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.
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978-1-119-38442-7 (9781119384427)
Schweitzer Klassifikation
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DNB DDC Sachgruppen
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BIC 2 Klassifikation
BISAC Klassifikation
Warengruppensystematik 2.0
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.
1 - Title Page [Seite 5]
2 - Copyright Page [Seite 6]
3 - Contents [Seite 9]
4 - List of Contributors [Seite 17]
5 - Chapter 1 Introduction to Biotherapeutics [Seite 21]
5.1 - 1.1 Introduction [Seite 21]
5.2 - 1.2 Types of Biotherapeutics and Manufacturing Systems [Seite 22]
5.3 - 1.3 Types of Analyses Performed [Seite 25]
5.4 - 1.4 Future perspectives [Seite 26]
5.5 - Acknowledgments [Seite 31]
5.6 - References [Seite 31]
6 - Chapter 2 Mass Spectrometric Characterization of Recombinant Proteins [Seite 35]
6.1 - 2.1 Introduction [Seite 36]
6.1.1 - 2.1.1 Ionization [Seite 36]
6.1.1.1 - 2.1.1.1 Matrix Assisted Laser Desorption Ionization [Seite 37]
6.1.1.2 - 2.1.1.2 Electrospray Ionization [Seite 39]
6.1.2 - 2.1.2 Mass Analyzers for Intact Molecular Weight Measurement of Biotherapeutics [Seite 40]
6.1.2.1 - 2.1.2.1 Time of Flight and Quadrupole Time of Flight Mass Spectrometers [Seite 40]
6.1.2.2 - 2.1.2.2 High-Resolution Intact Mass Measurement and Native MS [Seite 41]
6.1.2.3 - 2.1.2.3 Ion Mobility Spectrometry [Seite 42]
6.1.3 - 2.1.3 Software for the Analysis of Intact Molecular Weight Measurements [Seite 44]
6.1.4 - 2.1.4 Separation Devices for the Characterization of Biotherapeutics [Seite 45]
6.1.4.1 - 2.1.4.1 High-performance Liquid Chromatography [Seite 45]
6.1.4.2 - 2.1.4.2 Capillary Electrophoresis [Seite 46]
6.1.4.3 - 2.1.4.3 Microfluidic Chromatographic Devices [Seite 48]
6.2 - 2.2 Peptide Mass Fingerprinting [Seite 49]
6.3 - 2.3 Tandem Mass Spectrometric Characterization of Biomolecules [Seite 50]
6.3.1 - 2.3.1 Bottom-Up MS [Seite 53]
6.3.2 - 2.3.2 Proteoinformatic Analysis of Bottom-Up Proteomic Data Sets [Seite 54]
6.3.3 - 2.3.3 Top-Down MS [Seite 56]
6.4 - 2.4 Conclusions and Perspectives [Seite 57]
6.5 - References [Seite 57]
7 - Chapter 3 Characterizing the Termini of Recombinant Proteins [Seite 63]
7.1 - 3.1 Introduction [Seite 64]
7.2 - 3.2 Gel Electrophoresis and Edman Sequencing [Seite 66]
7.3 - 3.3 Mass Spectrometric Approaches for Characterizing True Starts of Proteins [Seite 69]
7.3.1 - 3.3.1 Top-Down Approaches [Seite 69]
7.3.2 - 3.3.2 Current Caveats in Mass Spectrometric Identification of Protein Termini [Seite 74]
7.3.3 - 3.3.3 Bottom-up Approaches for Identification of N- and C-Terminal Peptides [Seite 75]
7.3.4 - 3.3.4 Amino Terminal Orientated Mass Spectrometry [Seite 76]
7.3.5 - 3.3.5 Determining the True Start of Proteins from ATOMS LC-MS/MS Data [Seite 81]
