While significant progress has been made in the past decade, the current understanding of protein aggregation and its consequences is still immature. Aggregation of Therapeutic Proteins provides an up-to-date resource on protein aggregation and its consequences, and available methods to control or slow down the aggregation process.

Preface xvii Contributors xxi 1. Fundamental Structures and Behaviors of Proteins 1Jennifer S. Laurence and C. Russell Middaugh 1.1 The Problem of Protein Aggregation 1 1.2 Parallels to Protein Folding 11 1.3 Views of Protein Stability and Aggregation 12 1.4 Models of Aggregation 22 1.5 Models of Protein Folding 29 1.6 Influences of Chemical Alteration on Aggregation 40 1.7 Approaches to Predicting Aggregation 46 1.8 Conclusions 49 References 50 2. Protein Aggregation Pathways, Kinetics, and Thermodynamics 63Yi Li and Christopher J. Roberts 2.1 Introduction 63 2.2 Native and Nonnative Aggregation Pathways 66 2.3 Thermodynamics of Reversible Self-Association 69 2.4 Aggregation Kinetics and Distinguishing Kinetic Pathways 75 2.5 Chemical Modifications 80 2.6 Effects of Cosolvents or Cosolutes 82 Appendix—Derivation of „t32 for van der Waals (vdW) Mixture 94 Acknowledgments 97 References 97 3. Identification and Impact of Aggregation-Prone Regions in Proteins and Therapeutic Monoclonal Antibodies 103Sandeep Kumar, Xiaoling Wang, and Satish K. Singh 3.1 Introduction 103 3.2 Energy Landscapes, Protein Folding, and Aggregation 105 3.3 Prediction of APRs in Proteins and Biotherapeutics 106 3.4 Conclusions and Future Directions 114 Acknowledgments 115 References 115 4. External Factors Affecting Protein Aggregation 119Wei Wang, Ning Li, and Stan Speaker 4.1 Introduction 119 4.2 Protein Aggregation Pathways 120 4.3 Effects of Temperature 132 4.4 Effects of Solution Conditions and Composition on Protein Aggregation 136 4.5 Effects of Processing Steps on Protein Aggregation 164 4.6 Effects of Solid-State Condition and Composition on Protein Aggregation 174 4.7 Summary 177 Acknowledgment 178 References 178 5. Experimental Detection and Characterization of Protein Aggregates 205Vikas K. Sharma and Devendra S. Kalonia 5.1 Introduction 205 5.2 Aggregate Classifi cation 206 5.3 Analytical Tools for the Characterization of Aggregates 212 5.4 Summary 246 References 247 6. Approaches to Control Protein Aggregation during Bulk Production 257Linda O. Narhi, Yijia Jiang, Rohini Deshpande, Sohye Kang, and Joseph Shultz 6.1 Introduction 257 6.2 Candidate Selection 257 6.3 Protein Aggregation and Cell Culture 269 6.4 Protein Aggregation and Purifi cation 271 6.5 Summary 295 References 295 7. Protein Aggregation and Particle Formation: Effects of Formulation, Interfaces, and Drug Product Manufacturing Operations 301Hanns-Christian Mahler, Stefan Fischer, Theodore W. Randolph, and John F. Carpenter 7.1 Introduction 301 7.2 Roles of Conformational and Colloidal Stability in Reducing Rates of Aggregation 302 7.3 Effects of Interfaces on Protein Aggregation 305 7.4 Critical Processing Steps during Drug Product Manufacturing of Biopharmaceuticals 310 7.5 Particles in Parenteral Products and Visible Inspection 316 7.6 Summary and Outlook 324 References 325 8. Approaches to Managing Protein Aggregation in Product Development 333Wei Wang and Nicholas W. Warne 8.1 Introduction 333 8.2 Approaches in Formulation Development 334 8.3 Protection of Proteins in Various Processing Steps 345 8.4 Aggregation Control by Structural Modifi cations 351 8.5 Summary 353 References 354 9. Case Studies Involving Protein Aggregation 367Rahul S. Rajan, Tiansheng Li, and Tsutomu Arakawa 9.1 Introduction 367 9.2 Case Study 1: Aggregation in the Liquid State: The Role of Osmolytes in Stabilizing KGF toward Aggregation 368 9.3 Case Study 2: Aggregation in the Liquid State: Heterogeneity and Non-Linearity in IgG2 Aggregationduring Long-Term Storage 376 9.4 Case Study 3: Aggregation in the Frozen State: The Role of Excipient Crystallization 381 9.5 Case Studies 4 and 5: Aggregation in the Lyophilized State: Role of Residual Moisture and Mechanismsof Excipient Stabilization 385 9.6 Case Study 6: Protein Particulation Due to Nucleation by Foreign Material in Fill/Finish Manufacturing Operations 391 9.7 Overall Discussion 394 Acknowledgments 396 References 396 10. Aggregation and Immunogenicity of Therapeutic Proteins 403Vasco Filipe, Andrea Hawe, Huub Schellekens, and Wim Jiskoot 10.1 Introduction 403 10.2 Immunogenicity of Therapeutic Proteins 404 10.3 Immune Mechanisms Related to Protein Immunogenicity 409 10.4 Aggregates and Immunogenicity 415 10.5 Conclusions 427 References 428 11. Regulatory Perspective on Aggregates as a Product Quality Attribute 435Wendy C. Weinberg, Linan Ha, Susan L. Kirshner, and Daniela I. Verthelyi 11.1 Introduction 435 11.2 An Overview of the Regulatory Process 436 11.3 Product Aggregates and Safety Concerns 438 11.4 The Assessment of Aggregates: Regulatory Approaches to Controlling Product Aggregation 440 11.5 Future Challenges 446 11.6 Summary 447 Acknowledgments 447 Disclaimer 447 References 448 Index 453

Understanding and controlling protein aggregation The growth of therapeutic proteins as candidates in drug development pipelines is progressing at a record speed, outpacing significantly small-molecule drug development. Development of commercial protein drugs has been hampered by a variety of sources for instability, particularly the high tendency of protein molecules to aggregate under a wide range of processing and storage conditions. Aggregation of Therapeutic Proteins provides pharmaceutical scientists with a much-needed overview of the causes, consequences, characterization, and control of the aggregation of therapeutic proteins. Aggregation of Therapeutic Proteins summarizes current understanding and recent progress regarding protein aggregation in the context of biopharmaceutical products. The text: Explains methods to control or slow down the aggregation process—a major challenge for pharmaceutical and biotech companies Helps readers understand and apply principles to more rapid and predictable commercialization of protein drugs Explains and compares analytical methodologies for monitoring and/or characterizing protein aggregation in research and commercial development Covers control, inhibition, and monitoring of aggregation during processing, expression and purification, and product formulation Includes case studies with more detailed discussions to complement the more general presentations in previous chapters The text concludes with a discussion of the state of the science as well as key outstanding questions regarding immunogenicity issues for therapeutic proteins and protein aggregates, along with an overview of the regulatory process and considerations in developing protein drugs, with a focus on protein aggregation and stability issues. No text on the subject can pretend to be comprehensive, but this thorough, authoritative examination will facilitate and stimulate new and continued investigations into the principles of protein aggregation and the application of those principles to more rapid and predictable commercialization of protein drugs and effective protection of human lives in the future.