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 xv 1 Introduction to Biotherapeutics 1Jennie R. Lill 1.1 Introduction 1 1.2 Types of Biotherapeutics and Manufacturing Systems 2 1.3 Types of Analyses Performed 5 1.4 Future perspectives 6 Acknowledgments 11 References 11 2 Mass Spectrometric Characterization of Recombinant Proteins 15Corey E. Bakalarski, Wendy Sandoval, and Jennie R. Lill 2.1 Introduction 16 2.1.1 Ionization 16 2.1.1.1 Matrix Assisted Laser Desorption Ionization 17 2.1.1.2 Electrospray Ionization 19 2.1.2 Mass Analyzers for Intact Molecular Weight Measurement of Biotherapeutics 20 2.1.2.1 Time of Flight and Quadrupole Time of Flight Mass Spectrometers 20 2.1.2.2 High¿Resolution Intact Mass Measurement and Native MS 21 2.1.2.3 Ion Mobility Spectrometry 22 2.1.3 Software for the Analysis of Intact Molecular Weight Measurements 24 2.1.4 Separation Devices for the Characterization of Biotherapeutics 25 2.1.4.1 High¿performance Liquid Chromatography 25 2.1.4.2 Capillary Electrophoresis 26 2.1.4.3 Microfluidic Chromatographic Devices 28 2.2 Peptide Mass Fingerprinting 29 2.3 Tandem Mass Spectrometric Characterization of Biomolecules 30 2.3.1 Bottom¿Up MS 33 2.3.2 Proteoinformatic Analysis of Bottom¿Up Proteomic Data Sets 34 2.3.3 Top¿Down MS 36 2.4 Conclusions and Perspectives 37 References 37 3 Characterizing the Termini of Recombinant Proteins 43Nestor Solis and Christopher M. Overall 3.1 Introduction 44 3.2 Gel Electrophoresis and Edman Sequencing 46 3.3 Mass Spectrometric Approaches for Characterizing True Starts of Proteins 49 3.3.1 Top¿Down Approaches 49 3.3.2 Current Caveats in Mass Spectrometric Identification of Protein Termini 54 3.3.3 Bottom¿up Approaches for Identification of N¿ and C¿Terminal Peptides 55 3.3.4 Amino Terminal Orientated Mass Spectrometry 56 3.3.5 Determining the True Start of Proteins from ATOMS LC¿MS/MS Data 61 3.4 Conclusions 64 References 66 4 Assessing Activity and Conformation of Recombinant Proteins 73Diego Ellerman, Till Maurer, and Justin M. Scheer 4.1 Introduction 74 4.2 Circular Dichroism 75 4.2.1 Applications of CD 77 4.2.1.1 Thermal Stability Analysis 77 4.2.1.2 Characterization of the Effect of PEGylation 77 4.2.1.3 Formulation and Stability Studies 77 4.2.1.4 Analysis of Biosimilars 78 4.2.2 Technical Improvements 78 4.3 DSC and Isothermal Titration Calorimetry 79 4.3.1 Use of DSC and ITC in Therapeutics Discovery 80 4.3.2 Protein Conjugation 82 4.3.3 Formulation and Stability 82 4.3.4 Analysis of Biosimilars 83 4.4 Hydrogen-Deuterium Exchange-Mass Spectrometry 85 4.4.1 Applications of HDX 86 4.4.1.1 Ligand¿induced Conformational Changes and Mapping Interaction Sites 86 4.4.1.2 Applications in Protein Engineering 86 4.4.1.3 Comparability and Biosimilar Studies 88 4.4.1.4 Formulation and Aggregation Analysis 89 4.4.2 Technical Improvements and Challenges 89 4.5 Nuclear Magnetic Resonance 90 4.5.1 Applications of NMR 92 4.5.1.1 Flexible Proteins 92 4.5.1.2 Mapping Protein-Protein Interactions 93 4.5.1.3 Epitope Mapping 94 4.5.1.4 Protein Dynamics 94 4.5.1.5 Protein Conjugates and Complexes 94 4.5.1.6 Posttranslational Modifications 95 4.5.1.7 Biosimilars 95 4.6 Concluding Remarks 96 References 98 5 Structural Characterization of Recombinant Proteins and Antibodies 111Paola Di Lello and Patrick Lupardus 5.1 Introduction 112 5.2 