriptomic, metabolic and N-Glycosylation profiles of two CHO cell lines to integrate with a mechanistic kinetic metabolic model for the metabolic characterization of each cell line. The model incorporates mechanistic reactions of glycolysis, the pentose phosphate pathway, the citric acid cycle, redox balance, and anaplerosis and cataplerosis between compartments. The model also depicts the regulatory effects of signaling pathways such as AKT and AMPK. Transcriptional abundance of metabolic enzymes, including various isozymes, are used for specification of the model.

A detailed survey of nutrient and metabolite dynamics was performed. Stable isotope labeling experiments were performed to determine carbon flux at major branches of metabolism. The dynamics of flux distribution upon metabolic shifts from lactate production to lactate consumption were determined, and the model used to predict the dynamics of metabolic shift. Protein quality attributes, particularly N-glycosylation, present measurable changes over the course of the fed-batch process, which we evaluate in the context of metabolic behavior. The system engineering approach employed in this study is applicable to the rational design of bioprocesses through process and product characterization and metabolic modeling.

BIOT 591

13C metabolic flux analysis of pre-implantation bovine embryos: Surmounting challenges due to small sample size and complex media

Daniel Lugar2, [email protected], Sayanee Adhikari3, Carol L. Keefer4, Ganesh Sriram1. (1) University of Maryland, College Park, Maryland, United States (2) Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, United States (3) Chemical and Biomolecular Engineering, University of Maryland, College Park, College Park, Maryland, United States (4) Animal and Avian Sciences, University of Maryland, College Park, Maryland, United States

13C-assisted metabolic flux analysis (13C MFA) is a powerful methodology to quantify intracellular fluxes through primary and intermediary metabolism. 13C MFA relies on feeding a 13C-labeled carbon source to a cell culture, measuring the ensuing metabolite labeling patterns, principally by mass spectrometry, and employing isotopomer modeling to back-calculate the intracellular fluxes from the labeling data. We report the application of 13C MFA to preimplantation bovine embryos toward investigating carbon source usage and the effects of medium composition on metabolism. This problem presents unique challenges owing to the small size of the culture (< 200 cells) and the necessity to assess the metabolism of individual embryos. Previous studies have shown that the composition of culture medium strongly affects the viability of in-vitro fertilized embryos. Furthermore, such media are complex and contain multiple carbon sources, thus potentially obfuscating the labeling patterns of the 13C-labeled carbon source. Additionally, because of the small sample size, there is only a limited set of metabolites whose labeling patterns can be accurately measured. We have designed novel labeling experiments to surmount these challenges and obtain a metabolic flux map for this complex system. We will present MFA results comparing flux identifiability resulting from multiple types of 13C-glucose and 13C-fructose, and explain how these results may be collectively interpreted to provide snapshots of the metabolism of bovine embryos cultured in media of various compositions. For example, parallel labeling experiments that use differently labeled glucose and fructose have revealed that the embryos utilize substantially more glucose than fructose. Further experiments have been designed to probe the relative utilization of glycolysis and the pentose phosphate pathway, as well as the contributions of different carbon sources to metabolites originating at the pyruvate node. The methods developed by us demonstrate the extension of the MFA framework to complex mammalian systems.

BIOT 592

Feeding TCA cycle intermediates improves lactate consumption and antibody production in CHO cell cultures

Xiaolin Zhang, [email protected], Rubin Jiang, Sen Xu, Henry Lin. Merck & Co., Inc., Iselin, New Jersey, United States