7.4 - 3.4 Conclusions [Seite 84]
7.5 - References [Seite 86]
8 - Chapter 4 Assessing Activity and Conformation of Recombinant Proteins [Seite 93]
8.1 - 4.1 Introduction [Seite 94]
8.2 - 4.2 Circular Dichroism [Seite 95]
8.2.1 - 4.2.1 Applications of CD [Seite 97]
8.2.1.1 - 4.2.1.1 Thermal Stability Analysis [Seite 97]
8.2.1.2 - 4.2.1.2 Characterization of the Effect of PEGylation [Seite 97]
8.2.1.3 - 4.2.1.3 Formulation and Stability Studies [Seite 97]
8.2.1.4 - 4.2.1.4 Analysis of Biosimilars [Seite 98]
8.2.2 - 4.2.2 Technical Improvements [Seite 98]
8.3 - 4.3 DSC and Isothermal Titration Calorimetry [Seite 99]
8.3.1 - 4.3.1 Use of DSC and ITC in Therapeutics Discovery [Seite 100]
8.3.2 - 4.3.2 Protein Conjugation [Seite 102]
8.3.3 - 4.3.3 Formulation and Stability [Seite 102]
8.3.4 - 4.3.4 Analysis of Biosimilars [Seite 103]
8.4 - 4.4 Hydrogen-Deuterium Exchange-Mass Spectrometry [Seite 105]
8.4.1 - 4.4.1 Applications of HDX [Seite 106]
8.4.1.1 - 4.4.1.1 Ligand-induced Conformational Changes and Mapping Interaction sites [Seite 106]
8.4.1.2 - 4.4.1.2 Applications in Protein Engineering [Seite 106]
8.4.1.3 - 4.4.1.3 Comparability and Biosimilar Studies [Seite 108]
8.4.1.4 - 4.4.1.4 Formulation and Aggregation Analysis [Seite 109]
8.4.2 - 4.4.2 Technical Improvements and Challenges [Seite 109]
8.5 - 4.5 Nuclear Magnetic Resonance [Seite 110]
8.5.1 - 4.5.1 Applications of NMR [Seite 112]
8.5.1.1 - 4.5.1.1 Flexible Proteins [Seite 112]
8.5.1.2 - 4.5.1.2 Mapping Protein-Protein Interactions [Seite 113]
8.5.1.3 - 4.5.1.3 Epitope Mapping [Seite 114]
8.5.1.4 - 4.5.1.4 Protein Dynamics [Seite 114]
8.5.1.5 - 4.5.1.5 Protein Conjugates and Complexes [Seite 114]
8.5.1.6 - 4.5.1.6 Posttranslational Modifications [Seite 115]
8.5.1.7 - 4.5.1.7 Biosimilars [Seite 115]
8.6 - 4.6 Concluding Remarks [Seite 116]
8.7 - References [Seite 118]
9 - Chapter 5 Structural Characterization of Recombinant Proteins and Antibodies [Seite 131]
9.1 - 5.1 Introduction [Seite 132]
9.2 - 5.2 Antigens, Epitopes, and Paratopes [Seite 133]
9.2.1 - 5.2.1 Rationale for Structural Characterization of Epitopes [Seite 133]
9.3 - 5.3 Choice of Analytical Method for Epitope Mapping [Seite 137]
9.3.1 - 5.3.1 EM for Epitope Analysis [Seite 137]
9.3.2 - 5.3.2 Epitope and Paratope Mapping by NMR [Seite 138]
9.3.2.1 - 5.3.2.1 Epitope/Paratope Mapping by Chemical Shift Perturbations [Seite 139]
9.3.2.2 - 5.3.2.2 Final Considerations [Seite 142]
9.3.3 - 5.3.3 Epitope Mapping by X-ray Crystallography [Seite 142]
9.4 - 5.4 Recombinant Antigen Generation [Seite 143]
9.4.1 - 5.4.1 E. coli Expression of Antigens [Seite 144]
9.4.2 - 5.4.2 Insect Cell Expression of Antigens [Seite 145]
9.4.3 - 5.4.3 Mammalian Expression of Antigens [Seite 146]
9.5 - 5.5 N-linked Glycosylation [Seite 147]
9.5.1 - 5.5.1 E. coli Expression to Remove Glycosylation as a Factor [Seite 148]
