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Co- and Post-Translational Modifications of Therapeutic Antibodies and Proteins

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Co- and Post-Translational Modifications of Therapeutic Antibodies and Proteins

T. Shantha Raju

ISBN: 978-1-119-05336-1 March 2019 304 Pages

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Description

A Comprehensive Guide to Crucial Attributes of Therapeutic Proteins in Biological Pharmaceuticals

With this book, Dr. Raju offers a valuable resource for professionals involved in research and development of biopharmaceutical and biosimilar drugs. This is a highly relevant work, as medical practitioners have increasingly turned to biopharmaceutical medicines in their search for safe and reliable treatments for complex diseases, while pharmaceutical researchers seek to expand the availability of biopharmaceuticals and create more affordable biosimilar alternatives. 

Readers receive a thorough overview of the major co-translational modifications (CTMs) and post-translational modifications (PTMs) of therapeutic proteins relevant to the development of biotherapeutics. The majority of chapters detail individual CTMs and PTMs that may affect the physicochemical, biochemical, biological, pharmacokinetic, immunological, toxicological etc. properties of proteins. In addition, readers are guided on the methodology necessary to analyze and characterize these modifications. Thus, readers gain not only an understanding of CTMs/PTMs, but also the ability to design and assess their own structure-function studies for experimental molecules. Specific features and topics include: 

  • Discussion of the research behind and expansion of biopharmaceuticals
  • Twenty chapters detailing relevant CTMs and PTMs of proteins, such as glycosylation, oxidation, phosphorylation, methylation, proteolysis, etc.
  • Each chapter offers an introduction and guide to the mechanisms and biological significance of an individual CTM or PTM, including practical guidance for experiment design and analysis
  • An appendix of biologic pharmaceuticals currently on the market, along with an assessment of their PTMs and overall safety and efficacy

This volume will prove a key reference on the shelves of industry and academic researchers involved in the study and development of biochemistry, molecular biology, biopharmaceuticals and proteins in medicine, particularly as biopharmaceuticals and biosimilars become ever more prominent tools in the field of healthcare.

