Centre for Drug Delivery Research the School of Pharmacy University of London

CENTRE FOR DRUG DELIVERY RESEARCH THE SCHOOL OF PHARMACY UNIVERSITY OF LONDON

Poly sialic Acids: A Tool for the Optimisation of Peptide and Protein Therapeutics

Thesis submitted by Malini Mital In partial fulfilment for the degree of

Doctor of Philosophy,

University of London

October 2002 ProQuest Number: 10104164

INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.

In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest.

ProQuest 10104164

Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author.

ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 For

VIMAL MITAL

A lady of extraordinary strength and wisdom, who has not only believed in me throughout my life, but shown me that dreams can come true. Your compassion and love for me have known no bounds and are my foundations in life. You are more than a mother to me, you are an angel.

For

NARENDRA MITAL

The one and only man in my life, who told me repeatedly throughout my life, “There’s nothing to it,” “You don’t have to take No for an answer,” “You make your own luck,” and “There’s more where that came from,” Dad, you are my drive and inspiration. ______Index

Acknowledgments

First and foremost, I would like to acknowledge my supervisor, Professor Gregory Gregoriadis for providing me with the opportunity to pursue this project and for imparting his infinite expertise and knowledge in this novel and exciting field of drug delivery research.

I would also like to express my deepest gratitude to my colleagues in CDDR, who gave numerous invaluable suggestions and have contributed to the compilation of this thesis in particular, Mia Obrenovic, Ibrahim Zadi, Steve Yang, Kent Lau, Brenda McCormack and Yvonne Ferrie. In addition, a special thank you to Dr. Roshan Jumnah for his invaluable expertise with the chemistry and proof reading of this thesis. Also, many thanks to Wilfi*ed Baldeo, Mire Zloh and Mark Domin for their MS and NMR analysis and Dr. Simon Richardson for his help and guidance with my radiolabelling experiments.

I would also like to acknowledge the staff at the animal unit namely Steve Coppard, Dave Khan and Donna Howell who were always eager to help, good humoured and gave invaluable assistance with the animals. Many thanks also to Rob and Roy in stores and to Annie Cavanagh for her expert IT support and fiiendship, it is truly appreciated. I am also grateful to Concha Perring for her kind assistance and to the Registry for their continual support and reassurance throughout the course.

I am truly indebted to my parents for their immeasurable financial and emotional support and to my brother Anuj for his patience, care and understanding even in my darkest moments. I would equally like to thank my numerous fiiends whose contributions to my life are deeply grasped and appreciated.

Finally, I would like to gratefully acknowledge the School of Pharmacy, University of London for all its resources that were available to me over the many years of study. Index

Abstract

Colominic acid (CA) is a low molecular weight derivative of polysialic acid (PSA), which is a naturally occurring biodegradable, highly hydrophilic polymer of N- acetylneuraminic acid. CA was proposed as an alternative to non-biodegradable mPEG for the delivery of proteins with short plasma half-lives, poor stability and high immunogenicity, thus facilitating their therapeutic potential. Here we add to the repertoire of proteins modified and introduce three ‘new’ smaller therapeutic peptides for polysialylation with a view to improving their in vitro stability, biological potency and in vivo pharmacokinetic properties. The biotherapeutics employed included, bovine polyclonal IgG and monoclonal IgG2a antibodies, which were chosen to model therapeutic Mabs which have the potential in treating cancers, autoimmune diseases and infections. The serine protease inhibitor, aprotinin chosen for the potential treatment of pancreatitis and haematological problems, insulin for treating diabetes and somatostatin (SS) for the treatment of growth disorders i.e. acromegaly. In addition, a novel strategy was developed in an attempt to further improve the efficiency and therapeutic value of the established method of polysialylation. Periodate oxidised CA was first coupled via Schiff base reductive amination to IgG (4.43%), aprotinin (29.20%), and insulin (63.33%) and yielded neoglycoproteins with a poor to moderate degree of modification based on the percentage of available amine residues modified with CA. Only SS obtained a quantitative yield of polysialylation. Introduction of the anionic detergent sodium dodecyl sulphate (SDS) into the coupling reaction seemed to enhance polysialylation and afforded bioconjugates with 1.5-3.0 fold increase in CA content. Size exclusion chromatography and electrophoresis were used to characterise and indicate the emergence of the neoglycoproteins. In vitro biological potency was mostly preserved for catalase (63-66%), IgG2a (82-94%) and aprotinin (59-87%) modified by either method of polysialylation. Furthermore, in vivo mean residence times of polysialylated and SDS-modified polysialylated IgG (45.46- 51.81h), aprotinin (22.23-24.73h) and insulin (22.81-23.26h) were significantly increased in comparison with their native counterparts. Polysialylation appears to confer stability to the biotherapeutics, maintain potency, delay plasma clearance, thereby improving their therapeutic potential in vivo.

11 Index

List of Abbreviations

ANOVA One way analysis of variance AUC Area under the curve AIDS Acquired immunodeficiency syndrome p Terminal elimination rate constant BSA Bovine serum albumin CA Colominic acid Cl Clearance CNS Central nervous system DIVEMA Poly(divinyl ether maleic anhydride copolymer DNA deoxyribonucleic acid DNP Dinitrophenylhydrazine dpm Disintegrations per minute ELISA Enzyme-linked immunosorbant assay Fab Antigen-binding fi*agment PCS Foetal calf serum FT-IR Fourier transform-infi*ared spectroscopy FDA Food and Drug Administration HPMA N-(2-Hydroxypropyl)methacrylamide copolymers IgG Immunoglobulin G lA Intra-arterial IM Intramuscular IP Intraperitoneal IV Intravenous KDa KiloDaltons Km Michaelis Menten constant Mab Monoclonal antibody MPEG Monomethoxypolyethylene glycol Mol. wt Molecular weight MPS Mononuclear phagocyte system MRT Mean residence time

111 Index

MS Mass spectrometry N-CAM Neural cell adhesion molecule NeuSAc N-acetylneuraminic acid NMR Nuclear magnetic resonance spectroscopy PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PBS-T PBS containing 0.05% v/v Tween 20 PEG Polyethylene glycol PEGl 2-O-methoxypolyethylene glycol-4,6-dichloro-S-triazine PEG2 2,4-bis (O-methoxypolyethylene glycol)-6-chloro-S-triazine pi Isoelectric point PM13 Comb-shaped PEG copolymer (13 KDa) PMI 00 Comb-shaped PEG copolymer (100 KDa) PSA Polysialic acid PSAs Polysialic acids PVP Poly(N-vinylpyrrolidone) RES Reticuloendothelial system SC Subcutaneous SDS Sodium dodecyl sulphate SEC Size exclusion chromatography SMA Poly(styrene-co-maleic anhydride) SOD Sodium dismutase SS Somatostatin tl/2 p Terminal half-life TCA Trichloroacetic acid TEMED N,N,N ’ ,N ’-T etramethylethylenediamine TRIS Trizma base UHQ Ultra high quality

V e Elution volume Vd Volume of distribution

V o Void volume

^ m a x Maximum velocity

IV Index

Contents Page No.

Acknowledgements 1 Abstract ii List of Abbreviations iii

Contents V List of Figures xiv List of Tables xix List of Spectra xxiii

Chapter One: General introduction 1.1 The use of peptides and proteins as pharmaceuticals 1.2 Problems associated with the clinical use of peptide and protein Pharmaceuticals

1.3 Clearance mechanisms of peptide and protein pharmaceuticals 7 1.3.1 Distribution and catabolism of peptide and protein pharmaceuticals 7 1.4 Stabilisation of peptide and protein pharmaceutics: Novel approaches 10 1.4.1 Encapsulation methods for peptide and protein delivery 10 1.4.2 Polymer conjugation 10 1.4.2.1 Polymer conjugation for peptide and protein delivery 11 1.4.2.2 Proposed mechanisms for the improved biological characteristics of polymer protein conjugates 15

1.5 Soluble polymers for peptide and protein conjugation: Properties, activation methods and bioconjugate characteristics 17

1.5.1 Natural polymers for peptide and protein conjugation 17 1.5.1.1 Albumin 17 1.5.2.1 Dextran 19 1.5.2 Synthetic polymers for peptide and protein conjugation 23 1.5.2.1 Poly(ethylene glycol) (PEG) 23 1.5.2.2 Clinical applications of PEGylated peptides and proteins and products in development 29 Index

1.6 Polysialic acids: potential uses in drug delivery 32 1.6.1 Occurrence, structure and function of polysialic acids 32 1.6.2 Characteristics of polysialic acids relevant to their use in drug delivery 34 1.7 Aims and outline of thesis 35 1.7.1 Choice of polysialic acid: Colominic acid 36 1.7.2 Coupling strategy for polysialylation of peptides and proteins 36 1.7.3 Choice of peptides and proteins: Properties and clinical applications 37 1.7.3.1 Immunoglobulin G (IgG) 37 1.7.3.2 Aprotinin 38 1.7.3.3 Insulin 39 1.7.3.4 Somatostatin 40 1.7.3.5 Catalase 40

Chapter Two: General materials and methods 41 2.1 Materials 42 2.1.1 Polysialic acid, peptides and proteins 42 2.1.2 Deionised water 42 2.1.3 General materials 43 2.2 Methods 45 2.2.1 Quantitative estimation of total sialic acid 45 2.2.1.1 Resorcinol method 45 2.2.2 Quantitative estimation of total protein 45

2.2.2.1 Absorbance at 405nm {A405) 45 2 2 2 2 Dye-binding assay 46 2.2.3 Protein jfractionation 46 2.2.3.1 Size Exclusion Chromatography (SEC) 46 2.2.4 Analytical electrophoresis 48 2.2.4.1 SDS polyacrylamide electrophoresis (SDS-PAGE; denaturing conditions) 48

2.2.4.1.2 Equipment and procedures required for SDS polyacrylamide gel electrophoresis (SDS-PAGE) 49

VI Index

1.2A.2 Native PAGE (non-denaturing conditions) 53 2.2.5 Radiolabelling techniques 53 2.2.5.1 Tritiation of colominic acid with sodium boro[^H]hydride 55 2.2.5.2 radiolabelling of peptides and proteins by the Chloramine-T method 55

2.2.5.3 radiolabelling of peptides and proteins by the lodo-Gen method 56 2.2.5.4 Purification, labelling efficiency and specific activity of radiolabelled peptides and proteins 56

2.2.6 Handling and waste disposal 57 2.2.7 Dialysis and concentration of proteins 57 2.2.8 Lyophilization (Freeze-drying) 58

Chapter Three: Synthesis & Characterisation of polysialylated peptides and proteins 59 3.1 Introduction 60 3.2 Materials and Methods 62 3.2.1 Materials 62 3.2.2 Methods 63 3.2.2.1 Controlled periodate oxidation of colominic acid 63 3.2.2.2 Characterisation of colominic acid and periodate oxidised colominic acid by size exclusion chromatography (SEC) 63

3.2.2.3 Spectroscopic and analytical methods employed to identify the aldehyde fimctionality of oxidised colominic acid 64

3.2.2.4 Fluorescamine: A fluorogenic reagent for the detection and quantification of polysialylated L-lysine 65

3.2.2.5 Synthesis and size exclusion chromatographic (SEC) characterisation of polysialylated L-lysine 65

3.2.2.6 Oligomers of colominic acid prepared by hydrolysis 66 3.2.2.7 Synthesis of polysialylated peptide and protein therapeutics 67 3.2.2.7.1 Synthesis of polysialylated immunoglobulin G (IgG) 68

Vll Index

3.2.2.7.2 Synthesis of polysialylated aprotinin 69 3.2.2.7.3 Synthesis of polysialylated insulin 70 3.2.2.7.4 Synthesis of polysialylated somatostatin 71 3.2.2.S Ammonium sulphate fractionation: a method for isolating polysialylated peptides and proteins 72

3.2.2.8.1 Optimisation of the ammonium sulphate precipitation methodology 72 3.2.2.5.2 Application of ammonium sulphate to isolate polysialylated peptides and proteins 73

3.2.2.9 Evaluation of the properties of colominic acid and the polysialylated proteins 74

3.2.2.9.1 Effects of varying pH on colominic acid degradation 74 3.2.2.9.2 Effect of mouse plasma on colominic acid degradation 74 3.2.2.9.3 Freeze-drying and storage of native and polysialated peptides and proteins 74

3.3 Results and Discussion 75 3.3.1 Controlled periodate oxidation 75 3.3.2 Purification of oxidised colominic acid 76 3.3.3 Characterisation of colominic acid and the oxidised derivative by size exclusion chromatography (SEC) 76

3.3.4 Application of spectroscopic and analytical methods to identify the aldehyde functionality of oxidised colominic acid 79

3.3.4.1 FT-IR spectral interpretation of colominic acid and oxidised colominic acid derivative 80

3.3.4.2 Proton NMR spectral interpretation of colominic acid and oxidised colominic acid derivative 84

3.3.4.3 Electrospray-Mass spectrum interpretation of colominic acid and oxidised colominic acid derivative 86

3.3.4.4 The 2,4-dinitrophenylhydrazine test of colominic acid and the oxidised colominic acid derivative 87

V lll Index

3.3.4.5 Aldehyde determination: Qualitative and quantitative methods 92 3.3.5 Application of the fluorescamine reagent for the detection and quantification of polysialylated L-lysine 94

3.3.5.1 Detection and quantification of the amino acid L-lysine 94 3.3.5.2 Synthesis of polysialylated L-lysine 96 3.3.5.3 The purification and isolation of polysialylated L-lysine 98 3.3.5.4 Size exclusion chromatographic (SEC) characterisation of polysialylated L-lysine 98

3.3.6 Size exclusion chromatographic (SEC) characterisation of colominic acid oligomers produced by hydrolysis 100

3.3.7 Factors affecting the degree of modification of polysialylated IgG 103 3.3.8 Size exclusion chromatographic characterisation of polysialylated IgG 111 3.3.9 Validation of ammonium sulphate precipitation as a method suitable for isolating polysialylated IgG 114

3.3.10 SDS polyacrylamide gel electrophoretic (SDS-PAGE) characterisation of polysialylated IgG 115

3.3.11 Factors affecting the degree of modification of polysialylated aprotinin 118 3.3.12 Size exclusion chromatographic (SEC) characterisation of polysialylated aprotinin 121

3.3.13 Factors affecting the degree of modification of polysialylated insulin 124 3.3.14 Size exclusion chromatographic (SEC) characterisation of polysialylated insulin 126

3.3.15 Factors affecting the degree of modification of polysialylated somatostatin 129

3.3.16 Size exclusion chromatographic (SEC) characterisation of polysialylated somatostatin 131

3.3.17 Solubility of the native and polysialylated peptide and protein therapeutics 134

IX Index

3.3.18 Properties of colominic acid and the polysialylated proteins 135 3.3.18.1 Stability of colominic acid under varying pH 136 3.3.18.2 Stability of colominic acid in mouse plasma 137 3.3.18.3 Stability of native and polysialylated peptides and proteins upon lyophilisation and storage using size exclusion chromatography 138

3.4 Conclusion 139

Chapter Four: In vitro bioactivity of polysialylated peptides and proteins 142

4.1 Introduction 143 4.2 Materials and Methods 146 4.2.1 Materials 146 4.2.2 Methods 146 4.2.2.1 Synthesis and characterisation of polysialylated IgG2a and catalase 146 4.2.2.1.1 Polysialylated IgG2a 147 4.2.2.1.2 Polysialylated catalase 148 4.2.2.2 Catalase enzyme activity assay 148 4.2.2.3 Michaelis Menten: Efficacy of catalase and polysialylated constructs 149 4.2.2.4 Sodium dodecyl sulphate (SDS): an anionic surfactant used to reversibly unfold peptides and proteins 149

4.2.2.4.1 Optimisation of the sodium dodecyl sulphate (SDS) methodology 150 4.2.2.4.2 Analysis of the effect of sodium dodecyl sulphate (SDS) on peptide and protein conformation by size exclusion chromatography (SEC) 151

4.2.2.5 Synthesis of SDS-modified peptide and protein conjugates with colominic acid 152

4.2.2.6 In vitro stability and biological activity studies 153 4.2.2.6.1 Enzyme-Linked Immuno-Sorbent Assay (ELISA) 154 4.2.2.6.2 The trypsin-kallikrein inhibitory activity assay of aprotinin 156 4.2.2.7 Freeze-drying 158 4.2.2.8 Statistical analysis 158 Index

4.3 Results and Discussion 159 4.3.1 Preparation of polysialylated IgG2a 159 4.3.2 Size exclusion chromatographic characterisation of polysialylated IgG2a 161

4.3.3 Preparation of polysialylated catalase 163 4.3.4 Size exclusion chromatographic characterisation of polysialylated catalase 165

4.3.5 Catalase: Spectrophotometric characterisation and quantification 167 4.3.6 The influence of sodium dodecyl sulphate (SDS) on catalase inactivation and conformation of other peptides and proteins 170

4.3.6.1 Effects of varying concentrations of sodium dodecyl sulphate (SDS) on catalase activity 170

4.3.6.2 Determination of the optimal concentration of sodium dodecyl sulphate (SDS) resulting in the reversible inactivation of catalase 173

4.3.6.3 Application of size exclusion chromatography for the study of peptide and protein conformation 175

4.3.6.3.1 Analysis of the effect of sodium dodecyl sulphate (SDS) on the conformation of IgG by size exclusion chromatography 176

4.3.6.3.2 Analysis of the effect of sodium dodecyl sulphate (SDS) on the conformation of aprotinin by size exclusion chromatography 178

4.3.6.3.2 Analysis of the effect of sodium dodecyl sulphate (SDS) on the conformation of bovine serum albumin (BSA) by size exclusion chromatography 180

4.3.7 Degree of polysialylation of the SDS-treated peptides and proteins 184 4.3.8 Size exclusion chromatographic characterisation of polysialylated SDS-modified peptides and proteins 189

4.3.9 Electrophoretic characterisation of IgG and aprotinin modified by two methods of polysialylation 196

XI Index

4.3.9.1 SDS polyacrylamide gel electrophoresis of IgG modified by two methods of polysialylation 196

4.5.9.2 SDS polyacrylamide gel electrophoresis of aprotinin modified by two methods of polysialylation 198

4.3.10 In vitro stability and biological activity studies of catalase, IgG2a and aprotinin modified by two methods of polysialylation 200

4.3.10.1 Determination of catalase stability during the conjugation process 201 4.3.10.2 Michaelis Menten: Catalase enzyme kinetics 208 4.3.10.3 Antibody binding properties of native IgG2a and IgG2a modified by two methods of polysialylation 211

4.3.10.4 Inhibitory activity of aprotinin and aprotinin modified by two methods of polysialylation 214

4.3.11 Effect of fi*eeze-drying and storage on the biological properties of polysialylated peptides and proteins 217

4.3.11.1 Catalase 217 4.3.11.2 IgG2a 219 4.3.11.3 Aprotinin 219 4.4 Conclusions 221

Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates 224 5.1 Introduction 225 5.2 Materials and Methods 227 5.2.1 Materials 227 5.2.2 Animals 227 5.2.3 Methods 227 5.2.3.1 Radioiodination of peptides and proteins by the cbloramine-T method 227 5.232 Radioiodination of IgG by the lodo-Gen method 228 5.2.3.3 Synthesis of radioiodinated polysialylated and polysialylated SDS-modified bioconjugates 229

5.2.3.3.1 ^^^I-labelled polysialylated peptide and protein conjugates 229

X ll Index

5 2 3 3 2^^^I-labelled polysialylated SDS-modified peptide and protein conjugates 230

5.2.3.4 In vivo plasma clearance study of ^^^I-labelled native, polysialylated and polysialylated SDS-modified peptides and proteins 231

5.2.3.5 Estimation of pharmacokinetic parameters for native, polysialylated and polysialylated SDS-modified peptides and proteins after intravenous injection 231

5.2.3.6 Statistical analysis 233 5.3 Results and Discussion 235 5.3.1 Radioiodination of peptide and protein tracers for in vivo plasma clearance studies 235

5.3.2 Radioiodination of polysialylated and polysialylated SDS-modified peptide and protein conjugates 238

5.3.3 Size exclusion chromatographic (SEC) characterisation of radioiodinated polysialylated and polysialylated SDS-modified peptide and protein conjugates 240

5.3.4 Pharmacokinetic analysis 244 5.3.4.1 Immunoglobulin G (IgG) 244 5.3.4.2 Aprotinin 251 5.3.4.3 Insulin 256 5.4 Conclusions 262

Chapter Six: Discussion and Conclusions 264 6.1 General discussion 265 6.2 Conclusions 272 6.3 Further work 273 6.4 Future prospects 276

References 278 Publications 320 Appendices 322

X lll Index

List of Figures Page No.

Chapter One:

Figure 1,1 Transvascular exchange. Transport pathways in capillary endothelium 7

Figure 1.2 Proposed model to explain the biological properties and increased half-life of soluble polymer-protein adducts 15

Figure 1.3 Structures of polyethylene glycol (PEG) and some derivatives commonly used in peptide and protein modification 24

Figure 1.4 Structure of colominic acid (CA) 33

Figure 1.5 Two-step reaction scheme for the conjugation of peptides and proteins to colominic acid 36

Figure 1.6 Schematic model of an antibody molecule showing the two Identical light and heavy polypeptide chains linked together by disulphide bonds and the position of the antigen-binding sites 38

Figure 1.7 Structure of aprotinin showing active centre at Lysl5 and disulphide Bridges at cys5-55, cysl4-38 and cys30-51 39

Figure 1.8 Structure of bovine insulin indicating the two-polypeptide chains joined by disulphide bonds 39

Figure 1.9 Structure of somatostatin-14, showing disulphide bridge at cys3-14 and cyclic nature 40

Chapter Two:

Figure 2.1 Reduction of the aldehyde moiety at C7 via sodium boro[^H]hydride 54

Figure 2.2 Electrophilic addition of cationic iodine (I^) to tyrosine residues via Na^^^I to incorporate the isotope into the peptide or protein 54

Chapter Three:

Figure 3.1 Elution profile of CA and oxidised CA 78

Figure 3.2 Structure of colominic acid showing aldehyde fimctionality 79

XIV Index

Figure 3.3 Selective oxidation of the vicinal hydroxyl groups of CA to introduce aldehyde functionality 80

Figure 3.4 Rearrangement of the reactive aldehyde of CA showing the formation of a hemiacetal 83

Figure 3.5 A reaction of 2,4-dinitrophenylhydrazine with the aldehyde of periodate oxidised CA 88

Figure 3.6 2,4-dinitrophenylhydrazine assay of colominic acid and oxidised colominic acid derivative 89

Figure 3.7 Schematic diagrams representing the keto-enol equilibrium and the reaction of 2,4 dinitrophenylhydrazine reagent with colominic acid 90

Figure 3.8 Schematic diagrams showing the ‘naked’ reactive aldehyde of oxidised CA in equilibrium with the hemiacetal in solution and the reaction of 2,4 DNP reagent with the ‘naked’ reactive aldehyde (carbonyl) group 91

Figure 3.9 Reaction of fluorescamine with CA linked amino acid, L-lysine 94

Figure 3.10 Typical calibration curve obtained for L-lysine 96

Figure 3.11 Size exclusion chromatography of colominic acid and L-lysine 99

Figure 3.12 Elution profiles of CA and the depolymerised derivatives of CA, denoted as CA$, CAg and NeuSAc 101

Figure 3.13 Plot of the ratio Ve/Vo (elution volume (Ve) over (Vo) void volume) versus log molecular weight (Mr) for standard proteins 102

Figure 3.14 Molar ratios of colominic acid: IgG in the conjugates of IgG with different MWs of CA as a function of time 104

Figure 3.15 Molar ratios of non-oxidised colominic acid: IgG in the conjugates of IgG with different MWs of CA as a function of time 106

Figure 3.16 Size exclusion chromatography of colominic acid and IgG 112

Figure 3.17 Size exclusion chromatography of the oligomer colominic acid (CAs) and IgG 113

Figure 3.18 SDS-PAGE of the zero hour and native IgG reaction mixtures (controls) against the low molecular weight markers (BioRad) 116

XV Index

Figure 3.19 SDS-PAGE of the 0, 12, 24 and 48 hour polysialylated IgG aliquots from experiment 1.0, (section 3.2.2.7.1) against the low molecular weight markers (BioRad) 117

Figure 3.20 Colominic acid (CA): aprotinin molar ratios in the conjugates of aprotinin with CA 119

Figure 3.21 Size exclusion chromatography of colominic acid and aprotinin 123

Figure 3.22 Colominic acid (CA): insulin molar ratios in the conjugates of insulin with CA 124

Figure 3.23 Size exclusion chromatography of colominic acid and insulin 128

Figure 3.24 Colominic acid (CA): somatostatin (SS) molar ratios in the conjugates of somatostatin with CA 130

Figure 3.25 Size exclusion chromatography of colominic acid and somatostatin (SS) 132

Figure 3.26 Stability of CA after incubation in PBS (pH 7.4), 0.75M (K2HPO4) dipotassium hydrogen phosphate (pH 6.4) and 0.75M K2HPO4 (pH 9.0) 136

Figure 3.27 Stability of CA after incubation in PBS (pH 7.4) and fresh scintillation fluid and measured for radioactivity (tritium) 137

Chapter Four:

Figure 4.1 Size exclusion chromatography of colominic acid and IgG2a 162

Figure 4.2 Colominic acid (CA): catalase molar ratios in the conjugates of catalase with CA 163

Figure 4.3 Size exclusion chromatography of colominic acid and catalase 166

Figure 4.4 Calibration curve obtained for catalase standard solutions. 167

Figure 4.5 UV-visible spectra of native, polysialylated and SDS-modified polysialylated catalase. 168

Figure 4.6 Spectrum showing the decomposition of H 2 O2 per min by native catalase (control) diluted (0.03mg/ml) in 0.05M phosphate buffer, pH 7.0. 171

Figure 4.7 Catalase enzyme inhibition by different concentrations of SDS. 172

XVI Index

Figure 4.8 Comparison of the percentage of catalase enzyme retention during exposure to different concentrations of SDS and after extensive dialysis to remove SDS. 174

Figure 4.9 Chromatographic elution profiles for native IgG, IgG in 1 .OmM SDS and IgG in 3.12mM SDS, before and after dialysis. 176

Figure 4.10 Chromatographic elution profiles for native aprotinin, aprotinin in 1 .OmM SDS and aprotinin in 3.12mM SDS, before and after dialysis. 179

Figure 4.11 Chromatographic elution profiles for native BSA, BSA in 1 .OmM SDS and BSA in 3.12mM SDS before and after dialysis. 180

Figure 4.12 Elution profile of native CA av. mol. wt. lOKDa dissolved (0.4mg/ml) in 0.15M PBS, pH 7.4 and CA av. mol. wt. lOKDa dissolved in 0.15M PBS, pH 7.4 containing 1 .OmM SDS. 182

Figure 4.13 Size exclusion chromatography of colominic acid and SDS-modified IgG after A) zero time and B) 48 hours of reaction. 190

Figure 4.14 Size exclusion chromatography of colominic acid and SDS-modified IgG2a after A) zero time and B) 48 hours of reaction. 191

Figure 4.15 Size exclusion chromatography of colominic acid and SDS-modified catalase after A) zero time and B) 48 hours of reaction. 192

Figure 4.16 Size exclusion chromatography of colominic acid and SDS-modified aprotinin after A) zero time and B) 48 hours of reaction. 193

Figure 4.17 Size exclusion chromatography of colominic acid and SDS-modified insulin after A) zero time and B) 48 hours of reaction. 194

Figure 4.18 Size exclusion chromatography of colominic acid and SDS-modified somatostatin (SS) after A) zero time and B) 48 hours of reaction. 195

Figure 4.19 SDS-PAGE of native IgG and IgG modified by two methods of polysialylation against low molecular weight markers (BioRad). 197

Figure 4.20 SDS-PAGE of native aprotinin and aprotinin modified by two methods of polysialylation against a combination of low and ultra-low molecular weight markers (BioRad). 199

Figure 4.21 Spectra showing the decomposition of 30% H 2 O2 per min by catalase in the absence of CA Al) zero time (control) and Bl) after 48 hours reaction. 201

x v ii Index

Figure 4.22 Spectra showing the decomposition of 30% H 2 O2 per min by polysialylated catalase at A2) zero time (control) and B2) after 48 hours reaction. 202

Figure 4.23 Spectra showing the decomposition of 30% H 2 O2 per min by polysialylated SDS-modified catalase at A3) zero time (control) and B3) after 48 hours reaction. 202

Figure 4.24 Loss of enzyme activity of catalase and catalase conjugates during the coupling process. 204

Figure 4.25 Hanes-Woolf Plot for the H 2 O2 reaction catalysed by native catalase, polysialylated SDS-modified catalase and polysialylated catalase 209

Figure 4.26 Enzyme-linked immuno-sorbent assay (ELISA) titration curve 212

Chapter Five:

Figure 5.1 Elution profiles of radioiodinated IgG, aprotinin and insulin prepared by the chloramine-T method. 235

Figure 5.2 Elution profile of ^^^I-labelled IgG prepared by the lodo-Gen method. 236

Figure 5.3 Size exclusion chromatography of iodinated IgG compounds. 241

Figure 5.4 Size exclusion chromatography of iodinated aprotinin compounds. 242

Figure 5.5 Size exclusion chromatography of iodinated insulin compounds. 243

Figure 5.6 Clearance of chloramine-T labelled IgG and polysialylated conjugates from circulation of intravenously injected mice. 244

Figure 5.7 Clearance of lodo-Gen labelled IgG and polysialylated conjugates from circulation of intravenously injected mice. 245

Figure 5.8 Clearance of chloramine-T labelled aprotinin and polysialylated conjugates from circulation. 252

Figure 5.9 Clearance of chloramine-T labelled insulin and polysialylated conjugates from circulation. 257

XVlll ______Index

List of Tables Page No.

