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 365
Chemoproteomics Approaches 367
Probe-Based (Direct) Target Deconvolution 367
Target Deconvolution Without Chemical Probes 369
Proximity-Labeling Approaches 370
Mechanism-Centric (Indirect) Proteomics Methods 370
Covalent Drugs 370
Protein Degraders 373
Antibody-Drug Conjugates and Oligonucleotides 377
Concluding Remarks 378
Acknowledgments 379
Abbreviations 379
References 379
20 Drug-Delivery Modalities and Mechanisms 385
Yu Tong Tam, Madyson Migliozzi, Devin B. Tesar, Amin Famili, Aaron T. Wecksler, Sara Wichner, Alavattam Sreedhara, and Whitney Shatz-Binder
Introduction 385
Delivery of Proteins 387
Delivering Genes to the Immune System: Enabling Cell-Specific Targeting 399
Tissue-Specific Delivery: Targeting the Brain Parenchyma 402
Direct Injection 402
Next-Generation Approaches with Some Clinical Data 403
An Analytical Perspective to Drug-Delivery Technology Development 409
Conclusions/Future Perspectives 419
Abbreviations 421
References 422
Index 439
