About the Editors xvii List of Contributors xix Preface xxv 1 Introduction 1 Alissa D. Guarnaccia, Wendy Sandoval, and Jennie R. Lill Exploring a Diversity of Biotherapeutic Approaches 1 Delivering Diverse Biotherapeutics to Expand Patient Treatment 2 Unlocking Innovation: The Role of Biophysical Techniques in Drug Discovery and Development 2 Increasing Throughput: The Role of High-Quality Analytical Assays in Characterizing Biotherapeutics 3 The Next Frontier: Additional Bioanalytical Tools and Targets on the Horizon 3 Artificial Intelligence and Machine Learning: A Transformative Shift for Computational Tools 4 A Bioanalytical Call to Action! 4 References 5 2 Analytical Characterization of Bispecific Antibodies and Bispecific Molecules 7 Michael Dillon, T. Noelle Lombana, and Christoph Spiess Introduction 7 Bispecific Antibodies and Alternative Scaffolds with Tethered Domains 9 Identifying the Best Parental Antibody Pairs to Combine in a BsAb 13 Alternatives to Bispecific Antibodies: Antibody Mixtures 14 Characterization of the Bispecific Molecule 14 Characterization by Mass Spectrometry Methods 15 Conclusions 20 Abbreviations 20 References 21 3 Analytical Challenges in Analyzing IgM Biotherapeutics 29 Amber D. Rolland and Albert J.R. Heck Introduction 29 Characterization of Biotherapeutic mAbs 31 IgM Sequence and Structure 31 IgM Assembly Factors 35 IgM Glycosylation Patterns 38 IgM Disulfide Bond Networks 44 Conclusions 47 Acknowledgments 48 References 48 4 Mass Spectrometric Characterization of Therapeutic Recombinant Proteins 55 Alissa D. Guarnaccia, Corey E.

Bakalarski, Wendy Sandoval, and Jennie R. Lill Introduction 55 Instrumentation 55 Software for the Analysis of Intact Molecular Weight Measurements 60 Emerging Mass Spectrometric Approaches for Characterizing Molecular Heterogeneity in Biotherapeutics 61 Tandem Mass Spectrometric Characterization of Biomolecules 61 Data Acquisition Methods 63 Quantitative Mass Spectrometric Methods 63 Computational Analysis of Tandem Mass Spectra 64 Conclusions and Perspectives 65 Abbreviations 65 References 66 5 Therapeutic Proteins and Hyphenated Methods for Characterization 71 Dietmar Reusch and Markus Haberger Introduction 71 Methods 71 Considerations 81 Quality Control Feasibility 81 Conclusions 82 References 82 6 Methods for Characterization and Optimization of Peptide Therapeutics: Bioanalysis and Biotransformation 85 Phillip Chu, Suk-Joon Hyung, Robert S. Jones, S. Cyrus Khojasteh, Dennis H. Leung, Bin Ma, Huy Nguyen, Emile Plise, Ola M. Saad, John C. Tran, Qinying Yu, and Xing Zhang Introduction 85 Acknowledgment 104 References 104 7 Structural Characterization of Biotherapeutics and New Modalities 109 Paola Di Lello and Patrick Lupardus Introduction 109 Antigens, Epitopes, and Paratopes 109 Recombinant Antigen Generation 116 N-Linked Glycosylation 117 Manipulating N-Linked Glycans on Antigens 118 Antibody Generation for Crystallography 119 Crystallization of Antibody/Antigen Complexes 119 Consideration for New Modalities and New Targets 120 Conclusion 121 Abbreviations 121 References 122 8 Modern Methods for Characterizing the Higher-Order Structure (HOS) of Biotherapeutics 129 Felix Kuhne, Rachel P. Liu, Lingfei Wang, Aaron T.