Chapter One:

Table 1.1 Human recombinant proteins and vaccines approved by the Food and Drug Administration (FDA) for therapeutic use. 3

Table 1.2 Approved monoclonal antibodies. 4

Table 1.3 Clearance mechanisms for peptide and proteins as a function of molecular weight (MW). 9

Table 1.4 Examples of natural and synthetic polymer-protein conjugates. 12

Table 1.5 Examples of polyethylene glycol (PEG)-protein conjugates. 14

Table 1.6 Circulating half-lives of peptides and proteins modified with dextran of different molecular weights. 21

Table 1.7 Influence of PEGylation on pharmacokinetics and pharmacodynamics of therapeutic proteins. 27

Table 1.8 PEG-protein conjugates on the market or in clinical development. 30

Chapter Two:

Table 2.1 Properties and sources of polysialic acid, peptides and proteins utilised. 42

Table 2.2 General materials used in Chapter Two. 43

Table 2.3 Composition of SDS -poly acryl amide gels (Laemmli system). 50

Table 2.4 Composition of high molecular weight markers. 51

Table 2.5 Composition of low molecular weight markers. 52

Table 2.6 Composition of ultra-low molecular weight markers. 52

Chapter Three:

Table 3.1 Materials used in Chapter 3. 62

Table 3.2 The degree of polysialylation of L-lysine. 97

XIX ______Index

T able 3.3 The degree of polysialylation of IgG. 107

Table 3.4 The degree of polysialylation of aprotinin. 120

Table 3.5 The degree of polysialylation of insulin. 125

Table 3.6 The degree of polysialylation of somatostatin (SS). 130

Chapter Four:

Table 4.1 Materials used in Chapter 4. 146

Table 4.2 Degree of polysialylation of Ig02a. 159

Table 4.3 Degree of polysialylation of catalase. 164

Table 4.4 Kinetic data obtained from the decomposition of 30% H 2 O2 per min at 20°C by native catalase in the absence and presence of different concentrations of SDS. 171

Table 4.5 Degree of modification of six therapeutic peptides and proteins by polysialylation and a modified polysialylation procedure. 185

Table 4.6 Data derived from the decomposition of 30% H 2 O2 per min at 20°C by catalase reacted with non-oxidised CA taken at time intervals of the reaction. 203

Table 4.7 Data derived from the decomposition of 30% H 2 O2 per min at 20°C by polysialylated catalase taken at time intervals of the reaction. 203

Table 4.8 Data derived from the decomposition of 30% H 2 O2 per min at 20°C by polysialylated SDS modified catalase taken at time intervals of the reaction. 203

Table 4.10 Kinetic data determined from the decomposition of varying substrate [S] concentrations of 10, 20, 30 and 40 mM H 2 O2 per min at 20°C by catalase reacted with no CA after 48 hours. 208

XX Index

Table 4.13 Comparison between degree of modification and kinetic properties such as Km and Vmax of catalase conjugates prepared by two methods of polysialylation and native catalase. 210

Chapter Five:

Table 5.1 Materials used in Chapter 5. 227

Table 5.2 Specific activities of iodinated polysialylated and polysialylated SDS-modified IgG, aprotinin and insulin conjugates. 239

Table 5.3 Pharmacokinetic parameters derived using the MW/Pharm program for chloramine-T labelled native and polysialylated IgG conjugates after intravenous administration. 246

Table 5.4 Pharmacokinetic parameters derived using the MW/Pharm program for lodo-Gen labelled native and polysialylated IgG conjugates after intravenous administration. 247

Table 5.5 Pharmacokinetic parameters derived using the MW/Pharm program for native and polysialylated aprotinin conjugates after intravenous administration. 253

Table 5.6 Pharmacokinetic parameters derived using the MW/Pharm program for native and polysialylated insulin conjugates after intravenous administration. 258

XXI Index

Chapter Six:

Table 6.1 Physicochemical properties of the peptides and proteins used in this thesis. 267

Table 6.2 Degree of modification, residual biological activity and pharmacokinetic evaluation of peptide and protein conjugates prepared in this thesis via two methods of polysialylation. 271

xxii ______Index

List of Spectra Page No.

Chapter Three:

Spectrum 3.1 Fourier Transform-Infrared (FT-IR) Spectra of colominic acid oxidised colominic acid. 81

Spectrum 3.2 ^H-NMR of colomininc acid oxidised colominic acid. 85

Spectrum 3.3 Electrospray-Mass Spectra of colominic acid. 86

Spectrum 3.4 Electrospray-Mass Spectra of oxidised colominic acid. 87

X X lll Chapter One

General introduction Chapter One: General introduction

1.1 The use of peptides and proteins as pharmaceuticals

The potential value of peptides and proteins as pharmaceuticals has long been recognised. Historically, most therapeutic peptides and proteins were obtained from non-human sources, which produce adverse immunological responses if injected into humans (Inada et al., 1995). During the last two decades, the use of peptide, protein and antibody-based drugs as human therapeutics has flourished, mainly due to the advent of recombinant DNA (rDNA) and monoclonal antibody technology (Sheffield, 2001). This milestone in biotechnology has facilitated large-scale manufacturing of human recombinant proteins and led to the development of peptide and protein pharmaceuticals for clinical trials and ultimately as therapeutics approved by the Food and Drug Administration (FDA).

Peptide and protein pharmaceuticals currently occupy a unique place in the armamentarium of drugs available for the treatment of various human diseases (Latham, 1999). To date, more than 80 polypeptide drugs are marketed in the United States, (Milton Harris and Chess, 2003) and 500 more are undergoing clinical trials. (Roberts et al., 2002). Human insulin was the first FDA-approved peptide derived from rDNA techniques in 1982. It has since been used successfully in the treatment of insulin-dependent diabetes and shown to be less immunogenic than non-human derived insulin (Sindelar, 1997). Table 1.1 illustrates a few of the human recombinant proteins and vaccines that have already received approval by the FDA for clinical use. These protein pharmaceuticals include hormones, enzymes, cytokines, haematopoietic growth factors, blood clotting factors, and vaccines. Many possess a broad range of potential clinical benefits, with applications in some of the most dire and prevalent disease categories (Sindelar, 1997). For instance, cytokines such as interferons and interleukins are currently being used effectively for AIDS and cancer related treatments (Sindelar, 1997). Other interesting examples of rDNA-derived peptides and proteins under evaluation include, the antioxidant enzyme superoxide dismutase (SOD), which is potentially useful for the treatment of myocardial infarction, organ transplantation and stroke (Veronese et al., 2002). SOD is one among a repertoire of therapeutic enzymes that have been reviewed for their potential use in enzyme Chapter One: General introduction

Table 1.1 Human recombinant proteins and vaccines approved by the Food and Drug Administration (FDA) for therapeutic use. Updated from Sindelar (1997).

Protein Trade name (Company) Indication tPA Activase (Genentech) Myocardial infarction* Human Growth hormone (hGH) Humatrope (Eli Lilly) Growth deficiency* Insulin Humulin (Eli Lilly) Diabetes* Granulocyte colony- stimulating Neupogen (Amgen) Chemotherapy-induced factor (G-CSF) neutropenia* GM-CSF Leukine (Immunex) Bone marrow transplant* Interferon alpha-2a Roferon-A Leukaemia* and AIDS, (Hoffrnann-LaRoche) Kaposi’s sarcoma treatment Interferon alpha-2b Intron-A Leukaemia*, Genital warts. (Schering-Plough) Hepatitis B and C Interferon beta-lb Betaseron (Berlex) Multiple sclerosis* Interferon gamma-lb Actimmune (Genentech) Granulomatous disease* Interleukin-2 Proleukin (Chiron) Renal cell carcinoma* Antihaemophiliac factor VIII Recombinate (Baxter) Haemophilia A* Hepatitis B vaccine Energix-B Hepatitis B* (SmithKline-Beecham) Erythropoietin (EPO) Epogen (Amgen) Anaemia* Deoxyribonuclease I Pulmozyme (Genentech) Cystic fibrosis* * Denotes first approved indication(s); tPA, tissue-type plasminogen activator; GM-CSF, granulocyte macrophage colony-stimulating factor. deficiency syndromes (Veronese and Morpurgo, 1999). Haemoglobin is also under evaluation as a potential oxygen carrying blood substitute (Torres and Vazquez- Duhalt, 2000). To date, 20% of all biopharmaceuticals in clinical trials are monoclonal antibodies (Mabs), making this the second largest biopharmaceutical product category after vaccines (Chapman, 2002). Therapeutic Mabs function by three principle modes of action: blocking the action of specific molecules, targeting specific cells or acting as signalling molecules. There are approximately 200 humanised and chimeric (partially human) Mabs under development and several have been approved by the US FDA for the treatment of cancer (Farah et al., 1998), transplant rejection (Berard, 1999), rheumatoid arthritis, Crohn’s disease (Maini et al., 1999; Sandbom and Chapter One: General introduction

Table 1.2 Approved monoclonal antibodies. Most are humanised unless otherwise stated and *represents chimeric antibodies. Updated from Brekke and Sandlie (2003).

Year Product Type of molecule Indication Company approved Rituxan anti-CD20 * Non HL 1997 IDEC Pharma Corp Zenapax anti-IL-2 receptor Unstable angina 1997 Centocor Inc Herceptin anti-ERBB2 Breast cancer 1998 Genentech Inc Remicade anti-TNF-a * Crohn’s disease 1998 Centocor Inc Simulect anti-IL-2 receptor * Transplant rejection 1998 Novartis AG Synagis anti-F-protein RSVD 1998 Medimmune Inc Enbrel anti -TNF-a receptor RA, PA 1998 Amgen Mylotarg anti-CD33 Myeloid leukaemia 2000 Celltech Group pic Campath anti-CD52 B-cell CLL 2001 Millennium Pharma (Ilex Oncology Inc) Zevalin Mouse anti-CD20 B-cell Non HL 2002 IDEC Pharma Corp Humira anti-TNF-a receptor RA 2002 Abbott Laboratories Non-HL, Non-Hodgkin’s lymphoma; RSVD, respiratory syncitial viral disease; RA, rheumatoid arthritis; PA, psoriatic arthritis; B-cell CLL, B-cell chronic lymphocytic leukaemia; B-cell Non-HL, B-cell Non-Hodgkin’s lymphoma.

Hanauer, 1999), and antiviral prophylaxis (Saez-Llorens et al., 1998). Table 1.2 illustrates examples of the clinical successes, which underscore the new age of Mab therapeutics. Mabs and indeed smaller formats such as Fab or Fv fragments have been exploited for tumour targeting and their diagnostic potential. Indeed, Mab moieties have been coupled to radioisotopes (e.g. Zevalin and Mylotarg), cytotoxic drugs (Colcher et al., 1998) and drug delivery systems (Gregoriadis, 1981) on account of their versatile and highly specific nature.

In comparison with small chemical drugs, protein pharmaceuticals have high specificity and activity at low concentrations. These properties have made protein and antibody pharmaceuticals indispensable in combating human disease. Due to advances in analytical separation technology, recombinant proteins can now be purified to an unprecedented level thereby significantly reducing any potential side or toxic effects (Sheffield, 2001). Chapter One: General introduction

1.2 Problems associated with the clinical use of peptide and protein pharmaceuticals

Despite the tremendous advances made in the development of peptide and protein pharmaceuticals, these biopharmaceuticals posses several shortcomings that are typical of polypeptide therapeutics which limit their clinical usefulness (Milton Harris and Chess, 2003). Firstly, formulation of peptide and protein-based pharmaceuticals is largely hampered by their chemical and physical instability (Wang, 1999). Chemical instability results from peptide bond formation or cleavage via processes such as proteolysis, oxidation, racemization, deamination and disulphide exchange thereby yielding a new chemical entity. Physical instability involves changes in their secondary structure due to dénaturation, adsorption to surfaces, non-covalent self aggregation and precipitation. Changes caused by either type of instability often result in the consequent loss of biological activity. In an attempt to achieve an acceptable shelf life, peptide and protein pharmaceuticals are often stored under cold conditions in solution, suspension, dry powder state or frozen (Wang, 1999). A variety of stabilisation strategies including lyophilisation, spray-drying (Carpenter and Crowe, 1989) and use of cryo-and lyoprotectants e.g. sugars, polyols, polymers and proteins (Wang, 1999) exist, however owing to the unique characteristics of peptides and proteins these strategies are not universally applicable and need to be evaluated on an individual basis.

Secondly, administration of peptide and protein biopharmaceuticals via routes such as nasal, buccal, pulmonary, ocular, rectal, transdermal and vaginal are reported to be less than satisfactory on account of low bioavailability and high cost of polypeptide drugs (Lee, 1991). Consequently, most polypeptide drugs are administered by injection, either subcutaneously or intravenously (Roberts et al., 2002). Unfortunately, parenteral administration of therapeutic peptides and proteins is also sub optimal due to their susceptibility to proteolytic degradation, short circulating half-life, low solubility, rapid kidney clearance and propensity to generate neutralizing antibodies (Milton Harris and Chess, 2003). Furthermore, administration of large and frequent doses of polypeptide drugs, that are necessitated to maintain therapeutic efficacy, can Chapter One: General introduction result in potentially toxic effects (Bocci, 1990). Although it was anticipated that the production of human recombinant DNA-derived therapeutic peptides and proteins would alleviate the problems associated with immunogenicity and antigenicity, the problems of short circulating life and poor stability still existed thus resulting in the same vicious cycle ending in a lack of efficacy. Contaminants and different or unwanted glycosylation patterns arising from the host in which the recombinant peptides and proteins were produced in i.e. bacterial or mammalian cells and yeast cells are also capable of raising an immune response. Moreover, expression of a cloned human gene in a mammalian cell is no guarantee of homology with the human counterpart (Bocci, 1990; Goddard, 1991). Indeed, it has not been established beyond any reasonable doubt that human-derived peptides and proteins are themselves non- immunogenic in humans (Davis et al., 1991). It is imperative therefore that each recombinant peptide or protein is carefully screened for any toxic or immunogenic properties before it can be developed as a potential pharmaceutical candidate (Goddard, 1991). Finally, a further disadvantage of the parenteral route of administration of peptides and proteins is the systemic effect of the drug as opposed to localised action. These inadequacies constitute a major dilemma in the development of polypeptide drugs. Chapter One: General introduction

1.3 Clearance mechanisms of peptide and protein pharmaceuticals

As previously discussed (section 1.2), many peptide and protein therapeutics are removed from the circulation prematurely and before effective concentrations in the blood or target tissues can be achieved (Gregoriadis et al., 1999). In order to develop and improve polypeptide drug design and therapeutic index, knowledge of their clearance mechanisms are essential. Extensive reviews on the clearance mechanisms of therapeutic peptide and protein agents have been reviewed (Braeckman, 2000,

Kompella and Lee, 1991) and will be discussed briefly here.

1.3.1 Distribution and catabolism of peptide and protein pharmaceuticals The rapid clearance of proteins from plasma can be explained by a combination of transendothelial passage, proteolysis, receptor-mediated uptake, non-specific uptake by the mononuclear-phagocyte system (MPS) and glomerular filtration (Braeckman, 2000). Once in the systemic circulation, clearance of proteins generally begins

(1) (5a) (2) (3a)

Continuous Capillary m Muscles, CNS, lungs, subcutis and bones (1) (5aX5b)(2) (4a)

Fenestrated Capillary

Endocrine glands, kidney Intestinal villi, skin, (1) y 5a) (5b) (3b) synovial tissue

Discontinuous ^=f=vir~ Capillary

Liver, spleen, bone marrow and lymph nodes

Figure 1.1 Trans vascular exchange. Transport pathways in capillary endothelium. (1) endothelial cell, (2) lateral membrane diffusion, (3) interendothelial junctions: (a) narrow, (b) wide, (4) endothelial fenestrae: (a) closed, (b) open, (5) vesicular transport: (a) transcytosis, (b) transendothelial channels. Reproduced from Jain (1987). Chapter One: General introduction

with passage across the capillary endothelia (Kompella and Lee, 1991). The transendothelial passage of peptides and proteins is determined by the physiochemical properties of the molecule such as size, shape and charge (Brenner et al., 1978), and by the ultrastructure and physicochemical characteristics of the capillaries themselves (Kompella and Lee, 1991). Figure 1.1 shows the three types of capillary endothelia in increasing order of permeability: continuous (non-fenestrated), fenestrated and discontinuous (sinusoidal) and the organs they are typically associated with. The most permeable of all microvascular beds are found in the liver, spleen, bone marrow and postcapillary venules of lymph nodes, which possess capillaries with discontinuous endothelium allowing bulk transfer of peptides and proteins. Renal glomeruli, intestinal villi, skin, synovial tissue and endocrine glands have capillaries with a fenestrated endothelium characterised by the fenestrae, which are circular openings of about 60nm possibly closed by thin diaphragms allowing a more limited delay transfer of peptides and proteins. Finally, muscles, CNS, lungs, subcutis and bones have even less permeable capillaries because endothelial cells are in close contact and underlined by a continuous basement membrane. Besides these variable impediments, biodisposition of peptides and proteins depends upon the organ blood flow as well (Bocci, 1990).

The clearance mechanisms to which peptides and proteins are subjected in organisms are outlined in table 1.3. Small proteins (<70KDa) that are susceptible to renal clearance are eliminated via metabolism and/or excretion, whereas larger proteins (50- 400KDa) undergo processes such as receptor-mediated endocytosis, opsonisation and/or phagocytosis and elimination by the MPS in the liver and spleen. Factors such as molecular weight, charge, sugar recognition and susceptibility to proteases determine the removal of proteins from the circulation. In fact, the vast majority of therapeutic proteins are catabolised by proteolysis; the potential number of sites of catabolism is vast due to the ubiquitous nature of proteolytic enzymes (Bocci, 1987, 1990). Proteolysis can occur in plasma or at the cell surface by membrane bound proteases (Bocci, 1990). However, the most important aspect is intracellular catabolism after internalisation of the complex ligand-receptor (Langer and Pestka, 1988). Chapter One: General introduction

Table 1.3 Clearance mechanisms for peptide and proteins as a function of molecular weight (MW). Other determining factors are shape, charge, lipophilicity, functional groups, sugar recognition, vulnerability for proteases, aggregation to particles, formation of complexes with opsonisation factors, etc. The indicated mechanisms overlap and fluid-phase endocytosis can in principle occur across the entire MW range. Adapted from Meijer and Ziegler (1993).

Molecular weight Site of elimination Clearance mechanism Determinant Factor

<500 Blood Extracellular hydrolysis Liver Passive non-ionic diffusion

500-1,000 Liver Carrier-mediated uptake Structure Passive non-ionic diffusion Lipophilicity

1,000-30,000 Kidney Glomerular filtration MW

50,000-200,000 Kidney Receptor-mediated Sugar, charge Liver endocytosis 200,000-400,000 Opsonisation IgG >400,000 Phagocytosis Particle aggregation

The liver and kidney contribute significantly to the catabolism of therapeutic peptides and proteins (Bocci, 1990; Kompella and Lee, 1991). The liver is well perfused, possesses discontinuous capillaries and composed of several cell types, including hepatocytes, endothelial cells, Kupffer cells and fat-storing cells, which explains its significant contribution to the removal of macromolecules from the systemic circulation. Whilst the liver contributes significantly to the metabolism of protein therapeutics, the kidney plays a dominant role in the clearance of small proteins that are within the glomerular filtration range. Glomerular filtration and excretion is most efficient for peptides and proteins smaller than 30KDa (Kompella and Lee, 1991). Filtration of proteins is influenced by their size, shape and charge. Because the glomerular filtrate is negatively charged, anionic molecules are repelled and consequently do not filter as well as cationic ones. Once in the ultrafiltrate, peptides may be excreted unchanged in the urine or in the case of complex polypeptides and proteins actively reabsorbed by the proximal tubules by luminal endocytosis and then hydrolysed within the intracellular lysosomes to peptide fragments and amino acids (Wall and Maack, 1985). Chapter One: General introduction

1.4 Stabilisation of peptide and protein pharmaceutics: Novel approaches

It is generally accepted (Gregoriadis et al, 1999; Monfardini and Veronese, 1998) that the therapeutic and diagnostic potential of peptide and protein pharmaceuticals, could be optimised if factors responsible for their rapid clearance (described previously in section 1.3), were circumvented. To that end, a number of sophisticated drug delivery technologies are being developed to ameliorate these problems. Two of the most promising strategies involve encapsulation and covalent conjugation of peptides and proteins to hydrophilic polymers (Monfardini and Veronese, 1998).

1.4.1 Encapsulation methods for peptide and protein delivery

Therapeutic peptides and proteins have been successfully entrapped in microcapsules (Hildebrand and Tack, 2000), microspheres (Park et al., 1998) and, liposomes (Mastrobattista et al, 2002; Gregoriadis and Alison, 1974), hydrogels (Yang et al, 2002) or into biological carriers such as erythrocytes (Magnani et al, 2002). Whilst the half-life of the entrapped drug may be increased and the characteristics of the peptide or protein remain unchanged, the majority of these systems are limited as illustrated by studies of liposomes. For instance, drug delivery may not be directed to specific diseased tissues and tumours as desired, but rapidly enter the liver, spleen, kidneys and reticuloendothelial system (RES) (Milton Harris and Chess, 2003). Drug leakage whilst in the circulation (Allen, 1997) and pseudoallergic reactions that damage the heart and liver cells (Sebeni, 1998) are further concerns. Many of these deficiencies can be overcome by the potential of the so-called ‘stealth’ liposomes, other particles and discovery of new polymers (Monfardini and Veronese, 1998).

1.4.2 Polymer conjugation

The technique of coupling polymers to peptides and proteins originated in the 1950’s and 1960’s with investigations of protein structure and function by site-directed chemical modification (Davis et al, 1978). Ringsdorf (1975) made further proposals for using polymers as carriers in the mid 1970’s. Since then, numerous natural and

10 Chapter One: General introduction synthetic hydrophilic polymers have been covalently coupled to biologically active compounds such as polypeptides, enzymes and other small conventional drugs with a view to altering their pharmacokinetics, biodistribution, immunogenicity and toxicity (Veronese & Morpurgo, 1999). This approach may also beneficially alter peptide and protein solubility, stability, pH optimum, charge and susceptibility to proteolytic degradation (Monfardini and Veronese, 1998). Soluble polymer conjugates constitute a completely novel class of pharmacologically active agents which have only recently entered clinical practice. They can be subdivided into two main categories: polymer- drug and polymer-protein conjugates. To date, the latter conjugates are predominantly studied, and have been by far the most successful approach to enhance peptide and protein delivery (Veronese and Morpurgo, 1999). Whilst the development of polymer- drug conjugates is still in its infancy, much progress has been made in facilitating the controlled release and targeting of small conventional drug entities, particularly anti tumour agents (Duncan and Spreafico, 1994; Veronese and Morpurgo, 1999). In this thesis, discussion is limited to those peptide and protein conjugates under development for parenteral use.

1.4.2.1 Polymer conjugation for peptide and protein delivery To be therapeutically effective, polymer-protein conjugates should be water soluble, exhibit decreased to non-existent immunogenicity, have augmented circulatory half- lives whilst retaining a substantial portion of the original biological activity. Progress to that end has been made with differing degrees of success depending on the polymer. Table 1.4 lists examples of peptides and proteins that have been conjugated to natural polymers such as dextran, albumin, cellulose and synthetic, styrene-maleic anhydride (SMA), polyvinylpyrrolidone (PVP), poly(divinylether maleic anhydride (DIVEMA) and N-(2-Hydroxypropyl) methacrylamide copolymers (HPMA). (For reviews see Monfardini and Veronese, 1998). Interestingly, two polymer-protein conjugates have emerged as clinical products. They include, dextran-streptokinase, which has been marketed in Russia for thrombolytic therapy (Torchilin et al., 1982) and SMA-neocarzinostatin (SMANCS), which has been marketed in Japan for treating liver cancer (Maeda, 2001). The real breakthrough however in enhancing the pharmaceutical properties of peptides and proteins comes from the development of

11 Chapter One: General introduction

Table 1.4 Examples of natural and synthetic polymer-protein conjugates. Adapted from Duncan and Spreafico (1994).

Polymer Protein References Albumin Asparaginase Poznansky (1988) Dextran Aprotinin Larionova et al. (1985) Asparaginase Wileman (1991) Catalase Eremin (1996) Fab’ fragment Fagnani et al. (1995) Haemoglobin Caron e/a/. (1999) rhGH Battersby et al. (1996) Immunoglobulins Fagnani et al. (1990) Insulin Baudys et al. (1998) Soybean bowman-birk Gladysheva et al. (2001) inhibitor Streptokinase* Pautov et al. (1990) mEGF Zhao et al. (1999) Trichosanthin Chancr a/. (1999) Cellulose a -amylase Barker and Somers (1968) Polyvinylpyrrolidone (PVP) Superoxide dismutase Caliceti et al. (1995) Ribonuclease Veronese et al. (1997) N-(2-Hydroxypropyl) methacrylamide copolymers (HPMA) Acetylcholinesterase Laane et al. (1983) Chymotrypsin Lu et al. (1998) IgG Ulbrich (2000) Insulin Chytry er al. (1996) Transferrin Flanagan et al. (1992) Polydivinylether-maleic anhydride (DIVEMA) Superoxide dismutase Hirano et al. (1997) Neocarzinostatin Yamamoto et al. (1990) Styrene-maleic anhydride (SMA) Neocarzinostatin Maeda (2001) Superoxide dismutase Ogino et al. (1988) * Dextran-streptokinase has been manufactured on an industrial scale in Russia, previously the USSR, since 1980 and is approved in that country for the treatment of cardiovascular and opthalmological pathologies caused by thromboses.

12 Chapter One: General introduction

PEGylation by Davis, Abuchowski and colleagues in the 1970s. This widely acclaimed drug-delivery technology known as PEGNOLOGY^^, involves the covalent linkage of the hydrophilic polymer monomethoxypoly(ethylene glycol) (mPEG) to peptides and proteins. Polyethylene glycol (PEG) is the most widely studied polymer for peptide and protein conjugation owing to its unique set of properties (Harris and Chess, 2003), which will be discussed in detail in section 1.5.2.1. The FDA has approved PEG for various uses in pharmaceutical products and is therefore an important reason for its attractiveness. PEGylation is now well established and confers increased solubility, stability and reduced immunogenicity to the coupled peptide or protein (Harris et al., 2001). Moreover, by preventing rapid renal clearance of small proteins and receptor-mediated uptake via the RES, PEGylation can be used to augment plasma half-life (Duncan, 2003). Table 1.5 lists examples of peptides and proteins that have been modified by either first- or second- generation PEGylation methods. Whilst, the earlier first-generation PEGylation methods were fraught with difficulties (Harris and Chess, 2003), several PEGylated proteins still evolved into clinical products. The properties, chemistries and clinical applications of the first- and ‘newer’ second- generation PEG-protein conjugates will be discussed in detail in section 1.5.2.

Polymer conjugation of peptides and proteins offers numerous advantages over encapsulation techniques; however the concomitant loss of biological potency of the protein upon coupling is a major drawback. Therefore, during the last 10 years, the synthesis of polymer-protein conjugates has been optimised by the development and use of semi-telechelic polymers and employing very specific coupling chemistry and purification processes (Roberts et al., 2002). Semi-telechelic polymers posses a single reactive group at one terminal end providing a single attachment site, thus preventing cross-linking and a heterogeneous mixture of products (Duncan, 2003). Additionally, site-specific modification of proteins minimises the loss of biological activity and reduces immunogenicity (Harris and Chess, 2003). Currently, peptide and protein conjugates are not only being designed for a wide range of therapeutic uses, but also as diagnostics (Chapman, 2002) and biocatalysts (Inada et al., 1995).

13 Chapter One: General introduction Table 1.5 Examples of polyethylene glycol (PEG)-protein conjugates. Adapted from Duncan and Spreafico (1994). Class Protein Reference Enzymes Adenosine deaminase Hershfield (1997) Alkaline phosphatase Yoshinaga et al. (1987) Arginase Savoca et al. (1984) Asparaginase Soares et al. (2002) Bilirubin oxidase Kamisako et al. (1998) Catalase Lamka et al. (1995) Elastase Besson et al. (1995) a -Galactosidase Weider and Davis (1983) P - Glucuronidase Lisi et al. (1982) Lipase Hemaiz et al. (1999) Phenylalanine ammonia lyase Wieder et al. (1979) Purine nucleoside phosphorylase Hershfield et a/. (1991) Ribonuclease A Matousek et al. (2002) Streptokinase Fuke et al. (1994) Superoxide dismutase Veronese et al. (2002) Trypsin Zalipsky (1995) Urate oxidase Chen et al. (1981) Uricase Schiavon et al. (2000) Allergens Antigen E Norman et al. (1984) Ragweed allergen Wie et a/. (1981) Cardiovascular applications Haemoglobin Torres & Vazquez-Duhalt (2000) Urokinase Caliceti et al. (1994) t-Plasminogen activator Pizzo (1991) a -Thrombin Nakagomi and Ajisaka (1990) Cytokines or receptor antagonists G-CSF Kinstler et al. (2002) GM-CSF Smith et al. (1991) Interferon- a Michael et al. (2001) Interferon-2 Azanza (2001) Interleukin-1 receptor antagonist Thompson et al. (1992) Toxins Batroxobin Nishimura et al. (1985) Honeybee venom Zalipsky & Harris (1997) Other Monoclonal antibody A7 Kitamura et a/. (1991) Bovine serum albumin Abuchowski et al. (1997) Collagen Ito et al. (1997) Immunoglobulins Chapman (2002) a 2-Macroglobulin Beauchamp et al. (1983) Ovalbumin Saito et al. (1996) Insulin Hinds and Kim (2002) ______

14 Chapter One: General introduction

1.4.2.2 Proposed mechanisms for the improved biological characteristics of polymer-protein conjugates Peptides and proteins acquire many advantages after polymer derivatisation, including a plasma half-life increased by a factor of 5-500 (Monfardini and Veronese, 1998). In general, the more polymer chains attached per protein molecule, the greater the extension of the half-life (Inada et al., 1995), although specific activity may sometimes be proportionally reduced. The increased plasma half-life is due to several mechanisms. Figure 1.2 illustrates the mechanisms believed to be responsible for the improved circulatory half-life.

Active Site

Proteose

Antibody' 4 - Substrate

ENZYME r^ 4 — Product

Polymer

Antibody, Proteose,Cleoronce, or Immunogenic Recognition Sites

Figure 1.2 Proposed model to explain the biological properties and increased half-life of soluble polymer-protein adducts. Polymer modification may: (1) mask antigenic determinants; (2) mask immunogenic recognition sites; (3) mask protease susceptible sites; (4) mask clearance recognition signals; (5) allow free access of low molecular weight substrates; ( 6 ) maintain systemic injectability and (7) alter pH optimum by changing microenviroment. Adapted from Uren and Ragin (1979).

Primarily, grafting polymer chains to peptides and proteins increases the size of the conjugate. This may be beneficial for the circulatory survival of smaller peptides and proteins as their size increases beyond the glomerular filtration limit of approximately

70KDa (Harris et al., 2001). This in turn increases plasma residence time due to reduced renal excretion. In contrast, larger proteins which are above the range of renal

15 Chapter One: General introduction clearance would be expected to be cleared faster due to the probability of the conjugate being removed by the reticuloendothelial system (RES) (Monfardini and Veronese, 1998). Conjugation may decrease cellular protein clearance by reducing elimination through the reticulo-endothelial system or by specific cell-protein interactions (Brenner and Rector, 1996).

The coupled polymer chains form a ‘cloud’ around the protein, thereby shielding the epitopes and antigenic determinants thus potentially preventing the raising of antibodies against the conjugate. The polymer chains also sterically hinder the approach of pre-formed antibodies to the antigenic sites of the protein. Surface modification of peptides and proteins via polymer grafting creates a hydrophilic and hydrated surface that makes it energetically unfavourable for other molecules to approach (Tomlinson, 1991). Consequently, interactions with blood {i.e. fibrinogen, fibronectin), tissue components such as cellular receptors for carbohydrate moieties and plasma proteases that are responsible for their inactivation and removal fi*om the circulation are diminished. The net result is abrogated immunogenicity and antigenicity and diminished immune clearance of the conjugate.

Interestingly, the active sites of enzymes are still freely accessible to appropriate substrate molecules (unless the substrate is macromolecular, Abuchowski and Davis, 1979) and systemic injectabilty of the conjugate is maintained. Other new protein properties sometimes observed upon polymer modification include, increased activity, increased solubility in aqueous and organic solvents, and increased thermal stability (Funge^fl/., 1997).

