JENNIE R. LILL, PhD is Director of Proteomics at Genentech, where she is responsible for the management of Genentech's Microchemistry, Proteomics & Lipidomics Department. Dr. Lill has been involved in the experimental design and technological innovation of biological mass spectrometry for more than two decades. WENDY SANDOVAL leads the Applied Proteomics group at Genentech where she is responsible for the management and oversight of native mass spectrometry, antibody characterization, lipidomics and the core mass spectrometry group.

• List of Contributors xv1 Introduction to Biotherapeutics 1Jennie R. Lill1.1 Introduction 11.2 Types of Biotherapeutics and Manufacturing Systems 21.3 Types of Analyses Performed 51.4 Future perspectives 6Acknowledgments 11References 112 Mass Spectrometric Characterization of Recombinant Proteins 15Corey E. Bakalarski, Wendy Sandoval, and Jennie R. Lill2.1 Introduction 162.1.1 Ionization 162.1.1.1 Matrix Assisted Laser Desorption Ionization 172.1.1.2 Electrospray Ionization 192.1.2 Mass Analyzers for Intact Molecular Weight Measurement of Biotherapeutics 202.1.2.1 Time of Flight and Quadrupole Time of Flight Mass Spectrometers 202.1.2.2 High‐Resolution Intact Mass Measurement and Native MS 212.1.2.3 Ion Mobility Spectrometry 222.1.3 Software for the Analysis of Intact Molecular Weight Measurements 242.1.4 Separation Devices for the Characterization of Biotherapeutics 252.1.4.1 High‐performance Liquid Chromatography 252.1.4.2 Capillary Electrophoresis 262.1.4.3 Microfluidic Chromatographic Devices 282.2 Peptide Mass Fingerprinting 292.3 Tandem Mass Spectrometric Characterization of Biomolecules 302.3.1 Bottom‐Up MS 332.3.2 Proteoinformatic Analysis of Bottom‐Up Proteomic Data Sets 342.3.3 Top‐Down MS 362.4 Conclusions and Perspectives 37References 373 Characterizing the Termini of Recombinant Proteins 43Nestor Solis and Christopher M. Overall3.1 Introduction 443.2 Gel Electrophoresis and Edman Sequencing 463.3 Mass Spectrometric Approaches for Characterizing True Starts of Proteins 493.3.1 Top‐Down Approaches 493.3.2 Current Caveats in Mass Spectrometric Identification of Protein Termini 543.3.3 Bottom‐up Approaches for Identification of N‐ and C‐Terminal Peptides 553.3.4 Amino Terminal Orientated Mass Spectrometry 563.3.5 Determining the True Start of Proteins from ATOMS LC‐MS/MS Data 613.4 Conclusions 64References 664 Assessing Activity and Conformation of Recombinant Proteins 73Diego Ellerman, Till Maurer, and Justin M. Scheer4.1 Introduction 744.2 Circular Dichroism 754.2.1 Applications of CD 774.2.1.1 Thermal Stability Analysis 774.2.1.2 Characterization of the Effect of PEGylation 774.2.1.3 Formulation and Stability Studies 774.2.1.4 Analysis of Biosimilars 784.2.2 Technical Improvements 784.3 DSC and Isothermal Titration Calorimetry 794.3.1 Use of DSC and ITC in Therapeutics Discovery 804.3.2 Protein Conjugation 824.3.3 Formulation and Stability 824.3.4 Analysis of Biosimilars 834.4 Hydrogen–Deuterium Exchange–Mass Spectrometry 854.4.1 Applications of HDX 864.4.1.1 Ligand‐induced Conformational Changes and Mapping Interaction Sites 864.4.1.2 Applications in Protein Engineering 864.4.1.3 Comparability and Biosimilar Studies 884.4.1.4 Formulation and Aggregation Analysis 894.4.2 Technical Improvements and Challenges 894.5 Nuclear Magnetic Resonance 904.5.1 Applications of NMR 924.5.1.1 Flexible Proteins 924.5.1.2 Mapping Protein–Protein Interactions 934.5.1.3 Epitope Mapping 944.5.1.4 Protein Dynamics 944.5.1.5 Protein Conjugates and Complexes 944.5.1.6 Posttranslational Modifications 954.5.1.7 