DOCSLIB.ORG

Mab) Purification by Counter Current Chromatography (CCC

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mab) Purification by Counter Current Chromatography (CCC Monoclonal antibody (mAb) purification by Counter Current Chromatography (CCC) A thesis submitted to Brunel University for the degree of Doctor of Philosophy by Samantha Fernando Institute for Bioengineering, Brunel University, London December 2011 Abstract Counter current chromatography (CCC) is a form of liquid liquid chromatography, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. In this thesis, its application has been successfully extended to the rapid, scalable purification of monoclonal antibodies (mAb) from mammalian cell culture, using aqueous two-phase systems (ATPS) of inorganic salts and polymer. A polyethylene glycol (PEG) and sodium citrate system was found to be the most appropriate by robotic phase system selection. The search for an economical alternative to protein A HPLC is a substantial bioprocessing concern; in this work CCC has been investigated. Initial studies showed that unpredictably, despite separation from impurities being achieved, some loss in the IgG‘s ability to bind to Protein A was seen, as confirmed by Protein A BiaCore analysis. CCC machines were seen to adversely affect IgG functionality. This led to a systematic investigation of the effect of CCC phase mixing on IgG functionality in a number of different CCC instruments, allowing direct comparisons of modes of CCC (hydrodynamic and hydrostatic CCC) and their associated mixing (wave- like and cascade, respectively). The varying g forces produced within the CCC column were determined using a recently developed model to calculate g force range. The effect of interfacial tension was also studied using a custom built ‗g‘ shaker. The optimum CCC mode was identified to be the non synchronous CCC, operated in a hydrodynamic mode but allowing bobbin to rotor speed (Pr ratio) to be controlled independently. In a normal synchronous J type centrifuge a Pr of 1 is fixed, this is where the bobbin and rotor speed are identical I.e. one bobbin rotation (where mixing occurs) to one rotor revolution (where settling occurs). Constraints were seen with this 1:1 ratio and the separation of mAb using ATPS. This work has shown with the use of the non synchronous CCC at a Pr of 0.33, mixing is reduced and rotor rotations increased. Consequently the associated g force range is decreased. Furthermore, by the extension of settling time, the clear separation of the mAb from impurities has been achieved with retention of biological activity. This thesis demonstrates the importance of settling time for ATPS in phase separation and documents the fundamental requirements for the successful separation of biologics. Purified non synchronous CCC samples have additionally undergone rigorous quality control testing at Lonza Biologics by their purification 1 scientists. This work has ultimately showed that with optimisation, the non synchronous CCC can be used to produce biological samples that are of industry standard. 2 Table of contents 1) Introduction and literature review 10 1.1.1 Monoclonal antibody (mAb) discovery 11 1.1.2 mAb cell culture production 12 1.1.3 mAb structure 14 1.1.4 Monoclonal antibody market 15 1.1.5 Modes of action 17 1.1.6 Upstream/downstream processing of mAbs 18 1.1.7 Protein A: Structure 19 1.1.8 Protein A chromatography 20 1.1.9 Protein A purification issues 21 1.2 Chromatographic alterative to Protein A 22 1.2.1 Cation exchange chromatography 22 1.2.2 Hydrophobic interaction chromatography 24 1.2.3 Affinity chromatography 25 1.3 Non chromatographic alternatives 