Process Scale Purification of Antibodies

Uwe Gottschalk, PhD,is Chief Technology Officer at Lonza Pharma/Biotech, Switzerland. Previously, he served as Group Vice President at Sartorius Stedim Biotech (2004-2014) and in various development and manufacturing capacities at Bayer Health Care (1991–2004). Dr. Gottschalk received a doctorate in chemistry from the University of Münster (Germany) for work on antibody-drug conjugates at the Cancer Research Campaign Laboratories in Nottingham (UK). He is Head Lecturer at the University of Duisburg-Essen, Germany, and has written extensively in the areas of industrial biotechnology and somatic gene therapy.

• Preface xxiiiList of Contributors xxvii1 Downstream Processing of Monoclonal Antibodies: Current Practices and Future Opportunities 1Brian Kelley1.1 Introduction 11.2 A Brief History of Current Good Manufacturing Process mAb and Intravenous Immunoglobulin Purification 21.3 Current Approaches in Purification Process Development: Impact of Platform Processes 41.4 Typical Unit Operations and Processing Alternatives 71.5 VLS Processes: Ton‐Scale Production and Beyond 101.6 Process Validation 121.7 Product Life Cycle Management 131.8 Future Opportunities 161.9 Conclusions 18Acknowledgments 19References 192 The Development of Antibody Purification Technologies 23John Curling2.1 Introduction 232.2 Purification of Antibodies by Chromatography Before Protein A 252.3 Antibody Purification After 1975 282.4 Additional Technologies for Antibody Purification 312.5 Purification of mAbs Approved in North America and Europe 342.6 Current Antibody Process Technology Developments 40Acknowledgments 45References 463 Harvest and Recovery of Monoclonal Antibodies: Cell Removal and Clarification 55Abhinav A. Shukla and Eric Suda3.1 Introduction 553.2 Centrifugation 593.3 Microfiltration 623.4 Depth Filtration 673.5 Flocculation 703.6 Absolute Filtration 713.7 Expanded Bed Adsorption Chromatography 733.8 Harvesting in Single‐Use Manufacturing 743.9 Comparison of Harvest and Clarification Unit Operations 74References 764 Next‐Generation Clarification Technologies for the Downstream Processing of Antibodies 81Nripen Singh and Srinivas Chollangi4.1 Introduction 814.2 Impurity Profiles in Cell Cultures 834.3 Precipitation 844.4 Affinity Precipitation 894.5 Flocculation 904.6 Toxicity of Flocculants and Precipitants and Their Residual Clearance 964.7 Depth Filtration 974.8 Considerations for the Implementation of New Clarification Technologies 1024.9 Conclusions and Future Perspectives 103Acknowledgments 104References 1045 Protein A‐Based Affinity Chromatography 113Suresh Vunnum, Ganesh Vedantham and Brian Hubbard5.1 Introduction 1135.2 Properties of Protein A and Commercially Available Protein A Resins 1145.3 Protein A Chromatography Step Development 1185.4 Additional Considerations During Development and Scale‐Up 1235.5 Virus Removal/Inactivation 1275.6 Validation and Robustness 1285.7 Conclusions 129Acknowledgment 130References 1306 Purification of Human Monoclonal Antibodies: Non‐Protein A Strategies 135Alahari Arunakumari and Jue Wang6.1 Introduction 1356.2 Integrated Process Design for Human Monoclonal Antibody Production 1366.3 Purification Process Designs for HuMabs 1366.4 Conclusions 149Acknowledgments 151References 1527 Hydrophobic Interaction Chromatography for the Purification of Antibodies 155Judith Vajda and Egbert Muller7.1 Introduction 1557.2 HIC With mAbs 1567.3 HIC with Membrane Adsorbers 1737.4 Future Perspectives 174References 1758 Purification of Monoclonal Antibodies by Mixed‐Mode Chromatography 181Pete Gagnon8.1 Introduction 1818.2 A Brief History 1828.3 Prerequisites for Industrial Implementation 1838.4 Mechanisms, Screening, and Method Development 1858.5 Capture Applications 1928.6 Polishing Applications 1938.7 Sequential Capture/Polishing Applications 1938.8 Future Prospects 193Acknowledgments 194References 1949 Advances in Technology and Process Development for Industrial‐Scale Monoclonal Antibody Purification 199Nuno Fontes and Robert Van Reis9.1 Introduction 1999.2 Affinity Purification Platform 2009.3 Advances in the Purification of mAbs by CEX Chromatography 2019.4 High‐Performance Tangential Flow Filtration 2099.5 A New Nonaffinity Platform 211References 21310 Alternatives to Packed‐Bed Chromatography for Antibody Extraction and Purification 215Jorg Thommes, Richard M. Twyman and Uwe Gottschalk10.1 Introduction 21510.2 Increasing the Selectivity of Harvest Procedures: Flocculation and Filter Aids 21610.3 Solutions for Antibody Extraction, Concentration, and Purification 21810.4 