Medium components play an important role in modulating cell metabolism and improving product titer in mammalian cell cultures. To sustain cell productivity, a highly oxidative metabolism is necessary. Here we explored the effect of TCA cycle intermediates supplementation on lactate metabolism and productivity in Chinese hamster ovary (CHO) fed-batch cultures. Direct addition of alpha-ketoglutarate (α-KG), malic acid, or succinic acid in the basal medium did not have any significant impact on lactate metabolism and product titer. Further supplementation to a higher concentration in the basal media had detrimental effect on culture performance. Feeding α-KG, malic acid, and succinic acid, either as a single solution or as a mixture in the stationary phase, on the other hand, significantly improved lactate consumption and led to higher product titer (~35% higher for the best condition). Delivering those intermediates as an acidic solution for pH control avoided active CO2 sparging and eliminated high pCO2 accumulation. Feeding TCA cycle intermediates was also demonstrated to have higher production than feeding lactic acid or pyruvic acid. Taken together, feeding TCA cycle intermediates was effective in improving lactate consumption and increasing product titer, which is likely due to an enhanced oxidative TCA metabolism for an extended duration.

BIOT 593

Understanding the molecular mechanism governing the correlation between specific productivity and glycosylation

Sri Madabhushi, [email protected], Alexei Podtelezhnikov, Nicholas Murgolo, Sen Xu, Henry Lin. Merck & Co., Inc., Kenilworth, New Jersey, United States

The therapeutic efficacy of an antibody is influenced by the product quality attributes of the protein that include post-translational modifications such as glycosylation, charge variants, aggregates and fragments profile. One of the challenges in process development is to find a balance between increasing productivity while maintaining product quality of the protein. Across the various monoclonal antibody (mAb) programs at Merck & Co., Inc., we have observed that increasing the specific productivity (qP) of the mAb results in a decrease in the % galactosylation (%Gal) levels on the protein. Galactosylation levels may influence the mechanism of action (e.g., effector function) of the therapeutic mAbs. In order to understand the molecular mechanism governing the correlation between qP and %Gal, we cultured cells under conditions that exhibited a range of qP and N-glycan distribution and performed transcriptomics analysis. Results showed a correlation between specific productivity and expression levels of enzymes and solute transporters that are involved in the N-glycosylation pathway. Interestingly, based on the cell culture condition, different functional gene sets were either upregulated or downregulated which correlated with the productivity of the cells. These included pathways involved in cell cycle, cholesterol biosynthesis, apoptosis, and cell adhesion. Furthermore, we demonstrated that supplementation of potentially limiting cofactors increased the %Gal levels while maintaining qP for the high qP conditions. Together, the study suggests that the reduced galactosylation levels for higher productivity conditions is governed by the availability of enzymes, substrates and cofactors involved in the glycosylation pathways and not just the residence time of the protein in the cell. The insights from this study can be applied to modulate cell culture conditions to obtain the desired product quality thus producing mAb therapeutics with greater efficacy.

BIOT 594

Trace metal variability in culture media can impair CHO culture performance by enhancing mechanisms of oxidative stress

Ryan Graham1, [email protected], Ashli Polanco2, Seongkyu Yoon3. (1) Chemical Engineering, University of Massachusetts-Lowell, Cambridge, Massachusetts, United States (2) Chemical Engineering, University of Massachusetts Lowell, Lowell, Massachusetts, United States (3) University of Massachusetts Lowell, Lowell, Massachusetts, United States

The variability of trace and bulk metals in culture media may subsequently lead to unpredictable behavior in CHO cell culture. In the fields of human health and nutritional sciences, trace metal deficiencies and surpluses have shown to significantly impact mechanisms oxidative stress in mammalian cells. However, the relationship between cellular metal consumption, nutrient metabolism, and oxidative stress interplay in CHO cell culture remains unclear. In this study, the total intracellular and mitochondrial trace metal content of CHO cells supplied with various treatments of zinc, copper, and iron content in culture media were assessed via inductively coupled plasma mass spectrometry (ICP-MS). Mechanisms of oxidative stress—peroxidase activity, superoxide dismutase activity, and hydroxyl radical generation, as well as mitochondrial membrane potential were also surveyed. Current results indicate that intracellular metal acquisition is most active during lag phase, after which minimal differences in cellular metal consumption are noticed. Apoptotic cultures operating under either a trace metal deficiency or overabundance also exhibit higher specific production of both glutamate and ammonium comparatively. Peroxidase activity and total superoxide dismutase activity are also optimized by these trace metal treatments, demonstrating cellular capability to combat oxidative stress under varying metal conditions. Moreover, hydroxyl radical generation and membrane potential are significantly impacted by mitochondrial metal content as well, ultimately leading to lapses in cell growth and mAb productivity. Here, we offer new insight into the impact of trace metal variations in culture media on overall CHO culture performance.