27 1.3.1 Bulk separations 28 1.3.2 Field based separations 29 1.3.3 Absorptive separation 29 1.4 Aqueous two phase system (ATPS) extraction and Counter 30 current chromatography (CCC) 1.4.1 Monoclonal antibody purification by ATPS extraction 35 1.4.2 CCC machines for separation of proteins 42 1.4.3 CCC VS Protein A affinity chromatography 44 1.4.4 CCC column design for protein separation 44 1.5 Aims and objectives 57 2) Robotic solvent system selection 58 2.1 Summary 59 2.2 Introduction 60 2.3 Method and materials 61 2.3.1 Creation of solvent system table: obtained from Lukasz Grudzien 61 2.3.2 Preparation of solvents for the robot 64 2.3.3 Sample used 65 2.3.4 Stock solution preparation 65 2.3.5 Robot programming 66 2.3.6 Initial run with model proteins 67 2.3.7 Changes made to robotic handling due to the nature of the ATPSs 68 2.4 Results and discussion 69 2.4.1 Investigation of differing protein assays 69 2.4.2 Development of Protein A HPLC analytical method for two phase 76 samples 2.4.3 Robotic run using crude Cell Culture Supernatant (CCS), and 81 result analysis by developed protein A HPLC 2.5 Conclusions 89 3) Separation of monoclonal antibody from cell culture supernatant (CCS) using Centrifugal partitioning chromatography (CPC) 90 3.1 Summary 91 3.2 Introduction 92 3.3 Method and materials 94 3 3.3.1 Preparation of phase system 94 3.3.2 Sample preparation 94 3.3.3 CCC operation 95 3.4 Results and discussion 96 3.4.1 mAb separation from crude Cell Culture Supernatant (CCS) using CPC: CPC run 3-1 96 3.4.2 SDS PAGE results 99 3.4.3 Investigation of the formation Protein A non binding IgG 102 3.4.4 Study into specie interconversion 104 3.4.5 Sample loading capacity: CPC run 3-2 106 3.4.6 Stationary phase retention and stripping 107 3.4.7 Prediction of retention time 109 3.4.7 Precipitation issues associated with the ammonium sulphate system 109 3.4.8 Comparison of work with the Aires Barros group (Lisbon, Portugal): PEG 1000 14%/14% potassium phosphate system. 110 3.4.9 PEG 1000 17.5%/17.5% sodium citrate system vs. PEG 335011%/11% ammonium sulphate system 113 3.4.10 CPC run with the PEG 1000 17.5%/17.5% sodium citrate system 117 3.5 Conclusion 122 4) Separation of monoclonal antibody from cell culture supernatant (CCS) using toroidal coil CCC (TC CCC) centrifuge 123 4.1 Summary 124 4.2 Introduction 125 4.3 Method and materials 126 4.3.1 Preparation of phase system 126 4.3.2 TC CCC operation 126 4.4 Results and discussion 127 4.4.1 PEG 1000 17.5%/17.5% sodium citrate run on the TC CCC: Run 4-1 127 4.4.2 SDS PAGE results: silver stain 128 4.4.3 Determination of purity: host cell ELISA and concentration by stirred cell 130 4.4.4 Effect of flow rate, peak width and spin speed 133 4.4.5 Theoretical predictability: CCC2 modelling programme 135 4.4.6 Overview 136 4.5 Conclusions 138 5) Critical look at IgG functionality 140 5.1 Summary 141 5.2 Introduction 142 5.3 Method and materials 142 5.3.1 Aim of using Biacore from this work 142 5.3.2 Biacore theory: Surface Plasmon resonance 142 5.4 Results and discussion 143 5.4.1 Effect of phase system incubation on IgG functionality 145 5.4.2 Investigation of HPLC column saturation 148 5.4.3 Biacore analysis 149 5.4.4 Investigation of shaking vigour using a bench top centrifuge 154 5.4.5 Investigation of shaking vigour at one ‗g‘ using a custom built shaker 155 5.5 Conclusions 162 4 6) Separation of monoclonal antibodies using J-type CCC centrifuges: the need for gentler mixing 164 6.1 Summary 164 6.2 Introduction 165 6.3 Method and materials 166 6.3.1 Sample preparation 166 6.3.2 CCC processing 166 6.4 Results and discussion 167 6.4.1 Effect of rotational speed in IgG