Presents Practical Applications of Mass Spectrometry for Protein Analysis and Covers Their Impact on Accelerating Drug Discovery and Development * Covers both qualitative and quantitative aspects of Mass Spectrometry protein analysis in drug discovery * Principles, Instrumentation, Technologies topics include MS of peptides, proteins, and ADCs , instrumentation in protein analysis, nanospray technology in MS protein analysis, and automation in MS protein analysis * Details emerging areas from drug monitoring to patient care such as Identification and validation of biomarkers for cancer, targeted MS approaches for biomarker validation, biomarker discovery, and regulatory perspectives * Brings together the most current advances in the mass spectrometry technology and related method in protein analysis List of Contributors xiii Foreword xvii Preface xix 1 Contemporary Protein Analysis by Ion Mobility Mass Spectrometry 1Johannes P.C. Vissers and James I. Langridge 1.1 Introduction 1 1.2 Traveling-Wave Ion Mobility Mass Spectrometry 1 1.3 IM MS and LC IM MS Analysis of Simple and Complex Mixtures 2 1.4 Outlook 7 Acknowledgment 8 References 8 2 High-Resolution Accurate Mass Orbitrap and Its Application in Protein Therapeutics Bioanalysis 11Hongxia Wang and Patrick Bennett 2.1 Introduction 11 2.2 Triple Quadrupole Mass Spectrometer and Its Challenges 11 2.3 High-Resolution Mass Spectrometers 12 2.4 Quantitation Modes on Q Exactive Hybrid Quadrupole Orbitrap 13 2.5 Protein Quantitation Approaches Using Q Exactive Hybrid Quadrupole Orbitrap 14 2.6 Data Processing 16 2.7 Other Factors That Impact LC MS-based Quantitation 16 2.8 Conclusion and Perspectives of LC HRMS in Regulated Bioanalysis 18 References 18 3 Current Methods for the Characterization of Posttranslational Modifications in Therapeutic Proteins Using Orbitrap Mass Spectrometry 21Zhiqi Hao, Qiuting Hong, Fan Zhang, Shiaw-Lin Wu, and Patrick Bennett 3.1 Introduction 21 3.2 Characterization of PTMs Using Higher-Energy Collision Dissociation 23 3.3 Application of Electron Transfer Dissociation to the Characterization of Labile PTMs 26 3.4 Conclusion 31 Acknowledgment 32 References 32 4 Macro- to Micromolecular Quantitation of Proteins and Peptides by Mass Spectrometry 35Suma Ramagiri, Brigitte Simons, and Laura Baker 4.1 Introduction 35 4.2 Key Challenges of Peptide Bioanalysis 36 4.3 Key Features of LC/MS/MS-Based Peptide Quantitation 38 4.4 Advantages of the Diversity of Mass Spectrometry Systems 41 4.5 Perspectives for the Future 41 References 42 5 Peptide and Protein Bioanalysis Using Integrated Column-to-Source Technology for High-Flow Nanospray 45Shane R. Needham and Gary A. Valaskovic 5.1 Introduction LC MS Has Enabled the Field of Protein Biomarker Discovery 45 5.2 Integration of Miniaturized LC with Nanospray ESI-MS Is a Key for Success 46 5.3 Micro- and Nano-LC Are Well Suited for Quantitative Bioanalysis 47 5.4 Demonstrating Packed-Emitter Columns Are Suitable for Bioanalysis 49 5.5 Future Outlook 51 References 52 6 Targeting the Right Protein Isoform: Mass Spectrometry-Based Proteomic Characterization of Alternative Splice Variants 55Jiang Wu 6.1 Introduction 55 6.2 Alternative Splicing and Human Diseases 55 6.3 Identification of Splice Variant Proteins 56 6.4 Conclusion 64 References 64 7 The Application of Immunoaffinity-Based Mass Spectrometry to Characterize Protein Biomarkers and Biotherapeutics 67Bradley L. Ackermann and Michael J. Berna 7.1 Introduction 67 7.2 Overview of IA-MS Methods 69 7.3 IA-MS Applications Biomarkers 74 7.3.1 Peptide Biomarkers 74 7.4 IA-MS Applications Biotherapeutics 81 7.5 Future Direction 84 References 85 8 Semiquantification and Isotyping of Antidrug Antibodies by Immunocapture-LC/MS for Immunogenicity Assessment 91Jianing Zeng, Hao Jiang, and Linlin Luo 8.1 Introduction 91 8.2 Multiplexing Direct Measurement of ADAs by Immunocapture-LC/MS for Immunogenicity Screening, Titering, and Isotyping 93 8.3 Indirect Measurement of ADAs by Quantifying ADA Binding Components 95 8.4 Use of LC MS to Assist in Method Development of Cell-Based Neutralizing Antibody