Jennie R. Lill, PhD, is the Executive Director of the Proteomic and Genomic Technologies Department at Genentech, where she has worked since 2004. With over 90 peer-reviewed publications and three books, including Analytical Characterization of Biotherapeutics, her work focuses on cutting-edge analytical techniques in drug discovery and development. Wendy Sandoval was the Director of Translational Mass Spectrometry in Research at Genentech, where she had been a pivotal contributor for over 30 years. She has authored more than 100 scientific publications and co-authored two books, including Analytical Characterization of Biotherapeutics. She now runs a plant nursery and botanical garden in Northern California (https://botanicanurseryandgardens.com/) and enjoys playing in dirt.

• About the Editors xviiList of Contributors xixPreface xxv1 Introduction 1Alissa D. Guarnaccia, Wendy Sandoval, and Jennie R. LillExploring a Diversity of Biotherapeutic Approaches 1Delivering Diverse Biotherapeutics to Expand Patient Treatment 2Unlocking Innovation: The Role of Biophysical Techniques in Drug Discovery and Development 2Increasing Throughput: The Role of High-Quality Analytical Assays in Characterizing Biotherapeutics 3The Next Frontier: Additional Bioanalytical Tools and Targets on the Horizon 3Artificial Intelligence and Machine Learning: A Transformative Shift for Computational Tools 4A Bioanalytical Call to Action! 4References 52 Analytical Characterization of Bispecific Antibodies and Bispecific Molecules 7Michael Dillon, T. Noelle Lombana, and Christoph SpiessIntroduction 7Bispecific Antibodies and Alternative Scaffolds with Tethered Domains 9Identifying the Best Parental Antibody Pairs to Combine in a BsAb 13Alternatives to Bispecific Antibodies: Antibody Mixtures 14Characterization of the Bispecific Molecule 14Characterization by Mass Spectrometry Methods 15Conclusions 20Abbreviations 20References 213 Analytical Challenges in Analyzing IgM Biotherapeutics 29Amber D. Rolland and Albert J.R. HeckIntroduction 29Characterization of Biotherapeutic mAbs 31IgM Sequence and Structure 31IgM Assembly Factors 35IgM Glycosylation Patterns 38IgM Disulfide Bond Networks 44Conclusions 47Acknowledgments 48References 484 Mass Spectrometric Characterization of Therapeutic Recombinant Proteins 55Alissa D. Guarnaccia, Corey E. Bakalarski, Wendy Sandoval, and Jennie R. LillIntroduction 55Instrumentation 55Software for the Analysis of Intact Molecular Weight Measurements 60Emerging Mass Spectrometric Approaches for Characterizing Molecular Heterogeneity in Biotherapeutics 61Tandem Mass Spectrometric Characterization of Biomolecules 61Data Acquisition Methods 63Quantitative Mass Spectrometric Methods 63Computational Analysis of Tandem Mass Spectra 64Conclusions and Perspectives 65Abbreviations 65References 665 Therapeutic Proteins and Hyphenated Methods for Characterization 71Dietmar Reusch and Markus HabergerIntroduction 71Methods 71Considerations 81Quality Control Feasibility 81Conclusions 82References 826 Methods for Characterization and Optimization of Peptide Therapeutics: Bioanalysis and Biotransformation 85Phillip 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 ZhangIntroduction 85Acknowledgment 104References 1047 Structural Characterization of Biotherapeutics and New Modalities 109Paola Di Lello and Patrick LupardusIntroduction 109Antigens, Epitopes, and Paratopes 109Recombinant Antigen Generation 116N-Linked Glycosylation 117Manipulating N-Linked Glycans on Antigens 118Antibody Generation for Crystallography 119Crystallization of Antibody/Antigen Complexes 119Consideration for New Modalities and New Targets 120Conclusion 121Abbreviations 121References 1228 Modern Methods for Characterizing the Higher-Order Structure (HOS) of Biotherapeutics 129Felix Kuhne, Rachel P. Liu, Lingfei Wang, Aaron T. Wecksler, Manasi Gaikwad, Christine C. Jao, Alberto Estevez, Roshan M. Regy, and Saeed IzadiProtein Higher-Order Structure (HOS) 129Importance of HOS Characterization in Biotherapeutic Development 131Hydrogen/Deuterium Exchange Mass Spectrometry (H/DX-MS) 134Hydroxyl Radical Protein Footprinting Mass Spectrometry (HRPF-MS) 137Covalent Labeling Mass Spectrometry (CL-MS) 139X-Ray Crystallography 141Cryogenic Electron Microscopy (Cryo-EM) 145Multiscale in-silico Modeling of Biotherapeutics 149Conclusion 153References 1539 Target Binding Assays for Biotherapeutics 161Eric Janezic and Xiangdan WangIntroduction 161Assays to Determine Affinity of Biotherapeutics 161Cell-Based Affinity assays 166Conclusions 171References 17210 Functional Assays for Screening Protein Biotherapeutics 175Maureen Beresini and Kelly LoyetIntroduction 175Development of a Screening Cascade 175Biochemical Assays for Cellular Receptor-Binding or Extracellular Targets 180Cellular Assays for a Cell Signaling Readout 181Fundamentals of Assay Development, Validation, and