functionality 167 6.4.2 Investigation of IgG transit time of functionality 168 6.4.3 Investigation into sample loading capacity 169 6.4.4 Mini CCC separation using CCS 170 6.4.5 Investigation of model proteins 174 6.4.6 Conclusions drawn from the literature 177 6.4.7 Overview of results 178 6.5 Conclusions 182 7) Successful monoclonal antibody separation: Solution as provided by the non synchronous CCC 183 7.1 Summary 184 7.2 Introduction 185 7.3 Method and materials 189 7.4 Results and discussion 189 7.4.1 Investigation of differing Pr ratios using PA purified IgG: effect on recovery 189 7.4.2 Investigation of differing Pr ratios using CCS 191 7.4.3 Investigation of sample loading capacity using CCS 194 7.4.4 Increased fraction collection: effect on purity 196 7.4.5 Throughput: Studies into sample loading capacity 197 7.4.6 Effect of mass balance, purity and IgG recovery on throughput. 202 7.4.7 Investigation of associated g force with Non synchronous processing 204 7.5 Conclusions 207 8) Characterization of non synchronous CCC purified samples using Lonza quality control assays 209 8.1 Summary 210 8.2 Introduction 211 8.3 Method and materials 211 8.3.1 Investigation of sample clean up using PD 10 size exclusion columns 211 8.3.2 Investigation of ultrafiltration columns for sample clean up 213 8.3.3 Triplicate non synchronous CCC runs with sample clean up using a 5 kDa ultrafiltration column. 215 8.3.4 Child‘s assay for the removal of PEG from samples post ultrafiltration 218 8.3.5 QC assays conducted at Lonza by purification scientist Samit Patel 220 8.3.6 Summary of results 226 8.4 Conclusions 228 9) General discussion, conclusions and future work 231 5 Acknowledgements Firstly, I would like to acknowledge all my supervisors for giving me the opportunity to accomplish a personal ambition with the studying of a PhD.
Load more

Recommended publications
  • Protocols and Tips in Protein Purification
    Department of Molecular Biology & Biotechnology Protocols and tips in protein purification or How to purify protein in one day Second edition 2018 2 Contents I. Introduction 7 II. General sequence of protein purification procedures 9 Preparation of equipment and reagents 9 Preparation and use of stock solutions 10 Chromatography system 11 Preparation of chromatographic columns 13 Preparation of crude extract (cell free extract or soluble proteins fraction) 17 Pre chromatographic steps 18 Chromatographic steps 18 Sequence of operations during IEC and HIC 18 Ion exchange chromatography (IEC) 19 Hydrophobic interaction chromatography (HIC) 21 Gel filtration (SEC) 22 Affinity chromatography 24 Purification of His-tagged proteins 25 Purification of GST-tagged proteins 26 Purification of MBP-tagged proteins 26 Low affinity chromatography 26 III. “Common sense” strategy in protein purification 27 General principles and tips in “common sense” strategy 27 Algorithm for development of purification protocol for soluble over expressed protein 29 Brief scheme of purification of soluble protein 36 Timing for refined purification protocol of soluble over -expressed protein 37 DNA-binding proteins 38 IV. Protocols 41 1. Preparation of the stock solutions 41 2. Quick and effective cell disruption and preparation of the cell free extract 42 3. Protamin sulphate (PS) treatment 43 4. Analytical ammonium sulphate cut (AM cut) 43 5. Preparative ammonium sulphate cut 43 6. Precipitation of proteins by ammonium sulphate 44 7. Recovery of protein from the ammonium sulphate precipitate 44 8. Analysis of solubility of expression 45 9. Analysis of expression for low expressed His tagged protein 46 10. Bio-Rad protein assay Sveta’s easy protocol 47 11.
