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 xxiii

List of Contributors xxvii

1 Downstream Processing of Monoclonal Antibodies: Current Practices and Future Opportunities 1
Brian Kelley

1.1 Introduction 1

1.2 A Brief History of Current Good Manufacturing Process mAb and Intravenous Immunoglobulin Purification 2

1.3 Current Approaches in Purification Process Development: Impact of Platform Processes 4

1.4 Typical Unit Operations and Processing Alternatives 7

1.5 VLS Processes: Ton‐Scale Production and Beyond 10

1.6 Process Validation 12

1.7 Product Life Cycle Management 13

1.8 Future Opportunities 16

1.9 Conclusions 18

Acknowledgments 19

References 19

2 The Development of Antibody Purification Technologies 23
John Curling

2.1 Introduction 23

2.2 Purification of Antibodies by Chromatography Before Protein A 25

2.3 Antibody Purification After 1975 28

2.4 Additional Technologies for Antibody Purification 31

2.5 Purification of mAbs Approved in North America and Europe 34

2.6 Current Antibody Process Technology Developments 40

Acknowledgments 45

References 46

3 Harvest and Recovery of Monoclonal Antibodies: Cell Removal and Clarification 55
Abhinav A. Shukla and Eric Suda

3.1 Introduction 55

3.2 Centrifugation 59

3.3 Microfiltration 62

3.4 Depth Filtration 67

3.5 Flocculation 70

3.6 Absolute Filtration 71

3.7 Expanded Bed Adsorption Chromatography 73

3.8 Harvesting in Single‐Use Manufacturing 74

3.9 Comparison of Harvest and Clarification Unit Operations 74

References 76

4 Next‐Generation Clarification Technologies for the Downstream Processing of Antibodies 81
Nripen Singh and Srinivas Chollangi

4.1 Introduction 81

4.2 Impurity Profiles in Cell Cultures 83

4.3 Precipitation 84

4.4 Affinity Precipitation 89

4.5 Flocculation 90

4.6 Toxicity of Flocculants and Precipitants and Their Residual Clearance 96

4.7 Depth Filtration 97

4.8 Considerations for the Implementation of New Clarification Technologies 102

4.9 Conclusions and Future Perspectives 103

Acknowledgments 104

References 104

5 Protein A‐Based Affinity Chromatography 113
Suresh Vunnum, Ganesh Vedantham and Brian Hubbard

5.1 Introduction 113

5.2 Properties of Protein A and Commercially Available Protein A Resins 114

5.3 Protein A Chromatography Step Development 118

5.4 Additional Considerations During Development and Scale‐Up 123

5.5 Virus Removal/Inactivation 127

5.6 Validation and Robustness 128

5.7 Conclusions 129

Acknowledgment 130

References 130

6 Purification of Human Monoclonal Antibodies: Non‐Protein A Strategies 135
Alahari Arunakumari and Jue Wang

6.1 Introduction 135

6.2 Integrated Process Design for Human Monoclonal Antibody Production 136

6.3 Purification Process Designs for HuMabs 136

6.4 Conclusions 149

Acknowledgments 151

References 152

7 Hydrophobic Interaction Chromatography for the Purification of Antibodies 155
Judith Vajda and Egbert Muller

7.1 Introduction 155

7.2 HIC With mAbs 156

7.3 HIC with Membrane Adsorbers 173

7.4 Future Perspectives 174

References 175

8 Purification of Monoclonal Antibodies by Mixed‐Mode Chromatography 181
Pete Gagnon

8.1 Introduction 181

8.2 A Brief History 182

8.3 Prerequisites for Industrial Implementation 183

8.4 Mechanisms, Screening, and Method Development 185

8.5 Capture Applications 192

8.6 Polishing Applications 193

8.7 Sequential Capture/Polishing Applications 193

8.8 Future Prospects 193

Acknowledgments 194

References 194

9 Advances in Technology and Process Development for Industrial‐Scale Monoclonal Antibody Purification 199
Nuno Fontes and Robert Van Reis

