Protein Analysis using Mass Spectrometry

Dr. Mike S. Lee is a biotechnology entrepreneur and Founder and President of Milestone Development Services. He actively participates in the development of new technologies and their integration into industrial settings.  Dr. Lee is a founder of the Annual Symposium on Clinical and Pharmaceutical Solutions through Analysis (CPSA). These unique events, held in the US, China and Brazil, highlight industry-related applications and feature sessions promoting discussion on real-world experiences with the latest analytical technology and industry initiatives. Dr. Lee is the author or co-author of over 50 scientific papers and patents. He received his BS degree in Chemistry at the University of Maryland in 1982. In 1985 and 1987, he completed his MS and PhD, respectively, in Analytical Chemistry from the University of Florida under the direction of Professor Richard A. Yost.Dr. Qin C. Ji is a Research Fellow in the Department of Bioanalytical Sciences at Bristol-Myers Squibb, Princeton, New Jersey. His current job responsibilities include regulated bioanalytical support (with LC-MS/MS and ligand binding assays) for the development of biologic, new modality, and small molecule drugs in preclinical and clinical stages. He has authored and co-authored more than 60 peer reviewed articles and book chapters.  Prior to his current position, he held scientific and management positions at Abbott and Covance. Dr. Ji obtained his Ph.D. from Michigan State University and has completed Postdoctoral training at Mayo Clinic. He was awarded two President Awards and was an Associate Research Fellow in the prestigious Volwiler scientific society at Abbott Laboratories. He was also awarded a Chemistry Leadership Award at Bristol-Myers Squibb.

• List of Contributors xiiiForeword xviiPreface xix1 Contemporary Protein Analysis by Ion Mobility Mass Spectrometry 1Johannes P.C. Vissers and James I. Langridge1.1 Introduction 11.2 Traveling-Wave Ion Mobility Mass Spectrometry 11.3 IM–MS and LC–IM–MS Analysis of Simple and Complex Mixtures 21.4 Outlook 7Acknowledgment 8References 82 High-Resolution Accurate Mass Orbitrap and Its Application in Protein Therapeutics Bioanalysis 11Hongxia Wang and Patrick Bennett2.1 Introduction 112.2 Triple Quadrupole Mass Spectrometer and Its Challenges 112.3 High-Resolution Mass Spectrometers 122.4 Quantitation Modes on Q Exactive Hybrid Quadrupole Orbitrap 132.5 Protein Quantitation Approaches Using Q Exactive Hybrid Quadrupole Orbitrap 142.6 Data Processing 162.7 Other Factors That Impact LC–MS-based Quantitation 162.8 Conclusion and Perspectives of LC–HRMS in Regulated Bioanalysis 18References 183 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 Bennett3.1 Introduction 213.2 Characterization of PTMs Using Higher-Energy Collision Dissociation 233.3 Application of Electron Transfer Dissociation to the Characterization of Labile PTMs 263.4 Conclusion 31Acknowledgment 32References 324 Macro- to Micromolecular Quantitation of Proteins and Peptides by Mass Spectrometry 35Suma Ramagiri, Brigitte Simons, and Laura Baker4.1 Introduction 354.2 Key Challenges of Peptide Bioanalysis 364.3 Key Features of LC/MS/MS-Based Peptide Quantitation 384.4 Advantages of the Diversity of Mass Spectrometry Systems 414.5 Perspectives for the Future 41References 425 Peptide and Protein Bioanalysis Using Integrated Column-to-Source Technology for High-Flow Nanospray 45Shane R. Needham and Gary A. Valaskovic5.1 Introduction – LC–MS Has Enabled the Field of Protein Biomarker Discovery 455.2 Integration of Miniaturized LC with Nanospray ESI-MS Is a Key for Success 465.3 Micro- and Nano-LC Are Well Suited for Quantitative Bioanalysis 475.4 Demonstrating Packed-Emitter Columns Are Suitable for Bioanalysis 495.5 Future Outlook 51References 526 Targeting the Right Protein Isoform: Mass Spectrometry-Based Proteomic Characterization of Alternative Splice Variants 55Jiang Wu6.1 Introduction 556.2 Alternative Splicing and Human Diseases 556.3 Identification of Splice Variant Proteins 566.4 Conclusion 64References 647 The Application of Immunoaffinity-Based Mass Spectrometry to Characterize Protein Biomarkers and Biotherapeutics 67Bradley L. Ackermann and Michael J. Berna7.1 Introduction 677.2 Overview of IA-MS Methods 