16 Chapter One: General introduction

1.5 Soluble polymers for peptide and protein conjugation: Properties, activation methods and bioconjugate characteristics

The properties and activation methods of various soluble polymers (natural and synthetic) used for peptide and protein derivatisation and the bioconjugate characteristics are reviewed here. The emphasis is directed towards poly(ethylene glycol) (PEG) and its adducts, since it is the most widely used polymer for peptide and protein conjugation.

1.5.1 Natural polymers for peptide and protein conjugation

Natural polymers are easily available and biocompatible, although their preparation may be restricted by the need for several purification steps. Their use is also sometimes limited by their high immunogenicity. Nevertheless, polymers such as albumin and dextran (polysaccharide) are commonly used for the purpose of peptide and protein modification (Monfardini and Veronese, 1998).

1.5.1.1 Albumin Albumin is a natural constituent of plasma and exhibits a prolonged circulatory half- life (Davis et aL, 1991). It was thought that the conjugation of this naturally occurring protein to foreign peptides and proteins would result in conjugates that would be accepted as a normal component of the plasma (Poznansky, 1986). Albumin is an attractive choice for peptide and protein conjugation by virtue of its chemical and physical properties. For example, it is readily available, easily purified, exhibits great chemical and physical stability, is easily stored, lacks toxicity, exhibits a favourable degradation rate, shows lack of antigenicity and immunogenicity of homologous albumins and it possess at least five available reactive groups for modification. The latter include the epsilon amino groups of lysine, alpha amino groups, sulfhydryl groups of cysteine and imidazole groups of histidine. Peptide and protein modification by albumin is performed in aqueous buffer in the presence of cross-linking agents such as gluteraldehyde, carbodiimide and sodium periodate. Some loss in activity is anticipated following attachment of albumin to peptides and proteins. Whilst its

17 Chapter One: General introduction polyfunctionality allows a variety of derivatives to be prepared, it is also prone to forming intra- and intermolecular cross linkages thereby resulting in a heterogenous product.

A number of groups have reported on the successful use of albumin as a carrier. For example, albumin has been attached to uricase (Remy and Poznansky, 1978), superoxide dismutase (Wong et al., 1980), a- 1,4-glucosidase (Poznansky and Bhardwaj, 1980), asparaginase (Yagura et al, 1981; Poznansky et al, 1982), insulin (Poznansky et al, 1984), heparin (Hennink et al, 1984) and wasp venom (Gewurz et al, 1986). In each case, the attachment of albumin resulted in conjugates with improved therapeutic characteristics. For example, albumin-modified superoxide dismutase retained nearly 70% activity (Wong et al, 1980) and albumin-asparaginase retained 60% activity (Poznansky et al, 1982). The albumin conjugates collectively exhibited between 3 to 25-fold improved circulatory half-lives which more than offset the losses in activity following modification. Furthermore, cross-linked albumin conjugates were more resistant to proteolytic degradation and heat inactivation than the non conjugated protein. For example, albumin-modified uricase retained over 90% activity over a period of 8 days, while native uricase lost 40% of activity in the first 3 days (Poznansky, 1979). Albumin-asparaginase conjugates had an in vitro half-life of 20 hours in the presence of serum and 10 hours when incubated with a proteolytic enzyme. This was compared to half-lives of 1.5 and 0.5 hours respectively, for the native enzyme under similar conditions (Davis et al, 1991). The albumin conjugates did retain the ability to act in vivo on their specific substrates and repeated injections did not result in the development of hypersensitivity reactions (Davis et al, 1991). Cross-linked albumin-modified asparaginase conjugates were superior to their native counterparts in inhibiting the in vivo cell growth of the 6C3HED lymphosarcoma in mice. One hundred percent survival was increased fi*om 16 days for the native enzyme to approximately 23 days following injection of albumin-asparaginase (Poznansky et al, 1982). It should be noted that the source of albumin could alter the properties of the conjugate since antibodies are raised against the albumin moiety if the protein is of non-homologous origin (Poznansky and Remy, 1978).

18 Chapter One: General introduction

1.5.1.2 Dextran Dextran is an uncharged, hydrophilic glucose polymer, which has been used for more than 50 years as a blood plasma expander and reported to be of low toxicity (Mehvar, 2000). High molecular weight dextrans (40-110KDa) are an attractive potential macromolecular carrier for peptide and protein delivery owing to their favourable biological and physiochemical characteristics. For instance, they are biodegradable, posses a relatively narrow molecular distribution (Mehvar, 2000), exhibit long half- lives in the blood circulation (Mikolajczyk et ah, 1996), and contain a large number of hydroxyl groups available for derivatization with therapeutic peptides and proteins.

Conjugation of dextran to peptides and proteins often requires prior activation of the dextran moiety such that the reaction may proceed. One method of dextran activation involves introducing reactive imidocarbonate groups via alkylation with cyanogen bromide to yield an N-bromoacetylaminoethylamino-dextran (Chan et al., 1999). This method gained popularity for earlier conjugations of dextran with proteins such as insulin (Suzuki et al., 1972), aprotinin (Odya et al., 1978) and carboxypeptidase G 2 (Melton et al., 1987). Unfortunately, although imidocarbonate-dextran reacts in a one step reaction, it is unstable in aqueous solution and its reagents and by-products are toxic. The toxic by-products have been reported to be responsible for the great losses observed in enzyme activity (Mehvar, 2000). This process may also yield several protein-polymer conjugates with different structures (Larsen, 1989). Although these problems have in recent years lead to a decline in the use of this method, this technique was nevertheless used to successfully couple dextran to proteins such as glutathione (Kaneo et al., 1995) and more recently trichosanthin (Chan et al., 1999).

A more commonly used method for dextran activation involves periodate oxidation of the dextran vicinal diols to yield dialdehyde-dextran (Monfardini and Veronese, 1998). Examples of peptides and proteins coupled to dextran using this method include: soybean bowman-birk inhibitor (Gladysheva et al., 2001), epidermal growth factor (mEGF) (Zhao et al, 1999), Haemoglobin (Bonneaux et al., 1996), recombinant human growth hormone (rhGH) (Battersby et al., 1996), insulin (Mehvar, 1994), chymotrypsin (Torchilin et al., 1977), soybean trypsin inhibitor

19 Chapter One: General introduction

(Takakura, 1996), asparaginase (Wileman, 1991), monoclonal (Mab), polyclonal antibodies and Fab’ fragments (Fagnani et al., 1990; 1995). In contrast with imidocarbonate-dextran, dialdehyde-dextran is relatively stable in aqueous media. Although this procedure requires a later step for reductive amination, it is the preferred method for coupling dextran with proteins as the construct retains a significant percentage of its biological activity (Mehvar, 2000). More recently, the introduction of functionalised dextran derivatives such as carboxymethyl dextran (CMD) or diethylaminoethyl dextran coupled with site-specific attachment to peptides and proteins have resulted in bioconjugates with superior properties. For instance, insulin coupled to CMD in contrast with cyanogen bromide or periodate activated dextran exhibited superior bioactivity and pharmacokinetic data (Baudys et al., 1998). Aprotinin (Larionova et al., 1985) and superoxide dismutase (Fujita et al., 1992) coupled CMD also possessed increased residual activity and increased half-life in vivo.

During the last 3 decades, at least 20 or more peptides and proteins have been coupled to dextran (table 1.4). Indeed, clinical success with dextran-modified streptokinase saw the emergence of the first marketed pharmaceutical namely, Streptodekase® (Torchilin et al., 1982). A few representative examples of these biologically active conjugates with improved in vivo pharmacokinetics, increased stability and reduced immunogenic characteristics are discussed here in more detail.

Table 1.6 lists some of the peptides and proteins that have been coupled to dextran and compares their circulatory half-lives with their native counterparts. In most cases, the circulatory half-life of the peptide or protein was markedly improved upon modification with dextran. Similarly, dextran modified enzymes such as a-amylase (Marshall et al., 1977), asparaginase (Wileman et al., 1986) and catalase (Marshall et al., 1978) also exhibited increased circulatory half-lives in vivo. In some instances this was further improved by employing higher molecular weight dextran. However, a dextran of too high molecular weight may not further increase the circulatory half-life compared with a lower molecular weight dextran (Tabata et al., 1999). It is evident that modification of proteins with dextran can affect the characteristics of the adduct.

2 0 Chapter One: General introduction

Table 1.6 Circulatory half-lives of peptides and proteins modified with dextran of different molecular weights. Results in each case were obtained upon first intravenous injection. * Indicates percentage of protein remaining after 1 hour and ** indicates percentage of protein remaining after 4 hours when half-life is not reported.

MW Circulating half-life Protein Dextran Host Reference (KDa) Native Modified Trichosanthin 40 Rat 10.0 ± 1.0 min 275 ± 30 min Chan et a l (1999) mEGF 13 Rat 0.68 ±0.18 min 1.97 ± 0.37 min Zhao et al (1999) 46 0.68 ± 0.18 min 5.14 ± 1.15 min Mab (intact) 6.0 Mice 27 %** 33 %** Fagnani et a l (1990) Fab’ fi-agment 6.0 Mice 2.0 hr 15.0 hr Fagnani et a l (1995) Glutathione 40 Mice 5.0 ± 2.0 min 1.45 ±0.01 hr Kaneo et a l (1995) Insulin 51 Rat 12.4 ± 3.4 min 114.1±28.6min Baudys et a l (1998) Aprotinin 60 Rat 2-3 %* 55 %* Larionova et al (1985) TNF-a 74 Mice 5.70 ± 1.57%* 26.3 ± 1.51%* Tabata et a l (1999) 208 5.70 ± 1.57%* 26.8 ± 1.12%* mEGF, mouse epidermal growth factor; Mab, monoclonal antibody, TNF-a, tumour necrosis factor.

Indeed, some activity loss is noted following modification of proteins with dextran, the degree of which can depend on the molecular weight and amount of dextran coupled to the peptide or protein. For instance, dextran-modified trichosanthin (Chan et ai, 1999), aprotinin (Larionova et al, 1985), TNF-a (Tabata et al, 1999), IgG (Fagnani et al, 1990) and Fab’ fi-agment (Fagnani et al, 1995), retained 50%, 56%, 50%, 58% and 35% activity respectively. Conversely, the blood glucose lowering effect of dextran-modified insulin upon intravenous administration was found to be identical to its native counterpart (Baudys et al, 1998). Similarly, soybean bowman- birk inhibitor (BBI) coupled with dextran exhibited practically no decrease in the antiproteolytic activity of the inhibitor compared with free BBI (Gladysheva et al, 2001). Furthermore, mice injected with a hepatotoxic dose of acetaminophen exhibited a 46% survival rate after 30 days with co-administration of native glutathione, compared with an 83% survival rate with dextran-modified glutathione (Kaneo et al, 1994). Several studies (Larionova et al, 1985; Kaneo et al, 1994) revealed an anomalous relationship (if any) between molecular weight of dextran and

21 Chapter One: General introduction the degree of substitution on the residual activity of the construct. However, the coupling method used and the site of attachment i.e. whether at or near the active site, does affect the residual potency of the peptide or protein after modification with dextran (Chan et al., 1999; Larionova et al, 1985; Baudys et al, 1998).

Similarly, these factors were also found to influence the immunogenic properties of the final construct (Chan et al, 1999). Richter and Kagedal (1972) reported dextrans of high molecular weight to be immunogenic, especially when coupled to proteins. However, the immunogenicity of trichosanthin (TCS), a type I ribosome-inactivating protein was reduced by site-specific coupling to dextran (40KDa). Large discrepancies in activity and immunogenicity were reported between dextran-modified TCS conjugates prepared via non-or specific coupling, thus exemplifying the effect of modification at or near the active and antigenic site of a protein (Chan et al., 1999). Interestingly, Fagnani et al. (1990) coupled Mabs and polyclonal antibodies with low molecular weight (6KDa) dextran to generate conjugates of low or negligible immunogenicity. No measurable immune response was detected against either the antibody or dextran portion of these conjugates. Similarly, the immunogenic potential of the Fab’ fragment of a murine monoclonal anti-carcinoembryonic antigen (CEA) was reduced after coupling with low molecular weight dextran (Fagnani et al, 1995). Enzymes such as asparaginase (Wileman et al, 1986), a-amylase (Humphreys et al, 1977) and catalase (Marshall, 1978) were modified with dextran of molecular weights up to 250KDa and exhibited abrogated or reduced immunogenicity.

Naturally occurring glycoproteins are generally more stable than non-glycosylated proteins (Mehvar, 2000). Interestingly, the stability of dextran-modified enzymes was superior to their native counterparts and generally increased further with increased molecular weight dextran (Marshall, 1978; Wileman et al, 1986). They were also more resistant to heat, exogenous proteases such as trypsin and chymotrypsin and protein dénaturants e.g. urea (Marshall, 1978). The increased stabilisation of proteins after modification with dextran is believed to be due to changes in the degree of hydration of the protein and conformational stabilisation afforded by multipoint attachment of the protein to the same dextran molecule (Marshall, 1978).

2 2 Chapter One: General introduction

1.5.2 Synthetic polymers for peptide and protein conjugation

Synthetic polymers offer the opportunity for preparing tailor-made carriers, presenting the desired features and moieties such as targeting residues, peptidyl spacers as sites for enzymatic cleavage and pH-sensitive linkers. Furthermore, synthetically derived polymers allow one to reach the desired molecular weight and sufficiently low polydispersivity. Although the favourable biological properties of some polymers may sometimes be combined with the main use of the peptide or protein carried to create a synergic effect, the toxicity of others often strongly limits their in vivo use. For instance, poly(divinylether maleic anhydride) (DIVEMA) (Hirano et al., 1997) and poly-(styrene-co-maleic acid) (SMA) (Maeda, 2001) are examples of poly-functional synthetic polymers that have both gained popularity for peptide and protein conjugation due their intrinsic properties. It is noteworthy that in Japan a conjugate of SMA and the powerful antitumour neocarzinostatin (SMANCS) originally developed by Maeda et al. (1985) has received market approval for treating liver cancer. PolyfN- vinylpyrrolidone) (PVP), poly N-(acryloylmorpholine) (PAcM) (Veronese et al., 1997) and monomethoxypoly(ethylene glycol) (mPEG) (Roberts et al., 2002) are examples of semi-telechelic synthetic polymers. Their success among polymers for peptide and protein modification resides in their mono-functionality, which unlike poly-functional polymers prevents cross-linking and the formation of a complex mixture of conjugates (Veronese, 2001).

1.5.2.1 Poly(ethyIene glycol) (PEG) Polyethylene glycol (PEG) is an inert, uncharged, flexible amphiphilic polymer comprising of repeating ethylene glycol units. It is commercially available in a variety of configurations, including linear or branched and different molecular masses (Fig. 1.3). PEG lacks toxicity and is rapidly cleared from the body intact by either the kidneys (PEG < SOKDa) or in faeces (PEG > 20KDa) (Yamaoka et al., 1994). Unlike other polymers, PEG has a relatively narrow polydispersity (Mw/Mn < 1.1) and the unique ability to be soluble in both aqueous and organic media (Roberts et al., 2002). PEG exhibits low immunogenicity and antigenicity (Richter and Akerblom, 1984). In its most common form, PEG chains terminate with hydroxyl groups (Fig. 1.3a), which

23 Chapter One: General introduction

a) Polyethylene glycol (PEG) OH ■CHj— C H jO ^ H

b) Monomethoxypolyethylene glycol (mPEG) q H ------CHg— CHgO^ _CM

c) 2-0-methoxypolyethylene glycol-4,ô-dichloro-S-triazine

(activated mPEGl) O

N C l— ^ N N = < 01 d) 2,4-bis (0-methoxypolyethylene glycol)- 6-chloro-S-triazine

(activated mPEGl) O

01 N N O - C H ;— CHgO^^j-CH;

e) Comb-shaped copolymer of polyoxyethylene allyl methyl diether R and maleic anhydride (n= 33, m = 8 and R = H; MW=13KDa; PM13 0 — OHp— OH OH or OH,' À polyoxyethylene 2-methyl- 1 -2-propenyl methyl o o KO diether and maleic anhydride O n = 40, m = 50 and R = CH3; M W = lOOKDa; PMIOO C2H4O -OH,

Figure 1.3 Structures of polyethylene glycol (PEG) and some derivatives commonly used in peptide and protein modification. PEG is a bifimctional reagent while monomethoxypolyethylene glycol (mPEG) is monofunctional because a methoxyether linkage blocks one of the hydroxyl groups. Activation of mPEG by the coupling of one or two PEG chains (S.OKDa) to 2,4,6-trichloro-S-triazine (cyanuric chloride) yields mPEGl and mPEG2 respectively. Comb-shaped PEG copolymers are designated as PM 13 and PMIOO according to their molecular weight. Both linear and comb-shaped PEG are coupled to the primary amino groups of the peptide or protein.

24 Chapter One: General introduction are readily available for peptide and protein modification. These characteristics of PEG make it a suitable carrier for peptide and protein delivery. Indeed, PEGNOLOGY^^has been successfully applied to more than 40 different peptides and proteins to produce bioconjugates for a variety of disease states (table 1.5).

For the purpose of PEGylating proteins, it is common practice to first convert one of the terminal hydroxyl groups of PEG to a methoxy group thus yielding mono functional monomethoxy PEG (mPEG) (Fig. 1.3b). This minimises the heterogeneity of the conjugates that may arise fi*om protein cross-linking. In order for coupling to proceed, the mPEG molecule requires prior activation to yield a reactive functional group. This group is then commonly coupled to the e-amino group of lysine or N- terminal amino group of the protein. Roberts et al. (2002) and Harris et al. (2001) have exhaustively reviewed several methods of mPEG activation and PEGylation processes. Since the pioneering work of Frank Davis and his colleagues in the late 1970’s, PEGylated peptides and proteins have rapidly evolved fi"om the first- generation to the more superior second-generation bioconjugates, and will be reviewed here.

First-generation PEG chemistry First-generation PEGylation methods used mainly linear PEG derivatives (Fig. 1.3b) of 12KDa or less (Roberts et al., 2002). To maximise the pharmacological benefits of PEGylation, a stable bond between the PEG moiety and protein is required. Generally, protein PEGylation is achieved by formation of linkages between the amino group of the protein and an active carbonate, ester, aldehyde or tresylate derivative of PEG (Harris et al., 2001). For this purpose, the most common reagents used for the activation of mPEG are trichloro-s-triazine (cyanuric chloride) (Davis et al., 1978), carbonyldiimidazole (Beauchamp et al., 1983), succinic anhydride Abuchowski et al, 1984) and succinimidyl carbonate (Zalipsky, 1995). Examples of first-generation PEG derivatives suitable for amine modification include N-hydroxysuccinimidyl-activated esters (producing an amide linkage), PEG-epoxide, PEG-tresylate, PEG-aldehyde (producing amine linkages), PEG-carbonyl imidazole and PEG-nitrophenyl carbonate (producing urethane linkages).

25 Chapter One: General introduction

Initial work by Davis et al. (1978) used cyanuric chloride to prepare activated mPEGl (2-0-methoxypolyethylene glycol-4,6-dichloro-S-triazine) (Fig. 1.3c). Unfortunately, the second available chloride of activated mPEGl is capable of cross-linking with protein molecules. To circumvent this problem, Inada et al. (1995) developed the first branched structure, mPEG2 (2,4-bis (0-methoxypolyethylene glycol)-6-chloro-S- triazine) (Fig. 1.3d). The lower reactivity of the single chloride moiety translates into a more selective modification of lysine and cysteine residues without further side reactions. PEG derivatives possessing a comb-shaped conformation (activated PM, Fig. 1.3e) were synthesised by Kodera et al. (1992) and Sasaki et al. (1993). Such derivatives are copolymers of a PEG derivative and maleic anhydride; the reactive site of the derivative is multivalent and the molecular weight can vary widely (PM 13 and PM 100 corresponding to 13 and lOOKDa respectively were studied). Modification with PM introduces many more PEG chains per modified amino group than either mPEGl or mPEG2 (Inada et al., 1995). Comb-shaped PEG is thought to completely shield the protein to which it is coupled to via an amide linkage (anhydride + lysine residue) by hydrogen bonding between the side chains of the amino acid residues and the oxygen atoms of the PEG chains (Kodera et al., 1992). Interestingly, PM-modified asparaginase exhibited superior residual activity and immunogenicity compared with its chain-shaped counterpart (Kodera et al., 1992). Unfortunately, first-generation chemistries were generally plagued by PEG impurities (due to diol contamination), restriction to low molecular weight mPEG, unstable linkages, and lack of selectivity in modification (Milton Harris and Chess, 2003). However, despite these limitations PEGylated adenosine deaminase (Adagen®) and asparaginase (Oncaspar®) received regulatory approval and are used clinically today. More recently, improved conjugation techniques have been developed to ameliorate the early PEGylation problems and have resulted in the second-generation of PEG-protein therapeutics.

Second-generation PEG chemistry Researchers have developed a number of sophisticated second-generation chemistries. For instance, the introduction of PEG-propionaldehyde instead of PEG-acetalaldehyde prevents the formation of impurities in the PEG (by aldol condensation) and considerably reduces heterogeneity, which is frequently seen with lysine chemistry

26 Chapter One: General introduction

Table 1.7 Influence of PEGylation on pharmacokinetics and pharmacodynamics of therapeutic proteins. Both properties are compared against native protein (adapted from Harris et al., 2001).

Pharmacokinetic effect Pharmacodynamic effect References

Interferon-a -2a (INFa-2a) In vivo antiviral activity increased Algranati et al. (1999) Sustained absorption 12 to 135 times. Antitumour activity Half-life (from 3-8 to 65h)* increased 18-fold. Improved Chatelut et al. (1999) sustained response to chronic hepatitis C

Interleukin-6 (IL-6) Thrombopoietic potency increased Tsutsumi et al. (1997) Half-life (from 2.1 to 206min) 500-fold. Decreased IgGl production

Tumour necrosis factor Antitumour potency increased Tsutsumi et al. (1995) Half-life (from 3 to 45- 4 to 100-fold 136min)

Superoxide dismutase (SOD) 93-98% residual enzyme activity Nakadka et al. (1997) Half-life (from 3.5 to 540- 990min)

Streptokinase Decreased antigenicity Rajagopalan et al. (1985) Half-life (from 4 to 7-20min)

Mab intact Some loss of antigen binding Kitamura et a/. (1991) Half-life (from 3.6 to 5.4h)

Fab’ fragment Full antigen binding ability Chapman et al. (1999) Half-life (from 0.33 to 9.05h)

Insulin Half-life (from 4 to 15.99min) Preserved biological activity Hinds & Kim (2002)

(Bentley and Harris, 1999). An overall goal of second-generation PEGylation methods is to create larger PEG polymers to improve the pharmacokinetic and pharmacodynamic effects seen with lower molecular mass PEG’s. Table 1.7 summarises the effect of PEGylation on the pharmacokinetic and pharmacodynamic properties of some representative examples of PEGylated peptides and proteins. All gave rise to adducts with dramatically increased systemic exposure, however the most dramatic increase in blood residence time was noted for PEG-modified SOD compared with its native counterpart. Studies conducted on the influence of PEG size, percentage modification and clearance suggest that the mechanism predominantly

27 Chapter One: General introduction responsible for the reduced clearance of PEG-SOD is due to the increased hydrodynamic volume (above renal cut-off threshold) of the PEG conjugate (Veronese et al., 2002). It is noteworthy that the ‘effective’ molecular weight of PEG is greater than its apparent molecular weight on account of being heavily hydrated and in rapid kinetic motion (Zalipsky and Harris, 1997). PEG-modified streptokinase was cleared slower in contrast to the native enzyme via similar clearance mechanisms (Brucato and Pizzo, 1990). Unfortunately, whilst PEG-modified proteins exhibit superior half- lives in vivo, they tend to lose biological activity after excessive PEGylation (Olson et al., 1997). To that end, Nakadka et al. (1997) found that SOD was most effectively modified with a small number (2 to 3) of high molecular weight PEG molecules (41- 72KDa). These conjugates exhibited superior in vivo half-lives compared with the native enzyme, whilst retaining modest enzyme activity. Some researchers have found an inverse relationship between PEG mass and in vitro activity but a direct relationship between PEG mass and in vivo activity, on account of prolonged exposure of PEGylated biopharmaceutical. PEG-SOD conjugates prepared with too high molecular weight (lOOKDa) PEG did not further increase the circulatory half-life. Heavy losses of biological activity (>50%) were reported when an increasing number of PEG chains were attached (>6) and when PEG was activated with trichlorotriazine (Veronese et al., 2002).

Biological activity loss and immunogenicity of PEGylated proteins may also be minimised by PEGylating site-specifically. Thiol groups such as protein cysteine groups are ideal for such modifications, which can be moreover introduced by protein mutagenesis (Goodson and Katre, 1990) and modified by use of PEG-maleimide, vinyl sulphone and iodoacetamide. Indeed, monoclonal antibodies and monovalent antibody fragments (Fab’) have been site-specifically PEGylated. The latter conjugates prepared with large PEG molecules of up to 40KDa, retained antigen binding affinity, complement-fixing ability and exhibited increased circulatory half- lives in vivo (Kitamura et al., 1991; Chapman et al., 1999). Interestingly, site- specifically PEGylated insulin exhibited enhanced plasma residence time, improved aqueous solubility and favourable immunological properties (Hinds and Kim, 2002). The minimal antigenicities of these conjugates (as seen for PEG-IL-6) suggest that

28 Chapter One: General introduction clearance via opsonization and uptake by the RES mechanisms are hindered. PEGylation of proteins appears to lower the immunogenic response by steric masking of potential antigenic sites, thus preventing immune recognition of the therapeutic protein as foreign (see section 1.4.2.2). The development of immunogenicity occurs more frequently when the protein is administered subcutaneously and may be reduced or abolished depending on the molecular weight, extent of modification and shape of PEG coupled.

Another improvement in second-generation PEG polymers is the use of branched PEG structures. For instance, in contrast with linear-(5-12KDa)-PEGylated INFa-2a, branched-(40KDa)-PEGylated INFa-2a exhibited prolonged systemic exposure and higher rates of sustained virological response in patients treated with chronic hepatitis C virus infection (Algranati et al., 1999). Furthermore, branched-PEG-modified asparaginase exhibited greater residual activity (98%) after a 50-minute incubation period with trypsin compared with linear-PEG-asparaginase (25%) and the native enzyme (5%) (Monfardini et al., 1995). It is proposed that the “umbrella-like” structures of branched PEG are better at cloaking the attached polypeptide from the immune system and proteolytic enzymes, thereby reducing its antigenicity and likelihood of destruction (Veronese and Caliceti, 1997). Additionally, modification of the lysine residues with PEG, which are the sites of cleavage for trypsin accounts for the conjugates increased resistance to proteases (Monfardini et al., 1995). Finally, other advances in PEGylation chemistry include the design of degradable PEG-protein linkages to maximise the return of the protein bioactivity (Sato, 2002) and synthesis of heterobifunctional PEGs (e.g. PEGs derivatised with molecules such as biotin, fluorescein and phospholipids) (Bentley et al., 2001).

1.5.2.2 Clinical applications of PEGylated peptides and proteins and products in development The clinical value of PEGylation is now well established. Indeed, the FDA has approved several PEGylated polypeptides as therapeutics and more are undergoing clinical investigation (table 1.8). PEGylated adenosine deaminase (ADA) was the first PEGylated protein pharmaceutical to enter the market for a rare deficiency disorder of

29 Chapter One: General introduction

Table 1.8 PEG-protein conjugates on the market or in clinical development. Updated fi*om Duncan (2003).

PEGylated Name/ Company Year Indication Reference compound approved Adenosine Adagen®; Enzon 1990 SCID Levy et al. (1988) deaminase syndrome L-asparaginase Oncaspar®; Enzon 1994 ALL Soares et al. (2002) a-Interferon 2b PEG-Intron®; 2000 Hepatitis C Wang et al. (2002) Schering-Plough a-Interferon 2b PEG-Intron®; Various Cancer, MS Bukowski et al. (2002) Schering-Plough clinical fflV/AIDS trials a-Interferon 2a Pegasys®; 2002 Hepatitis C Reddy et al. (2002) Hoffinann-La Roche G-CSF Neulasta®; Amgen 2002 Prevent Kinstler et al. (2002) neutropenia rhGH antagonist Somavert®; 2003 Acromegaly Mukheijee et al. (2002) Pharmacia Anti-TNF-a Fab CDP870 Phase II RA Chapman et al. (1999) SCED, severe combined immunodeficiency; ALL, acute lymphoblastic leukaemia; MS, multiple sclerosis; HIV, human immunodeficiency virus; rhGH, recombinant human growth hormone; G-CSF, granulocyte colony-stimulating factor; TNF, tumour necrosis factor.

ADA. Other first-generation products to follow include, PEG-asparaginase and PEG- a-interferon 2b (PEG-Intron®). The latter conjugate is used for the treatment of chronic hepatitis C and is also under investigation for other indications including, cancer, multiple sclerosis and HIV/AIDS.

More recently, improved PEGylation techniques have resulted in the second- generation of clinical PEG-protein products.In addition to PEG-Intron®, PEG-a- interferon 2a (Pegasys®) has been approved for the treatment of hepatitis C infection.

Pegasys® and PEG-Intron® exhibit similar biological activities and differ only in respect of a single amino acid. However, Pegasys® is prepared with branched-

(40KDa) PEG and the linker employed is very different. Consequently, Pegasys® has

30 Chapter One: General introduction a higher specific activity in vitro and a longer plasma half-life than PEG-Intron®. PEGylated G-CSF (Neulasta®) is also approved and used clinically to prevent severe cancer chemotherapy-induced neutropenia. In the past year, the FDA has approved PEGylated rhGH (Somavert®) as an alternative treatment for acromegaly. The construct binds irreversibly to hGHs receptors and blocks the excessive effects of hGH. The clinical pipeline for PEGylated proteins is healthy and it is expected that several products currently in the late phase clinical trials will become product candidates in the next seven years (Roberts et al., 2002). For example, Pfizer/Pharmacia is continuing phase II clinical studies on a PEGylated anti TNF-a antibody fragment (CDP870) for the treatment of rheumatoid arthritis, while Celltech examine a similar conjugate (CDP571) for Crohn’s disease and another PEGylated antibody fragment for oncology. The clinical use of PEG-IL-2 has shown little advantage with cancer patients, but more promising results have been seen in terms of a prolonged immunostimulatoty effect in HIV-1-infected patients.

Other candidates include, PEGylated insulin with a lengthened circulation time and reduced immunogenicity (Hinds and Kim, 2002), PEGylated superoxide dismutase (SOD) for the treatment of ischaemia/reperfusion injury or bums (Rocca et al., 1996) and PEGylated haemoglobin for blood transfusion. The benefits of neocarzinostatin for cancer treatment, urokinase and streptokinase as antithrombotics are also under investigation. These PEGylated proteins all benefit from decreased immunogenicity, higher potency, augmented half-lives in vivo and require a reduced frequency of dosing thereby reducing toxicity and increasing patient compliance. The ability to significantly improve these properties translates these biotherapeutics into a viable commercial opportunity and reflects the interest in this new generation of peptide and protein pharmaceuticals.