9.1 Introduction 199

9.2 Affinity Purification Platform 200

9.3 Advances in the Purification of mAbs by CEX Chromatography 201

9.4 High‐Performance Tangential Flow Filtration 209

9.5 A New Nonaffinity Platform 211

References 213

10 Alternatives to Packed‐Bed Chromatography for Antibody Extraction and Purification 215
Jorg Thommes, Richard M. Twyman and Uwe Gottschalk

10.1 Introduction 215

10.2 Increasing the Selectivity of Harvest Procedures: Flocculation and Filter Aids 216

10.3 Solutions for Antibody Extraction, Concentration, and Purification 218

10.4 Antibody Purification and Formulation Without Chromatography 220

10.5 Membrane Adsorbers 223

10.6 Conclusions 225

References 226

11 Process‐Scale Precipitation of Impurities in Mammalian Cell Culture Broth 233
Judy Glynn

11.1 Introduction 233

11.2 Precipitation of DNA and Protein—Other Applications 235

11.3 A Comprehensive Evaluation of Precipitants for the Removal of Impurities 236

11.4 Industrial‐Scale Precipitation 241

11.5 Cost of Goods Comparison 243

11.6 Summary 244

Acknowledgments 244

References 244

12 Charged Ultrafiltration and Microfiltration Membranes for Antibody Purification 247
Mark R. Etzel and Abhiram Arunkumar

12.1 Introduction 247

12.2 Charged UF Membranes 248

12.3 Concentration Polarization and Permeate Flux 248

12.4 Stagnant Film Model 249

12.5 Sieving Coefficient 250

12.6 Mass Transfer Coefficient 251

12.7 Mass Balance Models 251

12.8 Scale‐Up Strategies and the Constant Wall Concentration (Cw) Approach 253

12.9 Membrane Cascades 255

12.10 Protein Fractionation Using Charged UF Membranes 256

12.11 Case Study 257

12.12 Charged MF Membranes 259

12.13 Virus Clearance 260

12.14 Salt Tolerance 261

12.15 Conclusions 264

Acknowledgments 264

References 264

13 Disposable Prepacked‐Bed Chromatography for Downstream Purification: Form, Fit, Function, and Industry Adoption 269
Stephen K. Tingley

13.1 Introduction 269

13.2 Development‐Scale Prepacked Column Applications 271

13.3 Process‐Scale Prepacked Column Applications 275

13.4 Basic Technical Datasets 278

13.5 Independent Industry Assessments of “Fit for Purpose” 285

13.6 Case Study 1: Cation‐Exchange Polishing Chromatography 285

13.7 Case Study 2: Prepacked Columns for Pilot‐/Large‐Scale Bioprocessing 287

13.8 Prepacked Columns—Fit 292

13.9 The Economics of Prepacked Column Technologies 295

13.10 The Implementation of Disposable Prepacked Columns 297

13.11 Conclusions 300

References 301

14 Integrated Polishing Steps for Monoclonal Antibody Purification 303
Sanchayita Ghose, Mi Jin, Jia Liu, John Hickey and Steven Lee

14.1 Introduction 303

14.2 Polishing Steps for Antibody Purification 304

14.3 Integration of Polishing Steps 316

14.4 Conclusions 320

Acknowledgment 320

References 320

15 Orthogonal Virus Clearance Applications in Monoclonal Antibody Production 325
Joe X. Zhou

15.1 Introduction 325

15.2 Model Viruses and Virus Assays 326

15.3 Virus Clearance Strategies at Different Development Stages 328

15.4 Orthogonal Virus Clearance During mAb Production 328

15.5 Conclusions and Future Perspectives 338

Acknowledgments 339

References 339

16 Development of a Platform Process for the Purification of Therapeutic Monoclonal Antibodies 343
Yuling Li, Min Zhu, Haibin Luo and Justin R. Weaver