Implementation 187Concluding Remarks 192References 19311 Immunogenicity Profiling of Biotherapeutics (Wiley Book – Bioanalytical Characterization of New Therapeutic Modalities) 197Qui T. Phung, M. Violet Lee, Ola Saad, Sivan Cohen, Yinyin Li, Mercedesz Balasz, Jieming Chen, Zhaojun Yin, Zhenru Zhou, and Jennie R. LillIntroduction 197The Anti-drug Antibody Response 199Clinical Monitoring of ADAs 200ADA Assay Development Challenges and Considerations for New Modalities 201Integrating Immunogenicity Assessments, Harmonization, and Incorporation of Clinically Relevant Information into Drug Product Labeling 201The Clinical Impact of an ADA 201Assays Employed to Assess Immunogenicity Risk 202In Silico Approaches for Immunogenicity Risk Assessment 202T-Cell and Other Cellular Assays 205MHC II Immunopeptidome 209Data Visualization and Curation 214Engineering Best Practices Post-immunogenicity Hotspot Data Elucidation 214Conclusion 215Abbreviations 215References 21612 High-Throughput Biophysical Assays for Developability Assessment 223Emma Pelegri-O’Day and Nithya SrinivasanIntroduction 223High-Throughput Intact Mass Spectrometry 223Aggregation 225Size Variants 226Hydrophobicity 227Charge Variants 227Viscosity 229Conformational Stability 230Chemical Stability Assessment 231Leveraging AI/ML for Molecule Assessment 233Future of Developability Assessments 234References 23513 Establishing Analytical Methods for RNA-Based Therapeutics Characterization 243Julien Camperi, Emily Freund, Carolina Galan, and Axel GuilbaudIntroduction to RNA-Based Therapeutics 243Applications of RNA-Based Drugs 243Quality Attributes and Techniques for the Characterization of mRNA 245RNA Integrity and Purity 247mRNA Quality Features 250Functionality Testing 252Conclusion 255References 25614 Analytical Methods Shaping Cell Therapy Characterization 261Amritha Lewis, Sadhu Sudeshna, Melmon Rebecca, Shaik Rahamthulla, Tutuncuoglu Egemen, and Gilbert AmyIntroduction 261Fundamentals of T-Cell Immunology in Cell Therapy 262Measuring Immune Receptor Expression 262Functional Cell-Based Assays for In Vitro Characterization 264Cell Phenotyping 265Immune Monitoring: BCR and TCR Sequencing 266Conclusions 267Acknowledgment 267References 26715 Characterizing Nucleic Acid-, Gene-, and Cell-Based Therapeutics with Sequencing Technologies 271Hayley M. Bennett, Ashley Byrne, William Stephenson, and Zora ModrusanIntroduction 271DNA Sequencing 271Nucleic-Acid-Based Therapeutics 274Gene-Based Therapies 277Cell-Based Therapies 279Immune Repertoire Sequencing Facilitates Therapeutic Advances 280Conclusions and Future Perspectives 282Abbreviations 282References 28316 Mass Spectrometry-Based Methods to Investigate Protein–Protein Interactions in Cells and Their Application in Drug Discovery 289Tess BranonIntroduction 289Affinity Purification Mass Spectrometry (AP-MS) 290Cross-Linking Mass Spectrometry (XL-MS) 293Proximity Labeling Mass Spectrometry (PL-MS) 296Concluding Remarks and Other Interactomic Technologies 302Abbreviations 307References 30817 Receptomics and Extracellular Interactomics for Biotherapeutic Characterization 321Naincy R. Chandan, Abel Ferrel, and Shengya CaoIntroduction 321Receptomic and Extracellular Interactomics Technology Overview 322Natural Products and Peptides 325Antibodies and Related Proteins 326Lipid Nanoparticles (LNPs), Extracellular Vesicles (EVs), Virus-Like Particles (VLPs) and Viruses 331Cell Therapies 332Summary 334Abbreviations 334References 33518 Membrane Proteins: Challenging Biotherapeutic Targets 341Hiruni S. Jayasekera, Farhana Afrin Mohona, and Michael T. MartyMembrane Proteins: Challenging Biotherapeutic Targets Families of Membrane Proteins 341Membrane Protein Solubilization Methods 344MS Analysis of Membrane Proteins 349Conclusion 353Acknowledgments 353References 35319 Chemoproteomics Approaches for Diverse Modalities 365H. Christian Eberl, Sascha Knecht, Markus A. Queisser, and Marcus BantscheffIntroduction 365Chemoproteomics Approaches 367Probe-Based (Direct) Target Deconvolution 367Target Deconvolution Without Chemical Probes 369Proximity-Labeling Approaches 370Mechanism-Centric (Indirect) Proteomics Methods 370Covalent Drugs 370Protein Degraders 373Antibody–Drug Conjugates and Oligonucleotides 377Concluding Remarks 378Acknowledgments 379Abbreviations 379References 37920 Drug-Delivery Modalities and Mechanisms 385Yu Tong Tam, Madyson Migliozzi, Devin B. Tesar, Amin Famili, Aaron T. Wecksler, Sara Wichner, Alavattam Sreedhara, and Whitney Shatz-BinderIntroduction 385Delivery of Proteins 387Delivering Genes to the Immune System: Enabling Cell-Specific Targeting 399Tissue-Specific Delivery: Targeting the Brain Parenchyma 402Direct Injection 402Next-Generation Approaches with Some Clinical Data 403An Analytical Perspective to Drug-Delivery Technology Development 409Conclusions/Future Perspectives 419Abbreviations 421References 422Index 439