Assays 96 8.5 Conclusion and Future Perspectives 97 References 97 9 Mass Spectrometry-Based Assay for High-Throughput and High-Sensitivity Biomarker Verification 99Xuejiang Guo and Keqi Tang 9.1 Background 99 9.2 Sample Processing Strategies 100 9.3 Advanced Electrospray Ionization Mass Spectrometry Instrumentation 102 9.4 Conclusion 105 References 105 10 Monitoring Quality of Critical Reagents Used in Ligand Binding Assays with Liquid Chromatography Mass Spectrometry (LC MS) 107Brian Geist, Adrienne Clements-Egan, and Tong-Yuan Yang 10.1 Introduction 107 10.2 Case Study Examples 114 10.3 Discussion 122 Acknowledgment 126 References 126 11 Application of Liquid Chromatography-High Resolution Mass Spectrometry in the Quantification of Intact Proteins in Biological Fluids 129Stanley (Weihua) Zhang, Jonathan Crowther, and Wenying Jian 11.1 Introduction 129 11.2 Workflows for Quantification of Proteins Using Full-Scan LC-HRMS 131 11.3 Internal Standard Strategy 133 11.4 Calibration and Quality Control (QC) Sample Strategy 135 11.5 Common Issues in Quantification of Proteins Using LC-HRMS 135 11.6 Examples of LC-HRMS-Based Intact Protein Quantification 137 11.7 Conclusion and Future Perspectives 138 Acknowledgment 140 References 140 12 LC MS/MS Bioanalytical Method Development Strategy for Therapeutic Monoclonal Antibodies in Preclinical Studies 145Hongyan Li, Timothy Heath, and Christopher A. James 12.1 Introduction: LC-MS/MS Bioanalysis of Therapeutic Monoclonal Antibodies 145 12.2 Highlights of Recent Method Development Strategies 146 12.3 Case Studies of Preclinical Applications of LC MS/MS for Monoclonal Antibody Bioanalysis 154 12.4 Conclusion and Future Perspectives 156 References 158 13 Generic Peptide Strategies for LC MS/MS Bioanalysis of Human Monoclonal Antibody Drugs and Drug Candidates 161Michael T. Furlong 13.1 Introduction 161 13.2 A Universal Peptide LC MS/MS Assay for Bioanalysis of a Diversity of Human Monoclonal Antibodies and Fc Fusion Proteins in Animal Studies 161 13.3 An Improved Dual Universal Peptide LC MS/MS Assay for Bioanalysis of Human mAb Drug Candidates in Animal Studies 165 13.4 Extending the Universal Peptide Assay Concept to Human mAb Bioanalysis in Human Studies 170 13.5 Internal Standard Options for Generic Peptide LC MS/MS Assays 173 13.6 Sample Preparation Strategies for Generic Peptide LC MS/MS Assays 175 13.7 Limitations of Generic Peptide LC MS/MS Assays 177 13.8 Conclusion 178 Acknowledgments 178 References 178 14 Mass Spectrometry-Based Methodologies for Pharmacokinetic Characterization of Antibody Drug Conjugate Candidates During Drug Development 183Yongjun Xue, Priya Sriraman, Matthew V. Myers, Xiaomin Wang, Jian Chen, Brian Melo, Martha Vallejo, Stephen E. Maxwell, and Sekhar Surapaneni 14.1 Introduction 183 14.2 Mechanism of Action 183 14.3 Mass Spectrometry Measurement for DAR Distribution of Circulating ADCs 186 14.4 Total Antibody Quantitation by Ligand Binding or LC MS/MS 189 14.5 Total Conjugated Drug Quantitation by Ligand Binding or LC MS/MS 193 14.6 Catabolite Quantitation by LC MS/MS 196 14.7 Preclinical and Clinical Pharmacokinetic Support 197 14.8 Conclusion and Future Perspectives 198 References 198 15 Sample Preparation Strategies for LC MS Bioanalysis of Proteins 203Long Yuan and Qin C. Ji 15.1 Introduction 203 15.2 Sample Preparation Strategies to Improve Assay Sensitivity 205 15.3 Sample Preparation Strategies to Differentiate Free, Total, and ADA-Bound Proteins 213 15.4 Sample Preparation Strategies to Overcome Interference from Antidrug Antibodies or Soluble Target 214 15.5 Protein Digestion Strategies 214 15.6. Conclusion 215 Acknowledgment 216 References 216 16 Characterization of Protein Therapeutics by Mass Spectrometry 221Wei Wu, Hangtian Song, Thomas Slaney, Richard Ludwig, Li Tao, and Tapan Das 16.1 Introduction 221 16.2 Variants Associated with Cysteine/Disulfide Bonds in Protein Therapeutics 221 16.3 N C-Terminal Variants 225 16.4 Glycation 226 16.5 Oxidation 226 16.6 Discoloration 228 16.7 Sequence Variants 230 16.8 Glycosylation 232 16.9 Conclusion 240 References 240 Index 251