31 Chapter One: General introduction

1.6 Polysialic acids: potential uses in drug delivery

Gregoriadis et al. (1993) demonstrated that polysialic acids (PSAs) exhibit long half- lives (e.g. up to 40h, depending on the size and type of PSA used) in the blood circulation of intravenously injected mice. Furthermore, a low molecular weight model drug (fluorescein) conjugated to PSA assumed the half-life of the latter. These findings provided the impetus to investigate the use of PSAs as carriers of short-lived proteins as well as small peptides, drugs and particulate drug delivery systems. Indeed, recent work showed significantly improved half-lives not only of proteins (Fernandes and Gregoriadis, 1996, 1997) and small drug molecules (Gregoriadis et al., 1993) when coupled to a low molecular weight PSA (colominic acid) but also of microparticles such as liposomes (Zhang, 1999). Moreover, CA-modified catalase and asparaginase lead to a considerable increase of enzyme stability in the presence of proteolytic enzymes or blood plasma enzymes (Fernandes and Gregoriadis, 1996, 1997). Several characteristics of PSAs that render them as ideal carrier molecules for the efficacious delivery of peptide and protein therapeutics will be discussed in the following sections.

1.6.1 Occurrence, structure and function of polysialic acids

PSA is a naturally occurring unique polysaccharide found in the capsules of neuroinvasive bacteria and as a highly regulated post-translational modification of the neural cell adhesion molecule N-CAM in vertebrates (Muhlenhoff et al., 1998). More than 36 derivatives of sialic acids, have been identified (Schauer et al., 1995) of which N-acetyl neuraminic acid (Neu5Ac) is the most common. Sialic acid is often found in glycoproteins and glycolipids at the terminal non-reducing end (Murray et al., 1989) and is one of the factors contributing to the survival of glycoproteins in the circulation (Morell et al., 1971).

PSAs are polymers of repeating 9-carbon NeuSAc units (Fig. 1.4). They include the serogroup B capsular polysaccharide (PSB) from Neisseria meningitidis B and Escherichia coli {E. coli) Kl; the serogroup C capsular polysaccharide C (PSC) from

32 Chapter One: General introduction

N. meningitidis C and the polysaccharide K92 (PSK92) from E.coli K92, as well as shorter chain derivatives. PSAs from N. meningitidis serogroup B and E. coli Kl are homopolymers of 199 and > 200 a-(2-8)- linked NeuSAc units respectively. PSC is a homopolymer of 74 a-(2-9)-linked NeuSAc units and PSK92 is a heteropolymer of 78 alternate a-(2-8), a-(2-9)-linked NeuSAc. PSB, PSC and PSK92 have a phospholipid moiety covalently attached through a phosphate group at their reducing end

(Gotschlich et al., 1981). Gregoriadis et al. (1993) showed that the in vivo clearance rate for a given PSA was dependant on the presence or absence of phospholipid acyl groups, molecular weight and type of internal linkages.

Group B meningococci and E. coli Kl have structurally identical capsules (Bhattacharjee et al., 197S) and the PSA derived from E. coli Kl is known as colominic acid (CA) on account of its origin. McGuire and Binkley (1964) described CA as a low molecular weight homopolymer of NeuSAc residues joined via a-(2-^8)- ketosidic linkages (Fig. 1.4). Low molecular weight CA was later shown to be a culture artefact (Troy II, 199S), derived from the acid hydrolysis of the long PSA chains that are found in the E.coli Kl cells. Structural analysis of CA demonstrated that It consists of at least 200 NeuSAc residues (Rohr and Troy, 1980).

O AcNH.7 AcNH AcNH COgH 3 Non-reducing end Figure 1.4 Structure of colominic acid (CA). N-acetylneuraminic acid units are linker via a-(2^8) glycosidic linkages (McGuire and Binkley, 1964). Carbon atom (C7) at the non-reducing end of the polysaccharide is where periodate oxidation introduces an aldehyde group. Adapted from Fernandes and Gregoriadis (1996).

In vertebrates, a-(2—>-8)-linked PSAs are found on neural cell adhesion molecules (N- CAMs) in the brain, and associated with normal morphogenesis and neural development (Rutishauser, 1989). They are also expressed on the surface of several human tumours (Rougon, 1993).

33 Chapter One: General introduction

1.6.2 Characteristics of polysialic acids relevant to their use in drug delivery

Unlike other hydrophilic polymers (e.g. dextran and mPEG), PSAs are biodegradable, and their catabolic products produced by sialidases (e.g. NeuSAc) are not known to be toxic (Saito and Yu, 1995) (section 6.3). In addition, PSAs are highly hydrophilic and hydrated (Rougon, 1993), and have no known receptors in the body (Troy II, 1995), which is pertinent to their long in vivo half-lives. The latter characteristic of PSAs was also found to be dependant upon molecular weight. Thus, it is envisaged that long chain PSAs (average molecular weight of 23 to 92KDa) could be coupled to small drugs and peptides in order to circumvent glomerular filtration, whilst shorter chain PSAs (average molecular weight of 22KDa or less) could be coupled to large proteins or liposomes (Gregoriadis et al., 1993).

PSAs are thymus-independent antigens and therefore do not induce immunological memory (Gregoriadis et al., 2000). However, PSC and PSK92 can be immunogenic in humans when their molecular weight exceeds 50KDa (average chain length greater than 170 Neu5Ac units). PSAs can become T-cell dependent antigens (with induction of memory), on coupling to proteins; nevertheless, PSA-induced immune responses are difficult to achieve. For instance, a-(2-8)-linked PSA coupled to tetanus toxoid (TT), failed to elicit antibodies against PSA (Jennings and Lugowski, 1981). More importantly, when selecting a PSA for peptide and protein delivery, antigenicity is a crucial concern. Interestingly, antibodies against the a-(2-8)-linked PSAs are generally of low affinity (Mandrell and Zollinger, 1982) thus limiting any immunological response (Finne, 1982). Whilst immunogenicity did not seem dependant upon molecular weight of a-(2-8)-linked PSAs (Wyle et al., 1972), antigenicity decreased with decreasing chain length (Lifely et al., 1988).

Finally, PSAs are chemically versatile polymers that possess numerous potential sites for conjugation to other molecules, either directly or after mild chemical modification. For instance, amino groups can be made available by deacetylation and a reactive aldehyde group can be introduced at the non-reducing end (C7) of the polymer by periodate oxidation. Carboxyl and hydroxyl groups are also potentially available.

34 Chapter One: General introduction

1.7 Aims and outline of the thesis

The principle aim of this research is to investigate the potential of PSA as a means to optimise the parenteral delivery of an assortment of different therapeutic peptides and proteins, thereby improving their efficacious use in long-term therapy. The biotherapeutics employed included, bovine polyclonal IgG, mouse monoclonal IgG2a, aprotinin, insulin, somatostatin and catalase. Individual peptides and proteins were coupled with hydrophilic CA (polysialylation) and their bioconjugate properties such as solubility, in vitro stability, biological potency and in vivo circulatory half-lives selectively investigated. In addition, a novel strategy was developed in an attempt to further improve the efficiency and therapeutic value of the established method of polysialylation. Key issues within each chapter pertinent to the aim of this thesis are outlined here. A detailed overview of the literature regarding the use of polymers for the delivery of therapeutic peptides and proteins is given in Chapter 1. Besides summarising the current status of peptide and protein pharmaceuticals and polymer conjugates in therapy, new techniques and developments of such delivery systems are also highlighted. Chapter 2 describes the general methods and materials applied in the thesis. In Chapter 3 challenges regarding the polysialylation of bovine immunoglobulin G (IgG), aprotinin, insulin and somatostatin are investigated. Initial tasks involved validating and optimising an established method of polysialylation, followed by the synthesis and in vitro characterisation of the polysialylated constructs. Chapter 4 is devoted to further improving the yield of conjugation obtained with the established coupling method used in chapter 3 and evaluating the effects of polysialylation on the in vitro biological activities of the coupled biotherapeutics. A novel modification of the conventional polysialylation process is developed using catalase as model protein. The effects of both coupling strategies are determined using polysialylated mouse anti bovine serum albumin IgG2a, aprotinin and calalase. The effects of polysialylation on the in vivo characteristics of IgG, aprotinin and insulin prepared by both coupling strategies are described in Chapter 5. Finally, a summary of the main findings and conclusions with their implications for further work and future prospects are reported in Chapter 6. The choice of PSA, therapeutic proteins and coupling strategy used in this thesis are presented in the following sections.

35 Chapter One: General introduction

1.7.1 Choice of polysialic acid: Colominic acid

In the present work, low molecular weight colominic acid (CA) (Fig. 1.4) was chosen mainly because of its poor immunogenicity (Wyle et aL, 1972) and antigenicity (Mandrell and Zollinger, 1982) in humans (see section 1.6.2). Moreover, CA is commercially available and is extracted from E. coli Kl, which is only marginally pathogenic (Sarff et al., 1975). CA exhibits an average molecular weight of lOKDa

(equivalent of 34-35 Neu5Ac residues per molecule) and is composed of a-(2—> 8 ) internal glycosidic linkages (McGuire and Binkley, 1964) which is a desirable advantage over other PSAs in light of the periodate oxidation step required for the coupling process (see section 1.7.2).

1.7.2 Coupling strategy for polysialylation of peptides and proteins

In this study, mild coupling conditions are required to preserve CA and the potency of the polypeptides, as they are both labile molecules. The methodology used by Jennings and Lugowski (1981) to couple polysialic acids to tetanus toxoid was adapted here to covalently couple CA to the peptides and proteins. This coupling method was used because it is highly selective, forms a stable covalent bond between

OH OH I I NalO, CA OH— OH— CHgOH CA —H

pH 9.0 Prot — NH, CA —H Prot CA 35 - 40“C NaCNBH.

Prot — N H -C H ,— CA

Figure 1.5 Two-step reaction scheme for the conjugation of peptides and proteins to colominic acid. A. derivatisation of colominic acid to introduce a free reactive aldehyde at the terminal non-reducing end of CA by periodate oxidation and B. reductive amination of the oxidised CA with the peptide or protein, forming an unstable intermediate imine. In the presence of NaBHgCN, the unstable intermediate is readily reduced to produce a secondary amine. Only the groups involved in the coupling reaction are depicted. CA and Prot represent the backbones of the protein and CA moieties respectively.

36 Chapter One: General introduction

PSA and the protein, is a gentle procedure and requires no linkers, which could present problems of immunogenicity (Delgado et al., 1992). The coupling strategy essentially involves a two-step process (Fig. 1.5). The first step involves controlled periodate oxidation of CA to introduce a single reactive aldehyde moiety at the terminal residue (Fig 1.5A). Single site attachment is preferential as it avoids protein cross-linking and a heterogeneous final product. The vicinal diols, located on adjacent carbon atoms at the non-reducing end of the polysaccharide are selectively derivatised as the internal a-(2 —>8 ) glycosidic linkages are not susceptible to periodate oxidation (Lifely et al., 1986). Although, the reducing end of CA exists in keto-enol equilibrium in solution, the (keto) form is thermodynamically unfavourable and less likely to couple with the protein (3.3.4.4). In the second step (Fig 1.5B) the newly introduced aldehyde group of CA typically reacts with the s-amino group of lysine residues and/or N-terminal amino group of the protein (Glazer et al., 1976). The imine or Schiff base formed is readily reduced in the presence of sodium cyanoborohydride (NaBHgCN) under alkaline conditions to a stable secondary amine. Even in the presence of the fi-ee aldehyde, NaBHgCN rapidly reduces the unstable intermediate imine to a secondary amine, thus shifting the equilibrium to the right and increasing the yield of the conjugate.

1.7.3 Choice of peptides and proteins: Properties and clinical applications

The properties and clinical applications of the chosen peptide and protein candidates are outlined in the following sections relevant to their use in this thesis.

1.7.3.1 Immunoglobulin G (IgG) Bovine polyclonal IgG was chosen in this study as a model for therapeutic Mabs as these agents have shown great promise in the treatment of cancer, autoimmune diseases and infection (section 1.1). Although bovine IgG is limited in that a pharmacological evaluation of the modified constructs could not be made, it is readily available and comparatively cheap, thus facilitating its extensive study. To ameliorate the shortcomings, mouse anti-bovine serum albumin Mab (IgG2a) was employed, as it is highly specific for the antigen bovine serum albumin (section 5.3.9.3). Both

37 Chapter One: General introduction

NMj

F ab y Light HOOC

I Constant

M w 150KDa

HOOCCOUH Figure 1.6 Schematic model of an antibody molecule showing the two identical light and heavy polypeptide chains linked together by disulphide bonds and the position of the antigen-binding sites. Mw = molecular weight. Adapted from Sindelar (1997).

antibodies possess similar structures and molecular weight but differ in the amino acid

sequence in the constant region of the heavy chain (Fig 1.6). IgG is a large, hydrophilic hi functional molecule presenting numerous available lysine residues for

coupling to CA. Therapeutic Mabs of non-human origin are unfortunately limited in treating chronic diseases due to short scrum half-lives and immunological problems. However, researchers have ameliorated these problems by developing chimeric and humanised antibodies (section 1.1). Furthermore, modification of antibodies and Fab

fragments such as Mab A7 (Kitamura et a/., 1991) and anti-TNF-a Fab (Chapman et a i, 1999) with soluble polymers such as mPEG resulted in constructs with reduced immunogenicity and increased circulatory half-lives in vivo.

1.7.3.2 Aprotinin Aprotinin is a small, single chain polypeptide of bovine origin (Fig 1.7). It was chosen

in this study because it represents a novel small therapeutic peptide for CA

conjugation. From a practical standpoint, it is cheap, readily available and its

pharmacological properties are easily determined (section 5.3.9.4). Furthermore,

aprotinin contains available lysine residues for coupling with CA. Indeed, these

reasons are also pertinent to the choice of studying insulin in this thesis (section

1.7.3.3). Aprotinin is a potent inhibitor of serine proteases such as kallikrein, plasmin,

trypsin and chymotrypsin. Thus aprotinin is clinically used (Trasylol®) for a variety

of human diseases (e.g., pancreatitis, septic and haemorrhagic shock and multiple

trauma). Moreover, it is also approved to reduce blood loss and transfusion

38 Chapter One: General introduction

15 Thr Gly Pro— Cys— Lys— Ala— Arg— lie— lie— Arg— Try— Phe Try— Asn— Ala I ® \s I Tyr Ala Arg— Cys— Gly—Gly— Tyr— Val— Phe—Thr— Gin Lys il I I p Lys-Arg-Asn-Asn-Phe—Lys-Ser-Ala-Glu Leu— Gly—Ala

P ro — Glu— Leu I /S I A sp — C ys C y s ------I I ^ ------C ys— T hr------Arg— Met P h e I I------Gly— Gly— Ala---- COOH Asp— Pro— Arg— NH 3 gg Mw6,512Da 1 Figure 1.7 Structure of aprotinin showing active centre at Lys 15 and disulphide bridges at cys5-55, cys 14-38 and cys30-51. Mw = molecular weight. Reproduced from Kassell and Laskowski (1965). requirements during cardiac surgery (Roberts et al., 1996). Unfortunately, aprotinin exhibits a short in vivo half-life and induces allergic reactions upon repeat intravenous administration (Boag et al, 1985), thereby limiting its clinical use. In an attempt to circumvent these problems, aprotinin has been covalently coupled to (carboxymethyl) cellulose and diethylaminoethyl dextran (Larionova et al., 1978), lactose (Larionova et al., 1984) and (carboxymethyl) dextran (Larionova et al., 1985).

1.7.3.3 Insulin

1 Gin— Glu— Val— He— Gly— NH^ Chain A

Cys S S \ I \ 21 Çys—Ala— Ser— Val— Cys -----Ser— Leu— Tyr— Gin— Leu— Glu— Asn— Tyr— Cys— Asn I I Cys Gly Ser His— Leu— Val— Glu— Ala— Leu— Tyr— Leu— Val ------Cys COOH

Leu Ala— Lys— Pro Thr— Tyr— Phe— Phe— Gly— Arg— Glu— Gly His I I COOH Gin— Asn— Val— Phe— NH^ Chain B Mw 5,733.5Da

Figure 1.8 Structure of bovine insulin indicating the two-polypeptide chains joined by disulphide bonds. Mw = molecular weight.

Insulin is a small polypeptide hormone composed of two peptide chains, which is poorly soluble at neutral pH. The primary reason for choosing insulin in this study is because there is a continued need for new soluble insulin forms to facilitate and

39 Chapter One: General introduction improve the treatment of insulin-dependent diabetes mellitus (IDDM). Parenteral administration of insulin is currently the only effective therapy for the treatment of IDDM (Hinds and Kim, 2002). Different varieties of insulin formulations are clinically available (e.g. short acting Humalog®, intermediate acting Humulin I® and long acting Human Monotard®), which provide a modulated absorption and permanence in the blood. Unfortunately, insulin therapy is limited owing to the inherent shortcomings of insulin such as short half-life in the circulation, self aggregation and immunogenicity (Baudys et al., 1998). To that end, researchers have coupled insulin to soluble polymeric carriers such as PVP (Hixson, 1973), PEG (Uchio et al, 1999; Hinds et al., 2000), carboxymethyldextran (Baudys et al., 1998) and dextran (Suzuki et al., 1972; Mehvar, 1994).

1.7.3.4 Somatostatin

1 HjN Ala— Gly— Cys— Lys— Asn— Phe— Phe— Try

S I s Mw 1, 638Da Cys— Ser— Thr— Phe— Thr— Lys 14 Figure 1.9 Structure of somatostatin-14, showing the disulphide bridge at cys 3-U and cyclic nature. (British Pharmacopoeia, 1999).

Somatostatin (SS) is a small cyclic peptide that belongs to a family known as a somatotropin-release inhibiting factors (SRIFs). It has a broad inhibitory effect on the secretion of hormones such as growth hormone, insulin and glucagon. Owing to its short in vivo half-life, long acting SRIF analogues i.e. Octreotide® have been developed for the treatment of acromegaly and endocrine tumours. SS is proposed for polysialylation on account of these findings. SS was not the subject of intense study in this thesis and is therefore only discussed in brief.

1.7.3.5 Catalase Catalase was used in this study as a model protein to optimise a novel polysialylation process and as such is only discussed in brief. Catalase (hydrogen peroxide; hydrogen peroxide oxidoreductase, EC 1.11.1.6) is a tetrameric haemprotein with a molecular weight of 24KDa. It is characteristic of the peroxisomes that catalyse the degradation of hydrogen peroxide to oxygen and water (Deisseroth and Dounce, 1970).

40 Chapter Two

General materials and methods

41 Chapter Two: General materials and methods

2.1 Materials

2.1.1 Polysialic acid, peptides and proteins

Table 2.1 Properties and sources of polysialic acid, peptides and proteins utilised Material Source**

Colominic acid (CA) sodium salt from E. coli Kl Sigma Chemical Company, UK (average mol. wt lOKDa)

Sialic acid monomer of CA from E. coli Kl Sigma Chemical Company, UK (NeuSAc, 309.3Da)

IgG lyophilised, reagent grade from bovine serum Sigma Chemical Company, UK (ISOKDa)

Catalase, twice crystallized from bovine liver (240KDa) Sigma Chemical Company, UK Specific activity 58000U/mg protein

Insulin, from bovine pancreas (ô.OKDa) Sigma Chemical Company, UK

Ig02a mouse anti-bovine serum albumin monoclonal Chemicon International Inc. USA antibody (ISOKDa)

Aprotinin, from bovine lung (6.SKDa) Fluka Biochemica, UK Specific activity 8200U/mg protein

Somatostatin lyophilised, from porcine hypothalamus Sigma Chemical Company, UK (1.64KDa)______** Manufacturers and suppliers full addresses may be found in Appendix 2.

2.1.2 Deionised water

Deionised water was obtained from an Elgastat Option 4 water purification unit (Elga Ltd, UK). The water was pre-treated in a reverse osmosis cartridge and then purified via an ion/organic removal cartridge, UV chamber and a 0.2pm filter. The resistivity of the purified water was above 5 MO cm at 25^0 and its pH was neutral. Ultra High Quality (UHQ) water was obtained from an Elgastat UHQ-PS unit (Ultra High Quality Polishing System; Elga Ltd, UK), using water pre-purified by deionisation as outlined above. The water was further purified by a combination of organic adsorption, deionisation, microfiltration and photo-oxidation. The resultant water had a resistivity above 18 MO cm at 25^C.

42 Chapter Two: General materials and methods

2.1.3 General materials

Table 2.2 General materials used in Chapter Two Material Source** Acetic acid glacial (100%) BDH, Laboratories Supplies, UK Acrylamide* Sigma Chemical Company, UK Ammonium sulfate Sigma Chemical Company, UK Ammonium persulphate (>98%)* Sigma Chemical Company, UK Blue dextran (2000 kDa) Sigma Chemical Company, UK Bovine serum albumin (98-99%) Sigma Chemical Company, UK Bromophenol blue* Pharmacia LKB Biotechnology, UK Chloramine T Sigma Chemical Company, UK Coomassie brilliant blue G-250 Sigma Chemical Company, UK Coomassie brilliant blue R-250 Sigma Chemical Company, UK Cupric sulphate (-98%) Sigma Chemical Company, UK Dialysis tubing Medicell International Ltd, UK Glycerol (99%)* BDH, Laboratories Supplies, UK Hydrochloric acid (HCl) BDH, Laboratories Supplies, UK High molecular weight markers Pharmacia LKB Biotechnology, UK Hydrogen peroxide 30% Sigma Chemical Company, UK lODO-GEN® Pre-coated lodination tubes Pierce Chemical Company, USA Liquid scintillation cocktail Optipbase ‘HiSafe’3 Fisons chemicals, UK Low range molecular weight markers BIO-RAD laboratories, UK Methanol BDH, Laboratories Supplies, UK p-Mercaptoethanol (> 98%)* Sigma Chemical Company, UK N,N'-Methylene-bis-acrylamide (> 98%)* Sigma Chemical Company, UK Orthophosphoric acid 85% BDH, Laboratories Supplies, UK PD-10 columns Pharmacia LKB Biotechnology, UK Polyethylene glycol (40 000 Da) Sigma Chemical Company, UK Potassium iodide BDH, Laboratories Supplies, UK Rapid silver staining kit Pharmacia LKB Bioteclmology, UK Resorcinol (1,3-Benzenediol) (99%) Sigma Chemical Company, UK Sephadex media Pharmacia LKB Biotechnology, UK Sodium acetate Sigma Chemical Company, UK Sodium azide Sigma Chemical Company, UK Sodium boro [^HJhydride (333 mCi/mg) Amersham Pharmacia Biotech, UK Sodium dodecyl sulphate (SDS) (-99%)* Sigma Chemical Company, UK Sodium iodide (1.0mCi/10|xl) Amersham Pharmacia Biotech, UK Sodium metabisulphite BDH, Laboratories Supplies, UK Trichloroacetic acid (TCA) Sigma Chemical Company, UK N,N,N’,N'-Tetramethylene diamine (TEMED) Sigma Chemical Company, UK Trizma base (Tris) (> 99%)* Sigma Chemical Company, UK Whatman No. 1 filter paper Whatman Scientific Ltd., UK * electrophoresis grade reagents. % purity denoted in parenthesis. ** Manufacturers and suppliers full addresses may be found in Appendix 2.

43 Chapter Two: General materials and methods

All other reagents were of analytical grade. A Wallac CompuSpec UV-visible spectrophotometer connected to a PC (Wallac UK Ltd, UK) was used for all spectrophotometric determinations.

44 Chapter Two: General materials and methods

2.2 Methods

This chapter briefly describes some general methods central to the quantification and characterisation of the polysialylated proteins referred to throughout the thesis. The composition of all buffers and reagent solutions employed can be found in Appendix 1.

2.2.1 Quantitative estimation of total sialic acid

Quantitative estimation of sialic acid with resorcinol reagent was performed by the method of Svennerholm (1957).

2.2.1.1 Resorcinol method A 0.5ml sample (diluted if necessary) containing 4-40pg/ml of sialic acid was added to 0.5ml of resorcinol reagent and the mixture heated in a boiling water bath for 30 minutes in sealed test tubes. After heating, the tubes were cooled for 20-30 minutes. The contents were transferred to SM cuvettes and the absorbance read at 570nm against a reagent blank. The sialic acid concentration was calculated by comparison with a calibration curve, prepared with standard solutions of sialic acid.

2.2.2 Quantitative estimation of total protein

Peptide and protein concentrations were either measured directly by their absorption of ultraviolet (UV) light or via colorimetric methods.

2.2.2.1 Absorbance at 405nm {Â4os) The absorbance A (extinction, optical density), for catalase was measured using a 1-cm path length cell. This enzyme exhibits a characteristic absorption maximum, the Soret band at 405nm (Aebi, 1983) (El%/405), which was used to determine its concentration.

45 Chapter Two: General materials and methods

222.2 Dye-binding assay Bradford (1976) introduced a protein assay based on the shift in absorbance maximum of Coomassie Brilliant Blue G, from 465 to 595nm, when it binds to protein. The procedure was as follows: a 0 .1 ml sample (diluted if necessary) containing 1 0 - lOOpg/ml of protein was added to 1.0ml of Bradford reagent (acid/dye solution) and mixed thoroughly. After 5 minutes and before 1 hour the absorbance at 595nm was read against a reagent blank. The protein concentration was calculated by comparison with a standard curve, prepared using a suitable protein. This method was applicable to soluble proteins only (Grossberg & Sedmark, 1977). Strongly alkaline buffers and detergents such as SDS and Triton X-100 were reported to reduce the colour yield.

2.2.3 Protein fractionation

2.2.3.1 Size exclusion chromatography (SEC) SEC was the method used most frequently for characterisation and fractionation of the native and polysialylated peptides and proteins studied throughout this thesis. Fractionation was based on the differential diffusion of molecules into the gel pores. Large proteins, above the ‘exclusion limit’ of the gel, could not enter the pores and so were eluted from the ‘void volume (Vo)’ of the column {i.e. the volume of the liquid between the beads; usually this is about 1/3 of the total column volume). Very small molecules entered the pores of beads fully and so had to pass through the total volume of the column before being eluted. Intermediate size proteins partially entered the pores and so were eluted between the void and total volume. Thus protein molecules eluted from the column in order of decreasing molecular weight.

Whitaker (1963) reported on molecular weight determinations of unknown proteins by size exclusion chromatography on a column that had been previously calibrated with protein standards of known molecular weight. However, it was also reported that there might be modifications introduced by deviations from a ‘normal’ globular shape. The procedures followed to prepare Sephadex gels are outlined below; they include gel preparation column preparation, packing and equilibration, void volume (Vo) determination, sample application and elution.

46 Chapter Two: General materials and methods

Gel preparation Sephadex gels were suspended in 0.15M sodium phosphate buffered saline pH 7.4 (PBS) and allowed to swell overnight at 20°C. The gel suspension was degassed before use.

Column preparation, packing and equilibration The columns were prepared from pipettes or burettes, whereby the bottom was plugged with glass wool. To fill the column, a slurry of approximately equal volumes of PBS and gel were poured into a vertical column that was one-third filled with PBS. This was left to settle for 15 minutes without flow, the excess buffer was then drained through the growing gel bed. The gel slurry was added continuously until the bed height fell 2cms from the top of the column. A buffer reservoir was connected to the top of the column and 2-3 column volumes of buffer were passed through the column.

Void volume (Vo) determination Freshly prepared Blue Dextran (MW ~ 2000KDa) dissolved in equilibration buffer at a concentration of 2 mg/ml was carefully applied to each column prior to any experimentation. This is performed to determine the void volume ( V o ) and to check column packing. The elution volume (Ve) of Blue Dextran was followed spectrophotometrically at 610nm by measuring the elution volume correspondent to the peak maximum. The void volume ( V o ) (for the column) was made by measuring the volume of effluent collected from the point of sample application to the centre of effluent peak. The ratio VgWo can be used to normalise the elution behaviour of a molecule, for each system.

Sample application Samples (less than 5% of the total column volume) were applied carefully to the top of the gel by allowing it to run slowly down the inside wall of the column so that the gel bed was not disturbed. Once the sample had penetrated the bed, the gel was carefully overlaid with a small volume of PBS to prevent dilution and the buffer reservoir reconnected to the top of the column. The column was checked regularly during sample elution to ensure that the gel did not dry out.

47 Chapter Two: General materials and methods

Sample elution The sample was eluted from the column by the gravitational flow from the reservoir and the vacuum conditions between the reservoir and column. For most gels, the maximum flow rate was governed by the operating pressure, which the gel could withstand, and by the resolution required. Eluate fractions of typically 1ml were collected with a fraction collector (Redifrac, Pharmacia LKB Biotechnology, UK) equipped with a drop counting device. The number of drops equivalent to 1ml was determined and the drop counting device calibrated for each individual system. Prior to the addition of any new samples, the column was washed thoroughly with at least 3 volumes of PBS (unless otherwise stated). When the columns were not in operation for a period of two or more days, they were equilibrated with PBS containing 0 .0 2 % (w/v) sodium azide to prevent microbial contamination.

2.2.4 Analytical electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is a powerful technique employed to separate proteins under the influence of an electric field. Proteins will migrate through polyacrylamide gels due to their amphoteric nature. At a protein’s isoelectric point (p/) the molecule is uncharged and will not migrate; at a pH above or below its p/ it acquires a net charge and will therefore migrate. The rate of migration depends upon the charge density (the ratio of charge to mass) of the protein concerned. Therefore, the application of an electric field to a protein mixture in solution will result in their separation due to both size and charge differences. PAGE techniques can be divided into denaturing and non-denaturing systems. In the former, proteins are treated with reagents such as sodium dodecyl sulphate (SDS), a powerful anionic detergent and/or urea. The denatured proteins are electrophoresed in the presence of the denaturing agent; in the latter, proteins are not subjected to any pre-electrophoresis treatment.

2.2.4.1 SDS polyacrylamide gel electrophoresis (SDS-PAGE; denaturing conditions) SDS-PAGE is the most widely used electrophoretic technique to assess protein purity and determine apparent molecular weight. All proteins are solubilised by heating in

48 Chapter Two: General materials and methods the presence of excess SDS and a reducing agent (usually 2-mercaptoethanol), which reduces the disulphide bonds. Under these conditions, most polypeptides will bind SDS avidly in a constant weight ratio such that they acquire essentially identical charge densities and migrate in polyacrylamide gels of correct porosity according to their size (Hames, 1990). Under these conditions, a plot of logio polypeptide molecular weight (MW) versus relative mobility {Rj) reveals a linear relationship.

The Laemmli system (Laemmli, 1970) a modification of those described by Omstein and Davis (1964) is a discontinuous SDS buffer system developed for SDS-PAGE. This system employed here consisted of a relatively high molarity (0.375M) Tris-HCL buffer pH 8 . 8 in the resolving gel and a low molarity (0.025M) Tris glycine reservoir buffer pH 8.3. A ‘stacking gel’ (a low porosity gel polymerised above the separating gel) was incorporated to increase the resolution. This consisted of a low molarity

(0.125 M) Tris-HCL pH 6 . 8 buffer. This resulted in concentrating the sample proteins into a narrow starting zone prior to separation, thereby ensuring very sharp protein bands during the separation process.

2.2.4.1.2 Equipment and procedures required for SDS polyacrylamide gel electrophoresis (SDS-PAGE) The equipment used and procedures for performing SDS polyacrylamide gel electrophoresis on peptide and protein samples are outlined below.

The Dual Cooled Mini Vertical Gel Unit (model MVl- DC), gel casting module (model CMl) and power supply (model PSU-400/200) was from Anachem, UK. 1.5mm combs and spacers. Glass plates and perspex backing plates were from Anachem, UK.