BIOT 595

13C metabolic flux analysis quantifies changes in metabolism during proliferation, differentiation, and under low oxygen conditions

Eleanor H. Oates, [email protected], Maciek R. Antoniewicz. University of Delaware, Newark, Delaware, United States

Intracellular metabolic fluxes provide important information about the internal operation of mammalian cells when exposed to different external conditions. In this work, we have quantified detailed metabolic flux profiles for proliferating, differentiating, and hypoxic mammalian cells. To accomplish this goal, we performed comprehensive stable-isotopic tracer experiments coupled with mass spectrometry and 13C metabolic flux analysis (13C-MFA). Specifically, we performed four parallel labeling experiments under each metabolic condition using the following optimal tracers: [1 -13C]glucose + [4,5,6 - 13C]glucose, [2 -13C]glucose + [4,5,6 - 13C]glucose, [3 -13C]glucose + [4,5,6 - 13C]glucose, and [U -13C]glutamine; subsequently, we collected intracellular mass isotopomer distributions as well as external growth and nutrient uptake and secretion rates and analyzed the data. With this information, we then applied 13C-MFA using the software METEAN to generate detailed metabolic flux maps of intracellular metabolism. Through these comprehensive investigations we identified how metabolic pathways were metabolically rewired in response to different growth phases and oxygenation conditions, including dramatic changes in glycolysis, TCA cycle, oxidative pentose phosphate pathway, and lipogenesis. For example, comparing proliferating to differentiating cellular metabolism, we observed reversal of glutamine flux (i.e. from net consumption to net production), more than 2-fold upregulation of glycolysis flux, more than 4-fold reduction in lactate secretion, and significant upregulation of lipogenesis. Comparing normoxic to hypoxic metabolism, we observed more than 4-fold increase in lactate production and more than 4-fold reduction in citric acid cycle activity. These results provide important new insights into the regulation of mammalian metabolism.

BIOT 596

Low-pass filtering effect of degradation-driven protein turnover

Bahareh Mahrou2, Azady Pirhanov2, Yong-Jun Shin2, Yongku Cho1,2, [email protected]. (1) Chemical and Biomolecular Engineering, University of Connecticut, Storrs, Connecticut, United States (2) Biomedical Engineering, University of Connecticut, Storrs, Connecticut, United States

A low-pass filter passes low-frequency signals while attenuating high-frequency ones. To remove high-frequency noise, low-pass filtering is widely used in science and engineering. Low-pass filtering behavior is also found in various biological systems, which allow robust adaptation to continuously changing environment. Since the intracellular environment is highly stochastic, reduction of high frequency noise is critical in maintaining homeostasis and on the other hand allowing efficient adaptation. Previous studies have reported low-pass filtering in the cellular transcription machinery, using fluctuating levels of galactose inducer with defined frequency. The other side of protein turnover is protein degradation, and continual balance between production and degradation results in observed protein levels in cells. Here, we hypothesized that protein degradation driven protein turnover also filters out high-frequency fluctuations. To experimentally validate this, we applied a light-activated protein degradation machinery, which enables fluctuations in protein degradation rate by delivering temporally controlled light stimulation. This optogenetic approach allowed us to observe degradation-driven fluctuations in fluorescent reporter protein levels in the yeast Saccharomyces cerevisiae. We show that under continuous transcription (as assessed by quantitative PCR of the transcript), the protein level fluctuates according to the light stimulation at frequencies (1/f) ranging from 100 min to 20 min. As stimulation frequency increases, the delay between input (light stimulation) and output (protein level) increases and the amplitude of fluctuation decreases. These characteristic behaviors of low-pass filtering indicate that the degradation machinery also filters out high frequency noise, but respond robustly to slow fluctuations with response times faster than that of the transcription machinery. Our results combined with previous studies conclude that both protein production and degradation enable protein turnover to exhibit low-pass filtering. BIOT 597

