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.