16.1 Introduction 343

16.2 Chromatography Steps in the Platform Process 345

16.3 Virus Inactivation 352

16.4 UF/DF Platform Considerations 352

16.5 Platform Development: Virus Filtration and Bulk Fill 354

16.6 Addressing Future Challenges in Downstream Processing 356

16.7 Representative Platform Processes 356

16.8 Developing a Virus Clearance Database Using a Platform Process 359

16.9 Summary 361

References 361

17 The Evolution of Platform Technologies for the Downstream Processing of Antibodies 365
Lee Allen

17.1 Introduction 365

17.2 The Definition of a Platform Purification Process 366

17.3 The Dominant Process Design 367

17.4 The Evolution of Unit Operations 372

17.5 Adapting the Platform Process for Product‐Specific Issues 382

17.6 Future Perspectives—Future Evolutionary Pathways 382

17.7 Concluding Remarks 383

Acknowledgments 384

References 384

18 Countercurrent Chromatography for the Purification of Monoclonal Antibodies, Bispecific Antibodies, and Antibody–Drug Conjugates 391
Thomas Muller‐Spath and Massimo Morbidelli

18.1 Introduction 391

18.2 Chromatography to Reduce Product Heterogeneity 392

18.3 Definition of Performance Parameters 394

18.4 Gradient Chromatography for Biomolecules 394

18.5 Continuous and Countercurrent Chromatography 395

18.6 Multicolumn Countercurrent Solvent Gradient Purification 397

18.7 Scalability of Multicolumn Countercurrent Chromatography 403

18.8 Online Process Monitoring for Multicolumn Countercurrent Chromatography 404

18.9 Outlook 405

References 405

19 The Evolution of Continuous Chromatography: From Bulk Chemicals to Biopharma 409
Marc Bisschops

19.1 Introduction 409

19.2 Continuous Chromatography in Traditional Process Industries 410

19.3 Continuous Chromatography in the Biopharmaceutical Industry 413

19.4 Advantages of Continuous Chromatography 420

19.5 Implementation Aspects of Continuous Chromatography 422

19.6 Regulatory Aspects 424

19.7 Conclusions 426

References 427

20 Accelerated Seamless Antibody Purification: Simplicity is Key 431
Benoit Mothes

20.1 Introduction 431

20.2 Accelerated Seamless Antibody Purification 432

20.3 Advantages of the ASAP Process 437

20.4 Scaling Up the ASAP Process 438

20.5 New Perspectives 440

20.6 Conclusion 442

Acknowledgments 442

Suggested Reading 443

21 Process Economic Drivers in Industrial Monoclonal Antibody Manufacture 445
Suzanne S. Farid

21.1 Introduction 445

21.2 Challenges When Striving for the Cost‐Effective Manufacture of mAbs 446

21.3 Cost Definitions and Benchmark Values 448

21.4 Economies of Scale 450

21.5 Overall Process Economic Drivers 453

21.6 DSP Drivers At High Titers 457

21.7 Process Economic Trade‐Offs for Downstream Process Bottlenecks 459

21.8 Summary and Outlook 461

References 462

22 Design and Optimization of Manufacturing 467
Andrew Sinclair

22.1 Introduction 467

22.2 Process Design and Optimization 468

22.3 Modeling Approaches 470

22.4 Process Modeling in Practice 481

22.5 Impact of the Process on the Facility 491

Acknowledgments 492

References 492

23 Smart Design for an Efficient Facility With a Validated Disposable System 495
Joe X. Zhou, Jason Li, Michael Cui and Haojun Chen

23.1 Design and Optimization of a Manufacturing Facility 495

23.2 Validation of a Disposable System 507

23.3 Conclusion 512

Acknowledgments 512

References 512

24 High‐Throughput Screening and Modeling Technologies for Process Development in Antibody Purification 515
Tobias Hahn, Thiemo Huuk and Jurgen Hubbuch