9.5.2 - 5.5.2 Manipulating N-linked Glycans on Antigens [Seite 148]
9.6 - 5.6 Antibody Generation for Crystallography [Seite 149]
9.7 - 5.7 Crystallization of Antibody/Antigen Complexes [Seite 150]
9.8 - 5.8 Conclusion [Seite 151]
9.9 - References [Seite 151]
10 - Chapter 6 Antibody de novo Sequencing [Seite 159]
10.1 - 6.1 Introduction [Seite 159]
10.2 - 6.2 Technical Details on Antibody de novo Sequencing [Seite 161]
10.2.1 - 6.2.1 Achieving Complete Protein Coverage [Seite 161]
10.2.2 - 6.2.2 Achieving High Sequencing Accuracy [Seite 162]
10.2.3 - 6.2.3 Handling Protein Modifications [Seite 163]
10.2.4 - 6.2.4 Handling Sample Purity [Seite 163]
10.3 - 6.3 Bioinformatics Workflow [Seite 166]
10.3.1 - 6.3.1 Spectral Preprocessing [Seite 166]
10.3.2 - 6.3.2 Spectral Alignment-based Approach [Seite 166]
10.3.3 - 6.3.3 Sequence Homology-based Approaches [Seite 167]
10.3.4 - 6.3.4 Semi-automated and Manual de novo Sequencing [Seite 169]
10.4 - 6.4 Sequence Validation [Seite 169]
10.4.1 - 6.4.1 Mass Spectrometry-based Statistics [Seite 169]
10.4.2 - 6.4.2 Intact Mass Comparison [Seite 170]
10.4.3 - 6.4.3 Synthetic Peptides [Seite 170]
10.5 - 6.5 Conclusions [Seite 170]
10.6 - References [Seite 171]
11 - Chapter 7 Characterization of Antibody-Drug Conjugates [Seite 175]
11.1 - 7.1 Introduction [Seite 176]
11.2 - 7.2 Characterization of DAR Utilizing MS [Seite 177]
11.2.1 - 7.2.1 The Stability of Conjugation Chemistry and the Cleavable Linker of ADC [Seite 177]
11.2.2 - 7.2.2 Historical Usage of Hydrophobic Interaction Chromatography in ADC Characterization [Seite 178]
11.2.3 - 7.2.3 Intact MS Detection under Denaturing Condition [Seite 179]
11.2.4 - 7.2.4 Intact MS Characterization under Native Conditions [Seite 179]
11.2.5 - 7.2.5 Middle-down and Bottom-up MS Approach in Mapping Drug Conjugates [Seite 181]
11.3 - 7.3 Structural Characterization of ADC [Seite 182]
11.3.1 - 7.3.1 Ion-Mobility Mass Spectrometry [Seite 182]
11.3.2 - 7.3.2 Hydrogen-Deuterium Exchange Mass Spectrometry [Seite 183]
11.4 - 7.4 Characterization of ADC Catabolism by MS [Seite 183]
11.5 - 7.5 Conclusions [Seite 184]
11.6 - References [Seite 185]
12 - Chapter 8 Characterization of Bispecific or Other Hybrid Molecules [Seite 189]
12.1 - 8.1 Introduction [Seite 190]
12.1.1 - 8.1.1 Bispecific Antibody Applications [Seite 190]
12.2 - 8.2 Overview of the Various Bispecific Formats [Seite 192]
12.2.1 - 8.2.1 Purification from Mixtures [Seite 195]
12.2.2 - 8.2.2 Bispecific Antibodies and Alternative Scaffolds with Tethered Domains [Seite 196]
12.2.3 - 8.2.3 Bispecific Molecules with Engineered Mutations [Seite 197]
12.2.4 - 8.2.4 Native Bispecific IgG with Dual Binding Behavior [Seite 198]
12.2.5 - 8.2.5 Bispecific Antibody Conjugates [Seite 199]
12.3 - 8.3 Alternatives to Bispecific Antibodies: Antibody Mixtures [Seite 199]