Antigens, Epitopes, and Paratopes 113 5.2.1 Rationale for Structural Characterization of Epitopes 113 5.3 Choice of Analytical Method for Epitope Mapping 117 5.3.1 EM for Epitope Analysis 117 5.3.2 Epitope and Paratope Mapping by NMR 118 5.3.2.1 Epitope/Paratope Mapping by Chemical Shift Perturbations 119 5.3.2.2 Final Considerations 122 5.3.3 Epitope Mapping by X¿ray Crystallography 122 5.4 Recombinant Antigen Generation 123 5.4.1 E. coli Expression of Antigens 124 5.4.2 Insect Cell Expression of Antigens 125 5.4.3 Mammalian Expression of Antigens 126 5.5 N¿linked Glycosylation 127 5.5.1 E. coli Expression to Remove Glycosylation as a Factor 128 5.5.2 Manipulating N¿linked Glycans on Antigens 128 5.6 Antibody Generation for Crystallography 129 5.7 Crystallization of Antibody/Antigen Complexes 130 5.8 Conclusion 131 References 131 6 Antibody de novo Sequencing 139Natalie Castellana and Adrian Guthals 6.1 Introduction 139 6.2 Technical Details on Antibody de novo Sequencing 141 6.2.1 Achieving Complete Protein Coverage 141 6.2.2 Achieving High Sequencing Accuracy 142 6.2.3 Handling Protein Modifications 143 6.2.4 Handling Sample Purity 143 6.3 Bioinformatics Workflow 146 6.3.1 Spectral Preprocessing 146 6.3.2 Spectral Alignment¿based Approach 146 6.3.3 Sequence Homology¿based Approaches 147 6.3.4 Semi¿automated and Manual de novo Sequencing 149 6.4 Sequence Validation 149 6.4.1 Mass Spectrometry¿based Statistics 149 6.4.2 Intact Mass Comparison 150 6.4.3 Synthetic Peptides 150 6.5 Conclusions 150 References 151 7 Characterization of Antibody-Drug Conjugates 155Yichin Liu 7.1 Introduction 156 7.2 Characterization of DAR Utilizing MS 157 7.2.1 The Stability of Conjugation Chemistry and the Cleavable Linker of ADC 157 7.2.2 Historical Usage of Hydrophobic Interaction Chromatography in ADC Characterization 158 7.2.3 Intact MS Detection under Denaturing Condition 159 7.2.4 Intact MS Characterization under Native Conditions 159 7.2.5 Middle¿down and Bottom¿up MS Approach in Mapping Drug Conjugates 161 7.3 Structural Characterization of ADC 162 7.3.1 Ion¿Mobility Mass Spectrometry 162 7.3.2 Hydrogen-Deuterium Exchange Mass Spectrometry 163 7.4 Characterization of ADC Catabolism by MS 163 7.5 Conclusions 164 References 165 8 Characterization of Bispecific or Other Hybrid Molecules 169T. Noelle Lombana and Christoph Spiess 8.1 Introduction 170 8.1.1 Bispecific Antibody Applications 170 8.2 Overview of the Various Bispecific Formats 172 8.2.1 Purification from Mixtures 175 8.2.2 Bispecific Antibodies and Alternative Scaffolds with Tethered Domains 176 8.2.3 Bispecific Molecules with Engineered Mutations 177 8.2.4 Native Bispecific IgG with Dual Binding Behavior 178 8.2.5 Bispecific Antibody Conjugates 179 8.3 Alternatives to Bispecific Antibodies: Antibody Mixtures 179 8.4 Characterization of the Bispecific Molecule 180 8.4.1 Characterization by Bioanalytical Methods 180 8.4.2 Characterization by Mass Spectrometry Methods 183 8.4.2.1 General Considerations 183 8.4.2.2 Purity Analysis of the Final Bispecific Antibody 183 8.4.2.3 Antibody Mixtures 184 8.4.2.4 Increasing Resolution 185 8.4.3 Characterization of Bispecific Antibodies by Binding Assays 185 8.4.4 Developability Assessment of the Bispecific Antibody 186 8.4.4.1 Expression 186 8.4.4.2 Physicochemical Properties 187 8.4.4.3 Chemical