Biosimilars 954.6 Concluding Remarks 96References 985 Structural Characterization of Recombinant Proteins and Antibodies 111Paola Di Lello and Patrick Lupardus5.1 Introduction 1125.2 Antigens, Epitopes, and Paratopes 1135.2.1 Rationale for Structural Characterization of Epitopes 1135.3 Choice of Analytical Method for Epitope Mapping 1175.3.1 EM for Epitope Analysis 1175.3.2 Epitope and Paratope Mapping by NMR 1185.3.2.1 Epitope/Paratope Mapping by Chemical Shift Perturbations 1195.3.2.2 Final Considerations 1225.3.3 Epitope Mapping by X‐ray Crystallography 1225.4 Recombinant Antigen Generation 1235.4.1 E. coli Expression of Antigens 1245.4.2 Insect Cell Expression of Antigens 1255.4.3 Mammalian Expression of Antigens 1265.5 N‐linked Glycosylation 1275.5.1 E. coli Expression to Remove Glycosylation as a Factor 1285.5.2 Manipulating N‐linked Glycans on Antigens 1285.6 Antibody Generation for Crystallography 1295.7 Crystallization of Antibody/Antigen Complexes 1305.8 Conclusion 131References 1316 Antibody de novo Sequencing 139Natalie Castellana and Adrian Guthals6.1 Introduction 1396.2 Technical Details on Antibody de novo Sequencing 1416.2.1 Achieving Complete Protein Coverage 1416.2.2 Achieving High Sequencing Accuracy 1426.2.3 Handling Protein Modifications 1436.2.4 Handling Sample Purity 1436.3 Bioinformatics Workflow 1466.3.1 Spectral Preprocessing 1466.3.2 Spectral Alignment‐based Approach 1466.3.3 Sequence Homology‐based Approaches 1476.3.4 Semi‐automated and Manual de novo Sequencing 1496.4 Sequence Validation 1496.4.1 Mass Spectrometry‐based Statistics 1496.4.2 Intact Mass Comparison 1506.4.3 Synthetic Peptides 1506.5 Conclusions 150References 1517 Characterization of Antibody–Drug Conjugates 155Yichin Liu7.1 Introduction 1567.2 Characterization of DAR Utilizing MS 1577.2.1 The Stability of Conjugation Chemistry and the Cleavable Linker of ADC 1577.2.2 Historical Usage of Hydrophobic Interaction Chromatography in ADC Characterization 1587.2.3 Intact MS Detection under Denaturing Condition 1597.2.4 Intact MS Characterization under Native Conditions 1597.2.5 Middle‐down and Bottom‐up MS Approach in Mapping Drug Conjugates 1617.3 Structural Characterization of ADC 1627.3.1 Ion‐Mobility Mass Spectrometry 1627.3.2 Hydrogen–Deuterium Exchange Mass Spectrometry 1637.4 Characterization of ADC Catabolism by MS 1637.5 Conclusions 164References 1658 Characterization of Bispecific or Other Hybrid Molecules 169T. Noelle Lombana and Christoph Spiess8.1 Introduction 1708.1.1 Bispecific Antibody Applications 1708.2 Overview of the Various Bispecific Formats 1728.2.1 Purification from Mixtures 1758.2.2 Bispecific Antibodies and Alternative Scaffolds with Tethered Domains 1768.2.3 Bispecific Molecules with Engineered Mutations 1778.2.4 Native Bispecific IgG with Dual Binding Behavior 1788.2.5 Bispecific Antibody Conjugates 1798.3 Alternatives to Bispecific Antibodies: Antibody Mixtures 1798.4 Characterization of the Bispecific Molecule 1808.4.1 Characterization by Bioanalytical Methods 1808.4.2 Characterization by Mass Spectrometry Methods 1838.4.2.1 General Considerations 1838.4.2.2 Purity Analysis of the Final Bispecific Antibody 1838.4.2.3 Antibody Mixtures 1848.4.2.4 Increasing Resolution 1858.4.3 Characterization of Bispecific Antibodies by Binding Assays 1858.4.4 Developability Assessment of the Bispecific Antibody 1868.4.4.1 Expression 1868.4.4.2 Physicochemical Properties 1878.4.4.3 Chemical Modifications 1878.4.4.4 Characterization of In Vivo Properties 1888.5 Conclusions 189References 1909 Bio‐Repository 199Anne Baldwin, Kurt