12.4 - 8.4 Characterization of the Bispecific Molecule [Seite 200]
12.4.1 - 8.4.1 Characterization by Bioanalytical Methods [Seite 200]
12.4.2 - 8.4.2 Characterization by Mass Spectrometry Methods [Seite 203]
12.4.2.1 - 8.4.2.1 General Considerations [Seite 203]
12.4.2.2 - 8.4.2.2 Purity Analysis of the Final Bispecific Antibody [Seite 203]
12.4.2.3 - 8.4.2.3 Antibody Mixtures [Seite 204]
12.4.2.4 - 8.4.2.4 Increasing Resolution [Seite 205]
12.4.3 - 8.4.3 Characterization of Bispecific Antibodies by Binding Assays [Seite 205]
12.4.4 - 8.4.4 Developability Assessment of the Bispecific Antibody [Seite 206]
12.4.4.1 - 8.4.4.1 Expression [Seite 206]
12.4.4.2 - 8.4.4.2 Physicochemical Properties [Seite 207]
12.4.4.3 - 8.4.4.3 Chemical Modifications [Seite 207]
12.4.4.4 - 8.4.4.4 Characterization of In Vivo Properties [Seite 208]
12.5 - 8.5 Conclusions [Seite 209]
12.6 - References [Seite 210]
13 - Chapter 9 Bio-Repository [Seite 219]
13.1 - 9.1 Introduction [Seite 219]
13.2 - 9.2 Large Molecule Repository Management [Seite 222]
13.2.1 - 9.2.1 Informatics [Seite 222]
13.2.2 - 9.2.2 Automation [Seite 226]
13.2.2.1 - 9.2.2.1 Automated Refrigerated or Freezer Stores [Seite 226]
13.2.2.2 - 9.2.2.2 Lab Automation [Seite 227]
13.3 - 9.3 Challenges and Future Perspectives for Working with Diverse Biological Reagent Types [Seite 228]
13.4 - References [Seite 229]
14 - Chapter 10 Characterization of Residual Host Cell Protein Impurities in Biotherapeutics [Seite 231]
14.1 - 10.1 Introduction [Seite 232]
14.2 - 10.2 HCP Measurement and Reporting [Seite 232]
14.2.1 - 10.2.1 Antibodies to HCPs [Seite 233]
14.2.2 - 10.2.2 Guidance on HCP Limits and Testing [Seite 235]
14.3 - 10.3 Methods to Characterize Host Cell Impurities [Seite 237]
14.3.1 - 10.3.1 HCP-ELISA [Seite 237]
14.3.2 - 10.3.2 SDS-PAGE and Western Blots [Seite 237]
14.3.3 - 10.3.3 MS Methods for HCP Analysis [Seite 239]
14.3.3.1 - 10.3.3.1 Gel Electrophoresis and MALDI or nanoLC-MS/MS [Seite 240]
14.3.3.2 - 10.3.3.2 Two Dimensional LC-MS/MS [Seite 241]
14.3.3.3 - 10.3.3.3 Targeted MS Analysis [Seite 243]
14.3.3.4 - 10.3.3.4 Ultrahigh-Resolution 1D LC-MS/MS [Seite 244]
14.3.3.5 - 10.3.3.5 Top-down Proteomics [Seite 247]
14.4 - 10.4 Use of HCP-ELISA and Orthogonal 1D LC-MS/MS in Practice [Seite 248]
14.4.1 - 10.4.1 Pros and Cons of MS for Orthogonal HCP Analysis [Seite 251]
14.4.2 - 10.4.2 Considerations and MS Evolution [Seite 252]
14.5 - 10.5 Risk of HCPs Present in Products [Seite 252]
14.6 - 10.6 Conclusions [Seite 253]
14.7 - References [Seite 254]
15 - Chapter 11 Analytical Tools for Biologics Molecular Assessment [Seite 259]
15.1 - 11.1 Introduction to Molecular Assessment [Seite 260]
15.2 - 11.2 Molecular Assessment [Seite 263]
15.3 - 11.3 Biotherapeutic Stability [Seite 264]
15.3.1 - 11.3.1 Deamidation and Isomerization of Asparagine [Seite 266]
15.3.2 - 11.3.2 Oxidation [Seite 266]
