Front Cover 1
Biophysical Characterization of Proteins in Developing Biopharmaceuticals 4
Copyright 5
Contents 6
List of Contributors 10
About the Editors 12
Preface 14
List of Abbreviations and Symbols 18
Chapter 1 - The Complexity of Protein Structure and the Challenges it Poses in Developing Biopharmaceuticals 24
1.1 THE BASICS OF PROTEIN HIGHER-ORDER STRUCTURE (HOS) 24
1.2 THE SEARCH FOR HOW PROTEINS ATTAIN THEIR CORRECT HOS: THE PROTEIN FOLDING PROBLEM 31
1.3 SURPRISES IN THE WORLD OF PROTEIN FOLDING: INTRINSICALLY DISORDERED OR UNSTRUCTURED PROTEINS (AN APPARENT CHALLENGE TO THE ... 35
1.4 PROTEINS AND THE BIOPHARMACEUTICAL INDUSTRY: PROBLEMS AND CHALLENGES 35
1.5 CONCLUSION 40
References 41
Further Reading 44
Chapter 2 - Biophysical Characterization and Its Role in the Biopharmaceutical Industry 46
2.1 DRUG DEVELOPMENT PROCESS 46
2.2 PROTEIN DRUGS (BIOPHARMACEUTICALS) 48
2.3 THE ROLE OF BIOPHYSICAL CHARACTERIZATION IN BIOPHARMACEUTICAL DRUG DEVELOPMENT 50
2.4 THE CHALLENGES IN CONDUCTING BIOPHYSICAL MEASUREMENTS TO DETECT CHANGES IN A PROTEIN DRUG'S HOS 62
2.5 REGULATORY NEEDS AND CONSIDERATIONS 65
References 66
Further Reading 70
Chapter 3 - Biopharmaceutical Industry's Biophysical Toolbox 72
3.1 ATTRIBUTES OF A SINGLE BIOPHYSICAL TOOL TO CHARACTERIZE AND DETECT CHANGES IN THE HIGHER ORDER STRUCTURE OF A BIOPHARMACEUTICAL 72
3.2 STUDYING THE BIOPHYSICAL PROPERTIES OF A BIOPHARMACEUTICAL AS AN INDIRECT APPROACH FOR CHARACTERIZING CHANGES IN ITS HOS 74
3.3 GENERAL CONSIDERATIONS IN ANALYZING THE BIOPHYSICAL PROPERTIES OF BIOPHARMACEUTICALS 77
3.4 THE UTILITY OF USING STRESS TO MONITOR CHANGES IN THE HOS PROFILE OF A PROTEIN DRUG 81
3.5 PRESENT BIOPHYSICAL TOOLBOX 82
3.6 CONCLUSION 97
References 97
Further Reading 101
Chapter 4 - An Introduction and Hierarchical Organization of the Biophysical Tool in Section II 102
4.1 INTRODUCTION 102
4.2 THE STANDARD CLASS OF BIOPHYSICAL TOOLS USED IN THE BIOPHARMACEUTICAL INDUSTRY 104
4.3 THE ADVANCED CLASS OF BIOPHYSICAL TOOLS USED IN THE BIOPHARMACEUTICAL INDUSTRY 105
4.4 AN OVERVIEW OF SECTION II 107
References 107
Further Reading 108
Chapter 5 - The Value of UV, Fluorescence, and FTIR Spectroscopy in Biopharmaceutical Development 110
5.1 INTRODUCTION 110
5.2 THE ORIGINS OF ELECTRONIC ABSORPTION, FLUORESCENCE, AND FT-IR SPECTROSCOPY 111
5.3 CONFORMATIONAL ANALYSIS OF PROTEINS IN SOLUTION 115
5.4 OPTICAL SPECTROSCOPY AND PRODUCT COMPARABILITY 118
5.5 OPTICAL SPECTROSCOPY AND HIGH-THROUGHPUT METHODS 122
5.6 SOLID-STATE MEASUREMENTS 125
5.7 CONCLUSIONS 127
References 128
Chapter 6 - Circular Dichroism Spectroscopy for Protein Characterization: Biopharmaceutical Applications 132
6.1 INTRODUCTION 132
6.2 INSTRUMENTATION 137
6.3 DATA GENERATED 138
6.4 GUIDE TO COLLECTING GOOD DATA 139
6.5 DATA PROCESSING AND ANALYSES 150
6.6 ROLE IN THE RESEARCH INDUSTRY 153
6.7 TECHNOLOGY AVAILABILITY 154
6.8 FUTURE DEVELOPMENTS 156
Acknowledgments 157
References 157
Further Reading 159
Chapter 7 - Size-Exclusion Chromatograph (SEC) in Biopharmaceutical Process Development 162
7.1 INTRODUCTION 162
7.2 BASIC THEORY OF NORMAL OR IDEAL SEC 163
7.3 MAXIMIZING SEC SEPARATION BY ENHANCING THE USAGE OF PORE VOLUME AND PORE STRUCTURE 167
7.4 CHARACTERISTICS OF PORE STRUCTURE 169