Medium as a message for metabolomic analysis. Metabolic footprinting: Tool to understand the effect of culture condition on production of lovastatin by Monascus purpureus

Seenivasan Ayothiraman, [email protected]. Department of Biotechnology , National Institute of Technology, Tadepalligudem, Andhra Pradesh, India

Lovastatin is the first commercialized drugs for hypercholesterolemia and still serving as a precursor for the next generation drugs, such as, wuxistatin and simvastatin. Lovastatin was produced as a secondary metabolite by Monascus purpureus. The present work is focused on studying the effect of culture conditions on the production of lovastatin by Monascus purpureus and predominant flux pattern of the organism to propose the robust biosynthesis pathway of lovastatin. Metabolic footprinting techniques is a quantitative analysis of exometabolites that was used to profile the extracellular metabolites of an organism. The same technique has been used to find the predominant metabolic pathway of the organism and the effect of culture conditions on the flux pattern of the organism under static and shake culture conditions. The flux pattern of the organism is different in those culture condition. By products, glycerol and ethanol was also produced along with TCA intermediates. The assimilation pattern of the secondary metabolites in the shake culture condition is different from that in static culture condition. It was found that the culture condition and the morphology of the organism has also played an important role in the different level of expression of lovastatin in static and shake cultures. It could also reason the possible choice of medium component of the fermentation. Further, the observation was developed into dynamic metabolic models to validate the observation.

BIOT 598

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria

Yasuo Yoshikuni, [email protected]. DOE Joint Genome Institute, Walnut Creek, California, United States

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products’ roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites. This work was published on Nature Microbiology (2019).

BIOT 599

From genotype to 13C-fluxome to a comprehensive kinetic model of Escherichia coli

Maciek R. Antoniewicz, [email protected]. University of Delaware, Newark, Delaware, United States

Metabolic and physiological responses to genetic perturbations are of fundamental interest to systems biology and metabolic engineering. They reveal underlying information about network structure including kinetics, regulation, and the presence of otherwise “hidden” reactions. Quantitative prediction of these responses is essential for rational strain design, but has been a persistent challenge in the field. There has been much work done to develop in silico predictive models of metabolism, most notably the constraint-based models. Progress in this area has been limited, however, by a lack of high-quality reproducible experimental metabolic flux (fluxomic) data of genetically perturbed strains.

In this study, 13C metabolic flux analysis (13C-MFA) was applied to 50 Escherichia coli knockout strains spanning the major pathways of central carbon metabolism (glycolysis, pentose phosphate pathway, TCA cycle). Moreover, a full physiological characterization of each strain was performed, including detailed biomass composition analysis. The flux and physiological responses of these strains collectively provide important new insights into the robustness of central carbon metabolic pathways, as well as areas of likely kinetic limitation. Severely growth-impaired knockout strains helped to point out the most important enzymes in central metabolism. Particularly surprising flux responses, including identification of novel enzymatic activities, will be emphasized in this presentation. Next, we used this comprehensive flux data set, combined with complementary transcriptomic and metabolomic data, to parametize a genome-scale kinetic model of E. coli metabolism. In addition to having significant value in advancing fundamental biological knowledge, this model provides a valuable new toolkit for industrial biotechnology.