    [Show full text]
  • PROTEIN a CHROMATOGRAPHY – the PROCESS ECONOMICS DRIVER in Mab MANUFACTURING
    PROCESS Application Note PROTEIN A CHROMATOGRAPHY – THE PROCESS ECONOMICS DRIVER IN mAb MANUFACTURING THE OPTIMIZATION OF THE PROTEIN A CAPTURE STEP IN DOWNSTREAM PROCESSING PLATFORMS CAN CONSIDER- ABLY IMPROVE PROCESS EFFICIENCY AND ECONOMICS OF INDUSTRIAL ANTIBODY MANUFACTURING. PARAMETERS LIKE RESIN REUSE AND ITS CAPACITY CONTRIBUTE CONSIDERABLY TO THE PRODUCTION COSTS. THE USE OF A HIGH CAPACITY PROTEIN A RESIN CAN IMPROVE THE PROCESS EFFICIENCY AND ECONOMICS. THIS PAPER PRESENTS THE KEY FEATURES OF A NEW CAUSTIC STABLE PROTEIN A RESIN PROVIDING EXTREMELY HIGH IgG BINDING CAPACITIES. Biopharmaceuticals represent an ever growing important Protein A affinity resins are dominating the Cost of Goods part of the pharmaceutical industry. The market for recom- (COGs) of mAb manufacturing. Bioreactors at the 10.000 L binant proteins exceeded $ 100 billion in 2011 with a scale operating at a titer of about 1g/L typically generate contribution of 45% sales by monoclonal antibodies (mAbs) costs of $ 4-5 million (2). Therefore the Protein A capturing (1). The introduction of the first mAb biosimilars in Europe step is the key driver to improve process economics. Besides raised the competitive pressure in an increasingly crowded the capacity of the resin, life time and cycle numbers market place. The industry faces challenges, such as patent significantly contribute to the production costs in mAb expirations accompanied by approvals of corresponding manufacturing. biosimilars, failures in clinical trials/rejections or the refusal of health insurers to pay for new drugs. Today, the IgG binding capacities of most Protein A resins are in the range of 30-50 g/L, offering significant advantages These challenges force the industry to minimize risk and time- for the processing of high-titer feedstreams when compared to-market and to proceed more cautiously.
    [Show full text]
  • Manufacturing of Agarose-Based Chromatographic Media with Controlled Pore and Particle Size
    MANUFACTURING OF AGAROSE-BASED CHROMATOGRAPHIC MEDIA WITH CONTROLLED PORE AND PARTICLE SIZE by Nicolas Ioannidis A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Chemical Engineering College of Engineering and Physical Sciences The University of Birmingham 2009 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Chromatography remains the most commonly employed method for achieving high resolution separation of large-sized biomolecules, such as plasmid DNA, typically around 150-250 nm in diameter. Currently, fractionation of such entities is performed using stationary phases designed for protein purification, typically employing pore sizes of about 40 nm. This results into a severe underexploitation of the porous structure of the adsorbent as adsorption of plasmid DNA occurs almost exclusively on the outer surface of the adsorbent. In this study, the effect of two processing parameters, the ionic strength of agarose solution and quenching temperature, on the structure of the resulting particles was investigated. Three characterization methods, Atomic Force and cryo-Scanning Electron microscopy, as well as mechanical testing of single particles where used to quantify the effect of these parameters on the pore size/size distribution and mechanical properties of the adsorbent.
    [Show full text]
  • EXPRESSION, PURIFICATION and CRYSTALLIZATION TRIALS of SMALL RUBBER PARTICLE PROTEIN (SRPP) from Hevea Brasiliensis
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repository@USM EXPRESSION, PURIFICATION AND CRYSTALLIZATION TRIALS OF SMALL RUBBER PARTICLE PROTEIN (SRPP) FROM Hevea brasiliensis SARANPAL SINGH A/L SATINDER SINGH UNIVERSITI SAINS MALAYSIA EXPRESSION, PURIFICATION AND CRYSTALLIZATION TRIALS OF SMALL RUBBER PARTICLE PROTEIN (SRPP) FROM Hevea brasiliensis by SARANPAL SINGH A/L SATINDER SINGH Thesis submitted in fulfillment of requirements for the degree of Master of Science February ACKNOWLEDGEMENT I owe my highest gratitude to Professor Dr. K. Sudesh Kumar and Dr. Teh Aik Hong for being my true mentors and for always being available when needed. My gratitude goes to them for their thoughtful insights, motivation, patience, professional rigour, and intellectual contributions. I could not have imagined having better advisors and mentors during the pursuit of my master’s degree. Their meticulous reading and critical comments on my drafts gave me the kind of feedback that always revitalized, encouraged, and propelled me forward with enthusiasm. It is an honor to have work with them. I am indebted and grateful for the encouragement and inspiration shared by my lab mates and post-docs at CCB: Chiam Nyet Cheng, Chung Corrine, Jess Loh Swee Cheng, Sam Ka Kei, Yue Keong Choon, Tengku Yasmin, Sim Pei Fang, Dr. Go Furusawa, Dr. Sheri-Ann Tan, Dr. Suganthi Appalasamy, Dr. Lau Nyok Sean, Dr.Farrukh Jamil, Dr. Abhilash Usharraj, and Dr. Gincy Paily Thottahil. I also appreciate the help of the administrative department of CCB for being helpful: Ms. Tengku Zalina Tengku Ahmad, Cik Nurul Farhana Che Hassan, and Ms.