697.3 IA-MS Applications – Biomarkers 747.3.1 Peptide Biomarkers 747.4 IA-MS Applications – Biotherapeutics 817.5 Future Direction 84References 858 Semiquantification and Isotyping of Antidrug Antibodies by Immunocapture-LC/MS for Immunogenicity Assessment 91Jianing Zeng, Hao Jiang, and Linlin Luo8.1 Introduction 918.2 Multiplexing Direct Measurement of ADAs by Immunocapture-LC/MS for Immunogenicity Screening, Titering, and Isotyping 938.3 Indirect Measurement of ADAs by Quantifying ADA Binding Components 958.4 Use of LC–MS to Assist in Method Development of Cell-Based Neutralizing Antibody Assays 968.5 Conclusion and Future Perspectives 97References 979 Mass Spectrometry-Based Assay for High-Throughput and High-Sensitivity Biomarker Verification 99Xuejiang Guo and Keqi Tang9.1 Background 999.2 Sample Processing Strategies 1009.3 Advanced Electrospray Ionization Mass Spectrometry Instrumentation 1029.4 Conclusion 105References 10510 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 Yang10.1 Introduction 10710.2 Case Study Examples 11410.3 Discussion 122Acknowledgment 126References 12611 Application of Liquid Chromatography-High Resolution Mass Spectrometry in the Quantification of Intact Proteins in Biological Fluids 129Stanley (Weihua) Zhang, Jonathan Crowther, and Wenying Jian11.1 Introduction 12911.2 Workflows for Quantification of Proteins Using Full-Scan LC-HRMS 13111.3 Internal Standard Strategy 13311.4 Calibration and Quality Control (QC) Sample Strategy 13511.5 Common Issues in Quantification of Proteins Using LC-HRMS 13511.6 Examples of LC-HRMS-Based Intact Protein Quantification 13711.7 Conclusion and Future Perspectives 138Acknowledgment 140References 14012 LC–MS/MS Bioanalytical Method Development Strategy for Therapeutic Monoclonal Antibodies in Preclinical Studies 145Hongyan Li, Timothy Heath, and Christopher A. James12.1 Introduction: LC-MS/MS Bioanalysis of Therapeutic Monoclonal Antibodies 14512.2 Highlights of Recent Method Development Strategies 14612.3 Case Studies of Preclinical Applications of LC–MS/MS for Monoclonal Antibody Bioanalysis 15412.4 Conclusion and Future Perspectives 156References 15813 Generic Peptide Strategies for LC–MS/MS Bioanalysis of Human Monoclonal Antibody Drugs and Drug Candidates 161Michael T. Furlong13.1 Introduction 16113.2 A Universal Peptide LC–MS/MS Assay for Bioanalysis of a Diversity of Human Monoclonal Antibodies and Fc Fusion Proteins in Animal Studies 16113.3 An Improved “Dual” Universal Peptide LC–MS/MS Assay for Bioanalysis of Human mAb Drug Candidates in Animal Studies 16513.4 Extending the Universal Peptide Assay Concept to Human mAb Bioanalysis in Human Studies 17013.5 Internal Standard Options for Generic Peptide LC–MS/MS Assays 17313.6 Sample Preparation Strategies for Generic Peptide LC–MS/MS Assays 17513.7 Limitations of Generic Peptide LC–MS/MS Assays 17713.8 Conclusion 178Acknowledgments 178References 17814 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 Surapaneni14.1 Introduction 18314.2 Mechanism of Action 18314.3 Mass Spectrometry Measurement for DAR Distribution of Circulating ADCs 18614.4 Total Antibody Quantitation by Ligand Binding or LC–MS/MS 18914.5 Total Conjugated Drug Quantitation by Ligand Binding or LC–MS/MS 19314.6 Catabolite Quantitation by LC–MS/MS 19614.7 Preclinical and Clinical Pharmacokinetic Support 19714.8 Conclusion and Future Perspectives 198References 19815 Sample Preparation Strategies for LC–MS Bioanalysis of Proteins 203Long Yuan and Qin C. Ji15.1 Introduction 20315.2 Sample Preparation Strategies to Improve Assay Sensitivity 20515.3 Sample Preparation Strategies to Differentiate Free, Total, and ADA-Bound Proteins 21315.4 Sample Preparation Strategies to Overcome Interference from Antidrug Antibodies or Soluble Target 21415.5 Protein Digestion Strategies 21415.6. Conclusion 215Acknowledgment 216References 21616 Characterization of Protein Therapeutics by Mass Spectrometry 221Wei Wu, Hangtian Song, Thomas Slaney, Richard Ludwig, Li Tao, and Tapan Das16.1 Introduction 22116.2 Variants Associated with Cysteine/Disulfide Bonds in Protein Therapeutics 22116.3 N–C-Terminal Variants 22516.4 Glycation 22616.5 Oxidation 22616.6 Discoloration 22816.7 Sequence Variants 23016.8 Glycosylation 23216.9 Conclusion 240References 240Index 251