Preparation of the resolving gel and stacking gel A variety of slab gels of varying acrylamide concentrations were prepared dependant upon the molecular weights of the proteins to be electrophoresed. Table 2.3 shows the compositions of the various SDS-polyacylamide gels that were prepared. All details on the preparation of the stock solutions can be found in Appendix 1.

49 Chapter Two: General materials and methods

Table 2.3 Composition of SDS-polyacrylamide gels (Laemmli system)

Stock solution Stacking gel: Resolving gel: 0.125 M Tris, pH 6.8 0.375 M Tris-HCL, pH 8.8 Final % concentration Final % concentration 5.0% 20% 17.5% 15.0% 12.5% 7.5%

Acrylamide-bisacrylamide 2.5 20.0 17.5 15.0 12.5 7.5 (30:0.8) (ml) (ml) (ml) (ml) (ml) (ml)

Stacking gel buffer stock: 5.0 ----- 0.5 M Tris, pH 6.8

Resolving gel buffer stock: - 3.75 3.75 3.75 3.75 3.75 3.0 M Tris, pH 8.8

10% SDS 0.2 0.30 0.30 0.30 0.30 0.30

1.5% ammonium persulphate 1.0 1.50 1.50 1.50 1.50 1.50

Water 11.3 4.45 6.95 9.45 11.95 16.95

TEMED 0.02 0.02 0.02 0.02 0.02 0.02

The glass plates were assembled using 1mm spacers into the vertical dual gel casting stand. The resolving gel: 3.0 M Tris-HCl, pH 8 . 8 was mixed with all the reagents in a side-arm vacuum flask, leaving out the ammonium persulphate and TEMED and deaerated for 1 0 minutes.

The TEMED (polymerisation catalyst) and ammonium persulphate were then added and the flask swirled gently so not to generate bubbles. The solution was poured down the spacer into the sandwich to a level about 4 cm from the top. Then, approximately 0.3ml of water-saturated n-butanol was run down each spacer to overlay the resolving gel. This formed an even layer over the surface. A very sharp liquid-gel interface was visible after 10-20 minutes when the gel had polymerised. The gel was allowed to fully polymerise for 1-2 hours at room temperature. After polymerisation, the n- butanol overlay was poured off and the surface of the gel rinsed thoroughly with deionised water. The gel surface was then overlaid with 1ml of deionised water and left to sit whilst the stacking gel: 0.125 M Tris-HCl, pH 6 . 8 was prepared. The method of preparation was as for the resolving gel. The deionised water overlay was oured off and the stacking gel added to the sandwich to polymerise above the resolving gel. A 5 well comb was inserted into the stacking gel, taking care not to trap

50 Chapter Two: General materials and methods any bubbles below the teeth of the combs. The gel was then allowed to polymerise for 60 minutes at room temperature. The sandwich was transferred over to the vertical gel unit where the comb was slowly removed from the stacking gel and the wells rinsed and filled with reservoir buffer.

Preparation of sample and molecular weight standards Samples containing a high level of salt or detergent that could interfere with electrophoresis were desalted on mini PD-10 columns or dialysed against deionised water. Equal parts of peptide or protein (at a suitable concentration) and sample solubilization buffer containing SDS and 2-mercaptoethanol were mixed and boiled for 1 minute. The various peptide and protein samples studied were electrophoresed against a range of molecular weight markers. The molecular weight standards were diluted 1: 20 with SDS Reducing Sample Buffer. The solution was heated at 95% for 2 minutes then cooled and 5-lOpl/well was loaded onto the gels. When the high molecular weight kit was used during SDS-PAGB, the number of reference standards increased to six; a result of the ferritin half unit (MW 220, 000) and ferritin subunit (MW 18, 500) which appeared in the presence of SDS-denaturing conditions. Tables

2.4-2. 6 show the compositions of the high, low and ultra-low molecular weight standards used.

Table 2.4 Composition of high molecular weight markers Polypeptide Mol. Wt. (Daltons) Source Native Sub-unit

Thyroglobulin 669,000 330, 000 hog thyroid Ferritin 440, 000 18, 500 horse spleen Catalase 232,000 60, 000 beef liver Lactate dehydrogenase 140,000 36, 000 beef heart Albumin 67, 000 67, 000 bovine serum Recommended gel percentage 7.5%

51 Chapter Two: General materials and methods

Table 2.5 Composition of low molecular weight markers

Polypeptide Mol. Wt. (Daltons) Source Native

Phosphorylase b 97,400 rabbit muscle Albumin 66, 200 bovine serum Ovalbumin 45, 000 hen egg white Carbonic anhydrase 31,000 bovine Trypsin inhibitor 21,500 soybean Lysozyme 14,400 hen egg white Recommended gel percentage 12.5%

Table 2.6 Composition of ultra-low molecular weight markers Polypeptide Mol. Wt. (Daltons) Source Native

Triosephosphate Isomerase 26, 600 rabbit muscle Myoglobin 17, 000 horse heart a-Lactalbumin 14, 200 bovine milk Aprotinin 6,500 bovine lung Insulin Chain B, oxidized 3,496 bovine Bradykinin 1,060 bovine Recommended gel percentage 15-20%

Loading and running the gel The samples were carefully loaded into the wells. The gels were run at a constant current of 15mA per slab gel until the samples reached the resolving gel interface, whereby the current was increased to 25mA per gel. When the tracking dye had reached 1 cm from the bottom of the gel the power supply was disconnected.

Staining and destaining the gel The reservoir buffer was removed, the sandwich disassembled and the gels incubated in 3-5 volumes of Coomassie blue staining solution. The gels were stained in a glass dish for 1 hour at room temperature on a shaker. The stain was then discarded, the gel quickly rinsed in water and destained with 30% methanol-10% acetic acid. This was left shaking for a further hour. Finally, the gel was destained overnight with 10%

52 Chapter Two: General materials and methods methanol-10% acetic acid until a clear background was obtained. Some gels were silver stained using a rapid silver staining kit according to the manufacturer’s instructions for mini gels (10x8, 1.0mm thick). This method of staining is much more sensitive than Coomassie blue staining, however it is prone to artefacts and often produced gels with unacceptably high background staining.

Storing and drying polyacrylamide gels The gels were stored dry. To air dry, the gels were sandwiched between two sheets of porous cellophane and locked into a drying ft-ame. The fi*ame was inserted into an air dryer and left to dry naturally overnight. Moisture evaporated through the cellophane leaving a flat, easy to-store gel with a clear background. The percentage acrylamide and gel thickness influenced gel cracking during the drying process therefore, gels up to 12.5% acrylamide were dried without any prior treatments and those greater than 12.5% had 1 to 2 % glycerol added to the final destain solution prior to drying.

2.2.4.2 Native PAGE (non-denaturing conditions) Under the conditions of native PAGE, the proteins were electrophoresed unaltered i.e. they were neither disrupted nor denatured and separation was largely due to the inherent charge of the components. Essentially the native page system was as described by Omstein and Davis (1964) for the standard Laemmli SDS-PAGE protocol, however SDS was omitted from all solutions. The samples were dialysed against electrode buffer and then sucrose (final 1 0 % w/v) and bromophenol blue (final 0.005%) were added before loading. The samples were not heated. The stock electrode buffer was diluted 40 or 80-fold rather than 10-fold as used for SDS-PAGE.

2.2.5 Radioiabeliing techniques

Radioactive labelling of colominic acid and the native peptides and proteins studied in this thesis was crucially important to the study of the in vitro characterisation and stability of CA (section 3.3.13) and in following the fate of the polysialylated peptide and protein constructs in vivo (Chapter 5).

53 Chapter Two: General materials and methods

In terms of isotopic labelling of oxidised CA, Lifely et al. (1986) reported on a method of tritiation, whereby tritiated hydrogen (^H) was introduced into the molecular structure of oxidised CA by chemical modification. Essentially, this process involved reducing the aldehyde moiety (introduced at Cl at the non-reducing end of CA via periodate oxidation) back to an alcohol on exposure to sodium boro[^H]hydride to incorporate the tritium label (Fig. 2.1)

COOHCOOH AcNH AcNH

Figure 2.1 Reduction of the aldehyde moiety at Cl via sodium boro[ HJhydride at A) the terminal N-acetylneuraminic acid residue at the non-reducing end of CA back to B) an alcohol to introduce the tritium label (^H).

Iodine-125 was chosen as the isotope for labelling the water-soluble peptides and proteins. This method was preferred because this isotope (^^^1) is relatively cheap, easily detected, has a half-life of 60 days and it is possible to obtain preparations with high specific activity. Essentially, labelling occurs by electrophilic addition of the cationic iodine (1^) to tyrosine (Fig. 2.2) residues and to a lesser extent, to histidine and tryptophan. The methods of radioiodination reported here include the chloramine- T method and the mild solid-phase reagent, lodo-Gen.

OH OH

125, N a‘“ l

OH'2

000- 000 - Tyrosine residue lodinated tyrosine residue Figure 2.2 Electrophilic addition of cationic iodine (1^) to tyrosine residues via Na to incorporate the isotope ^^^1 into the peptide or protein.

54 Chapter Two: General materials and methods

2.2.5.1 Tritiation of colominic acid with sodium boro[^H]hydride

In brief, NaB[^H]4 (2.92 |iCi/|il) was mixed with sodium hydroxide (lOjil, O.IM) and added to a solution of CA dissolved in distilled water (lOOjil, l.Omg/l.Oml). This mixture was incubated in a water bath at 30°C for 24 hours. Once the tritium label was incorporated into CA, the reaction was terminated by the addition of acetic acid (50pl,

O.IM) to destroy any unreacted NaB[^H] 4 . The final volume of the solution was made up to 0.5ml with distilled water. The purification of tritiated CA involved applying the mixture to a Sephadex G-25, 1.0 x 10.0cm (PD-10) column pre-equilibrated with distilled water or PBS, pH 7.4 whereby 1.0ml fractions were collected. From each fraction, lOOpl was transferred to beta vials and 4.0ml scintillation fluid added. The fractions were counted for 60 seconds in a Wallac, 1409 Scintillation P-counter. Thus, the fractions corresponding to the tritiated CA peak were pooled and dialysed (4 x 2L;

24h) against distilled water to remove any unbound NaB[^H] 4 . Once the extent of tritium incorporated into CA had been established, this was used to determine the specific activity of CA (pCi/mg of CA).

2.2.5.2 radioiabeliing of peptides and proteins by the Chloramine-T method The native peptides and proteins studied in this thesis were iodinated to a very high specific activity by a modification of the method by Greenwood and Hunter (1963). In short, carrier free Na ^^^I (50pCi, 2pl) was mixed with phosphate buffer (lOpl, 0.25M, ph7.5) to which a solution of peptide or protein dissolved in PBS (lOOpl, 0.5mg/ml) was added. Chloramine-T dissolved in 0.05M phosphate buffer (lOpl, 2mg/ml, pH 7.5) was then added to the mixture and incubated at 20°C for 10 min. Once iodine had been incorporated into the tyrosine residues of the peptide or protein the reaction was terminated by the addition of sodium metabisulphite (20pl, 1.2mg/ml in 0.05M phosphate buffer, pH 7.5). The final volume of the solution was made up to 0.5ml using potassium iodide (1 mg/ml in 0.05M phosphate buffer, pH 7.5). The radiolabelled peptides or proteins were purified and subsequently the labelling efficiency and the specific activity were determined as outlined in section 2.2.5.4.

55 Chapter Two: General materials and methods

2.2.5.3 radioiabeliing of peptides and proteins by the lodo-Gen method lodo-Gen reagent (l,3,4,6-tetrachloro-3a, 6 a-diphenylglycoluril was described by Franker and Speck (1978) as an effective and mild solid-phase reagent for the radioiodination of peptides and proteins. This reagent was well established in the literature as being milder towards protein molecules causing limited oxidative damage as compared with the chloramine-T method (Franker and Speck, 1978). Thus, a modification of this method was employed comparatively. In brief, a solution of peptide or protein dissolved in PBS (lOOpl, 0.5mg/ml) was added to the lODO-GEN® pre-coated iodination tube followed by the addition of sodium acetate (lOOpl, 0.35M, pH 4.0). Carrier fi*ee Na (100 pCi, 2pl) was mixed with phosphate buffer (lOpl, 0.25M, Ph7.5) and also added into the tube. The mixture was incubated in a water bath at 40°C for 30 min with occasional agitation. The tube was then chilled on an ice- water bath whereby sodium hydroxide (lOOpl, l.OM) was added to quench the reaction. The final volume of the solution was made up to 0.5ml using PBS, pH 7.5). The radiolabelled peptides or proteins were purified and subsequently the labelling efficiency and the specific activity were determined as outlined in section 2.2.5.4.

2.2.5.4 Purification, labelling efficiency and specific activity of radiolabelled peptides and proteins The labelled peptides or proteins were purified by application to a Sephadex G-25, 1.0

X 10.0cm (PD-10) column pre-equilibrated with 0.05M sodium phosphate buffer, pH 7.5 whereby 1.0ml fractions were collected. From each fraction, 5.0pl was transferred to gamma vials and counted for 60 seconds in a Wallac, 1275 Minigamma y-counter. The percentage labelling efficiency was determined fi*om the plot of counts per minute (CPM) versus elution volume (ml), where the area under the curve (AUG) for the first peak (labelled protein) was divided by the total AUG (both peaks) and multiplied by 100. The fi^actions corresponding to the peptide or protein radioactivity peak were pooled and dialysed (4 x 2L; 24h) against sodium phosphate (0.05M, pH 7.5) to remove any unbound The purity of the radiolabelled peptides and protein was assessed by trichloroacetic acid (TGA) precipitation. A sample of labelled peptide or protein (5pi) was mixed with BSA (lOOpl, 10%) in an eppendorf tube and TGA (1.25ml, 20%) was added to the mixture. After incubation at 4°G for 1.0 hour.

56 Chapter Two: General materials and methods the solution was centrifuged at 3000xg for lOmin. Then the supernatant and the pellet recovered at the bottom of the tube were transferred separately into y-vials and their radioactivity content determined (60s). Thus, the ratio of counts in the pellet to those of the total radioactivity, multiplied by 1 0 0 gave the percentage of radioactivity of peptide or protein in the pellet. Radiolabelled peptides or proteins giving a percentage of less than 90% were subjected to dialysis again until a ratio of over 90% was achieved. Once the extent of radioactivity incorporated into the peptide or protein had been established, this was used to determine the specific activity of the protein (pCi/mg protein).

2.2.6 ^^^1 Handling and waste disposal

All procedures involving the handling of at the doses we required were conducted in the designated supervised areas behind a lead shield. Under the COSHH regulations these experiments were considered to involve low exposure thus rated as medium hazard with risk assessment 2. All liquid waste generated firom our experiments was designated to the appropriate radioactive waste sink and flushed with copious amounts of water. The solid waste including contaminated magnetic stirrer bars and reaction vessels were soaked in Decon and stored behind lead shielding for the decomposition of The columns were flushed with phosphate buffer and stored behind lead also until safe to dispose. All paper waste was monitored for contamination and thrown away accordingly.

2.2.7 Dialysis and concentration of proteins The principle of dialysis is the exchange of small molecules by diffusion across a semi-permeable membrane into a reservoir of a given buffer until equilibrium is reached. At equilibrium there should be no change in sample volume (unless caused by an incidental osmotic pressure difference). This principle allowed us to desalt, purify samples and perform buffer exchanges. Samples were placed in secured semi- permeable cellulose visking tubing of nominal molecular weight cut-off 12-14kDa or 3.5kDa, depending upon the solute molecular dimensions within the bag. This was dialysed extensively (usually 3 x 2L; 24h) at 4°C against the appropriate buffer

57 Chapter Two: General materials and methods system, which was changed repeatedly throughout this procedure. A variation called reverse dialysis was used to concentrate the sample within the dialysis tubing. The filled bag was concentrated by laying the bag on the bottom of a flat tray, onto which

dry water-soluble polyethylene glycol 8 kDa was sprinkled. This was left until the sample had concentrated to the required volume, which was usually within 2 hours.

2.2.8 Lyophilisation (Freeze-drying)

Freeze-drying (lyophilisation) was the most commonly used method for long term storage of peptides and proteins. This method involved the drying of sample directly by evaporation of the frozen sample solution under vacuum (termed ‘freeze-drying’ or ‘lyophilization’). The average moisture content of freeze-dried proteins is of the order of 3%. The peptide and protein conjugates (except catalase) and oxidised CA prepared in this thesis were frozen to approximately -40°C and freeze-dried using the Edwards Modulyo to yield a fluffy white lyophilised plug that was stored at approximately 4°C until further required.

58 Chapter Three

Synthesis & characterisation of polysialylated peptides and proteins

59 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.1 Introduction

Hitherto, the only proteins to be modified by the technique of polysialylation are the therapeutic enzymes, catalase and asparaginase (Fernandes and Gregoriadis, 1996, 1997). In light of this, this chapter aimed to add to the repertoire of proteins modified by this technique and introduce three ‘new’ smaller therapeutic peptides for polysialylation. To that end, bovine polyclonal antibody immunoglobulin G (IgG), peptide hormones insulin and somatostatin and the serine protease inhibitor, aprotinin were all introduced. Table 6.1 summarises their physico-chemical properties for comparison. The coupling strategy chosen to covalently link CA to the individual peptides and proteins was the two-step process previously described by Jennings and Lugowski (1981) in section 1.7.2. Indeed, Fernandes and Gregoriadis (1996; 1997) employed this technique for the polysialylation of catalase and asparaginase. In essence, this reaction involved reductive amination in the presence of sodium cyanoborohydride, following controlled periodate oxidation of the polysaccharide. The procedure involves mainly the e-amino groups of the lysine residues of proteins, although the terminal amino groups are also known to possess reactivity for this reaction to proceed, (Glazer et al, 1976). Four key challenges deemed pertinent to the polysialylation of the four ‘new’ peptide and protein biotherapeutics were investigated here. They included: validation of the chosen two-step coupling strategy. Optimisation of the polysialylation process with respect to the individual peptide and protein therapeutics. Synthesis and characterisation of the polysialylated constructs. Finally, an evaluation of the physico-chemical, stability and storage properties of the native biotherapeutics, CA and the polysialylated constructs.

Initial research embarked with an investigation of the periodate oxidation process for its suitability in fiinctionalising CA to introduce an aldehyde moiety at the non reducing end of the polysaccharide and its necessity for subsequent protein linkage to occur. Size exclusion chromatography (SEC) was employed initially to compare the chromatographic profiles of CA with the oxidised derivative to elucidate whether the oxidative process had caused diminution of the CA molecule. The oxidative method employed in this thesis, was utilised previously by Fernandes and Gregoriadis (1996)

60 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins for the derivatisation of CA however, no attempts were made to identify the ‘newly’ introduced aldehyde functionality. To that end, this study introduced various spectroscopic and chemical techniques including mass spectrometry (MS); nuclear magnetic resonance spectroscopy (NMR), Fourier transform-infrared spectroscopy (FT-IR) and the 2,4-dinitrophenylhydrazine (DNP) test in an attempt to identify the aldehyde functionality of periodate oxidised CA. In a further attempt to validate the Schiff base reductive amination reaction, the primary CA bioconjugate synthesised employed L-lysine as a synthetic protein model. It was chosen as it contains the minimum available representative groups of a protein {i.e. e-amino lysine and N- terminal amino groups) that could be coupled to CA. A novel fluorogenic assay was developed and optimised for the detection and quantification of L-lysine. The molar yield of conjugation was determined and SEC was employed to characterise the L- lysine-CA conjugate.

On completing the preliminary somewhat artificial tests with L-lysine, the emphasis of this chapter involved optimising the yield of polysialylation with respect to the four therapeutic peptides and proteins introduced in this thesis. Process variables such as starting molar ratios of reactants, size of CA and proteins, time and temperature of incubation were altered and their effect on the degree of polysialylation evaluated. Initial experiments employed IgG to establish the optimal reaction conditions. Subsequently, aprotinin, insulin and somatostatin were polysialylated under the conditions optimised for IgG and modified accordingly to achieve maximal degrees of polysialylation. A non-invasive method was optimised and validated for the separation of the polysialylated constructs from un-reacted components and reviewed against other techniques. The polysialylated constructs were characterised utilising SEC and polysialylated IgG was also characterised by employing the method of SDS polyacrylamide gel electrophoresis (SDS-PAGE). Further studies highlighted the water solubility of the native and polysialylated conjugates. An evaluation of the stability of CA in the presence of varying pH, blood plasma and stability of the native and polysialylated peptides and proteins upon lyophilisation and storage were also performed. The strategies involved here were considered to lay the foundations upon which future chapters were built thus prompting our research.

61 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.2 Materials and Methods

This section details specific materials and methods used to provide data to investigate the research proposals of this chapter.

3.2.1 Materials

Table 3.1 Materials used in Chapter 3 Material Source Ammonium carbonate Sigma Chemical Company, UK Aprotinin (bovine lung)* BioRad Laboratories Ltd, UK Bradykinin* BioRad Laboratories Ltd, UK Colominic acid (average mol. wt 30KDa) EY Laboratories, San Mateo, USA Dialysis tubing Medicell International Ltd, UK

2 , 4-Dinitrophenylhydrazine Aldrich Chemical Company, UK Dioxan Fluka BioChemica, UK Ethylene glycol Lancaster Chemical Company, UK Fluorescamine Sigma Chemical Company, UK Insulin chain (3 (bovine pancreas)* BioRad Laboratories Ltd, UK a-Lactalbumin* Sigma Chemical Company, UK L-Lysine hydrate, 97% (146.19Da) Aldrich Chemical Company, UK Myoglobin* BioRad Laboratories Ltd, UK Sodium meta-periodate Sigma Chemical Company, UK Sodium cyanoborohydride (>98%) Sigma Chemical Company, UK Triosephosphate isomerase* BioRad Laboratories Ltd, UK * Components of an ultra low molecular marker calibration kit. ** Manufacturers and suppliers full addresses may be found in Appendix 2.

The composition of reagent solutions and buffers can be found in Appendix 1. All other reagents were of analytical grade and may be found in previous chapters.

62 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.2.2 Methods

3.2.2.1 Controlled periodate oxidation of colominic acid Colominic acid (average mol. wt. lOKDa) was oxidised by employing the controlled periodate oxidation technique. Freshly prepared O.IM sodium periodate (NaI 0 4 ) solution was mixed with CA (lOmg CA/lml NaI 0 4 ) at 20°C. The reaction mixture was stirred magnetically for 15 min in the dark. Periodate decomposes under the influence of light, generally to produce more reactive species (Dyer, 1956). Therefore, to maintain reproducible results it was important to keep solutions and reaction mixtures in the dark. Following this period, ethylene glycol (2ml)/ NaI 0 4 solution

(1 ml) was added to the reaction mixture to expend the excess NaI 0 4 , and the mixture was left to stir at 20°C for a further 30 min. The oxidised colominic acid was dialysed (12-14kDa mol. wt. cut-off) extensively against a 0.01% ammonium carbonate buffer

(3 X 2L; 24h) at 4°C. The dialysate was lyophilised and stored at -40°C until further required.

3.2.2.2 Characterisation of colominic acid and periodate oxidised colominic acid by size exclusion chromatography (SEC) A Sephadex G-75 column (45.0 x 1.1cm) was prepared to compare the chromatographic behaviour of CA (average mol. wt. lOKDa), with the oxidised derivative of CA (average mol. wt lOKDa), prepared in section 3.2.2.1.

Following that, CA (average mol. wt. lOKDa), the monomer unit of CA i.e. sialic acid

(Neu5Ac) mol. wt. 309.3Da and bovine serum albumin (BSA), mol. wt. 6 6 KDa, were all applied to the same column, respectively. In each case, 0.5ml samples of each of the above in PBS (2mg/ml) were applied to the column and eluted with 0.15M PBS at pH 7.4, (flow rate l.Oml/min). Sialic acid in the eluted fractions was measured spectroscopically (A570nm) using the resorcinol method (Svennerholm, 1957) and protein (A595nm) using the dye-binding assay (Bradford, 1976).

63 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.2.2.3 Spectroscopic and analytical methods employed to identify the aldehyde functionality of oxidised colominic acid In an attempt to identify the newly formed aldehyde functional group introduced in section 3.2.2.1, the following spectroscopic and analytical methods were employed.

Fourier Transform-Infrared (FT-IR) Spectroscopy Fourier transform-infrared (FT-IR) spectroscopy is a simple and rapid method of identifying selected functional groups in organic compounds. Molecular vibrations of chemical bonds caused by the absorption of infrared wavelengths will produce a characteristic spectrum indicating the presence of discrete functional groups. FT-IR spectra were obtained using the Avatar 360 E.S.P. FT-IR spectrometer, employing approximately Img of lyophilised CA and oxidised CA.

Proton Nuclear Magnetic Resonance (NMR) Spectra NMR spectra were obtained for CA and oxidised CA using the Bruker AM 500 instrument operating at 500 MHz. CA and oxidised CA were dissolved in D 2 O solvent, and 1% trimethylsilane (TMS) was added as an internal reference. Chemical shifts (ô^H) were reported in ppm (parts per million) downfteld from the internal TMS reference.

Electrospray-Mass Spectrometry Electrospray- mass spectrometry is a “soft” ionisation technique whereby samples are analysed in a chosen solvent to produce primarily the molecular ion and also offer identifiable fragments. The mass spectrometer measures the mass-to-charge (m/z) ratio. Mass spectra were obtained for CA and oxidised CA using the Finnigan LCQ^^^ ThermoQuest spectrometer. Solid samples were submitted and analysed in methanol solvent.

2.4-Dinitrophenylhydrazine test (2,4-DNP) 2.4-Dinitrophenylhydrazine (2,4-DNP) is a reagent often used to characterise compounds containing aldehydes or ketones. It yields sparingly soluble 2,4- dinitrophenylhydrazones with carbonyl compounds. CA and oxidised CA (10-30mg)

64 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins respectively, were added to 2,4-DNP reagent (1.0ml) and shaken. The mixture was allowed to stand until a crystalline precipitate was observed (usually within 30 min). Occasionally the precipitate was oily at first, but this did become crystalline upon standing.

3.2.2.4 Fluorescamine: A fluorogenic reagent for the detection and quantification of polysialylated L-lysine. This section involved the development and validation of a novel fluorogenic assay for the detection and quantification of the L-lysine-CA conjugate.

The novel reagent fluorescamine, (4-phenylspiro [furan-2 (3H), T-phthalan]-3,3'- dione) was synthesised by Weigele et al (1972). This reagent reacts with primary amines (including amino acids, peptides and proteins) under alkaline conditions to yield highly fluorescent products. The reaction is complete within a few seconds at room temperature. The product is optimally excited at 390nm with emission at 490nm. The resulting fluorescence is proportional to amine concentration and the fluorophors are stable over several hours.

The reaction with fluorescamine was used to detect and quantify the amino acid L- lysine in the L-lysine-CA conjugate by comparison with L-lysine standards. L-lysine (1.575mg/ml, 0.01 InM) in distilled water was used as the amino group standard and fluorescamine (7.2mg, 0.0259 mmole) in dioxan (24ml) as the amino group fluorophore reagent. L-lysine solutions were made up (2.5-lOOpL, 0.012nM-0.48nM) to 1.75 mL with PBS and fluorescamine (500|iL) added to each cuvette. Fluorescence at 390nm (excitation wavelength) and 475nm (emission wavelength) were read immediately using the Perkin Elmer LS-5 Luminescence spectrometer.

3.2.2.5 Synthesis and size exclusion chromatographic (SEC) characterisation of polysialylated L-lysine Colominic acid (average mol. wt. lOKDa) was activated by controlled periodate oxidation as described by Jennings and Lugowski (1981) and subsequently coupled to L-lysine (mol. wt. 146.19). This reaction involved a 50:1 L-lysine:CA molar ratio and

65 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins proceeds by reductive amination in the presence of sodium cyanoborohydride

(NaBHsCN) in 0.75M dipotassium hydrogen phosphate (K 2 HPO4 ), (3.0ml) at pH 9.0. A control of L-lysine in the presence of non-oxidised CA and all reagents as above was included. L-lysine hydrate (3.7mg, 25|imol) was reacted with oxidised CA

(IS.Omg, 0.5nmol) or non-oxidised CA (control) in 0.75M (K 2HPO4 ), (3.0ml) at pH 9.0 in the presence of NaBHgCN (20.0mg, 0.32mmol). The sealed reaction vials were magnetically stirred at 35-40°C in an oil bath. After 48 hours, the reaction vials were cooled over ice to terminate the reaction. The reaction mixtures were then exhaustively dialysed against PBS at 4°C (3.5KDa mol. wt. cut-off, 3 x 2L; 24h) to remove free, non-covalently linked L-lysine. The mixtures were further fi*actionated by size exclusion chromatography on a Sephadex G-25 (25.0 x 1.0cm in PBS) column.

Assuming that all the CA molecules had been modified and the fi*ee L-lysine had been removed by dialysis, the extent of the reaction was estimated by assaying the products for sialic acid, (Svennerholm, 1957) and amino groups, (Weigele et al, 1972).

3.2.2.6 Oligomers of colominic acid prepared by hydrolysis In an attempt to study the effect of polymer size on the degree of polysialylation of IgG, CA (average mol. wt lOKDa) was dissolved in PBS (lOmg CA/lml PBS) and oligomers prepared by hydrolysis in a boiling water bath.

CA samples were boiled for 5 and 8 hours, respectively and passed through a Sephadex G-50 column (45.0 x 1.0cm) to reveal the products of hydrolysis. Multiple peaks represented a series of oligosaccharides, whereby the major peaks were isolated and characterised by size exclusion chromatography (SEC) on a Sephadex G-75 column (45.0 x 1.0cm) against CA (average mol. wt lOKDa) and N-acetylneuraminic acid (Neu5Ac) 309.3Da (Fig. 3.12). The major peaks hrom each hydrolysed sample represented the ‘new’ molecular weight derivatives of CA, denoted as CA5 & CAg. Molecular weight estimations were made for CA5 & CAg via application to a Sephadex G-50 column (45.0 x 1.0cm) pre-calibrated with protein standards of known molecular weight (Fig. 3.13). Protein standards were chosen in preference of

66 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins polysaccharides as the latter are known to behave abnormally in SEC. Based on the differences in shape between the protein standards and CA; it was realised that this method is not ‘ideal’ for determining the molecular weights of the oligomers of CA (i.e. CAg and CAg), however it was nevertheless hoped that an order of size of the oligomers relative to CA could be obtained. Thus, 0.5ml (2mg/ml) of the standard protein solution was applied to the column and eluted with PBS (flow rate 1 .Omlmin' ^). The composition of the protein standards was as follows: triosephosphate isomerase, 26.6KDa; myoglobin, 17KDa; a-Lactalbumin, 14.2KDa; aprotinin, 6.0KDa; insulin chain P, oxidised, 3.5KDa and bradykinin, 1.06KDa. The eluted fractions were assayed for protein (A595nm) and sialic acid (570nm) content. Under these conditions CA5 & CAg were estimated to exhibit molecular weights of 6 and 3KDa respectively. CA$ & CAg were oxidised as described for CA (section 3.2.2.1) and then coupled to IgG as in (section 3.2.2.7.1, experiment 3).