Modifications 187 8.4.4.4 Characterization of In Vivo Properties 188 8.5 Conclusions 189 References 190 9 BiöRepository 199Anne Baldwin, Kurt Schroeder, Lovejit Singh, and Karen Billeci 9.1 Introduction 199 9.2 Large Molecule Repository Management 202 9.2.1 Informatics 202 9.2.2 Automation 206 9.2.2.1 Automated Refrigerated or Freezer Stores 206 9.2.2.2 Lab Automation 207 9.3 Challenges and Future Perspectives for Working with Diverse Biological Reagent Types 208 References 209 10 Characterization of Residual Host Cell Protein Impurities in Biotherapeutics 211Denise Krawitz, Jason C. Rouse, Justin B. Sperry, Wendy Sandoval, and Martin Vanderlaan 10.1 Introduction 212 10.2 HCP Measurement and Reporting 212 10.2.1 Antibodies to HCPs 213 10.2.2 Guidance on HCP Limits and Testing 215 10.3 Methods to Characterize Host Cell Impurities 217 10.3.1 HCP¿ELISA 217 10.3.2 SDS¿PAGE and Western Blots 217 10.3.3 MS Methods for HCP Analysis 219 10.3.3.1 Gel Electrophoresis and MALDI or nanoLC¿MS/MS 220 10.3.3.2 Two Dimensional LC¿MS/MS 221 10.3.3.3 Targeted MS Analysis 223 10.3.3.4 Ultrahigh¿Resolution 1D LC¿MS/MS 224 10.3.3.5 Top¿down Proteomics 227 10.4 Use of HCP¿ELISA and Orthogonal 1D LC¿MS/MS in Practice 228 10.4.1 Pros and Cons of MS for Orthogonal HCP Analysis 231 10.4.2 Considerations and MS Evolution 232 10.5 Risk of HCPs Present in Products 232 10.6 Conclusions 233 References 234 11 Analytical Tools for Biologics Molecular Assessment 239Wilson Phung, Wendy Sandoval, Robert F. Kelley, and Jennie R. Lill 11.1 Introduction to Molecular Assessment 240 11.2 Molecular Assessment 243 11.3 Biotherapeutic Stability 244 11.3.1 Deamidation and Isomerization of Asparagine 246 11.3.2 Oxidation 246 11.4 Physical Degradation 248 11.5 Yield and Structural Stability 249 11.6 Posttranslational Modifications 250 11.7 Analytical Techniques 251 11.8 Summary 252 References 254 12 Glycan Characterization: Determining the Structure, Distribution, and Localization of Glycoprotein Glycans 257John B. Briggs 12.1 Introduction 258 12.2 Glycan Labeling 264 12.3 Compositional Analysis 266 12.3.1 Neutral Sugar Analysis 267 12.3.2 Sialic Acid Analysis 269 12.4 Glycan Release 272 12.4.1 Release of N¿linked Glycans 272 12.4.2 Release of O¿linked Glycans 274 12.5 Determining Sites of Glycosylation 276 12.5.1 MS¿Based Screening for Glycopeptides 278 12.5.2 Identification of Glycosylation Sites by Analysis of Native Glycopeptides 279 12.5.3 Identification of N¿linked Glycosylation Sites by Enzymatic Labeling of Glycosylation Sites 281 12.5.4 Identification of O¿linked Glycosylation Sites by Chemical Labeling of Glycosylation Sites 283 12.5.5 Identification of Glycosylation Sites by Edman Degradation 285 12.6 Determining N¿linked Glycan Distribution 286 12.6.1 Assessing Glycan Distribution by MS 287 12.6.1.1 Assessing Glycan Distribution by Mass Spectrometric Analysis of Glycoproteins 287 12.6.1.2 Assessing Glycan Distribution by Mass Spectrometric Analysis of Glycopeptides 294 12.6.1.3 Determining Glycan Distribution by Mass Spectrometric Analysis of Native Glycans 294 12.6.1.4 Determining Glycan Distribution by Mass Spectrometric Analysis of Derivatized Glycans 298 12.6.2 Assessing Glycan Distribution by Chromatography and CE 300 12.6.2.1 Analysis of N¿linked Glycans by CE 300 12.6.2.2 Analysis of N¿linked Glycans by HILIC 303...