BIOT 600 Desktop digital genome engineering: CRISPR editing of microbes on a massively parallel scale

James J. Lalonde, [email protected], Richard Fox, Nandini Krishnamurthy, Michael Clay. Microbial Genome Engineering, Inscripta, Inc., Pleasanton, California, United States

To fuel the pace of discovery through CRISPR editing, the research community requires tools to deliver genome-scale edits at the gene, pathway, and genome level. This would enable testing thousands, perhaps hundreds of thousands, of individual ideas rapidly and efficiently. The most urgent need in current CRISPR pipelines is a significant improvement in the ability to generate diversity in a scalable manner, which includes introducing both large numbers of edits as well as a broad variety of edit types. Achieving high edit efficiencies is a serious challenge in high throughput editing workflows. We have developed a platform which dramatically increases the scale of Digital Genome Engineering, making it possible to easily perform CRISPR-based forward engineering experiments through high-throughput diversity generation in E. coli and yeast. The platform simplifies the complex editing workflow solution from design to diversity generation – including software for oligo design, reagents, benchtop instrument and analytics. The platform enabled high-throughput diversity generation with up to 200,000 edits across the genome in both coding and non-coding regions to improve lysine production in E. coli. Similar experiments have been conducted in yeast to improve tyrosine production. This technology has also fueled large-scale discovery of gene-phenotype relationships in antibiotic resistance and abiotic stress responses. Examples of unprecedented genome wide studies performed on tens of thousands of different microbial strains containing a spectrum of gain and loss of function edits spanning a variety of environmental conditions.

BIOT 601

Scaling up synthetic biology: Engineering synthetic genetic systems with 100s to 1000s of non-repetitive genetic parts Howard Salis, [email protected]. Pennsylvania State University, University Park, Pennsylvania, United States

New challenges arise when scaling up the engineering of synthetic genetic systems to express many sensors, regulators, and enzymes. Here, we tackle Synthetic Biology's "Repeat Problem" whereby designers accidentally introduce repetitive DNA sequences when they are forced to re-use similar genetic parts within large genetic systems. Repetitive genetic parts confound several steps of the design-build-test-learn cycle by creating undesired products during DNA fragment synthesis and assembly, by triggering homologous recombination and creating genetic instability, and by preventing the correct alignment of next-generation sequencing reads, thereby reducing our ability to troubleshoot genetic system function. To successfully scale up Synthetic Biology, we must engineer large genetic systems without introducing repetitive DNA sequences.

In this talk, we first present a novel algorithm, the Non-Repetitive Parts Calculator, that rapidly designs thousands of highly non-repetitive genetic parts from specified constraints. We demonstrate its utility by designing and experimentally characterizing a toolbox of 4350 highly non-repetitive bacterial promoters with transcription rates that varied across a 1150000-fold range as well as another toolbox of 1917 highly non- repetitive yeast promoters with transcription rates that varied across a 25,000-fold range. Altogether, these genetic parts can all be used simultaneously to express over a thousand RNAs or proteins without introducing more than a 15 bp repeat sequence.

In the second part of the talk, we demonstrate how to co-express 20 CRISPR single- guide RNAs within a single compact and highly non-repetitive DNA cassette, called an Extra Long sgRNA Array (ELSA), utilizing the toolboxes of non-repetitive genetic parts as well as several system design rules. Across several examples, we illustrate how ELSAs are used in CRISPR interference mode to knock-down the expression of several proteins and thereby reprogram organism function. In particular, we show that a single 20-sgRNA ELSA can significantly rewire metabolic fluxes and increase succinic acid production by 150-fold, while maintaining evolutionary robustness across many-day adaptation and production cultures.

BIOT 602

Characterizing the DNA-cleaving and gene-editing abilities of NgAgo and other programmable endonucleases