Antibody Purification and Formulation Without Chromatography 22010.5 Membrane Adsorbers 22310.6 Conclusions 225References 22611 Process‐Scale Precipitation of Impurities in Mammalian Cell Culture Broth 233Judy Glynn11.1 Introduction 23311.2 Precipitation of DNA and Protein—Other Applications 23511.3 A Comprehensive Evaluation of Precipitants for the Removal of Impurities 23611.4 Industrial‐Scale Precipitation 24111.5 Cost of Goods Comparison 24311.6 Summary 244Acknowledgments 244References 24412 Charged Ultrafiltration and Microfiltration Membranes for Antibody Purification 247Mark R. Etzel and Abhiram Arunkumar12.1 Introduction 24712.2 Charged UF Membranes 24812.3 Concentration Polarization and Permeate Flux 24812.4 Stagnant Film Model 24912.5 Sieving Coefficient 25012.6 Mass Transfer Coefficient 25112.7 Mass Balance Models 25112.8 Scale‐Up Strategies and the Constant Wall Concentration (Cw) Approach 25312.9 Membrane Cascades 25512.10 Protein Fractionation Using Charged UF Membranes 25612.11 Case Study 25712.12 Charged MF Membranes 25912.13 Virus Clearance 26012.14 Salt Tolerance 26112.15 Conclusions 264Acknowledgments 264References 26413 Disposable Prepacked‐Bed Chromatography for Downstream Purification: Form, Fit, Function, and Industry Adoption 269Stephen K. Tingley13.1 Introduction 26913.2 Development‐Scale Prepacked Column Applications 27113.3 Process‐Scale Prepacked Column Applications 27513.4 Basic Technical Datasets 27813.5 Independent Industry Assessments of “Fit for Purpose” 28513.6 Case Study 1: Cation‐Exchange Polishing Chromatography 28513.7 Case Study 2: Prepacked Columns for Pilot‐/Large‐Scale Bioprocessing 28713.8 Prepacked Columns—Fit 29213.9 The Economics of Prepacked Column Technologies 29513.10 The Implementation of Disposable Prepacked Columns 29713.11 Conclusions 300References 30114 Integrated Polishing Steps for Monoclonal Antibody Purification 303Sanchayita Ghose, Mi Jin, Jia Liu, John Hickey and Steven Lee14.1 Introduction 30314.2 Polishing Steps for Antibody Purification 30414.3 Integration of Polishing Steps 31614.4 Conclusions 320Acknowledgment 320References 32015 Orthogonal Virus Clearance Applications in Monoclonal Antibody Production 325Joe X. Zhou15.1 Introduction 32515.2 Model Viruses and Virus Assays 32615.3 Virus Clearance Strategies at Different Development Stages 32815.4 Orthogonal Virus Clearance During mAb Production 32815.5 Conclusions and Future Perspectives 338Acknowledgments 339References 33916 Development of a Platform Process for the Purification of Therapeutic Monoclonal Antibodies 343Yuling Li, Min Zhu, Haibin Luo and Justin R. Weaver16.1 Introduction 34316.2 Chromatography Steps in the Platform Process 34516.3 Virus Inactivation 35216.4 UF/DF Platform Considerations 35216.5 Platform Development: Virus Filtration and Bulk Fill 35416.6 Addressing Future Challenges in Downstream Processing 35616.7 Representative Platform Processes 35616.8 Developing a Virus Clearance Database Using a Platform Process 35916.9 Summary 361References 36117 The Evolution of Platform Technologies for the Downstream Processing of Antibodies 365Lee Allen17.1 Introduction 36517.2 The Definition of a Platform Purification Process 36617.3 The Dominant Process Design 36717.4 The Evolution of Unit Operations 37217.5 Adapting the Platform Process for Product‐Specific Issues 38217.6 Future Perspectives—Future Evolutionary Pathways 38217.7 Concluding Remarks 383Acknowledgments 384References 38418 Countercurrent Chromatography for the Purification of Monoclonal Antibodies, Bispecific Antibodies, and Antibody–Drug Conjugates 391Thomas Muller‐Spath and Massimo Morbidelli18.1 Introduction 39118.2 Chromatography to Reduce Product Heterogeneity 39218.3 Definition of Performance Parameters 39418.4 Gradient Chromatography for Biomolecules 39418.5 Continuous and Countercurrent Chromatography 39518.6 Multicolumn Countercurrent Solvent Gradient Purification 39718.7 Scalability of Multicolumn Countercurrent Chromatography 40318.8 Online Process Monitoring for Multicolumn Countercurrent Chromatography 40418.9 Outlook 405References 40519 The Evolution of Continuous Chromatography: From Bulk Chemicals to Biopharma 409Marc Bisschops19.1 Introduction 40919.2 Continuous Chromatography in Traditional Process Industries 41019.3 Continuous Chromatography in the Biopharmaceutical Industry 41319.4 Advantages of Continuous Chromatography 42019.5 Implementation Aspects of Continuous Chromatography 42219.6 Regulatory Aspects 42419.7 Conclusions 426References 42720 Accelerated Seamless Antibody Purification: Simplicity is Key 431Benoit