3.2.2.7 Synthesis of polysialylated peptide and protein therapeutics The bovine polylonal antibody IgG (ISOKDa), peptide hormones insulin (5.7KDa), somatostatin (1.6KDa) and the serine protease inhibitor, aprotinin (6.5KDa) were all covalently attached to oxidised CA (average mol. wt lOKDa) via the method of Schiff base, reductive amination in the presence of sodium cyanoborohydride. All the conjugation reactions were carried out for 48 hours in sealed vessels at 35-40°C, whereby aliquots (1.0ml) of the mixture were removed at time intervals of 0, 6 , 12, 24 and 48 hours to study the effect of reaction time on molar yield of conjugation. In this thesis, the degree of conjugation was expressed as a molar yield as a ‘standard’ method previously employed by Jennings and Lugowski (1981) and Fernandes and Gregoriadis (1996, 1997). Additionally, the percentage of modified amino groups was also determined for each polysialylated peptide and protein based on the number of available e-lysine and N-terminal amino residues of the native protein (table 6.1). The relationship between starting molar ratios of the reactants {i.e. proteins and CA) and the degree of polysialylation was also investigated for each peptide and protein where appropriate. Starting molar ratios of 100:1, 50:1 and 10:1 (CA:Protein) where chosen in this study as previously employed by Fernandes and Gregoriadis (1996, 1997), such that comparisons could be made. In each case CA was present in excess.

67 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Controls included reacting the native proteins with non-oxidised CA and in the absence of CA under the same reaction conditions described. Stirring was kept to a minimum to avoid concomitant dénaturation of the proteins.

The neoglycoproteins were isolated from individual reactions via ammonium sulphate precipitation and were characterised using techniques such as size exclusion chromatography (SEC) and SDS polyacrylamide gel electrophoresis (SDS-PAGE).

3.2.2.7.1 Synthesis of polysialylated immunoglobulin G (IgG) A series of reactions were performed here to study the correlation between the starting molar ratios of CA: IgG and the yield of conjugation achieved. The effects of reaction time and CA size on the molar yield of conjugation were also investigated. All the reactions involved covalently coupling immunoglobulin G (mol. wt ISOKDa) to CA (average mol. wt lOKDa) or a smaller mol. wt. oligomer of CA. The experimental procedures were as follows:

Experiment 1: A 50:1 starting molar ratio of oxidised CA (SOmg, S.Opmol) was reacted with IgG (ISmg, O.lpmol) in 0.75M dipotassium hydrogen phosphate (5ml) at pH 9.0 in the presence of sodium cyanoborohydride (NaBHgCN) (20mg, 0.32mmol). CA was in excess. Controls included IgG (15mg, O.lpmol) in the presence of non-oxidised CA (SOmg, S.Opmol) and IgG alone under the reaction conditions above.

Experiment 2: A 100:1 starting molar ratio of oxidised CA (lOOmg, lO.Opmol) was reacted with IgG (ISmg, O.lpmol) in 0.75M dipotassium hydrogen phosphate (5ml) at pH 9.0 in the presence of sodium cyanoborohydride (NaBHgCN) (20mg, 0.32mmol). CA was in much greater excess. The control included was as in experiment 1.

Experiment 3: This involved reacting a 100:1 starting molar ratio of oxidised CAg & CAg prepared in section 3.2.2.6 (estimated average mol. wt 6 and 3KDa, respectively) and the oxidised

68 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

CA monomer unit NeuSac (mol. wt 309.3Da) with IgG. IgG (15mg, O.lgmol) was reacted with CA5 (60mg, lO.Ogmol), CAg (30mg, lO.Ogmol) and NeuSac (l.SSmg, S.Ofimol). The controls included reacting non-oxidised CA5, CAg and NeuSac with IgG under the same reactions conditions above.

Experiments 1, 2 and 3 were conducted simultaneously whereby 1.0ml aliquots were withdrawn over 0-48 hours from these experiments with IgG, then all subjected to ammonium sulphate precipitation (section 3.2.2.S.2) and dialysed to isolate the IgG conjugates. After centrifugation, the pellets (re-suspended in PBS) and supernatants were assayed for sialic acid (Svennerholm, 1957) and IgG (Bradford, 1976) content. The conjugation yields were expressed in terms of CA (sialic acid): IgG molar ratios found in the pellets (Fig. 3.14) and controls (3.15).

Size exclusion chromatography (SBC) was utilised to characterise the zero hour (control) and 48-hour aliquots from experiment 1 with IgG. A Sephadex G-lOO column (40.0 x 1.1cm) was prepared and the eluted fractions (1.0ml) were assayed for CA and IgG content as above (Fig. 3.16). The neoglycoproteins isolated from the aliquots (0-48 hours) removed from the reaction mixture in experiment 1 together with native IgG (control) were all subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and reviewed in section 3.3.10.

3.2.2.7.2 Synthesis of polysialylated aprotinin In this section an attempt was made to investigate the correlation between the starting molar ratio of CA: aprotinin and reaction time on molar yield of conjugation obtained. Aprotinin (mol. wt 6.5KDa) was covalently conjugated to oxidised CA (average mol. wt lOKDa) at varying molar ratios of CA: aprotinin. The experimental procedures were as follows:

Experiment 1 : A 50:1 starting molar ratio of oxidised CA (50mg, 5.0pmol) was reacted with aprotinin (0.65mg, O.lpmol) in 0.75M dipotassium hydrogen phosphate (5ml) at pH 9.0 in the presence of sodium cyanoborohydride (NaBHsCN) (20mg, 0.32mmol). CA

69 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins was in excess. Controls included aprotinin (0.65mg, O.lpmol) in the presence of non- oxidised CA (50mg, S.Opmol) and aprotinin alone under the same reaction conditions above.

Experiment 2: A 10:1 starting molar ratio of oxidised CA (lOmg, l.Opmol) was reacted with aprotinin (0.65mg, O.lpmol) in 0.75M dipotassium hydrogen phosphate (5ml) at pH 9.0 in the presence of sodium cyanoborohydride (NaBHgCN) (20mg, 0.32mmol).

The controls included were as in experiment 1. The 1.0ml aliquots withdrawn over 0- 48 hours from the two experiments with aprotinin were all subjected to ammonium sulphate precipitation (section 3.2.2.8.2) and dialysed to isolate the aprotinin conjugates. After centrifugation, pellets (re-dissolved in PBS) and supernatants were assayed for sialic acid (Svennerholm, 1957) and aprotinin (Bradford, 1976) content. The conjugation yields were expressed in terms of CA (sialic acid): aprotinin molar ratios found in the pellets (Fig. 3.20).

Zero hour (control) and 48 hour aliquots from experiment 1 with aprotinin were characterised using size exclusion chromatography (SEC). A Sephadex G-50 column

(35.0 X 1.0cm) was prepared and the eluted fractions (1ml) were assayed for CA and content as above (Fig. 3.21). The zero hour (control) and 48 hour neoglycoprotein isolated from the time study in experiment 1 (section 3.2.2.7.2) were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and results reviewed in Chapter 4.

3.2.2.7.3 Synthesis of polysialylated insulin An attempt was made to investigate the correlation between reaction time and molar yield of polysialylated insulin achieved. This involved reacting insulin (5.7KDa) with oxidised CA (average mol. wt lOKDa). The experimental procedures were as follows:

Experiment 1: A 50:1 molar ratio of oxidised CA to insulin was employed. Insulin (0.60mg, O.lpmol) was reacted with oxidised CA (50mg, 5.0jimol) in 0.75M dipotassium

70 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins hydrogen phosphate (5.0ml) at pH 6.4 in the presence of NaBHgCN (20mg, G.32mmol). Controls included insulin (0.60mg, O.lpmol) in the presence of non- oxidised CA (50mg, S.Opmol) and insulin alone under the same reaction conditions as above.

The 1.0ml aliquots withdrawn over 0-48 hours from the reactions with insulin were all subjected to ammonium sulphate precipitation (section 3.2.2.8.2) and dialysed to isolate the insulin conjugates. After centrifugation, pellets (re-dissolved in PBS) and supernatants were assayed for sialic acid (Svennerholm, 1957) and insulin (Bradford, 1976) content. The conjugation yields were expressed in terms of CA (sialic acid): insulin molar ratios found in the pellets (Fig. 3.22).

Zero hour (control) and 48 hour aliquots (1.0ml) from the reaction mixture were chromatographed using a Sephadex G-50 column (40.0 x 1.1cm) and the eluted fractions (1.0ml) were assayed for CA and insulin content as above (Fig. 3.23).

3.2.2.7.4 Synthesis of polysialylated somatostatin Somatostatin was the final peptide polysialylated in this chapter. An attempt was made to study the relationship between reaction time and molar yield of polysialylated somatostatin obtained. Somatostatin (1.64KDa) was reacted with oxidised CA (average mol. wt lOKDa). The experimental procedures were as follows:

Experiment 1: A 10:1 starting molar ratio of oxidised CA (50mg, 5.0pmol) was reacted with somatostatin (0.82mg, 0.5pmol) in 0.75M dipotassium hydrogen phosphate (5.0ml) at pH 9.0 in the presence of NaBHgCN (20mg, 0.32mmol). Controls included somatostatin (0.82mg, 0.5pmol) in the presence of non-oxidised CA (50mg, 5.0pmol) and somatostatin alone under the same reaction conditions as above.

The 1.0ml aliquots withdrawn over 0-48 hours from the reactions with somatostatin were all subjected to ammonium sulphate precipitation (section 3.2.2.8.2) and dialysed to isolate the somatostatin conjugates. Post centrifugation, pellets (re-dissolved in

71 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

PBS) and supernatants were assayed for sialic acid (Svennerholm, 1957) and somatostatin (Bradford, 1976) content. The conjugation yields were expressed in terms of CA (sialic acid): somatostatin molar ratios found in the pellets (Fig. 3.24).

Zero hour (control) and 48 hour aliquots (1.0ml) of the reaction mixture were chromatographed using a Sephadex G-50 column (45.0 x 1.1cm) and the eluted fractions (1.0ml) were assayed for CA and somatostatin content as above (Fig. 3.25).

3.2.2.S Ammonium sulphate fractionation: a method for isolating polysialylated peptides and proteins Peptides and proteins differ in their solubility in concentrated salt solution and hence can be separated from one another by precipitation at high ionic strength (Hofrneister,

1888). In light of this, ammonium sulphate (NH 4 )2 S0 4 was chosen for the purpose of precipitating (salting-out) the protein-CA conjugates thus isolating them from the unreacted starting materials. (NH 4 )2 S0 4 was preferred over other salts, as it is known not to significantly affect pH; it is inexpensive, very soluble and known not to destabilise proteins. It has been suggested that the NH 4"^ ion stabilises proteins (Hofineister, 1888).

3.2.2.8.1 Optimisation of the ammonium sulphate precipitation methodology The method of ammonium sulphate fractionation of proteins was optimised with respect to IgG, aprotinin, insulin and somatostatin. The individual proteins were dissolved 1 mg/ml in (0.15M) PBS (5.0ml) and subjected to varying amounts of solid

ammonium sulphate (NH 4 )2 S0 4 to achieve a range from 20-80% ammonium sulphate saturation. The procedure was as follows:

Solid ammonium sulphate was added slowly to each protein solution to achieve the required percentage ammonium sulphate saturation i.e. 20, 30, 40, 50, 60, 70 and 80%. The samples were left stirring for 1.0 hour at 4°C to allow complete equilibration between dissolved and precipitated proteins. The solutions were then centrifuged (Megaftige 1.0, Haraeus Equipment Ltd, UK). Generally, 4500xg for IgG or 6000xg for aprotinin, insulin and somatostatin for 40 min was found sufficient for

72 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins complete precipitation. The supernatants were decanted and the pellets drained and redissolved into (0.15M) PBS (5.0ml). Both supernatants and redissolved pellets were dialysed extensively (3 x 2L; 24 h) at 4°C against the same PBS and then assayed for total protein content to quantitate complete precipitation (Bradford, 1976). The percentage of ammonium sulphate saturation that led to optimal protein precipitation was employed to isolate the polysialylated peptides and protein respectively

(3.2.2.S.2). It was confirmed that CA did not precipitate with (NH 4 )2 S0 4 .

3.2.2.S.2 Application of ammonium sulphate to isolate polysialylated peptides and proteins The concentration of ammonium sulphate that caused optimal precipitation of IgG, aprotinin, insulin and somatostatin was then applied as the procedure for “salting-out” the polysialylated peptides and proteins and was as follows:

The peptide or protein conjugate mixtures (5.0ml) were pre cooled on ice, whereby

(NH2 )2 S0 4 (440mg/ml) was added slowly whilst continually stirring to achieve 70% (^/v) saturation. The suspensions were stirred for 1.0 hour at 4°C, centrifuged for 40 min (as described in section 3.2.2.8.1), the supernatants decanted and the pellets containing the polysialylated peptide or protein washed twice with 70% (NH 4 )2 S0 4 to remove any free, non-covalently conjugated CA. The solutions were centrifuged again

(as described in section 3.2.2.8.1) for 10 min dissolved in 70% (NH 4 )2 S0 4 . The precipitates recovered were redissolved in (0.15M) PBS (5.0ml) and dialysed extensively (3 x 2L; 24 h) at 4°C against the same PBS buffer.

73 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.2.2.9 Evaluation of the properties of colominic acid and the polysialylated proteins This section aimed to evaluate the in vitro properties of colominic acid such as its stability under different pH, exposure to mouse plasma and also the stability of the native and polysialylated peptides and proteins upon freeze-drying and storage at 4°C. Throughout this thesis, native proteins and polysialylated protein constructs were often lyophilised and stored at 4°C until further required. Thus, SEC was employed in an attempt to investigate the affect of lyophilisation and storage at 4°C on the structures of the native peptides and proteins and the integrity of the covalent bond proposed between CA and the coupled peptide or protein in this chapter.

3.2.2.9.1 Effects of varying pH on colominic acid degradation

CA (2.0mg/ml) was dissolved in (0.15M) PBS (pH 7.4), 0.75M K 2 HPO4 (pH 4.5) and

0.75M K2HPO4 (pH 9.0). Each solution was incubated at 37°C for 48 hours. Aliquots (SOOpl) were individually applied to a Sephadex G-25 (25.0 x 1.0cm) column and the eluted fractions were assayed for sialic acid content (Svennerholm, 1957).

3.2.2.9.2 Effect of mouse plasma on colominic acid degradation Tritiated (^H) CA (l.Omg) was mixed with 1.0ml of fresh mouse plasma and incubated at 37°C for 24 hours. A 500pi aliquot was applied to a Sephadex G-25 (25.0

X 1.0cm) column whereby lOpl aliquots were mixed with 4.0ml scintillation fluid and measured for radioactivity in a Wallac 1409 beta-counter (Wallac UK Ltd, UK). The control included tritiated CA mixed with (0.15M) PBS (pH 7.4).

3.2.2.9.3 Freeze-drying and storage of native and polysialylated peptides and proteins Samples (l.Omg) of freeze-dried native and polysialylated peptides and proteins (synthesised in section 3.2.2.7), were reconstituted in (0.15M) PBS (1.0ml) either immediately or upon 3 months storage at 4°C. Aliquots (500pl) from each solution of native and polysialylated protein were applied sequentially to a Sephadex G-50 column (35.0 x 1.0cm) and the eluted fractions (1ml) assayed for protein (Bradford, 1976) and (for conjugates) CA content (Svennerholm, 1957).

74 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.3 Results and Discussion

Having posed our research aims in section 3.1 of this chapter and applying the relevant methodologies in section 3.2 to investigate them, this section reports on the results obtained. It discusses the methodologies in terms of their suitability and practicality in providing data to substantiate our research aims/hypotheses.

3.3.1 Controlled periodate oxidation

The periodate oxidation reaction was exploited for the strategic introduction of a free reactive aldehyde group preferentially at C7 of the non-reducing terminus of colominic acid. Sodium periodate was chosen as it is known to selectively oxidise carbohydrate molecules containing hydroxyl groups on adjacent carbon atoms (vicinal diols) thereby cleaving the carbon-carbon bond and generating an aldehyde group at the site of each hydroxyl group (Fleury and Lange, 1932). Lifely et al. (1986) reported that periodate oxidation of polysaccharides should be carefully controlled to avoid over oxidation and consequent changes in the physicochemical properties.

In this study, exposure of CA to the oxidant was limited to 15 min with lOOmM periodate, as proposed by Jennings and Lugowski (1981). The potential cleavage of the internal a- (2—>8 )-linked NeuSAc residues of CA due to oxidation was determined using size exclusion chromatography (SEC). Fortunately, no significant diminution of the molecular weight of CA was seen. Indeed, almost identical elution profiles resulted for both intact CA and periodate oxidised CA (Fig. 3.1 A). The hydrodynamic properties of CA appear to have been preserved and no lower molecular weight products were detected. These results were anticipated as Lifely et al (1986) showed that the internal a- (2—>8 )-linked NeuSAc residues of E. coli K1 polysaccharide were not susceptible to periodate oxidation.

75 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.3.2 Purification of oxidised colominic acid

Several methods for the purification of oxidised CA were tried. Firstly, direct applications of periodate treated CA solution to GPC columns, pre-equilibrated with de-ionised water were attempted. Although termination of the oxidation reaction and purification are simultaneous, repeat applications were time-consuming and the time of CA exposure to periodate whilst eluting fi*om the column could not be exactly reproduced. The columns were eluted with de-ionised water to circumvent salt contamination of the lyophilised product. This complicated matters further as aqueous solutions of CA are extremely acidic, (pH 2.6). The internal a- (2—>- 8 )-linked N- acetylneuraminic acid residues of CA are acid sensitive (McGuire and Binkley, 1964) and therefore autohydrolysis of the carbohydrate is possible (Kundig et al, 1971). Hence, this method of purification was not adopted.

Instead, the periodate oxidation reaction was supplemented with ethylene glycol

(2 ml)/NaI0 4 solution (1ml) to expend the excess periodate and terminate the reaction. This process yields a large volume of highly viscous liquid, fi*om which the oxidised CA was purified. Purification of such a product would require a very large GPC column and hence extensive dialysis against a 0 .0 1 % ammonium carbonate buffer at 4°C (12-14KDa mol. wt cut-off, 3 x 2L; 24h) was chosen as an alternative purification method. The recovery of the dialysed oxidised CA was in the range of 95-97%, compared with 85-95% obtained by GPC.

3.3.3 Characterisation of colominic acid and the oxidised derivative by size exclusion chromatography (SEC)

Size exclusion chromatography (SEC) was chosen in this thesis as a practical and simple technique for the characterisation of CA, CA derivatives and later CA- modified peptides and proteins. At the outset of the work, it became apparent that CA behaved abnormally in SEC i.e. its elution volume did not reflect its average molecular weight (lOKDa as determined by low angle laser light scattering; data obtained from Sigma Chemical Company). On the contrary, ‘globular’ proteins are

76 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins known to exhibit a relationship between their elution volume (Ve) and log molecular mass (Mr) (Andrews, 1962). It was therefore decided to compare the chromatographic behaviour of CA (average mol. wt. lOKDa) and sialic acid (NeuSAc) the monomer unit of CA (mol. wt. 309.3Da) with a standard ‘globular’ protein (BSA, 6 6 KDa) of known molecular weight. Additionally, SEC was employed to characterise both intact CA and periodate oxidised CA (prepared in section 3.2.2.1) and confirm the integrity of the internal a- (2^8)-linked N-acetylneuraminic acid residues of CA post periodate exposure (see section 3.3.1).

Results fi"om the latter experiment (Fig. 3.1 A) show that intact CA and periodate oxidised CA have very similar elution profiles, which confirms that the oxidative process did not caused any significant reduction in the molecular weight of the oxidised derivative of CA. The broad elution profiles of CA are due to its polydispersity. As expected. Fig. 3.IB confirmed that the hydrodynamic properties of the native ‘globular’ protein and CA are in fact different. It would appear that CA is retained less efficiently, an effect that may be attributed to the extensive hydration of the carbohydrate moiety. The result is a more expanded overall structure of CA and would explain why its elution volume does not reflect its average molecular weight, i.e. lOKDa (as determined by low angle laser light scattering; obtained by Sigma Chemical Company). Interestingly, Andrews (1965) revealed that certain glycoproteins (containing carbohydrate moieties) deviated from the ‘normal’ model making accurate molecular weight determinations difficult. It follows that it would be unsuitable to calibrate the GPC columns with standard ‘globular’ proteins to estimate the molecular weight of polysaccharide-protein conjugates on account of the shape differences between the two moieties. Indeed, fi"om the elution values (Ve) obtained for CA (16ml) and BSA (14) it is evident that determination of molecular mass of CA would be erroneously overestimated from the ‘normal’ model.

No attempts were made to estimate the molecular weights of the novel neoglycoconjugates prepared in this thesis by SEC, due to the abnormal behaviour of CA in gel permeation chromatography.

77 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

1.00n E c o N 0.75- m 0) o c 0.50- s L_ 8 0.25-

0.00 20 30 40 Elution volume (ml)

1 . 00-1 rl.O O E c R 0.75- -0.75 I“1 lO cr fi) 3 0.50- -0.50 S

Q iUl W 0.25- -0.25 3 3

0.00 ■0.00 0 10 20 30 40 50 Elution volume (ml)

Figure 3.1 Elution profile of A) CA av. mol. wt. lOKDa (•) and oxidised CA lOKDa (□) and B) Bovine serum albumin (BSA), 66KDa (□), CA av. mol. wt. lOKDa (A) and NeuSAc 309.3Da (T) on a Sephadex G-75 (45.0 x 1.0cm; sample volume 0.5ml; PBS eluent; flow rate, 1 .Omlmin'*) column.

78 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

In conclusion, the integrity of the internal a- (2-^8)-linked N-acetylneuraminic acid residues of CA were maintained post periodate exposure thus suggesting that the periodate process had not caused any significant reduction in the molecular weight of the oxidised derivative of CA. The broad elution profile of CA is due to its polydispersity and the deviations from normal behaviour arise due to the size, shape and charge of CA. It was reported (Jennings and Lugowski, 1981) that CA exists in a cyclic configuration in solution, thus CA has a larger effective volume than globular proteins. This phenomenon will be noted in later chapters.

3.3.4 Application of spectroscopic and analytical methods to identify the aldehyde functionality of oxidised colominic acid

The introduction of aldehyde functionality preferentially at the non-reducing end of colominic acid via controlled periodate oxidation is documented as an essential step in the coupling of colominic acid to amine moieties (Fernandes and Gregoriadis, 1996, 1997). In recognition of this, an attempt was made to identify the aldehyde moiety of the newly derivatised colominic acid (i.e. aldehyde containing CA) by its spectral properties and reaction with 2, 4-dinitrophenylhydrazine and compared to colominic acid. Fig. 3.2 shows the structure of colominic acid.

-OH -OH HO OH OH OH OH AcNH AoNH

HO COoHj HO

Non-reducing

Figure 3.2 Structure of colominic acid. Sialic acid (Neu5Ac) units are linked via a- (2—>8) glycosidic linkages. Cl at the non-reducing end of the sugar indicates where periodate oxidation introduces aldehyde functionality.

79 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

COOH COOH 5 0

Figure 3.3 Selective oxidation of the vicinal hydroxyl groups located on adjacent carbon atoms (Cl and C8) via sodium periodate at a) the terminal N-acetylneuraminic acid residue at the non-reducing end of CA to introduce aldehyde functionality at Cl forming b) the oxidised derivative of CA.

3.3.4.1 FT-IR spectral interpretation of colominic acid and oxidised colominic acid derivative Fourier transform-infrared (FT-IR) spectroscopy was the spectroscopic method employed initially in the identification of the newly formed aldehyde functional group at the non-reducing end of periodate oxidised CA (Fig. 3.3B). This method allowed us to study the presence of discrete functional groups in both CA and the oxidised derivative. The FT-IR spectral characteristics of CA and oxidised CA are reported in this section with attempts made to the interpretation of the spectra (see Spectrum 3.1 A and 3.IB).

FT-IR characteristics of colominic acid FT-IR was used in an attempt to identify the O-H stretch of the carboxylic acid and ring hydroxyl moieties of colominic acid (Fig. 3.3A). This is characterised by a very broad intense absorption in the region of 2500-3500cm \ The carbonyl group (C=0 stretch) of the carboxylic acid moiety is typically observed in the region of 1700- 1725cm'\ Conjugation shifts this absorption to lower frequencies (1680-1700cm'^). Other C=0 absorptions, can be found in the region of 1680-1630cm'^ which are indicative of the amide group in the AcNH moiety of the carbohydrate. Conjugation may also shift this absorption to lower frequencies.

80 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

S 9S lO U

in o in o CM o

aouBqjosqv aoueqjosqv Spectrum 3.1 Fourier Transform-Infrared (FT-IR) Spectra of (A) colominic acid anc (B) oxidised colominic acid, (brs, broad strong singlet; s, strong singlet).

81 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

A very broad and intense peak was observed in the region of 3283cm'\ which can be assigned to the O-H stretching of the carboxylic acid and ring hydroxyl moieties. Unfortunately, no peak was identified at 1700-1725cm'*, however a strong peak was identified in the region of 1619 cm'*, which may be indicative of the carbonyl group (C=0) of the carboxylic acid and amide functionality of CA. Other strong absorptions in the FT-IR spectrum can be observed in the fingerprint region e.g. 1040-1200cm * and 1367-1400 cm *. However the absorption bands cannot be correlated to specific functional groups.

FT-IR characteristics of the oxidised colominic acid derivative FT-IR was used to detect the emergence of a new carbonyl group (C=0 stretch) due to the aldehyde moiety introduced via periodate oxidation of CA (Fig. 3.3B). The C=0 absorption is typically characterised by a strong absorption in the region of 1720- 1740cm *. Conjugation to a double bond or benzene ring shifts this absorption to a lower fi"equency (1675cm *). Identification of the C=0 stretch alone is not necessarily evidence for an aldehyde, as carboxylic acids and amides possess this functionality too. The C-H stretching of the aldehyde group (CHO) is also characterised by two peaks at 2700-2900cm'*, although in FT-IR this is displayed as a much weaker band. Single absorptions in the region of 2950-2750cm * are generally assignable to aliphatic C-H stretching.

Unfortunately, the characteristic double peak representing the C-H stretching of the aldehyde group (CHO) at 2700-2900cm * could not be positively identified nor a new absorption peak at 1720-1740cm'* of the aldehyde carbonyl group (C=0 stretch). However, a strong band was observed in the region of 1603cm'*, which may be attributed to the carbonyl group (C=0) of the carboxylic acid and amide functionality as were present in the FT-IR spectra for CA. The O-H stretching of the carboxylic acid (COOH) and ring hydroxyl (OH) groups were seen in the region of 3272cm'* as for CA.

In conclusion, similar functional groups were identified in the FT-IR spectra for both CA and oxidised CA in the region of 1400-4000cm'*. This came as no surprise since

82 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins both compounds contain similar functional groups. However, the complex region of the FT-IR spectra (in the region of 1400-800cm-l), namely the fingerprint region, was found to be unique for CA and oxidised CA. Unfortunately, correlations between absorption peaks and specific functional groups cannot be made with accuracy in this region. It was hoped that the FT-IR spectra for the oxidised derivative of CA would have identified the emergence of a new absorption peak. Ideally identification of a peak corresponding to the aldehyde carbonyl group (C=0 stretch) at 1720-1740cm" ^ and a peak at (2700-2900cm"^) representing the C-H stretching of the aldehyde moiety. Unfortunately, these new peaks were not definitively identified. This phenomenon may be explained by considering the structural conformation adopted by both (CA and oxidised CA) compounds in solution. The predominant form of the NeuSAc residue at the reducing end of CA is cyclic (Jennings and Lugowski, 1981). The C-2 keto group in the open chain form of the NeuSAc residue at the reducing end of CA reacts with the C-6 hydroxyl group to form an intramolecular hemiketal. Similarly, the oxidised derivative of CA containing the new reactive aldehyde moiety at the non-reducing end is prone to attack via a water molecule to form a hemiacetal, Fig. 3.4.

AcNH AcNH AcNH CO.H Non-reducing end HP

OH p O—- ' OH AcNH. / \ I AcNH AcNH CO.H HO CO.HI HO CO„H Non-reducing end Figure 3.4 Rearrangement of the reactive aldehyde introduced at Cl of the NeuSAc residue at the non-reducing end of CA via periodate-oxidation showing the formation of a hemiacetal.

83 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Rowan et al (1951) observed that periodate-oxidised cellulose showed no infrared absorbance characteristics of aldehyde groups and proposed that this was due to: 1) intramolecular formation of stable cyclic hemiacetals via attack of a hydroxyl group on the aldehyde and 2) attack on the second aldehyde group by water.

3.3.4.2 Proton NMR spectral interpretation of colominic acid and oxidised colominic acid derivative Further evidence to corroborate the synthetic conversion of the vicinal diols of CA (Fig. 3.3A) to generate the aldehyde moiety (Fig. 3.3B), was sought for in the NMR spectra. Spectrum 3.2 shows the results for both CA and oxidised CA.

Proton NMR characteristics of colominic acid In ’H-NMR spectroscopy, the acidic proton of a carboxylic acid is seen as a broad singlet far downfield at 510-13. There is no unique splitting pattern associated with the carboxylic acid group because the carboxyl proton has no neighbouring protons. The ^H-NMR spectra for CA showed no signal in the 510-13 region (Spectrum 3.2A).

Proton NMR characteristics of the oxidised colominic acid derivative The carbonyl group (C=0) has a profound effect on protons spectroscopy in NMR. The aldehyde proton is shifted far downfield to a position in the region of 59.3-9.7. ^H-NMR was utilised in an attempt to confirm the synthetic conversion of the diol fragment of CA to the reactive aldehyde of the oxidised derivative of CA by identifying the emergence of a new peak in the region of 59.3-9.7. The *H-NMR spectra for oxidised CA (Spectrum 3.2B) revealed a strong signal at 58.46 and a weak signal at 59.18. These signals could be seen more clearly in the expanded ^H-NMR spectra for oxidised CA (not shown).

In conclusion, no definitive elucidation with respect to the introduction of the aldehyde moiety at Cl of the non-reducing end of oxidised CA could be made using the ’H-NMR technique due to the absence of a new peak in the region of 59.3-9.7. In spite of the fact that both CA and oxidised CA are structurally identical, these compounds did yield slightly different *H-NMR spectra in the region of 51.0- 54.0.

84 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

SIS Colominic acid

52.00 (s, N H C O -C H 3) 53.25-4.68 (m , C H and C H 2 )

5 2 . 5 8

( b r s , O H ) 51.65 (brs, O H )

7 .00 6 . 00 5 .00 3 .00 2.00

Oxidised Colominic acid 53.21-4.73 (m , C H

5 1 . 7 7 ( b r s , O H )

9.00 5 00 4.00 3.00 2 .00 1,00

Spectrum 3.2 H -N M R of (A ) colom ininc acid (B) oxidised colom inic acid, (brs, broad strong singlet; s, strong singlet; m , m ed iu m singlet).

85 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

The factors that could have contributed to the difference of appearance include external factors such as D 2O exchange rate, temperature, solvent concentration and mechanical parameters.

3.3.4.3 Electrospray-Mass spectrum interpretation of colominic acid and oxidised colominic acid derivative Mass spectrometry was the final spectroscopic method employed in the elucidation of the aldehyde carbonyl functionality introduced at the non-reducing end of the oxidised derivative of CA. Spectrum 3.3 shows the results for CA and Spectrum 3.4 for oxidised CA.