Kok Zhi Lee1, [email protected], Michael Mechikoff1, Archana Kikla2, Arren Liu2, Paula Pandolfi2, Kevin Fitzgerald1, Zachary Hartley3, Tyler Rankin2, Frederick Gimble4, Kevin Solomon1. (1) Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States (2) Biological Sciences, Purdue University, West Lafayette, Indiana, United States (3) PLANT GENETICS, BREEDING, AND BIOTECHNOLOGY, Purdue University, West Lafayette, Indiana, United States (4) Biochemistry, Purdue University, West Lafayette, Indiana, United States Prokaryotic Argonautes (pAgos) have been proposed as more flexible gene-editing tools as they do not require a sequence motif adjacent to their targets for activity, unlike popular CRISPR/Cas systems. One promising candidate from the halophilic archaeon Natronobacterium gregoryi (NgAgo), however, has been subject to intense debate regarding its endonuclease activity and gene-editing potential in eukaryotes. Here, we develop methods and characterize NgAgo in vitro and in vivo, which revealed several unrecognized features that may explain the inconsistent results in the literature. First, NgAgo expresses poorly in non-halophilic hosts with the majority of protein being insoluble and inactive even after refolding, similar to other halophilic proteins. While our data suggest that soluble NgAgo does indeed act as a DNA endonuclease, it does not display significant levels of targeted activity in vitro. Second, structure homology modeling revealed that NgAgo not only shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Phylogenetic analysis showed that this repA domain is a hallmark of halophilic pAgos, which form a unique clade that is distinct from other characterized pAgos. We showed that both repA and conserved PIWI domains are required for DNA cleavage in vitro and in vivo. In vivo, these domains can be programmed with guides to induce targeted double-stranded DNA breaks that reduce survival ~51% when targeted to loci. In the absence of such loci, survival remains unaffected relative to unguided controls. Similarly, this targeted cleavage enhances homologous recombination, or gene-editing, in E. coli by 107%. In summary, our results provide new insight into poorly characterized NgAgo for subsequent gene-editing tool development and shed new light on seemingly contradictory reports. Meanwhile, the methods we develop can be applied to other endonucleases, providing new tools for screening potential programmable endonucleases.

Prokaryotic Argonautes cut DNA without sequence-specific motifs

BIOT 603

Expanding the capabilities of the Cas6 based dynamic high affinity scaffold for spatial and temporal co-localization of proteins

Alexander A. Mitkas1, [email protected], Wilfred Chen2. (1) Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States (2) Chemical Engineering, University of Delaware, Newark, Delaware, United States

In modern biotechnology, platform organisms such as E. coli and S. cerevisae are used produce small chemicals of interest through non-native enzymatic pathways. Traditional approaches for optimizing nonnative pathway productivity involve directed evolution of the key enzymes of the pathway and manipulation of the cell’s native metabolism through deletions or overexpression of enzymes todrive product formation. These approaches require multiple iterations and do not always yield the desired results. An alternative approach involves clustering nonnative enzymes together in the cell in order to maximize metabolic flux through the nonnative pathway. If the clustering is not dynamic though, metabolite imbalances can occur which can prove detrimental to maximizing product titer. To address this issue a dynamic high affinity scaffold to colocalize enzymes dynamically was developed. The scaffold utilizes small RNA sequences and orthogonal Cas6 proteins taken from the CRISPR/Cas Type I systems. The Cas6 proteins bind with high affinity and sequence specificity to short RNA hairpin sequences. By designing the remaining RNA sequences appropriately, assembly of the scaffold through base pair hybridization has been demonstrated. Furthermore, the scaffold can be disassembled through the addition of a short 6-18 nt toehold sequence on one of the hybridized strands. Upon expression of a third RNA strand, the trigger, the scaffold quickly disassociates via toehold mediated strand displacement (TMSD). In previous work, successful scaffold assembly and disassembly via trigger strand expression has been demonstrated using the split luciferase reporter system by correlating increased and decreased luminescence to scaffold assembly and disassembly. Scaffold assembly has been demonstrated to occur only when all correct scaffold components are expressed. So farr scaffold disassembly was only demonstrated using a synthetic trigger. Recently scaffold disassembly using native sRNA sequences as the trigger (RyhB)has been succesfully demonstrated. Furthermore, a turn ON system which relies on the expression of the trigger to assemble the scaffold has also been developed using the split luciferase system. Finally, the toolkit has been applied to increase the product yield of model metabolic pathways. The expanded capabilities of this scaffold toolkit increase the scope of its application and allows users more freedom to design a scaffold that directly fits their needs.