    [Show full text]
  • WO 2015/094804 Al 25 June 2015 (25.06.2015) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/094804 Al 25 June 2015 (25.06.2015) P O P C T (51) International Patent Classification: (74) Agent: HEMENWAY, Carl; The Dow Chemical Com C13B 50/00 (201 1.01) BOW 61/14 (2006.01) pany, Intellectual Property, P.O. Box 1967, Midland, BOW 61/02 (2006.01) Michigan 48641-1967 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US20 14/069248 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (22) International Filing Date: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, ' December 2014 (09.12.2014) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (26) Publication Language: English MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (30) Priority Data: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 61/917,508 18 December 201 3 (18. 12.2013) US SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants: DOW GLOBAL TECHNOLOGIES LLC [US/US]; 2040 Dow Center, Midland, Michigan 48674 (84) Designated States (unless otherwise indicated, for every (US).
    [Show full text]
  • Differences Between Homogeneous Spermidine Synthases Isolated
    J. Biochem. 96, 1273-1281 (1984) Differences between Homogeneous Spermidine Synthases Isolated from Rat and Pig Liver1 Banri YAMANOHA,* Keijiro SAMEJIMA,*,z Terumi NAKAJIMA,** and Tadashi YASUHARA** *Tokyo Biochemical Research Institute , Takada, Toshima-ku, Tokyo 171, and **Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, Kandasurugadai, Chiyoda-ku, Tokyo 101 Received for publication, May 25, 1984 Spermidine synthase was purified to homogeneity from rat and pig liver by a method modified from a previously reported one using DEAE-Sepharose, S-adenosyl(5•Œ)- 3-thiopropylamine-Sepharose affinity chromatography, Sephacryl S-300 gel filtration and polyacrylamide gel electrophoresis. No apparent difference between the two enzymes was observed in specific activity, molecular weight (74,000), or subunit composition (two subunits). However, significant differences were observed in their pI values, which were 5.16 for the pig enzyme and 5.34 for the rat enzyme, and their peptide maps. Amino acid compositions of the two enzymes were closely related, but differed significantly in some amino acids. In addition, the rat enzyme was more sensitive to inhibition by S-adenosyl-1,8-diamino-3-thiooctane than the pig enzyme. Spermidine synthase [EC 2.5.1.16] catalyzes the On the other hand, a recent report (4) has shown transfer of the propylamine moiety from S-adeno that there were significant differences among sper syl(5•Œ)-3-methylthiopropylamine (decarboxy Ado midine synthases from different bacterial species Met) to putrescine to form spermidine. The en and rat ventral prostate in their responses to two zyme has been purified to homogeneity from potent spermidine synthase inhibitors, S-adenosyl- Escherichia coli (1), rat prostate (2), and bovine 1,8-diamino-3-thiooctane (AdoDATO) (5, 6) and brain (3); each has shown very similar properties dicyclohexylamine (7).
    [Show full text]
  • Affinity Separation 3
    Sepsci*1*TSK*Venkatachala=BG I / AFFINITY SEPARATION 3 AFFINITY SEPARATION K. Jones, Affinity Chromatography Ltd, Freeport, Separation and puriRcation methods for biological Ballsalla, Isle of Man, UK macromolecules vary from the very simple to the esoteric. The type of technique adopted is basically Copyright ^ 2000 Academic Press a function of source, the fragility of the molecule and the purity required. Traditionally, high purity protein Introduction pharmaceuticals have used multistage processing, but this is very inefRcient as measured by the well- Of the collection of separation technologies known documented fact that 50}80% of total production as ‘afRnity’, afRnity chromatography is by far the costs are incurred at the separation/puriRcation stage. most widely used variant. AfRnity chromatography is In contrast, the highly selective indigenous properties becoming increasingly important as the speed of the of the afRnity method offer the alternative of revolution taking place in biotechnology processing very elegant single-step puriRcation strategies. The increases. The concept of an ‘afRnity’ separation re- inherent simplicity and universality of the method has sults from a naturally occurring phenomenon existing already generated a wide range of separation tech- within all biological macromolecules. Each biological nologies, mostly based upon immobilized naturally macromolecule contains a unique set of intermolecu- occurring proteinaceous ligands. By comparing the lar binding forces, existing throughout its internal ‘old’ technologies of ‘natural’ ligands or multistage and external structure. When alignment occurs be- processing with the ‘new’, exempliRed by synthetic tween a speciRc site of these forces in one molecule designed ligands, the most recent advances in af- with the site of a set of forces existing in another Rnity processing can be described.
