The definitive guide to the myriad analytical techniques available to scientists involved in biotherapeutics research Analytical Characterization of Biotherapeutics covers all current and emerging analytical tools and techniques used for the characterization of therapeutic proteins and antigen reagents. From basic recombinant antigen and antibody characterization, to complex analyses for increasingly complex molecular designs, the book explores the history of the analysis techniques and offers valuable insights into the most important emerging analytical solutions. In addition, it frames critical questions warranting attention in the design and delivery of a therapeutic protein, exposes analytical challenges that may occur when characterizing these molecules, and presents a number of tested solutions. The first single–volume guide of its kind, Analytical Characterization of Biotherapeutics brings together contributions from scientists at the leading edge of biotherapeutics research and manufacturing. Key topics covered in–depth include the structural characterization of recombinant proteins and antibodies, antibody de novo sequencing, characterization of antibody drug conjugates, characterization of bi–specific or other hybrid molecules, characterization of manufacturing host–cell contaminant proteins, analytical tools for biologics molecular assessment, and more. Each chapter is written by a recognized expert or experts in their field who discuss current and cutting edge approaches to fully characterizing biotherapeutic proteins and antigen reagents Covers the full range of characterization strategies for large molecule based therapeutics Provides an up–to–date account of the latest approaches used for large molecule characterization Chapters cover the background needed to understand the challenges at hand, solutions to characterize these large molecules, and a summary of emerging options for analytical characterization Analytical Characterization of Biotherapeutics is an up–to–date resource for analytical scientists, biologists, and mass spectrometrists involved in the analysis of biomolecules, as well as scientists employed in the pharmaceuticals and biotechnology industries. Graduate students in biology and analytical science, and their instructors will find it to be fascinating and instructive supplementary reading. INDICE: 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 Bio–Repository 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 257 John 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 .12.6.2.3 Determining Glycan Distribution by HPAEC 305 .12.7 Comparison of Methods Used in Determining Glycan Distribution 307 .12.8 Assessing N–linked Glycan Structure 309 .12.8.1 Characterization of Glycan Structure Using Standards and Enzymatic Studies 309 .12.8.2 Characterization of Glycan Linkage by Methylation Analysis 310 .12.8.3 Characterization of Glycan Structure by MS2 312 .12.8.4 Characterization of Glycan Structure by NMR 317 .References 320 .Index 333 .
- ISBN: 978-1-119-05310-1
- Editorial: Wiley–Blackwell
- Encuadernacion: Cartoné
- Páginas: 368
- Fecha Publicación: 03/10/2017
- Nº Volúmenes: 1
- Idioma: Inglés