24.1 Introduction 515

24.2 Adsorption Isotherms 516

24.3 Batch Chromatography 519

24.4 Column Chromatography 524

References 532

25 Downstream Processing of Monoclonal Antibody Fragments 537
Mariangela Spitali

25.1 Introduction 537

25.2 Production of Antibody Fragments for Therapeutic Use 538

25.3 Downstream Processing 539

25.4 Improving the Pharmacological Characteristics of Antibody Fragments 552

25.5 Conclusions 553

Acknowledgments 555

References 555

26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody–Drug Conjugates 559
Abhinav A. Shukla and Carnley L. Norman

26.1 Introduction 559

26.2 Biochemical Properties 562

26.3 Purification From Mammalian Expression Systems 576

26.4 Purification From Microbial Production Systems 585

26.5 Future Innovations 587

Acknowledgment 589

References 589

27 Manufacturing Concepts for Antibody–Drug Conjugates 595
Thomas Rohrer

27.1 Introduction 595

27.2 Targeting Components 596

27.3 Cytotoxic Drugs 600

27.4 Chemically Labile Linkers 602

27.5 General Process Overview 602

27.6 Facility Design and Supporting Technology 604

27.7 Single‐Use Equipment 607

27.8 Manufacturing ADCs 608

27.9 Analytical Support for ADC Manufacturing 609

27.10 Raw Materials Supply Chain 611

27.11 Conclusion 611

Acknowledgments 613

References 613

28 Purification of IgM and IgA 615
Charlotte Cabanne and Xavier Santarelli

28.1 Introduction 615

28.2 Purification of IgM 616

28.3 Purification of IgA 621

28.4 Conclusion 623

Acknowledgments 623

References 623

29 Purification of Monoclonal Antibodies From Plants 631
Zivko L. Nikolov, Jeffrey T. Regan, Lynn F. Dickey and Susan L. Woodard

29.1 Introduction 631

29.2 Antibody Production in Plants 632

29.3 Downstream Processing of Antibodies Produced in Plants 636

29.4 Purification of Plant‐Derived Antibodies Using Protein A Resins 641

29.5 Purification of Plant‐Derived Antibodies Using Non‐Protein A Media 642

29.6 Polishing Steps 643

29.7 Conclusions 645

Acknowledgment 645

References 645

30 Very‐Large‐Scale Production of Monoclonal Antibodies in Plants 655
Johannes F. Buyel, Richard M. Twyman and Rainer Fischer

30.1 Introduction 655

30.2 Process Schemes for mAb Production in Plants 656

30.3 Scalable Process Models 661

30.4 Process Adaptation for VLS Requirements 663

30.5 Translation into VLS Applications 666

References 667

31 Trends in Formulation and Drug Delivery for Antibodies 673
Hanns‐Christian Mahler and Roman Mathas

31.1 Introduction 673

31.2 Degradation Pathways 674

31.3 Physical Instability 674

31.4 Chemical Instability 676

31.5 How to Achieve Product Stability 678

31.6 Developability: Molecule Selection and Elimination of Degradation Hotspots 679

31.7 Stabilizing an Antibody in a Liquid Formulation 679

31.8 Stabilizing an Antibody by Drying 681

31.9 Choice of Adequate Primary Packaging 682

31.10 Minimizing Stress During Drug Product Processing 683

31.11 Implementation of a Formulation Strategy 685

31.12 Hot Topics 685

31.13 Summary 689

References 690

32 Antibody Purification: Drivers of Change 699
Narahari Pujar, Duncan Low and Rhona O’Leary

32.1 Introduction 699

32.2 The Changing Regulatory Environment—Pharmaceutical Manufacturing for the 21st Century 701

32.3 Technology Drivers—Advances and Innovations 707

32.4 Economic Drivers 708

32.5 Conclusions 711

Acknowledgment 712

References 713

Index 717