Preface xv

About the Author xix

Abbreviations xxi

1 Introduction to Co- and Post-translational Modifications of Proteins 1

Brief Introductions to Individual Chapters 8

Chapter 2: Acetylation of Proteins 8

Chapter 3: C-Terminal Lys or Arg Clipping of Proteins 8

Chapter 4: Cysteinylation of Proteins 8

Chapter 5: Deamidation of Proteins 8

Chapter 6: Glycation of Proteins 9

Chapter 7: Glycosylation of Proteins 9

Chapter 8: N-glycosylation of Proteins 9

Chapter 9: O-glycosylation of Proteins 10

Chapter 10: Hydroxylation of Proteins 10

Chapter 11: Methylation of Proteins 10

Chapter 12: Oxidation of Proteins 11

Chapter 13: Phosphorylation of Proteins 11

Chapter 14: Prenylation of Proteins 11

Chapter 15: Proteolysis of Proteins 11

Chapter 16: Selenylation of Proteins 12

Chapter 17: Signal Peptides of Proteins 12

Chapter 18: Sulfation of Proteins and Glycoproteins 12

Chapter 19: SUMOylation 12

Chapter 20: Ubiquitination 13

Chapter 21: Other PTMs 13

References 13

2 Acetylation of Proteins 17

Introduction 17

Mechanism of N-acetylation at the N-termini of Proteins 19

Mechanism of N-acetylation and N-deacetylation of Lysine Residues 21

Mechanism of O-acetylation of Sugar Residues 21

Biological Significance of Protein Acetylation 22

Acetylation in Recombinant Therapeutic Proteins 23

Methods to Analyze Acetylation in Proteins and Carbohydrates 23

References 24

3 C-terminal Lys or Arg Clipping in Proteins 31

Introduction 31

Biological Significance of C-terminal Lys or Arg Clipping in Proteins 31

Analysis of C-terminal Lys or Arg Clipping in Proteins 32

References 32

4 Cysteinylation of Proteins 35

Introduction 35

Biological Significance of Cysteinylation of Proteins 35

Cysteinylation and Trisulfide Bonds in Recombinant Therapeutic Proteins 36

Analysis of Cysteinylation of Proteins 36

References 37

5 Deamidation of Proteins 39

Introduction 39

Mechanism of Deamidation of Proteins 40

Physicochemical Characteristics of Deamidated Proteins 42

Biological Significance of Deamidation of Proteins 43

Deamidation and Immunogenicity 43

Deamidation and Pharmacokinetics Properties of Proteins 44

Deamidation in Recombinant Therapeutic Proteins 44

Methods for the Analysis of Deamidation in Proteins 44

References 45

6 Glycation of Proteins 51

Introduction 51

Mechanism of Protein Glycation 52

Glycation of Proteins in Human 54

Protein Glycation and Human Diseases 55

Glycation in Recombinant Therapeutic Proteins 56

Methods to Analyze Protein Glycation 57

References 58

7 Glycosylation of Proteins 63

Introduction 63

Glycans and Aglycans 64

Glycosidic Bonds 64

Aldoses and Ketoses 66

Anomeric Groups: α- and β-Configurations 69

Natural Diversity of Glycans 70

Glycans and Enzymes 71

N-glycosylation 71

O-glycosylation 72

Phospho-Serine Glycosylation 72

GPI-Anchors (Glypiation) 72

C-mannosylation 73

References 73

8 N-glycosylation of Proteins 77

Introduction 77

Mechanism of N-glycosylation of Proteins 81

Biosynthesis of N-Glycans 81

Biosynthesis of Lipid-linked Precursor Oligosaccharide 81

En Bloc Transfer of the Precursor Oligosaccharide to Nascent Polypeptide Chain 82

Processing of the Glycan 82

Additional Processing of Oligosaccharide Unit for Chain Elongation and/or Modifications 83