Mass spectrum characteristics of colominic acid The mass spectrum for CA (Spectrum 3.3) showed a characteristic pattern, which differed from that of oxidised colominic acid (Spectrum 3.4). The parent ion was shown at m/z 974.5. Prominent peaks were separated by a mass of 136, however no fragment could be attributed to this mass differential.

C:\XcaiiburVJata\OOtrap458 C o I O m i n i C a C i d

00trap45«#5-7 RT 010-015 AV 3 SB 18 0 01-0 08,0 22-0 48 NL 8 25E5 T .CES...,..,.0000. 00000, Parent ion

8 97.2 918.2

1346 1 1478 4 1270.8 I 1430.2 1655.2 1919 7 '1 9 2 7 .3 I t l : "W 0!| I , I ! W ; I !j , i l (I ! I I ,1 1656.1 i-il 1800 1900 Spectrum 3.3 Electrospray-Mass Spectra of colominic acid

86 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

c vxcafibijndata\ootrap459 Oxjdised Colominic acId

00trat>459#2-10 RT 0.03-0 21 AV 9 NL: 2 S7E6 P aTGIlt 1011 T, + c ESI Full m s I 400.0 0 -2 0 0 0 .0 0 )

1 2 8 3 7 10228 1177.1

I , ; 1 4 M . 1 l! ” 30,5 ,258 6

4 8 5 2 840 4

6 0 4 3 6 0 1806 1 8 01.8

7 2 4 ,3 538 8 702-4 5 9 S 4

627.1

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

Spectrum 3.4 Electrospray-Mass Spectra of oxidised colominic acid

Mass spectrum characteristics of the oxidised colominic acid derivative Direct comparisons between the mass spectra for periodate oxidised CA and CA showed obvious differences. However, the characteristic mass fragment of the carbonyl group (C=0) of the newly generated aldehyde at the terminal non-reducing end of oxidised CA was not identified. Ideally, this would have appeared as a strong peak corresponding to a fragment mass of 28 represented as m/z 28 in the spectrum.

3.3.4.4 The 2,4-dinitrophenylhydrazine test of colominic acid and the oxidised colominic acid derivative The spectroscopic methods employed previously did not definitively characterise the aldehyde functional group of the oxidised CA. Hence, our attention was turned to a commonly used qualitative method of chemical analysis specific for aldehydes and ketones. A number of reagents exist which react with aldehydes and/or ketones such as Schiff s reagent or Fehling’s solution. However, the preferred reagent was 2,4- dinitrophenylhydrazine (2,4-DNP), which forms sparingly soluble phenylhydrazones with carbonyl compounds (Fig. 3.5).

87 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

0-lh H N-NH p' RT H AcNH^^C°7 -H2O OgN ^ HO AcNH. '

oxidised CA 2,4dmitrophenylhydrazone (2,4-DNP) non-reducing end CA derivative Figure 3.5 A reaction of 2,4-dinitrophenylhydrazine with the aldehyde (carbonyl) group introduced at the non-reducing end of periodate oxidised CA.

The sparingly soluble phenylhydrazone can be identified as a bright orange/yellow precipitate, which forms immediately or within 5-10 minutes if allowed to stand. It was home in mind that colominic acid and the oxidised derivative possessed carbonyl functionality and therefore both compounds could potentially react with 2,4-DNP to form an insoluble phenylhydrazone however, it was rationalised that this reaction may be used to differentiate between CA and the oxidised CA derivative on the basis of intensity of the reaction. It was hoped that the ‘naked’ reactive aldehyde group of the oxidised derivative of CA would form a dinitrophenylhydrazone precipitate more efficiently than CA, which does not possess the aldehyde functionality. However, CA does possess a ‘masked’ ketone functionality, which is in equilibrium with the more thermodynamically favoured ring structure of CA. Therefore, some reaction of CA with 2,4-DNP reagent was expected.

The 2,4-dinitrophenylhydrazine test of colominic acid and oxidised colominic acid On addition of 2,4-DNP reagent to a sample of CA the pale yellow reagent remained pale yellow. The reaction was followed over 1 hour and the time required for a precipitate to form was noted. A faint yellow precipitate formed within 15 minutes for CA and developed marginally over the hour. These observations are seen in Fig. 3.6 Bl, Cl and Dl. 2,4-DNP reagent was also added to a sample of the lyophilised oxidised derivative of CA. The pale yellow reagent turned orange instantly. The reaction was followed over 1 hour and the time required for a precipitate to form was Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins noted. An intense orange precipitate formed within 10 minutes for the oxidised derivative of CA and continued to develop further over the hour (Fig. 3.6, B2, C2 and

D2).

i 9 . J

Figure 3.6 2,4-dinitrophenylhydrazine assay of colominic acid and oxidised colominic acid derivative: (A) 2,4-DNP reagent only; (B) reaction at t = l.Omin, (1) 2,4-DNP and CA (2) 2,4-DNP and oxidised CA (3) 2,4-DNP reagent only; (C) reaction at t = 10 min, (1) 2,4-DNP and CA (2) 2,4-DNP and oxidised CA (3) 2,4- DNP reagent only; (D) reaction at t = 15 min, (1) 2,4-DNP and CA (2) 2,4-DNP and oxidised CA (3) 2,4-DNP reagent only.

These observations can be correlated to the structural conformation that both compounds (CA and oxidised CA) adopt in solution as described in section 3.3.4.1.

Firstly, considering the reaction of 2,4-DNP reagent with CA, the faint yellow precipitate that developed may be due to the formation of an insoluble dinitrophenylhydrazone with the C-2 keto group in the open chain form of the

Neu5Ac residue at the reducing end of CA. In solution it is unlikely that the reducing terminal sialic acid residue would oxidise to any great extent under these oxidative conditions because CA exists mainly in its ring form (Bhattacherjee et a!., 1975) and as such should behave similarly to an interchain residue. The open ring (keto) form exists in a keto-enol equilibrium (Fig. 3.7) whereby, the thermodynamic stability

89 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

AcNH AcNH AcNH CO.H HO CO„HI HO CO„H

Reducing end end form

-OH -OH -OH HO OH OH OH AcNH AcNH AcNH OH COgH HO CO,H HO CO,

Reducing end keto form

2,4 - DNP reagent

NO2

-OH -OH -OH NOg HO OH OH OH AcNH AcNH AcNH OH

HO CO,H HO

Reducing end 2,4-DNP derivative

Figure 3.7 Schematic diagrams representing the keto-enol equilibrium at the reducing end of colominic acid and the reaction of 2,4 dinitrophenylhydrazine reagent with the C-2 keto (carbonyl) group. The C-2 (keto) open form of CA exists in smaller quantities than the thermodynamically more favourable (enol) cyclic form (Jennings and Lugowski, 1981).

90 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

-OH -OH OH OH OH AcNH AcNH AcNH

HO CO,H| HO

Non-reducing end ‘naked’ aldehyde

H2 O

-OH HO -OH OH OH OH OH AcNH AcNH AcNH

Non-reducing end hemiacetal 2,4-DNP reagent

OH OH O-*— X OH O— ' OH AcNH AcNH AcNH

CO,H HO COgHI HO CO„H

Non-reducing end 2,4-DNP derivative

Figure 3.8 Schematic diagrams showing the ‘naked’ reactive aldehyde introduced at Cl of periodate oxidised CA in equilibrium with the hemiacetal in solution and the reaction of 2,4 dinitrophenylhydrazine reagent with the ‘naked’ reactive aldehyde (carbonyl) group.

91 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins acquired by the cyclic (enol) conformation means that it is less inclined to exist in the open ring (keto) form. This results in the equilibrium shifting towards the more thermodynamically favourable cyclic enol form. This would account for the presence of a much smaller amount of the reactive C-2 keto (carbonyl) group available to react with 2,4-DNP.

Considering the reaction of 2,4-DNP reagent with oxidised CA, the intense orange precipitate that formed instantly with the oxidised CA might suggest the formation of the insoluble dinitrophenylhydrazone with the ‘naked’ aldehyde (carbonyl) group introduced possibly at C7 at the non-reducing end of oxidised CA. The ‘naked’ reactive aldehyde should be very susceptible to 2,4-DNP nucleophilic attack even though it exists in equilibrium as a hemiacetal in solution (Fig. 3.8). Thus, the naked reactive aldehyde of periodate oxidised CA should be readily available to react with 2,4-DNP in contrast to the masked keto (carbonyl) group of CA. Furthermore, the lack of steric hindrance around the carbonyl group of oxidised CA could also account for its increased reactivity with 2,4 DNP reagent compared with the C-2 keto group of CA.

3.3.4.S Aldehyde determination: Qualitative and quantitative methods The 2,4-dinitrophenylhydrazine test of colominic acid and colominic aldehyde was a simple and convenient method that successfully qualitatively identified aldehyde formation of periodate oxidised CA by comparison of the two samples as per our objective. It was however realised that, a quantitative determination of aldehyde content of periodate oxidised CA was important as well as the location of aldehyde introduction. Although the 2,4-DNP test is a colorimetric method, no attempts were made to prepare a standard calibration curve for the quantification of aldehyde as the strongly acidic nature of the 2,4-dinitrophenylhydrazine reagent decomposes CA over time. A quantitative study is highly recommended for future work.

Interestingly, Zhang (1999) reported on a method to quantitatively determine the aldehyde content of periodate oxidised CA (average mol. wt. lOKDa) prepared under the same conditions as in our study. The degree of oxidation of CA was ascertained

92 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins via a radiolabelling method, whereby the aldehyde (CHO) was reduced to the hydroxyl (Orf) under the treatment with NaB[^H ] 4 (Zhang, 1999). Controlled periodate oxidation of CA was performed under similar reaction conditions reported in section 3.2.2.1 and the yield of colominic aldehyde was estimated at ~ 82-92%.

Colominic aldehyde (0.1 mg) was labelled with NaB[^H ] 4 and the molar ratio of aldehyde introduced per 1.0 mole of CA was determined. Results showed that ~1.0 mole of aldehyde was introduced per 1.0 mole CA in the reaction mixture {i.e. 1: 1 aldehyde: CA molar ratio). Assuming that only the non-reducing end residues of CA were preferentially oxidised, the equimolar ratio of aldehyde to CA might suggest that 100% of the non-reducing end CA residues were converted to the aldehyde (Zhang, 1999). To date, the location of aldehyde introduction is hypothetical and it is strongly suggested that further work is conducted to substantiate the position of the ‘newly’ introduced aldehyde functionality.

Interestingly, Wileman et al. (1986) reported on the periodate oxidation of polyaldehyde dextran. The aldehyde content of the polymer (600mg) was determined using hydroxylamine reagent (British Pharmacopoeia, 1973). Hydroxylamine hydrochloride reacts stoichiometrically with aldehydes to yield hydrochloric acid; the production of acid was determined by volumetric titration using potassium hydroxide. The results showed that 0.86 moles of aldehyde were introduced for each mole of glucose present in the reaction mixture. Thus ~ 40% of the glucose residues were converted to the dialdehyde.

To summarise, based on the unique structure of CA, sodium periodate was employed in an attempt to preferentially cleave only the terminally located non-reducing end vicinal hydroxyl groups of CA via controlled periodate oxidation to introduce a free reactive aldehyde for the subsequent conjugation to a protein. Although very specific spectroscopic techniques such as MS, ^H-NMR and FT-IR were used to identify the ‘newly’ introduced aldehyde moiety, the results obtained were unfortunately insufficient to substantiate the hypothesis. Fortunately, the 2,4-DNP chemical analysis method did qualitatively indicate the introduction of aldehyde functionality of periodate oxidised CA in comparison with non-oxidised CA. Unfortunately, the

93 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins percentage aldehyde content and location was not verified and is strongly suggested for further work. SEC results offered further characterisation of the oxidised derivative of CA and suggested that the conditions of periodate oxidation used in this study do not cause diminution of the CA molecule.

3.3.5 Application of the fluorescamine reagent for the detection and quantification of polysialylated L-lysine Having characterised the oxidised derivative of CA for carbonyl functionality via the 2,4-DNP method, it was our intention to subsequently conjugate it to the amino acid L-lysine. However, before we could proceed with the covalent conjugation of L-lysine to oxidised CA, our primary consideration at this juncture was choosing a method suitable for the detection and quantification of the L-lysine amino acid in the L-lysine- CA conjugate. In this instance, fluorescamine, (4-phenylspiro [furan-2 (3H), 1’- phthalan]-3,3'-dione) was chosen as the most suitable reagent for the assay of L- lysine. It was proposed that fluorescamine should reacted directly with CA linked L- lysine as shown in Fig. 3.9.

Ph NH, Ph HOOC COOH OH CA linked Lysine COOH

Fluorescamine Fluorophor

Figure 3.9 Reaction of fluorescamine with CA linked amino acid, L-lysine.

3.3.5.1 Detection and quantification of the amino acid L-lysine The fluorescamine assay was adopted, as it offered a technique sensitive enough to monitor dilute samples of the single amino acid L-lysine in the L-lysine-CA conjugate from GPC effluent. Various other methods of amino acid, peptide and protein determination were also considered for this purpose and are discussed in this section.

94 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

For instance, the Lowry method of protein determination was considered however; a comparative study conducted by Lowry et al. (1951) on the fluorescamine technique with the standard Lowry procedure for the monitoring of protein in column effluent found the fluorescamine assay a much more sensitive and accurate method of amino acid determination. The Lowry reaction involves the whole peptide chain, whereby colour development is enhanced in those proteins consisting of amino acid residues containing aromatic rings. This procedure was also subject to a wide range of interferences from substances including carbohydrates and buffer salts (Bradford, 1976). Alternative methods also considered for amino acid determination included measurement of absorbance in the far-UV (205nm) region and the dye-binding method of protein estimation (Bradford, 1976). However, the disadvantage of operating at such a low wavelength (far-UV region) was the high risk of interference from other non-protein species (e.g. CA absorbs strongly in this region).

The Bradford method principally involves the interaction between the anionic form of the dye and basic amino acid residues as well as those consisting of aromatic groups (Compton and Jones, 1985). This method had several advantages over that of Lowry in that it offered a fourfold improvement in protein detection (Bradford, 1977), was more simplistic, less time consuming, more reproducible and had a better overall resistance to interference (Bradford, 1976; Spector, 1978; Sedmark and Grossberg, 1977). However, in this instance this method could not be employed for the detection of L-lysine in the L-lysine-CA conjugate as the colour obtained with L-lysine (a basic amino acid with an aliphatic structure) was very faint. Thus, the fluorescamine assay was adopted as the method for amino acid determination in this chapter. Fig. 3.10 shows a typical calibration curve obtained for the estimation of the amino acid L- lysine concentration by the fluorescamine method. Two sets (n=2) of L-lysine standards were used in the preparation of the standard curve. The L-lysine-CA conjugate (0.1-0.5ml) was mixed with 500pl of the fluorescamine solution and 1.75ml of PBS and measured by the same method.

95 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

250-1

E 200-

150 -

P 100-

r2 = 0.9983

0 10 20 30 40 50 60 70 80 Concentration (ng/ml) Figure 3.10 Typical calibration curve obtained for L-lysine in 500|il of fluorescamine. (excitation wavelength (Ex) set at 390nm, emission wavelength (Em) set at 475nm)

3.3.S.2 Synthesis of polysialylated L-lysine The technique by which periodate oxidised CA (average mol. wt. lOKDa) was coupled to L-lysine (mol. wt. 146.19) was described in section 3.2.2.5. This reaction involved an initial molar ratio of 50:1 (L-lysine: CA), whereby L-lysine was in excess. A control experiment was conducted simultaneously such that all the experimental conditions were the same as for above. The degree of polysialylation was estimated post extensive dialysis and GPC purification (section 3.3.5.3) of the reactants. The yield of conjugation obtained in both experiments was expressed in terms of L-lysine: CA molar ratio and the results can be seen in table 3.2.

The direct method of covalent coupling L-lysine to periodate oxidised CA via reductive amination, in aqueous solution, yielded a molar conjugation ratio of approximately one mole CA to 1.00 ±0.1 moles of L-lysine at the end of the 48 hour reaction. In the control experiment the experimental conditions were kept identical except that non-oxidised CA was employed in the conjugation reaction. This yielded a conjugate containing approximately 3-fold less sialic acid coupled to L-lysine, which was estimated as one mole CA to 0.35 ± 0.04 moles of L-lysine.

96 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Table 3.2 The degree of polysialylation of L-lysine under the reaction conditions described and expressed as moles L-lysine per one mole CA (L-lysine: CA molar ratio). Non-oxidised CA was included as the control. Colominic acid Initial Time of Molarity of Molar ratio of average mol. wt molar ratio of reaction phosphate buffer CA: L-lysine lOKDa CA: L-lysine (hours) at pH 9.0 in conjugate CA 50: 1 48 0.75M 1 .0 : 1 . 0 0 ± 0 .1 (Oxidised) Non-oxidised CA 50:1 48 0.75M 1.0: 0.35 ±0.04 (Control)

By comparison of the data, the results suggest that modification of the coupling procedure by first introducing a functional aldehyde group (preferably at the non reducing end) of CA is beneficial to couple L-lysine to CA. The proposed mechanism by which the control reaction may have occurred is via the direct linkage of L-lysine to the hemiketal (C=0) group of CA at its reducing end. This process does not require prior periodate activation of CA and would appear to be a more simple approach, however the poor molar yield of conjugation obtained discredits this approach. By considering the structural conformation that CA adopts in solution (section 3.3.4.1) it was expected that conjugation through the C-2 keto-group of the terminal sialic acid residue at the reducing end of CA would yield diminished reactivity and a low molar conjugation ratio. Jennings and Lugowski (1981) reported similar findings for the conjugation of the same polysaccharide to (TT) tetanus toxoid. It was also considered that weak electrostatic forces of attraction between positively charged L-lysine and negatively charged CA may contribute to the molar conjugation yield however, a high molarity buffer was incorporated into the coupling reaction to counteract these effects.

To summarise, polysialylation of L-lysine with activated CA led to an apparent 3-fold increase in the degree of conjugated CA (1.14 ± 0.1 moles of L-lysine per mole of CA) compared with the control experiment (0.35 ± 0.04 moles of L-lysine per mole of CA). Results (table 3.2) suggest that the degree of polysialylation may be directly dependant on the prior introduction of the aldehyde functional group of CA.

97 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.3.5.3 The purification and isolation of polysialylated L-lysine Purification and isolation of polysialylated L-lysine was attempted by first extensively dialysing the reaction mixture to remove fi*ee non-covalently linked L-lysine. Then, the conjugate constructs were isolated from the reaction mixture by fractionation using a Sephadex G-25 (25.0 x 1.0cm in PBS) column. This method was employed to isolate the polysialylated L-lysine conjugate from any un-reacted L-lysine, CA and smaller molecular weight reagents.

The eluted fractions were subjected to sialic acid assay and amino group assay (Fig. 3.11 A and 3.1 IB). Fig. 3.11 A represents the elution profile of an aliquot of the reaction mixture that was taken immediately after adding all the reagents (zero time) and was regarded as the control. Fig. 3.1 IB shows the elution profile of a sample of the dialysed reaction mixture post 48 hours reaction. The amino and sialic acid positive fractions from Fig. 3.1 IB were pooled and the degree of polysialylation estimated by assaying the isolated product for sialic acid and L-lysine (amino group). The isolated product was lyophilised and stored at -40°C.

3.3.5.4 Size exclusion chromatographic (SEC) characterisation of polysialylated L-lysine Two partially overlapping peaks are observable in Fig. 3.11 A, which in theory should have been completely resolved due to the differences in their molecular weights i.e. CA (average mol. wt. lOKDa) and L-lysine (146.19Da). The incomplete resolution may be attributed to the polydispersed nature of CA. This highly sialylated macromolecule is known to behave abnormally in gel filtration as shown earlier in section 3.3.3. Nonetheless these peaks are sufficiently resolved and indicate the presence of un-reacted CA and L-lysine respectively.

Comparisons with the elution profile of Fig. 3.1 IB (48 hours reaction) show a very obvious shift of CA and the L-lysine peak into a higher molecular weight, which incidentally have co-incident elution profiles. Fig. 3.1 IB showed no observable peak due to ‘free’ un-reacted Lysine, which would suggest that dialysis, had removed the excess L-lysine. The absence of a distinct ‘free’ CA peak in Fig. 3.1 IB may also

98 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

1.00 -, r30

c o 0 .7 5 - o m -20 8 S 0 .5 0 - 3 S - 1 0 0 .2 5 - Î

0.00 0 10 20 30 Elution volume (ml)

r1 5 E c c o o N lO - 1 0 8 8 3 O (D 8 Î

0.0 0 10 20 30 Elution volume (ml)

Figure 3.11 Size exclusion chromatography of colominic acid (À) and L-lysine (■) after A) zero time and B) after 48 hours of reaction. Starting molar ratio 50:1 L-lysine: CA. Samples were chromatographed on a Sephadex G-25 (25 x 1.0cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'*) column. Arrows indicate the co elution volume of the conjugate (Ve = 9.0ml) and the void volume (Vo = 2.0ml) of the column.

99 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins suggest that the technique of ammonium sulphate precipitation was appropriate to separate the proposed neoglycoprotein from un-reacted components.

The eluted CA fractions were also subjected to the fluorescamine amino acid assay and found to be negligible i.e. fluorescamine did not react with CA. Thus the co elution of CA and L-lysine in the same elution volume (Ve) of the column might suggest that a covalent link existed between the two molecules, however further studies are warranted to rule out other types of bonding.

Native CA and L-lysine were mixed together and applied to the column to rule out the possibility of an ionic interaction between these oppositely charged molecules. This was verified as the elution profile obtained (data not shown) assumed that of Fig. 3.11 A (zero time).

3.3.6 Size exclusion chromatographic (SEC) characterisation of colominic acid oligomers produced by hydrolysis

Lifely et al. (1986) reported on the instability of CA under high temperature. By varying the time of heating CA in PBS (pH 7.4) at 100°C, this yielded oligomers of sialic acid, thus offering a method for the controlled hydrolysis of the polysaccharide.

In this study, CA was boiled for 5 and 8 hours respectively and produced two smaller molecular weight derivatives of CA denoted as CA 5 and CAg respectively. Fig. 3.12 shows the elution profiles of each new derivative CA 5 and CAg with respect to CA intact and N-acetylneuraminic acid (NeuSAc) (309.3 Da), following their application to a Sephadex G-75 (45.0 x 1.0cm) column.

Indeed, the elution profiles demonstrate that over the time course of boiling CA there was definitely the emergence of two new derivatives CA 5 and CAg with a shift into a lower molecular weight. From the elution profiles in Fig. 3.12 it would appear that the order of size was, CA> CA$> CAg> NeuSAc. In order to make the correlation between degree of polysialylation of IgG and CA size, the smaller oligomers CA 5 and CAg derived from CA hydrolysis above were chosen for conjugation to IgG.

100 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

fbbuuu Muvb 10 20 30 40 Elution volume (ml)

Figure 3.12 Elution profiles of: (A) CA (av. mol. wt lOKDa) and the oligomers of CA (prepared by boiling CA at 100°C for 5 and 8 hours respectively) denoted as (X) CAs, (T) CAg and (A) NeuSAc (309.3 Da). A Sephadex G-75 (45.0 x 1.0cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^) column.

Thus, aliquots (SOOpl) of the sialic acid positive fractions (Fig. 3.12) for both CA$ and CAg were applied to a Sephadex G-50 column (45.0 x 1.0cm) pre-calibrated with proteins (Fig. 3.13) to estimate their molecular weights respectively. By this comparison, the estimated molecular weights of CA 5 and CAg exhibited were 6 and 3 KDa respectively. However, it was borne in mind that this method might lead to erroneous overestimations of the molecular weights of CAg and CAg as CA behaves “abnormally” in gel filtration chromatography as established in section 3.3.3. These results do however, support the order of size as CA> CA$> CAg >Neu5Ac. Interestingly, Lifely et al. (1986) reported on chain length determination of oligomers and polymers of sialic acid by several methods, one of which involved the radiolabelling of the reducing end residue by reduction with borotritiide. Zhang (1999) adapted this method for the molecular weight determinations of CA (average mol. wt lOKDa) post controlled hydrolysis. The analytical method was based on the theory that one atom could be gained by the reducing end of CA upon exposure to

NaBH3(H^). By measuring the radioactivity per mole CA, the number of end groups

101 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

10®-g

- Triosephosphate isomerase

- Myoglobin u-Lactalbumin Aprotinin CAs % ^^^'mksulin chain 3 , oxidised - CAg 10®- Bradykinin

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ' 1 1 1 ' 1 n 1 1 1 1 ' r' 1 ' 1 1 0. 5 1.0 1.5 2.0 2.5 3.0 3.5

Figure 3.13 Plot of the ratio VJVo (elution volume (Ve) over (Vo) void volume) versus log molecular weight (Mr) for standard proteins on a Sephadex G-50; column, 45.0 X 1.0cm; sample volume 500pl; PBS; flow rate, 1.0mlmin'\ Standards were: triosephosphate isomerase, 26.6KDa; myoglobin, 17KDa; a- lactoalbumin, 14.2KDa; aprotinin, 6.5KDa; insulin chain p, oxidised, 3.5KDa and bradykinin, 1060Da. Void volume (Vo) = 13ml. was identified and thus the molecular weights were estimated. The findings (Zhang, 1999) revealed that CA chain length decreased with increased heating time, whereby the molecular weights of the derivatives from CA boiled for 3 and 9 hours were estimated to be 6.3KDa and 3.9KDa, respectively.

Having attempted to estimate the molecular weights of CAg and CAg {i.e. 6 and 3 KDa respectively), CAg and CAg were subsequently periodate oxidised and chromatographed using a Sephadex G-75 (45.0 x 1.0cm) column. Their elution profiles were similar to non-oxidised CAg and CAg chromatographed in Fig. 3.12 (results not shown). The sialic acid positive fractions for both CAg and CAg were pooled and dialysed extensively (3 x 2L; 24h) against (0.15M) PBS at 4°C utilising dialysis tubing of nominal molecular weight cut-off, 3.5 KDa. Retention of CAg (estimated mol. wt 3KDa) using a membrane with a mol. wt cut-off of 3.5KDa may be explained on the basis that fractionation of CA was dependant upon its shape as well

102 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins as its size. As established in section 3.3.3, CA adopts an increased hydrodynamic size in solution. This was reflected in the 92% of CAg retentate recovery from the dialysis bag post dialysis. In order to ascertain the effect of polymer size on the degree of modification of polysialylated IgG, the hydrolysed derivatives (CAg and CAg) were subsequently oxidised and coupled to IgG. The results can be seen in section 3.3.7.

To conclude, CA (av. mol. wt lOKDa) yielded two lower mol. wt oligomers denoted as CAs and CAg when hydrolysed in a water bath at 100°C for 5 and 8 hours respectively (Fig. 3.12). These oligomers were characterised by employing the technique of size exclusion chromatography (SEC) (Fig. 3.12). Although smaller oligomers were obtained they were still fairly disperse owing to the polydisperse nature of the polymer CA. An attempt was nevertheless made to estimate the molecular weight of the CAs and CAg oligomers by application to a SEC column pre calibrated with protein standards. Whilst it is acknowledged that differences due to shape of these molecules makes this a ‘non-ideaT method, it was proposed that an order of size of the oligomers relative to CA could be determined.

The molecular weight determinations of CAs and CAg were estimated at 6 and 3 KDa respectively (Fig. 3.13). Interestingly, these findings were similar to the findings of Zhang (1999) although different methodologies were applied in the analysis.

3.3.7 Factors affecting the degree of modification of polysialylated IgG

In an attempt to review the factors that affected the degree of modification of polysialylated IgG, this section aimed to identify the correlation between starting molar ratio of CA: IgG, reaction time and CA size on the molar yield of polysialylated IgG obtainable. A series of experiments (1, 2 and 3) were conducted (section 3.2.2.7.1) and the results are discussed here. Experiments (1 and 2) involved reacting periodate oxidised CA (av. mol. wt lOKDa) with IgG (mol. wt ISOKDa) at starting molar ratios of 50:1 and 100:1 (CA: IgG). The process of reaction was via reductive amination in the presence of NaBHsCN at 35-40°C. The control experiments were conducted simultaneously whereby all the experimental conditions were kept the same

103 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

7.5n -r (100: 1) (100: 1) i (100: 1) 5.0-

O Œ Q) Ô 2.5- l i 0 .0 -<’

1 (100: 1)

(50: 1)

2.5

(control) (50: 1)

0.0 0 6 12 18 24 30 36 42 48 Time (h)

Figure 3.14 Molar ratios of colominic acid (CA): IgG in the conjugates of IgG with different MWs of CA, (isolated by ammonium sulphate precipitation) as a function of time. (A) oxidised CAg (~ 6 KDa): IgG (100: 1) (•); oxidised CAg (-3KDa): IgG (100: 1) (o) and oxidised NeuSAc (309.3Da): IgG (100: 1), (T). (B) Non-oxidised CA (av. mol. wt. lOKDa): IgG (50: 1), control (□); oxidised CA: IgG (50: 1); (A) oxidised CA: IgG (100: 1% (■). Values are a mean ± s.d of 3 experiments. CAg and CAg denote oligomers of CA, with an order of size CA> CAs> CAg >Neu5Ac. See Fig. 3.15 for controls of non-oxidised CAg, CAg and NeuSAc with IgG (100: 1).

104 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins except that either non-oxidised CA was included instead of oxidised CA or CA was omitted altogether. Essentially, a time study was performed whereby aliquots (500|il) of the reaction mixture were removed at intervals of 0, 6 , 12, 24 and 48 hours. The polysialylated conjugates were isolated from the reactants and free, non-covalently linked CA by ammonium sulphate precipitation. The results were expressed in terms of CA: IgG molar ratios found in the precipitates and presented in Fig. 3.14.

Both ratios (50: 1 and 100: 1) shown in Fig. 3.14B showed an initial rapid rate of reaction, reaching a plateau at around 12 hours, lasting for at least 48 hours. In contrast, the control (non-oxidised CA) exhibited (Fig. 3.14B) a slower initial reaction, reaching a plateau around 6.0 hours and lasting again for at least 48 hours. The reaction kinetics exhibited in Fig. 3.14B was reflected in the reaction mechanism for the coupling of colominic acid to proteins (Fig. 1.5). This reaction proceeds by the formation of an unstable Schiff base which is initially in equilibrium with the reactants i.e. CA and protein. Thus, in the presence of NaBHgCN, the Schiff base is readily reduced and the equilibrium quickly shifts to the right thus increasing the yield of conjugation until saturation is achieved and no more conjugate is made. The reactions were terminated after 48 hours as earlier conjugation experiments (results not shown) with CA and IgG (50: 1) conducted over 72 hours showed no significant increase or reduction in the molar yield of conjugation.

The reaction kinetics for the three controls prepared in experiment 3 (section

3.2.2.7.1) employing smaller oligomers of non-oxidised CA i.e. CAg (~ 6 KDa), CAg (~3KDa) and Neu5Ac (309.3Da) are exhibited in Fig. 3.15. Again these controls show a slower initial reaction compared with their oxidised CA counterparts (Fig. 3.14A), reaching a plateau around 1.2 hours and lasting at least 48 hours. The final conjugation yield obtained after 48 hours with the non-oxidised derivatives of CA seem independent of their size.