7.5 NONIDEAL SEC CHROMATOGRAPHY 170
7.6 ASSESSING AND MAINTAINING AN OPTIMUM SEC CHROMATOGRAPHY METHOD 174
7.7 DETECTORS 176
7.8 MULTIDETECTOR SEC 183
7.9 AGGREGATION 187
7.10 TECHNOLOGY ADVANCES 188
7.11 CONCLUSION 188
References 189
Chapter 8 - Scattering Techniques for the Characterization of Biopharmaceuticals 194
8.1 INTRODUCTION 194
8.2 INTENSITY- AND TIME-DEPENDENT LIGHT SCATTERING 195
8.3 GENERAL COMMENT CONCERNING SLS AND DLS 208
8.4 THE “DUST PROBLEM” IN SLS AND DLS 209
8.5 X-RAY SCATTERING: CHARACTERIZATION OF PROTEINS IN SOLUTION USING SMALL-ANGLE X-RAY SCATTERING 210
References 229
Chapter 9 - Characterizing Biopharmaceuticals using Analytical Ultracentrifugation 234
9.1 INTRODUCTION 234
9.2 UNIQUE FEATURES OF THE ANALYTICAL ULTRACENTRIFUGE THAT MAKE IT DIFFERENT FROM OTHER CENTRIFUGES 235
9.3 THEORY 236
9.4 UTILITY OF AUC IN THE BIOPHARMACEUTICAL INDUSTRY 240
9.5 BOUNDARY SV-AUC 242
9.6 BAND SV-AUC 262
9.7 SEDIMENTATION EQUILIBRIUM, SE-AUC 264
9.8 DENSITY-GRADIENT SE-AUC 266
9.9 AUC DETECTORS 268
9.10 MISCELLANEOUS HELPFUL INFORMATION ABOUT CONDUCTING AUC EXPERIMENTS 275
9.11 CONCLUSION 277
References 278
Further Reading 282
Chapter 10 - Subvisible and Visible Particle Analysis in Biopharmaceutical Research and Development 284
10.1 INTRODUCTION 284
10.2 OVERVIEW OF ANALYTICAL METHODS 285
10.3 GENERAL RECOMMENDATIONS AND PITFALLS FOR PARTICLE ANALYSIS 302
10.4 OUTLOOK AND CONCLUSIONS 305
References 306
Chapter 11 - Differential Scanning Calorimetry in the Biopharmaceutical Sciences 310
11.1 BACKGROUND 310
11.2 DSC INSTRUMENTS 316
11.3 PRACTICAL CONSIDERATIONS FOR DSC USE 318
11.4 DATA ANALYSIS 322
11.5 APPLICATIONS OF SOLUTION DSC IN BIOPHARMACEUTICAL DISCOVERY AND DEVELOPMENT 323
11.6 APPLICATIONS OF SOLID-SAMPLE DSC IN BIOPHARMACEUTICAL DISCOVERY AND DEVELOPMENT 325
11.7 CONCLUSIONS 327
Acknowledgments 327
References 327
Chapter 12 - Biophysical Mass Spectrometry for Biopharmaceutical Process Development: Focus on Hydrogen/Deuterium Exchange 330
12.1 INTRODUCTION 330
12.2 SYNOPSIS OF THE TECHNIQUE 334
12.3 MECHANISM OF EXCHANGE 335
12.4 ADVANCES IN THE TECHNIQUE 337
12.5 COMMERCIALIZATION 345
12.6 APPLICATIONS IN THE BIOPHARMACEUTICAL INDUSTRY 346
12.7 FUTURE PERSPECTIVE 353
Acknowledgments 354
References 354
Chapter 13 - One- and Two-Dimensional NMR 
364 
13.1 PHYSICAL BASIS OF THE TECHNIQUE 364
13.2 THE APPROPRIATE TECHNIQUE FOR A PARTICULAR PROBLEM 379
13.3 METHOD REQUIREMENTS AND PERFORMANCE 384
13.4 DATA PROCESSING (PROCEDURES) 396
13.5 ROLE IN RESEARCH VS PROCESS DEVELOPMENT 398
13.6 TECHNOLOGY UPDATE: RECENT AND FUTURE ADVANCES AND UNIQUE APPLICATIONS 400
References 401
Further Reading 405
Chapter 14 - Biophysical Characterization: An Integral Part of the “Totality of the Evidence” Concept 408
14.1 BIOPHARMACEUTICAL DEVELOPMENT 408
14.2 AN INTRODUCTION TO THE “TOTALITY OF THE EVIDENCE” AND ITS MORE GLOBAL MEANING IN DEVELOPING BIOPHARMACEUTICALS 409
14.3 BIOPHYSICAL CHARACTERIZATION IN DEVELOPING PROTEIN BIOPHARMACEUTICALS 411
14.4 BUILDING A BIOPHARMACEUTICAL'S BIOPHYSICAL FINGERPRINT 412
14.5 DETECTING SMALL DIFFERENCES IN BIOPHARMACEUTICALS VIA BIOPHYSICAL CHARACTERIZATION MEASUREMENTS 416
14.6 CONCLUSION 418
References 418
Index 420