Mothes20.1 Introduction 43120.2 Accelerated Seamless Antibody Purification 43220.3 Advantages of the ASAP Process 43720.4 Scaling Up the ASAP Process 43820.5 New Perspectives 44020.6 Conclusion 442Acknowledgments 442Suggested Reading 44321 Process Economic Drivers in Industrial Monoclonal Antibody Manufacture 445Suzanne S. Farid21.1 Introduction 44521.2 Challenges When Striving for the Cost‐Effective Manufacture of mAbs 44621.3 Cost Definitions and Benchmark Values 44821.4 Economies of Scale 45021.5 Overall Process Economic Drivers 45321.6 DSP Drivers At High Titers 45721.7 Process Economic Trade‐Offs for Downstream Process Bottlenecks 45921.8 Summary and Outlook 461References 46222 Design and Optimization of Manufacturing 467Andrew Sinclair22.1 Introduction 46722.2 Process Design and Optimization 46822.3 Modeling Approaches 47022.4 Process Modeling in Practice 48122.5 Impact of the Process on the Facility 491Acknowledgments 492References 49223 Smart Design for an Efficient Facility With a Validated Disposable System 495Joe X. Zhou, Jason Li, Michael Cui and Haojun Chen23.1 Design and Optimization of a Manufacturing Facility 49523.2 Validation of a Disposable System 50723.3 Conclusion 512Acknowledgments 512References 51224 High‐Throughput Screening and Modeling Technologies for Process Development in Antibody Purification 515Tobias Hahn, Thiemo Huuk and Jurgen Hubbuch24.1 Introduction 51524.2 Adsorption Isotherms 51624.3 Batch Chromatography 51924.4 Column Chromatography 524References 53225 Downstream Processing of Monoclonal Antibody Fragments 537Mariangela Spitali25.1 Introduction 53725.2 Production of Antibody Fragments for Therapeutic Use 53825.3 Downstream Processing 53925.4 Improving the Pharmacological Characteristics of Antibody Fragments 55225.5 Conclusions 553Acknowledgments 555References 55526 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody–Drug Conjugates 559Abhinav A. Shukla and Carnley L. Norman26.1 Introduction 55926.2 Biochemical Properties 56226.3 Purification From Mammalian Expression Systems 57626.4 Purification From Microbial Production Systems 58526.5 Future Innovations 587Acknowledgment 589References 58927 Manufacturing Concepts for Antibody–Drug Conjugates 595Thomas Rohrer27.1 Introduction 59527.2 Targeting Components 59627.3 Cytotoxic Drugs 60027.4 Chemically Labile Linkers 60227.5 General Process Overview 60227.6 Facility Design and Supporting Technology 60427.7 Single‐Use Equipment 60727.8 Manufacturing ADCs 60827.9 Analytical Support for ADC Manufacturing 60927.10 Raw Materials Supply Chain 61127.11 Conclusion 611Acknowledgments 613References 61328 Purification of IgM and IgA 615Charlotte Cabanne and Xavier Santarelli28.1 Introduction 61528.2 Purification of IgM 61628.3 Purification of IgA 62128.4 Conclusion 623Acknowledgments 623References 62329 Purification of Monoclonal Antibodies From Plants 631Zivko L. Nikolov, Jeffrey T. Regan, Lynn F. Dickey and Susan L. Woodard29.1 Introduction 63129.2 Antibody Production in Plants 63229.3 Downstream Processing of Antibodies Produced in Plants 63629.4 Purification of Plant‐Derived Antibodies Using Protein A Resins 64129.5 Purification of Plant‐Derived Antibodies Using Non‐Protein A Media 64229.6 Polishing Steps 64329.7 Conclusions 645Acknowledgment 645References 64530 Very‐Large‐Scale Production of Monoclonal Antibodies in Plants 655Johannes F. Buyel, Richard M. Twyman and Rainer Fischer30.1 Introduction 65530.2 Process Schemes for mAb Production in Plants 65630.3 Scalable Process Models 66130.4 Process Adaptation for VLS Requirements 66330.5 Translation into VLS Applications 666References 66731 Trends in Formulation and Drug Delivery for Antibodies 673Hanns‐Christian Mahler and Roman Mathas31.1 Introduction 67331.2 Degradation Pathways 67431.3 Physical Instability 67431.4 Chemical Instability 67631.5 How to Achieve Product Stability 67831.6 Developability: Molecule Selection and Elimination of Degradation Hotspots 67931.7 Stabilizing an Antibody in a Liquid Formulation 67931.8 Stabilizing an Antibody by Drying 68131.9 Choice of Adequate Primary Packaging 68231.10 Minimizing Stress During Drug Product Processing 68331.11 Implementation of a Formulation Strategy 68531.12 Hot Topics 68531.13 Summary 689References 69032 Antibody Purification: Drivers of Change 699Narahari Pujar, Duncan Low and Rhona O’Leary32.1 Introduction 69932.2 The Changing Regulatory Environment—Pharmaceutical Manufacturing for the 21st Century 70132.3 Technology Drivers—Advances and Innovations 70732.4 Economic Drivers 70832.5 Conclusions 711Acknowledgment 712References 713Index 717