15.4 - 11.4 Physical Degradation [Seite 268]
15.5 - 11.5 Yield and Structural Stability [Seite 269]
15.6 - 11.6 Posttranslational Modifications [Seite 270]
15.7 - 11.7 Analytical Techniques [Seite 271]
15.8 - 11.8 Summary [Seite 272]
15.9 - References [Seite 274]
16 - Chapter 12 Glycan Characterization: Determining the Structure, Distribution, and Localization of Glycoprotein Glycans [Seite 277]
16.1 - 12.1 Introduction [Seite 278]
16.2 - 12.2 Glycan Labeling [Seite 284]
16.3 - 12.3 Compositional Analysis [Seite 286]
16.3.1 - 12.3.1 Neutral Sugar Analysis [Seite 287]
16.3.2 - 12.3.2 Sialic Acid Analysis [Seite 289]
16.4 - 12.4 Glycan Release [Seite 292]
16.4.1 - 12.4.1 Release of N-linked Glycans [Seite 292]
16.4.2 - 12.4.2 Release of O-linked Glycans [Seite 294]
16.5 - 12.5 Determining Sites of Glycosylation [Seite 296]
16.5.1 - 12.5.1 MS-Based Screening for Glycopeptides [Seite 298]
16.5.2 - 12.5.2 Identification of Glycosylation Sites by Analysis of Native Glycopeptides [Seite 299]
16.5.3 - 12.5.3 Identification of N?linked Glycosylation Sites by Enzymatic Labeling of Glycosylation Sites [Seite 301]
16.5.4 - 12.5.4 Identification of O?linked Glycosylation Sites by Chemical Labeling of Glycosylation Sites [Seite 303]
16.5.5 - 12.5.5 Identification of Glycosylation Sites by Edman Degradation [Seite 305]
16.6 - 12.6 Determining N-linked Glycan Distribution [Seite 306]
16.6.1 - 12.6.1 Assessing Glycan Distribution by MS [Seite 307]
16.6.1.1 - 12.6.1.1 Assessing Glycan Distribution by Mass Spectrometric Analysis of Glycoproteins [Seite 307]
16.6.1.2 - 12.6.1.2 Assessing Glycan Distribution by Mass Spectrometric Analysis of Glycopeptides [Seite 314]
16.6.1.3 - 12.6.1.3 Determining Glycan Distribution by Mass Spectrometric Analysis of Native Glycans [Seite 314]
16.6.1.4 - 12.6.1.4 Determining Glycan Distribution by Mass Spectrometric Analysis of Derivatized Glycans [Seite 318]
16.6.2 - 12.6.2 Assessing Glycan Distribution by Chromatography and CE [Seite 320]
16.6.2.1 - 12.6.2.1 Analysis of N-linked Glycans by CE [Seite 320]
16.6.2.2 - 12.6.2.2 Analysis of N-linked Glycans by HILIC [Seite 323]
16.6.2.3 - 12.6.2.3 Determining Glycan Distribution by HPAEC [Seite 325]
16.7 - 12.7 Comparison of Methods Used in Determining Glycan Distribution [Seite 327]
16.8 - 12.8 Assessing N-linked Glycan Structure [Seite 329]
16.8.1 - 12.8.1 Characterization of Glycan Structure Using Standards and Enzymatic Studies [Seite 329]
16.8.2 - 12.8.2 Characterization of Glycan Linkage by Methylation Analysis [Seite 330]
16.8.3 - 12.8.3 Characterization of Glycan Structure by MS2 [Seite 332]
16.8.4 - 12.8.4 Characterization of Glycan Structure by NMR [Seite 337]
16.9 - References [Seite 340]
17 - Index [Seite 353]
18 - Supplemental Images [Seite 364]
19 - EULA [Seite 372]
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