    [Show full text]
  • Aqueous Two-Phase System (ATPS): an Overview and Advances in Its
    Iqbal et al. Biological Procedures Online (2016) 18:18 DOI 10.1186/s12575-016-0048-8 REVIEW Open Access Aqueous two-phase system (ATPS): an overview and advances in its applications Mujahid Iqbal1, Yanfei Tao1*, Shuyu Xie1, Yufei Zhu1, Dongmei Chen1, Xu Wang1, Lingli Huang1, Dapeng Peng1, Adeel Sattar1, Muhammad Abu Bakr Shabbir2, Hafiz Iftikhar Hussain2, Saeed Ahmed2 and Zonghui Yuan1,2* Abstract Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications. Keywords: Aqueous two-phase system (ATPS), Biomolecule separation, Solvent extraction, Veterinary drug residues History and background hydrophobic groups [5] Interested readers about In 1896, Martinus Willem Beijerinck accidently found aqueous two-phase affinity partitioning (ATPAP) are the ATPS while mixing an aqueous solution of starch referred to an excellent review by Ruiz-Ruiz et al. [6]. and gelatin. However, its real application was discovered Water as the main component of both phases in ATPS by Per-Åke Albertsson.
    [Show full text]
  • Development of Aqueous Two-Phase Separations by Combining High-Throughput Screening and Process Modelling
    Development of aqueous two-phase separations by combining high-throughput screening and process modelling A thesis submitted to University College London for the degree of DOCTOR OF ENGINEERING by Nehal Patel Friday 21st July 2017 The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Gower Street, London, WC1E 6BT, UK 1 I, Nehal Patel, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Separation based on aqueous two-phase extraction (ATPE) is a promising downstream separation technology for the production of biological products. The advantages of using aqueous two-phase systems include but are not limited to easy scalability, ease of continuous operation and a favourable environment for biological compounds. One of the main challenges associated with aqueous two-phase systems is process development. This is in part due to the many factors which influence the separation of biological materials in such systems such as polymer and salt type, pH and charge. The large number of factors to consider makes the development of aqueous two-phase systems challenging due to the need to find a robust and efficient separation in a large experimental space. This work addresses this issue by considering the use of dynamic process models and high-throughput experimentation for the development of aqueous two-phase extraction processes for biological products. The use of a dynamic equilibrium stage process model to simulate aqueous two-phase extraction is considered in Chapter 3. The process model is capable of simulating various modes of operation; and both multi-cycle batch and continuous counter-current modes of operation are considered.