Microheterogeneity of N-Glycans 84

Species-Specific N-glycosylation 85

Functions of N-glycans 87

Physicochemical Functions of N-glycans 87

Biological Functions of N-glycans 88

Impact of N-Glycans on Pharmacokinetic Properties of Proteins 88

N-Glycans and Human Diseases 89

N-glycosylation of RTPs 89

Methods to Analyze N-Glycans 90

References 92

9 O-glycosylation of Proteins 101

Introduction 101

Biosynthesis of O-Glycans 103

Biosynthesis of Mucin Type O-Glycans 103

Biosynthesis of O-linked GlcNAc on Proteins 105

Biosynthesis of O-linked Fucose on Proteins 106

Biosynthesis of O-linked Glc Residues 107

Biosynthesis of O-linked Gal Residues 107

Biosynthesis of O-linked Man Residues 107

O-Glycans on Hydroxyproline Residues 108

Physicochemical Properties of O-Glycosylated Proteins 108

Biological Functions of O-Glycans 108

O-glycosylation in RTPs 110

Analysis of O-Glycans 111

References 113

10 Hydroxylation of Proteins 119

Introduction 119

Mechanism of Hydroxylation 120

Mechanism of Hydroxylation in Organic Molecules 120

Mechanism of Hydroxylation in Biomolecules 121

Prolyl 4-Hydroxylase 123

Prolyl 3-Hydroxylase 123

Lysyl 5-Hydroxylase 123

Phenylalanine Hydroxylase 123

Tyrosine Hydroxylase 124

Biological Significance of Hydroxylation 124

Hydroxylation in RTPs 126

Analysis of Hydroxylation 126

References 127

11 Methylation of Proteins 133

Introduction 133

Mechanism of Protein Methylation 135

Chemical Methylation Reactions 135

Biological Methylation Reactions 135

Methylation of Arg Residues 135

Methylation of Lys Residues 137

Methylation of Prenylcysteine Residues 137

Methylation of Protein Phosphatase 2A 137

Methylation of Isoaspartyl Residues 138

O-Methylation of Sugar Residues 138

Physicochemical and Biological Significance of Methylation of Proteins 139

Methylation in RTPs 140

Methods to Analyze Methylation in Proteins and Glycoproteins 140

References 141

12 Oxidation of Proteins 147

Introduction 147

Mechanism of Protein Oxidation 149

Methionine (Met) Oxidation 149

Cysteine (Cys) Oxidation 150

Tryptophan (Trp) Oxidation 151

Tyrosine (Tyr) Oxidation 153

Oxidation of Other Amino Acid Residues and Protein Backbone 155

Oxidation in RTPs 155

Met Oxidation in mAbs 157

Analytical Methods to Measure Protein Oxidation 157

References 158

13 Phosphorylation of Proteins 163

Introduction 163

Mechanism of Protein Phosphorylation in Living Cells 164

Mg2+ Mediated Mechanism of Protein Phosphorylation 165

Protein Kinase-Based Mechanism of Protein Phosphorylation 166

Mechanism of Dephosphorylation of Proteins by Phosphatases 166

Protein Kinases 167

Serine Kinases 167

Tyrosine Kinases 168

Physicochemical and Biological Functions of Protein Phosphorylation 169

Phosphorylation in RTPs 169

Methods to Analyze Protein Phosphorylation 170

Gel Electrophoresis 170

Mass Spectrometry 171

Enrichment of Phosphoproteins 171

Enrichment of Phosphorylated Peptides 171

References 171

14 Prenylation of Proteins 177

Introduction 177

Mechanism of Protein Prenylation 177

Biological Significance of Prenylation of Proteins 179

Analysis of Prenylation of Proteins 179

References 179

15 Proteolysis of Proteins 183

Introduction 183

Chemical Proteolysis 184

Enzymatic Proteolysis 185

Biological Significance of Proteolysis 187

Post-translational Processing of Proteins by Proteolysis 187

Intracellular and Extracelluar Degradation of Proteins by Proteases 189

Proteolysis and Food Digestion 190

Role of Proteases in Apoptosis 190

Role of Proteolysis in Human Diseases 191

Use of Proteases in Laboratories 191

Sources of Proteases 193

Proteolysis in RTPs 193

Chemical Proteolysis in RTPs 193

Enzymatic Proteolysis in RTPs 194

Analytical Methods for the Detection of Proteolysis of Proteins 195

References 196

16 Selenylation 203

Introduction 203

Biological Significance of Selenylation of Proteins 203

References 205

17 Signal Peptides 207

Introduction 207

Biological Significance of Signal Peptides 207

Signal Peptides in RTPs 208

References 208

18 Sulfation of Proteins and Glycoproteins 211

Introduction 211

Biosynthesis of PAPS 212

Sulfation Reactions in the Cytosol 213

Sulfation in the Golgi Compartments 215

Sulfation of Tyrosine Residues 215

Mechanism of Tyrosine Sulfation 216

Biological Functions of Tyr Sulfation 216

Sulfation of Glycosaminoglycans (GAGs) and Proteoglycans 217

Biological Functions of GAGs 219

Sulfation in RTPs 220

Analysis of Sulfation in Biomolecules 221

Analysis of Tyr Sulfation 222

Analysis of Sulfated Glycoconjugates 222

Colorimetric Methods to Analyze Sulfated Glycoconjugates 222

Electrophoretic Methods to Analyze Sulfated Glycoconjugates 223

Chromatographic Methods to Analyze Sulfated Glycoconjugates 223

Analysis of Sulfated Glycoconjugates by Mass Spectrometry 224

References 224

19 SUMOylation 231

Introduction 231

Mechanism of SUMOylation 232

Biological Significance of SUMOylation 232

References 232

20 Ubiquitination 235

Introduction 235

Mechanism of Ubiquitination 235

Biological Significance of Ubiquitination 236

References 236

21 Other CTMs and PTMs of Proteins 239

Adenylylation or AMPylation 239

ADP-Ribosylation 239

Amidation 239

Arginylation 240

Butyrylation 240

Carbamylation 240

Carbonylation 240

γ-Carboxylation 241

Citrullination 241

Diphthamide 241

Formylation 241

Glypiation 242

Hypusine Formation 242

Iodination 242

Lipoylation 243

Malonylation 243

Myristoylation 243

Neddylation 243

Palmitoylation 244

Polyglutamylation 244

Polyglycylation 244

Propionylation 245

Pupylation 245

Pyroglutamate Formation 245

S-Glutathionylation 245

S-Nitrosylation 247

References 247

Appendix A 253

Index 271