Wecksler, Manasi Gaikwad, Christine C. Jao, Alberto Estevez, Roshan M. Regy, and Saeed Izadi Protein Higher-Order Structure (HOS) 129 Importance of HOS Characterization in Biotherapeutic Development 131 Hydrogen/Deuterium Exchange Mass Spectrometry (H/DX-MS) 134 Hydroxyl Radical Protein Footprinting Mass Spectrometry (HRPF-MS) 137 Covalent Labeling Mass Spectrometry (CL-MS) 139 X-Ray Crystallography 141 Cryogenic Electron Microscopy (Cryo-EM) 145 Multiscale in-silico Modeling of Biotherapeutics 149 Conclusion 153 References 153 9 Target Binding Assays for Biotherapeutics 161 Eric Janezic and Xiangdan Wang Introduction 161 Assays to Determine Affinity of Biotherapeutics 161 Cell-Based Affinity assays 166 Conclusions 171 References 172 10 Functional Assays for Screening Protein Biotherapeutics 175 Maureen Beresini and Kelly Loyet Introduction 175 Development of a Screening Cascade 175 Biochemical Assays for Cellular Receptor-Binding or Extracellular Targets 180 Cellular Assays for a Cell Signaling Readout 181 Fundamentals of Assay Development, Validation, and Implementation 187 Concluding Remarks 192 References 193 11 Immunogenicity Profiling of Biotherapeutics (Wiley Book - Bioanalytical Characterization of New Therapeutic Modalities) 197 Qui T. Phung, M. Violet Lee, Ola Saad, Sivan Cohen, Yinyin Li, Mercedesz Balasz, Jieming Chen, Zhaojun Yin, Zhenru Zhou, and Jennie R. Lill Introduction 197 The Anti-drug Antibody Response 199 Clinical Monitoring of ADAs 200 ADA Assay Development Challenges and Considerations for New Modalities 201 Integrating Immunogenicity Assessments, Harmonization, and Incorporation of Clinically Relevant Information into Drug Product Labeling 201 The Clinical Impact of an ADA 201 Assays Employed to Assess Immunogenicity Risk 202 In Silico Approaches for Immunogenicity Risk Assessment 202 T-Cell and Other Cellular Assays 205 MHC II Immunopeptidome 209 Data Visualization and Curation 214 Engineering Best Practices Post-immunogenicity Hotspot Data Elucidation 214 Conclusion 215 Abbreviations 215 References 216 12 High-Throughput Biophysical Assays for Developability Assessment 223 Emma Pelegri-O''Day and Nithya Srinivasan Introduction 223 High-Throughput Intact Mass Spectrometry 223 Aggregation 225 Size Variants 226 Hydrophobicity 227 Charge Variants 227 Viscosity 229 Conformational Stability 230 Chemical Stability Assessment 231 Leveraging AI/ML for Molecule Assessment 233 Future of Developability Assessments 234 References 235 13 Establishing Analytical Methods for RNA-Based Therapeutics Characterization 243 Julien Camperi, Emily Freund, Carolina Galan, and Axel Guilbaud Introduction to RNA-Based Therapeutics 243 Applications of RNA-Based Drugs 243 Quality Attributes and Techniques for the Characterization of mRNA 245 RNA Integrity and Purity 247 mRNA Quality Features 250 Functionality Testing 252 Conclusion 255 References 256 14 Analytical Methods Shaping Cell Therapy Characterization 261 Amritha Lewis, Sadhu Sudeshna, Melmon Rebecca, Shaik Rahamthulla, Tutuncuoglu Egemen, and Gilbert Amy Introduction 261 Fundamentals of T-Cell Immunology in Cell Therapy 262 Measuring Immune Receptor Expression 262 Functional Cell-Based Assays for In Vitro Characterization 264 Cell Phenotyping 265 Immune Monitoring: BCR and TCR Sequencing 266 Conclusions 267 Acknowledgment 267 References 267 15 Characterizing Nucleic Acid-, Gene-, and Cell-Based Therapeutics with Sequencing Technologies 271 Hayley M. Bennett, Ashley Byrne, William Stephenson, and Zora Modrusan Introduction 271 DNA Sequencing 271 Nucleic-Acid-Based Therapeutics 274 Gene-Based Therapies 277 Cell-Based Therapies 279 Immune Repertoire Sequencing Facilitates Therapeutic Advances 280 Conclusions and Future Perspectives 282 Abbreviations 282 References 283 16 Mass Spectrometry-Based Methods to Investigate Protein-Protein Interactions in Cells and Their Application in Drug Discovery 289 Tess Branon Introduction 289 Affinity Purification Mass Spectrometry (AP-MS) 290 Cross-Linking Mass Spectrometry (XL-MS) 293 Proximity Labeling Mass Spectrometry (PL-MS) 296 Concluding Remarks and Other Interactomic Technologies 302 Abbreviations 307 References 308 17 Receptomics and Extracellular Interactomics for Biotherapeutic Characterization 321 Naincy R. Chandan, Abel Ferrel, and Shengya Cao Introduction 321 Receptomic and Extracellular Interactomics Technology Overview 322 Natural Products and Peptides 325 Antibodies and Related Proteins 326 Lipid Nanoparticles (LNPs), Extracellular Vesicles (EVs), Virus-Like Particles (VLPs) and Viruses 331 Cell Therapies 332 Summary 334 Abbreviations 334 References 335 18 Membrane Proteins: Challenging Biotherapeutic Targets 341 Hiruni S.

Jayasekera, Farhana Afrin Mohona, and Michael T. Marty Membrane Proteins: Challenging Biotherapeutic Targets Families of Membrane Proteins 341 Membrane Protein Solubilization Methods 344 MS Analysis of Membrane Proteins 349 Conclusion 353 Acknowledgments 353 References 353 19 Chemoproteomics Approaches for Diverse Modalities 365 H. Christian Eberl, Sascha Knecht, Markus A. Queisser, and Marcus Bantscheff Introduction 3.