    [Show full text]
  • Protein L—More Than Just an Affinity Ligand
    processes Review Protein L—More Than Just an Affinity Ligand Stefan Kittler 1,2, Mihail Besleaga 1, Julian Ebner 1,2 and Oliver Spadiut 1,* 1 Research Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorfer Strasse 1a, 1060 Vienna, Austria; [email protected] (S.K.); [email protected] (M.B.); [email protected] (J.E.) 2 Alfred Gruber GmbH, Nordstrasse 6, 5301 Eugendorf, Austria * Correspondence: [email protected]; Tel.: +43-1-58801-166473 Abstract: In the past 30 years, highly specific drugs, known as antibodies, have conquered the biopharmaceutical market. In addition to monoclonal antibodies (mAbs), antibody fragments are successfully applied. However, recombinant production faces challenges. Process analytical tools for monitoring and controlling production processes are scarce and time-intensive. In the downstream process (DSP), affinity ligands are established as the primary and most important step, while the application of other methods is challenging. The use of these affinity ligands as monitoring tools would enable a platform technology to monitor process steps in the USP and DSP. In this review, we highlight the current applications of affinity ligands (proteins A, G, and L) and discuss further applications as process analytical tools. Keywords: monoclonal antibodies; antibody fragments; affinity ligands; process analytical technol- ogy; protein A; protein G; protein L Citation: Kittler, S.; Besleaga, M.; Ebner, J.; Spadiut, O. Protein L—More 1. Introduction Than Just an Affinity Ligand. Processes 2021, 9, 874. https:// In 1986, a new type of drug was approved and introduced to the biopharmaceutical doi.org/10.3390/pr9050874 market—the first monoclonal antibody (mAb) with the trade name Orthoclone (OKT3), preventing rejection after kidney transplantation [1].
    [Show full text]
  • Residual on Column Host Cell Protein Analysis During Lifetime Studies Of
    Journal of Chromatography A, 1461 (2016) 70–77 Contents lists available at ScienceDirect Journal of Chromatography A jo urnal homepage: www.elsevier.com/locate/chroma Residual on column host cell protein analysis during lifetime studies of protein A chromatography a b c c b Katherine Lintern , Mili Pathak , C. Mark Smales , Kevin Howland , Anurag Rathore , a,∗ Daniel G. Bracewell a Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom b Department of Chemical Engineering, IIT Delhi, Hauz Khas, New Delhi, Delhi 110016, India c Centre for Industrial Biotechnology, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, United Kingdom a r t i c l e i n f o a b s t r a c t Article history: Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic anti- Received 9 February 2016 body manufacture is well documented. Identification of which residual proteins remain from previous Received in revised form 16 July 2016 cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing Accepted 20 July 2016 process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liq- Available online 21 July 2016 uid chromatography mass spectrometry (LC–MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, Keywords: revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell Protein A affinity chromatography proteins (HCPs) by LC–MS/MS present on the resin is shown to increase 10-fold between 50 and 100 Host cell proteins LC–MS/MS cycles.
    [Show full text]
  • Affinity Chromatography Principles and Methods GE Healthcare GE
    GE Healthcare Affinity Chromatograpy Handbook – Principles and Methods GE, imagination at work and GE Monogram are trademarks of General Electric Company. ÄKTA, ÄKTAexplorer, ÄKTAFLPC, ÄKTAprime, ÄKTApurifier, Biacore, BioDirectory, BioProcess, ECL, ECL Plus, ExcelGel, FPLC, GSTPrep, GSTrap, HisTrap, HiPrep, HiTrap, Hybond, MAbTrap, MabSelect, MicroSpin, Microplex, Multiphor, STREAMLINE, Sepharose, Percoll, PhastSystem, PhastGel, Sephadex, Superdex, and Tricorn are trademarks of GE Healthcare companies. Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US pat 5,284,933 and US pat 5,310,663, including corresponding foreign patents (assigne: Hoffman La Roche, Inc). A license for commercial use of GST gene fusion vectors must be obtained from Chemicon International, Incorprated, 28820 Singel Oak Drive, Temecula, California 92590 USA. The Tricorn column and components are protected by US design patents USD500856, USD506261, USD500555, USD495060 and their equivalents in other countries. All third party trademarks are the property of their respective owners. © 1988–2007 General Electric Company – All rights reserved. Affinity Chromatography First published 1988. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare that supplies them. A copy of these terms and Principles and Methods For local office contact information, conditions is available on request. Contact your local GE Healthcare representative please visit www.gelifesciences.com/contact for the most current information. GE Healthcare Europe GmbH Munzinger Strasse 5 GE Healthcare Bio-Sciences AB D-79111 Freiburg, Germany GE Healthcare UK Limited Björkgatan 30 Amersham Place Little Chalfont 751 84 Uppsala Buckinghamshire, HP7 9NA, UK Sweden GE Healthcare Bio-Sciences Corp.
    [Show full text]