Schroeder, Lovejit Singh, and Karen Billeci9.1 Introduction 1999.2 Large Molecule Repository Management 2029.2.1 Informatics 2029.2.2 Automation 2069.2.2.1 Automated Refrigerated or Freezer Stores 2069.2.2.2 Lab Automation 2079.3 Challenges and Future Perspectives for Working with Diverse Biological Reagent Types 208References 20910 Characterization of Residual Host Cell Protein Impurities in Biotherapeutics 211Denise Krawitz, Jason C. Rouse, Justin B. Sperry, Wendy Sandoval, and Martin Vanderlaan10.1 Introduction 21210.2 HCP Measurement and Reporting 21210.2.1 Antibodies to HCPs 21310.2.2 Guidance on HCP Limits and Testing 21510.3 Methods to Characterize Host Cell Impurities 21710.3.1 HCP‐ELISA 21710.3.2 SDS‐PAGE and Western Blots 21710.3.3 MS Methods for HCP Analysis 21910.3.3.1 Gel Electrophoresis and MALDI or nanoLC‐MS/MS 22010.3.3.2 Two Dimensional LC‐MS/MS 22110.3.3.3 Targeted MS Analysis 22310.3.3.4 Ultrahigh‐Resolution 1D LC‐MS/MS 22410.3.3.5 Top‐down Proteomics 22710.4 Use of HCP‐ELISA and Orthogonal1D LC‐MS/MS in Practice 22810.4.1 Pros and Cons of MS for Orthogonal HCP Analysis 23110.4.2 Considerations and MS Evolution 23210.5 Risk of HCPs Present in Products 23210.6 Conclusions 233References 23411 Analytical Tools for Biologics Molecular Assessment 239Wilson Phung, Wendy Sandoval, Robert F. Kelley, and Jennie R. Lill11.1 Introduction to Molecular Assessment 24011.2 Molecular Assessment 24311.3 Biotherapeutic Stability 24411.3.1 Deamidation and Isomerization of Asparagine 24611.3.2 Oxidation 24611.4 Physical Degradation 24811.5 Yield and Structural Stability 24911.6 Posttranslational Modifications 25011.7 Analytical Techniques 25111.8 Summary 252References 25412 Glycan Characterization: Determining the Structure, Distribution, and Localization of Glycoprotein Glycans 257John B. Briggs12.1 Introduction 25812.2 Glycan Labeling 26412.3 Compositional Analysis 26612.3.1 Neutral Sugar Analysis 26712.3.2 Sialic Acid Analysis 26912.4 Glycan Release 27212.4.1 Release of N‐linked Glycans 27212.4.2 Release of O‐linked Glycans 27412.5 Determining Sites of Glycosylation 27612.5.1 MS‐Based Screening for Glycopeptides 27812.5.2 Identification of Glycosylation Sites by Analysis of Native Glycopeptides 27912.5.3 Identification of N‐linked Glycosylation Sites by Enzymatic Labeling of Glycosylation Sites 28112.5.4 Identification of O‐linked Glycosylation Sites by Chemical Labeling of Glycosylation Sites 28312.5.5 Identification of Glycosylation Sites by Edman Degradation 28512.6 Determining N‐linked Glycan Distribution 28612.6.1 Assessing Glycan Distribution by MS 28712.6.1.1 Assessing Glycan Distribution by Mass Spectrometric Analysis of Glycoproteins 28712.6.1.2 Assessing Glycan Distribution by Mass Spectrometric Analysis of Glycopeptides 29412.6.1.3 Determining Glycan Distribution by Mass Spectrometric Analysis of Native Glycans 29412.6.1.4 Determining Glycan Distribution by Mass Spectrometric Analysis of Derivatized Glycans 29812.6.2 Assessing Glycan Distribution by Chromatography and CE 30012.6.2.1 Analysis of N‐linked Glycans by CE 30012.6.2.2 Analysis of N‐linked Glycans by HILIC 30312.6.2.3 Determining Glycan Distribution by HPAEC 30512.7 Comparison of Methods Used in Determining Glycan Distribution 30712.8 Assessing N‐linked Glycan Structure 30912.8.1 Characterization of Glycan Structure Using Standards and Enzymatic Studies 30912.8.2 Characterization of Glycan Linkage by Methylation Analysis 31012.8.3 Characterization of Glycan Structure by MS2 31212.8.4 Characterization of Glycan Structure by NMR 317References 320Index 333