The degree of modification of polysialylated IgG obtained in experiments 1, 2 and 3 (section 3.2.2.7.1) post 48 hours reaction was expressed in molar terms of CA: IgG and the results can be seen in table 3.3. Essentially, the results (table 3.3) indicate that

105 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

■o Î 0 3 O o 09 TJ 10 z (0 o % < o 3 1 -) o oZ Ü

0.0 0 6 12 18 24 30 36 42 48 Time (h) Figure 3.15 Molar ratios of non-oxidised colominic acid (CA): IgG in the conjugates of IgG with different MWs of CA, (isolated by ammonium sulphate precipitation) as a function of time. (*) Non-oxidised CAg: IgG (100: 1), (0); non-oxidised CAg: IgG (100: 1) and (X) non-oxidised NeuSAc: IgG (100: 1). Values are a mean ± s.d of 3 experiments. CAg and CAg denote depolymerised derivatives of CA, with an order of size CA> CAs> CAg >Neu5Ac. covalent coupling of periodate oxidised CA to IgG (50: 1) via reductive amination, in aqueous solution yielded an apparent molar conjugation ratio of approximately 4.25 ± 0.20 moles of CA to 1.0 mole IgG. Considering that each immunoglobulin G (IgG) molecule consisted of 1320 amino acids of which 92 were lysine residues, (Edelman et al., 1969), this ratio corresponded to the polysialylation of an average of 4.62% of the available lysine groups.

In contrast, the direct coupling of non oxidised CA to IgG (control) by reductive amination, in aqueous solution, yielded an apparent conjugate containing approximately 3-fold less CA coupled to IgG. Thus, the molar ratio of CA coupled to

IgG was estimated at 1.25 ± 0.36: 1.0. Similarly, non-oxidised CA 5, CAg and Neu5Ac (controls) reacted with IgG (100: 1) yielded conjugates containing 4.5 to 5.5-fold less coupled CA compared with the oxidised counterparts. The differences in molar yields of conjugation observed for activated CA-modified IgG and non-activated CA- modified IgG conjugates (controls) may be reflected by the structural conformation

106 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Table 3.3 The degree of polysialylation of IgG under the reaction conditions described and expressed as moles CA per one mole IgG (CA: IgG molar ratio). Non- oxidised CA, CAs CAg and NeuSAc were included as controls. Colominic acid Initial Time of Molarity of Molar ratio of molar ratio of reaction phosphate buffer CA: IgG (CA) CA : IgG (hours) at pH 9.0 in conjugate CA(lOKDa) SO: 1 48 0.7SM 4.2S±0.20: 1.0 (Oxidised) CA(lOKDa) 1 0 0 : 1 48 0.7SM 6.70 ±0.20: 1.0 (Oxidised) CAs (6 KDa) 1 0 0 : 1 48 0.7SM S.36 ± 0.28: 1.0 (Oxidised) CAg(3KDa) 1 0 0 : 1 48 0.7SM 6.61 ±0.2S: 1.0 (Oxidised) NeuSAc (309.3Da) 1 0 0 : 1 48 0.7SM 7.01 ±0.08: 1.0 (Oxidised) Non-oxidised CA SO: 1 48 0.7SM 1.2S±0.36: 1.0 (control) Non-oxidised CAs 1 0 0 : 1 48 0.7SM 1 .2 0 ± 0 .2 1 : 1 .0 (control) Non-oxidised CAg 1 0 0 : 1 48 0.7SM 1.24 ±0.12: 1.0 (control) Non-oxidised 1 0 0 : 1 48 0.7SM 1.26 ±0.09: 1.0 NeuSAc (control) that CA and non-oxidised CA adopt in solution (see section 3.3.4.4). Whilst the thermodynamically favoured form of non-oxidised CA at the reducing end is in the cyclic (enol) form, a small amount of CA that exists in the C-2 (keto) form may be able to react with the peptide or protein thus producing a low conjugation yield. In comparison, the highly available reactive naked aldehyde introduced preferentially at the non-reducing end of activated CA possibly accounts for the higher molar yields of conjugation observed for activated CA-modified IgG.

A higher degree of polysialylation was achieved by increasing the starting molar ratio of reactants i.e. CA to IgG (100: 1) to yield 6.70 ± 0.20: 1.0 moles CA: IgG. This ratio corresponded to the polysialylation of an average of 7.3% of the available lysine groups. Although different coupling chemistries were employed, Suzuki et al. (1984) found that increasing the starting molar ratio of mPEG to IgG resulted in increased molar yield conjugates.

107 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Jennings and Lugowski (1981) attempted to covalently couple the group B polysaccharide (equivalent to CA) to bovine serum albumin (ESA) at a 6 8 : 1 (CA: ESA) molar ratio via the method of Schiff base reductive amination. Ey first incorporating a terminally located aldehyde in the polysaccharide and then coupling this more reactive group in the reductive amination procedure yielded a conjugate with 4-fold more sialic acid. In parallel with our experiments, this methodology proved to be more successful and was thus employed to couple oxidised CA and the smaller oligomers of CA le. CAg, CAg and NeuSAc to IgG (100: 1). It was proposed that CA could be more efficiently coupled to IgG by using CA of reduced size. Thus, CA oligomers were prepared as described in section 3.2.2.6 of two smaller derivatives denoted as CA 5 and CAg. The monomer unit of CA (NeuSAc) was also included in this study (order of size CA> CAs> CAg> NeuSAc as established in section 3.3.6).

Experiment 3 (section 3.2.2.7.1) involved reacting periodate oxidised CA 5 and CAg

(estimated mol. wt 6 and 3 KDa, respectively) and oxidised NeuSAc (309.3 Da) with IgG at a 100:1 starting molar ratio. The time study and controls were performed as in experiments 1 and 2 (section 3.2.2.7.1). Fig. 3.14A showed that the initial rate of reaction was faster with the smaller oligomers of CA, whereby the reactions reached a peak around 6 hours and a plateau was observed over 12-48 hours. These results may be attributed to the increased mobility of the smaller CA molecules and increased accessibility to the binding sites of IgG. The degree of modification of IgG obtained after 48 hours reaction with oxidised CA 5 , CAg (estimated mol. wt 6 and 3 KDa, respectively) and Neu5Ac (309.3 Da) was marginally improved compared with that seen with oxidised intact CA (table 3.3). However, further comparison with the molar conjugation ratio of CA: IgG (100: 1), whereby oxidised CA (average mol. wt 1 OKDa) was employed, yielded a degree of coupling similar to that of CAg coupled to IgG (100: 1). The results suggest that the molar conjugation yield of IgG with CA is independent of the size of CA however; a larger molecular weight range of CA should be employed to definitively prove this relationship.

Similarly, Mehvar (1994) reported that the degree of modification of insulin with dextran was independent of its size. The studies involved preparing insulin-dextran conjugates, whereby periodate oxidised dextran, (mol. wt 40KDa, 7OKDa and

108 Chapter Three: Synthesis &. characterisation ofpolysialylated peptides and proteins

500KDa, respectively) were conjugated to insulin via the method of reductive amination in the presence of sodium borohydride. The reaction of insulin with dextran aldehyde was monitored by measurement of the conjugate at 24, 48 and 72 hours after the start of the reaction.

It was considered that chemical modification of IgG at 35-40°C might result in a concomitant loss of its biological activity, thus an attempt was made to conduct the reductive amination reaction at a temperature low enough to minimise the loss of biological activity. However, when CA was incubated with IgG (50: 1) at 4°C the degree of polysialylation of IgG was found to be disappointingly poor. The reaction yielded 1.44 ± 0.05 moles CA to 1.0 mole IgG, thus excluding the possibility of operating at this temperature.

In an attempt to justify the use of the reducing agent sodium cyanoborohydride for the stabilisation of the Schiff base linkage to produce a stable protein conjugate, Fernandes and Gregoriadis (1996) reported on the conjugation of periodate oxidised CA to catalase (50: 1) incubated at 35-40°C (pH 9.0) in the absence of the reducing agent. This reaction yielded 0.65 ± 0.04 moles CA to 1.0 mole catalase; however, the extent of polysialylation of catalase was approximately 4-fold more in the presence of sodium cyanoborohydride. It was hypothesised that the proposed Schiff base formed between the aldehyde moiety introduced in CA and the reactive e-lysine of catalase was to some extent stable. Therefore, the conjugate can exist albeit to a lesser extent as a Schiff base conjugate without reductive amination.

Other key factors in addition to the reaction conditions that could have contributed to the degree of sialylation of IgG included: the availability of surface reactive 8 -lysine and amino residues in the protein. Edelman et al. (1969) reported on the entire amino acid sequence of immunoglobulin G molecule (150KDa). The molecule was found to consist of 1320 amino acids of which 92 were identified as e-lysine residues with a potential for conjugation to CA. However, the poor degree of polysialylation of IgG reflected not only the need for a more efficient conjugation but also the importance of structural conformation of the protein and thus surface proximity of the reactive

109 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

8 -lysine and amino residues. Roy et al. (1994) postulated that low incorporation of sialic acid into proteins was attributed to the increasing negative charge of the growing neoglycoprotein, thus preventing incorporation beyond a certain point. In the case of CA, this effect could have been exacerbated by its polymeric nature. A higher degree of polysialylation was achieved with polylysine as compared with IgG, whereby the extent of sialylation was estimated as 6.76 ± 0.31 moles CA to 1.0 mole of polylysine (Fernandes et al., 1996). It was proposed that this was not only due to the higher availability of amino groups but also a counteracting effect of the increase of negative charge in the forming cationic neoglycoprotein. Further limitations to the degree of polysialylation could have occurred because of the steric hindrance offered by polysialic acid.

To summarise, it was established that the extent of polysialylation of IgG was dependent upon the starting molar ratios of CA: IgG used in the coupling reaction (Fig. 3.14 and table 3.3). The introduction of carbonyl functionality (C=0) of CA prior to conjugation was also found to yield higher molar ratios of IgG: CA (table 3.3) than direct coupling with non-oxidised CA (control). This phenomenon was also observed in work conducted by Fernandes and Gregoriadis (1996) and Jennings and Lugowski (1981). The degree of modification of IgG was found to be independent of the size of CA coupled as seen in table 3.3 which, is in agreement with polysialylated catalase (Fernandes and Gregoriadis, 1996) and insulin-dextran conjugates, (Mehvar, 1994).

The reaction conditions found most favourable for optimal conjugation yields with IgG required temperatures of 35-40°C at pH 9.0 in the presence of sodium cyanoborohydride and 48 hours to complete. Although conjugation did occur in the absence of sodium cyanoborohydride (NaBHsCN), the extent of sialylation of catalase was approximately 4-fold more in the presence of NaBHsCN, (Fernandes and Gregoriadis, 1996). Thus NaBHgCN was employed in all our conjugation reactions.

110 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.3.8 Size exclusion chromatographic characterisation of polysialylated IgG

Size exclusion chromatography (SEC) was performed in an attempt to characterise the conjugate and provide evidence to suggest that CA had covalently coupled to IgG thus forming a stable conjugate. Essentially, samples from the zero and 48 hour aliquots obtained from the time study in experiment 1.0 (section 3.2.2.7.1), were applied to a Sephadex G-lOO column. The zero hour sample was regarded as the control, whereby the aliquot was removed from the reaction mixture immediately after adding all the reagents (Fig. 3.16A). The eluted fractions were assayed for protein (A595nm) and total sialic acid (A570nm) content. Figs. 3.16A and 3.16B show the elution profiles of the zero time and 48 hours samples respectively.

The two partially overlapping peaks observable in Fig. 3.16A (zero time) should have been completely resolved in light of the differences in their molecular weights i.e. CA (average mol. wt lOKDa) and IgG (ISOKDa). This poor resolution may be explained by the polydisperse nature of CA, discussed in section 3.3.3. However, the peaks were sufficiently resolved to indicate the presence of unreacted CA and IgG at zero time. Conversely, both peaks in Fig. 3.16B were shifted to the left and both CA and IgG were co-eluting in elution volume (Ve) 12ml of the column. It was hypothesised therefore that the emergence of a ‘new’ derivative with an increased molecular weight might represent a covalent link existed between CA and IgG. It was thus proposed that the Schiff base reductive amination reaction might have yielded polysialylated IgG. Further evidence to corroborate the synthesis of the conjugate was sought for in SDS- polyacrylamide gel electrophoresis (SDS-PAGE) experiments (see section 3.3.10). In the case of the smaller oligomers of CA from experiment 3.0 (section 3.2.2.7.1), complete resolution resulted when the zero time sample from the reaction mixture consisting of IgG and the oligomer of CA (CA 5) was applied to the column. Indeed, the differences in molecular weights were sufficient for these components to be completely resolved by the technique of SEC (Fig. 3.17A). Fig. 3.17B shows the proposed conjugate co-eluting in fraction 1 2 ml of the column, thus suggesting the synthesis of polysialylated IgG, even with the low molecular weight polysialic acid, CAg. Further experiments are warranted to corroborate the synthesis of the conjugate.

Ill Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

r1.5

(A Ou O' - 1.0 fi> 0) 3 o c S (Ü SI cn (O O 0.5- -0.5 cn U) 3 3

0.0 -0.0 0 10 20 30 40 Elution volume (ml)

B I.OOn i r2.0 E c (A o 0.75- -1.5 O IT) O' fi> 0 ) 3 O O C 0.50- - 1.0 (D (0 SI cn L. o cnCO U) 0.25- -0.5 3 5 3 0.00 0.0 20 30 40 Elution volume (ml)

Figure 3.16 Size exclusion chromatography of colominic acid (À) and IgG (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: IgG was (50: 1). Samples were chromatographed on a Sephadex G-lOO column (40.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^). Arrows indicate the co-elution volume of the conjugate (Ve = 12ml) and the void volume (Vo = 7.0ml) of the column.

112 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

r1.5 E c o N o If) O' - 1.0 fi) 0) 3 u O c (D (Q SI cn (O o 0.5- -0.5 cn (/> 3 € 3

0.0 ■0.0 0 10 20 30 40 Elution volume (ml)

0 .2 -, r1.5 E c o N o lO - 1.0 % 3 8 O c (D cn L. to O -0.5 cn (0 3 3

0.0 ■0.0 0 10 20 30 40 Elution volume (ml)

Figure 3.17 Size exclusion chromatography of the oligomer colominic acid (CAg) (A) and IgG (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA 5 : IgG was (100: 1). Samples were chromatographed on a Sephadex G-lOO column (45.0 xl.lcm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'*). Arrows indicate the co-elution volume of the conjugate (Ve = 12ml) and the void volume (Vo = 8 .0 ml) of the column.

113 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.3.9 Validation of ammonium sulphate precipitation as a method suitable for isolating polysialylated IgG

The methodology established in section 3.2.2.8.2 to isolate peptides and proteins via ammonium sulphate precipitation was adapted for the isolation of polysialylated IgG. No observable peak was detected at the elution volume (Ve =17) due to ‘free’ unreacted CA (Fig. 3.16B) and SDS-PAGE for polysialylated IgG showed the absence of any free IgG (Fig. 3.19). This suggests that the technique of ammonium sulphate precipitation suitably isolated the neoglycoprotein and that all the IgG molecules were polysialylated.

Alternative methods also considered for the purification and isolation of the neoglycoprotein, included, trichloroacetic acid (TCA) precipitation, dialysis, and ultrafiltration. However, Fernandes and Gregoriadis (1996, 1997) reviewed these methods independently for the separation of CA (average mol. wt lOKDa) from the proteins catalase (240KDa) and asparaginase (135 KDa) and found them to be poor methods of separation. For example, the method of TCA precipitation was found satisfactory in the separation of polysialylated catalase from initial starting reactants, however the catalase enzyme was irreversibly denatured. Further characterisation of polysialylated catalase necessitated its prior purification whilst retaining enzymic activity. Thus, milder methods of purification of the neoglycoprotein were sought after. Dialysis was considered for the separation of low molecular weight reactants as well as CA from polysialylated proteins. However, extensive dialysis using a 12- 14KDa molecular weight cut-off tubing retained 91% of CA (average mol. wt lOKDa), thus our attention was turned to ultrafiltration. A wide range of ultrafiltration cut-off membranes (10, 30, 50 and lOOKDa) were employed in the separation of CA from catalase and asparaginase. Unfortunately, neither protein could be retained quantitatively, with ‘free’ CA passing out in the filtrate. Essentially, the latter two methods of separation were both size exclusion techniques, where separation is dependant upon differences of size and shape of the molecules under separation. Thus, the poor resolution of molecules may once again be attributed to the hydrodynamic properties of CA adopted in solution.

114 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

The ‘abnormal’ behaviour of CA was displayed in SEC and explained in section 3.3.3. Thus, these inadequate separation techniques were not employed for the isolation of polysialylated IgG, and ammonium sulphate precipitation was adopted as the main method for neoglycoprotein isolation in this thesis.

3.3.10 SDS polyacrylamide gel electrophoretic (SDS-PAGE) characterisation of polysialylated IgG

In the previous section (3.3.8), size exclusion chromatography (SEC) was employed for the characterisation of polysialylated IgG. It was also used in an attempt to corroborate the synthesis of polysialylated IgG. In this section, an attempt was made to further corroborate these findings, by utilising SDS-PAGE. This technique essentially separates proteins on the basis of molecular weight. Thus, SDS-PAGE was employed to detect the changes in molecular weight of the proposed IgG-CA conjugate, compared with the starting materials. Two SDS-PAGE gels were run simultaneously. The first included the two controls i.e. the zero hour and native IgG samples fi"om experiment one (section 3.2.2.7.1). In the second, the neoglycoproteins isolated fi*om the 12, 24 and 48 hour aliquots fi*om experiment one (section 3.2.2.7.1) were used together with the zero hour (control). All the samples were run utilising a discontinuous SDS buffer system developed for SDS-PAGE employing a 12.5% polyacrylamide gel. They were all run under denaturing conditions following the procedures outlined in section 2.2.4.1. The samples were all calibrated against low molecular weight markers; the composition of which may be seen in table 2.3. The details of the buffer composition and gel mixture preparations are presented in table

2 . 1.

The low molecular weight markers (lane M) were run initially against the controls i.e. the zero hour sample (lane 1) and native IgG sample (lane 2) (Fig. 3.18). Analysis of the SDS-PAGE gel (Fig. 3.18) revealed two well resolved bands for both controls that had migrated approximately the same distances through the gel. Under the denaturing conditions described, it was anticipated that IgG (present in both controls) would dissociate into four peptide sub-units, as SDS reduced each disulphide bond

115 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

(Edelman, 1969). The sub-units of IgG (Fig. 1.6) consisted of two light chains with a molecular weight of ~ 25KDa and two heavy chains with a molecular weight of ~

50KDa. This was consistent with the position of the bands seen in Fig. 3.18 (lanes 1 and 2).

The control results showed that the zero hour sample consisted of un-reacted IgG and that IgG incubated for 48 hours under the reaction conditions was neither polymerised nor degraded into lower molecular weight derivatives.

Figure 3.18 SDS-PAGF of the zero hour and native IgG reaction mixtures (controls) from experiment 1.0, (section 3.2.2.7.1) against the low molecular weight markers (BioRad). SDS-PAGF was conducted on a 12.5% polyacrylamide gel under reducing conditions. Lane M: low molecular weight markers comprising of, phosphorylase b (97.4KDa), albumin (66.2KDa), ovalbumin (45.0KDa), carbonic anhydrase (3 LOKDa), trypsin inhibitor (21.5KDa) and lysozyme (14.4KDa). Lane 1: zero time reaction mixture (control). Lane 2: native IgG (control). The gel was stained with Coomassie blue. For other details refer to Chapter two.

The second SDS-PAGF gel (Fig. 3.19) consisted of polysialylated IgG isolated from the 12, 24 and 48 hour incubation aliquots of the time study performed in experiment one (section 3.2.2.7.1), together with the zero hour sample. These samples were calibrated against low molecular weight markers as in Fig. 3.18. Results (Fig. 3.19) showed a similar pattern of the dissociated sub-units of IgG (as seen in Fig. 3.18) in lanes 1,2,3, and 4 (representing the 0, 12, 24 and 48 hour incubation samples).

16 Chapter Three: Synthesis & characterisation of polysialylated peptides and proteins

However, each band in successive lanes appeared to migrate slower than the control, thereby suggesting an increase in molecular weight perhaps due to an increasing degree of polysialylation of IgG over time. The 48 hour sample (lane 4) yielded a broadened band shape and multiple diffused bands. This suggested (Carlsson, 1993) microheterogeneity as a result of the polysialic acid chains thus, further suggesting the presence of a neoglycoprotein.

Figure 3.19 SDS-PAGE of the 0, 12, 24 and 48 hour polysialylated IgG aliquots from experiment 1.0, (section 3.2.2.7.1) against the low molecular weight markers (BioRad). SDS-PAGE was conducted on a 12.5% polyacrylamide gel under reducing conditions. Lane M: low molecular weight markers comprising of, phosphorylase b (97.4KDa), albumin (66.2KDa), ovalbumin (45.OKDa), carbonic anhydrase (3 LOKDa), trypsin inhibitor (21.5KDa) and lysozyme (14.4KDa). Lane 1: zero time reaction mixture (control). Lane 2: 12h reaction mixture. Lane 3: 24h reaction mixture. Lane 4: 48h reaction mixture. The gel was stained with Coomassie blue. For other details refer to Chapter two.

Fernandes and Gregoriadis (1996) reported similar findings for the broad diffuse and multiple bands observed with catalase and asparaginase enzymes after modification with polysialic acid. Although SDS-PAGE is a commonly used electrophoretic technique that separates peptides and proteins with differing molecular weights, there are some exceptions to this, which cause anomalous migration. For example carbohydrate residues have a weak affinity for SDS and therefore heavily glycosylated proteins migrate slower than non-glycosylated proteins of the same molecular weight

117 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

(Segrest and Jackson, 1972). Thus the estimation of their molecular weight by SDS- PAGE is not accurate. However, it was proposed (Leach et al, 1980) that the negatively charged sialic acid residues would partially compensate for the reduced SDS affinity, thus rendering the measurement of molecular weight more precise. Based on the sum of the molecular weights determined fi*om the bands in lane 4 (Fig. 3.19) for the proposed polysialylated IgG conjugate, the molecular weight was estimated to be 194KDa.

PEG-derivatised proteins also showed abnormal migration on SDS-PAGE. Kunitani et al. (1991) attributed these observations to the highly heterogeneous nature of pegylated proteins, not only in terms of molecular weight (dependent on the degree of modification and on the polydispersity of PEG) but also the charge (different numbers of derivatised lysine residues result in charge heterogeneity) and shape (site specific attachment of PEG to distinct locations of the protein leads to erratic SDS binding). The latter point was exemplified by the comparison of randomly modified PEG with a Fab’ fragment and site-specific attachment by SDS-PAGE (Chapman et al., 1999). In parallel, the multiple migration pattern observed with the IgG-CA conjugate (48 h sample, lane 4, Fig. 3.19) on SDS-PAGE, may be attributed to the involvement of the ketonic group at the reducing end of CA with the amino group of IgG as well as the proposed single terminal aldehyde group introduced at Cl of the non-reducing end of CA. The ketonic group of CA (although less reactive because of its involvement in a keto-enol equilibrium) may react with the e-amino groups of IgG producing a conjugate of low yield, thus introducing another attachment site of CA.

3.3.11 Factors affecting the degree of modification of polysialylated aprotinin

Having optimised the method of polysialylation with IgG in section 3.3.7, this method was employed in an attempt to polysialylate the peptide aprotinin. Periodate oxidised CA (average mol. wt lOKDa) was coupled to aprotinin (6.5KDa) via the method of reductive amination in the presence ofNaBHgCN, (pH 9.0) at 35-40°C. Starting molar ratios of 50: 1 and 10: 1 (CA: aprotinin) were employed as in experiments 1 and 2 of section 3.2.2.7.2. The control experiments were conducted simultaneously such that

118 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins all the experimental conditions were maintained as above, except that non-oxidised CA was included instead of oxidised CA, or CA omitted altogether. The reactions were followed over 48 hours whereby aliquots (1.0ml) of the reaction mixtures were removed at intervals of 0, 6 , 12, 24 and 48 hours. The proposed polysialylated constructs were isolated from each aliquot by ammonium sulphate precipitation and the results expressed in terms of CA: aprotinin molar ratios found in the precipitates (Fig. 3.20).

(50: 1)

2 8 ? 1 0 - (10: 1)

(control) (50: 1)

—I------1----- r 12 18 24 30 36 42 48 Time (h)

Figure 3.20 Colominic acid (CA): aprotinin molar ratios in the conjugates of aprotinin with CA (average mol. wt lOKDa), estimated by ammonium sulphate precipitation as a function of time. (▼) non-oxidised CA: aprotinin (50: 1) control, (À) oxidised CA: aprotinin (10: 1) and (■) oxidised CA: aprotinin (50: 1). Values are a mean ± s.d of three experiments.

Results (Fig. 3.20) showed a steady initial increase in the rate of reaction for both ratios (50: 1 and 10: 1), whereby the reactions reached a peak around 12 hours and continued to increase gradually over the period of 12-48 hours. In contrast, the control

(non-oxidised CA) exhibited a much slower initial reaction, reaching a peak around 1 2 hours and a plateau observed over 12-48 hours.

The degree of modification of the polysialylated aprotinin preparations prepared in experiments 1 and 2 (section 3.2.2.T.2) post 48 hours reaction were expressed in terms

119 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Table 3.4 The degree of polysialylation of aprotinin under the reaction conditions described and expressed in terms of moles CA per one mole aprotinin (CA: aprotinin

Initial Time of Molarity of Molar ratio of Colominic acid molar ratio of reaction phosphate buffer CA: aprotinin (CA) CA: aprotinin (hours) at pH 9.0 in conjugate CA(lOKDa) 50: 1 48 0.75M 1.46 ±0.05: 1.0 (Oxidised) CA(lOKDa) 1 0 : 1 48 0.75M 0.74 ±0.06: 1.0 (Oxidised) Non-oxidised CA 50: 1 48 0.75M 0.39 ±0.03: 1.0 (control) of CA: aprotinin molar ratios found in the precipitates and are presented in table 3.4. It appears that the extent of polysialylation of aprotinin was dependent upon the starting molar ratio of CA: aprotinin used in the coupling reaction, i.e. a 50: 1 excess of CA: aprotinin yielded a conjugate estimated to consist of 1.46 ± 0.05 moles CA to 1.0 mole aprotinin. Thus, approximately 1.5-fold more CA coupled to aprotinin in comparison with a 10:1 excess of CA: to aprotinin, where the extent of polysialylation was estimated at 0.74 ± 0.06 moles CA to 1.0 mole aprotinin.

Direct coupling of non-oxidised CA to aprotinin (control) via reductive amination in aqueous solution yielded a conjugate containing approximately 3-fold less CA coupled to aprotinin estimated at 1.0 mole aprotinin to 0.39 ± 0.03 moles of CA when compared to coupling of oxidised CA: aprotinin (50: 1). This result was consistent with the control experiment of polysialylated IgG in section 3.3.7. Considering the covalent coupling of CA to aprotinin (50: 1) it was estimated that an average of 36.5% of the available lysine residues could be polysialylated. Calculations were derived on the basis that each aprotinin molecule consisted of 58 amino acids of which 4 are lysine residues, (Roberts et al, 1996). A lower degree of polysialylation was achieved in the 10: 1 reaction, where 0.74 ± 0.06 moles of CA reacted with 1.0 mole aprotinin. Based on the calculations derived previously, this ratio would correspond to the polysialylation of an average of 18.5% of the available lysine groups.

These findings highlighted key issues pertaining to the effect of peptide reactivity on the molar yield of conjugation. As seen with polysialylated IgG (3.3.7), the reactivity

120 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins of the protein was not simply a measure of the number of reactive e-lysine residues in the molecule but more importantly their availability. Aprotinin comprises a single polypeptide chain, highly folded and held together by 3 disulphide bridges (Fig. 1.7). The structural conformation of aprotinin seems to leave LyslS and Lys26 more accessible to react with CA, however Lys41 and Lys46 appear concealed within the folded tertiary structure of aprotinin. The protein conformation will also have affected the reactivity of the N-terminal amino group.

Other aprotinin-polymer conjugates have been synthesised in order to solve the problem of native aprotinins rapid excretion, for example Larionova et al (1984) reported on aprotinin coupled to D-lactose and also aprotinin coupled to (carboxymethyl) dextran derivatives of D-galactose (Larionova et al, 1985). Although direct comparisons could not be made between the degree of polysialic acid modified aprotinin and D-lactose modified aprotinin, it was interesting to note that 1.0 mole aprotinin reacted with 2.0 moles D-lactose.

3.3.12 Size exclusion chromatographic (SEC) characterisation of polysialylated aprotinin

The ammonium sulphate pellets of polysialylated aprotinin isolated fi"om the reaction mixture in experiment 1 (section 3.2.2.7.2) at zero and 48 hours were re-dissolved in (0.15M) PBS, dialysed extensively (3 x 2L; 24h) against the same PBS buffer at 4°C and chromatographed using SEC (Fig. 3.21). The zero hour (control) and 48 hours sample were applied to a Sephadex G-50 column. The eluted fi*actions were assayed for protein (A595nm) and total sialic acid (A570nm) content. Fig. 3.21A represents the elution profile of the zero time (control) sample after application to the column and Fig. 3.2IB represents the elution profile of a sample of the reaction mixture post 48 hours.

The two peaks observable in Fig. 3.21 A were almost completely resolved due to their differences in molecular weight i.e. CA (av. mol. wt lOKDa) and aprotinin (6.5KDa). Thus, the almost completely resolvable peaks indicated the presence of unreacted CA

121 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins and aprotinin at zero time. Conversely, after 48 hours reaction (Fig. 3.2IB), aprotinin and CA showed co-incident elution profiles, with a definite shift to the left. This perhaps suggests that covalent coupling may have occurred with the emergence of a

‘new’ derivative of increased molecular weight eluting in fi*action 1 1ml of the column. The elution profiles also suggest (Fig. 3.2IB) that ammonium sulphate precipitation and concomitant dialysis of the proposed polysialylated aprotinin conjugate may have completely isolated the neoglycoprotein from un-reacted CA due to the absence of an extra peak at the elution volume (Ve = 13ml) for fi-ee CA. Whilst it is borne in mind that further experiments are warranted to corroborate these findings, the zero and 48 hour samples were applied to SDS-PAGE gels in an attempt to gain more insight into the synthesis of polysialylated aprotinin. These results are discussed later in Chapter 4.

To summarise, under the conditions described, the highest molar ratio of CA: aprotinin was achieved using a 50-fold excess of oxidised CA in the reaction mixture (Fig. 3.20 and table 3.4). The degree of modification achieved was 1.0: 1.46 ± 0.05 moles of aprotinin to CA, compared with 1.0: 0.74 ± 0.06 for the (10: 1) reaction of CA to aprotinin. This suggests that the extent of polysialylation is probably dependent upon the starting molar ratio of CA: aprotinin used in the coupling reaction. Interestingly, the results also suggest that the extent of polysialylation of aprotinin may also be dependant upon the availability of reactive amino groups of the protein as well as the number present. It was simultaneously established that prior oxidation of CA was a prerequisite for an apparent higher molar yield of conjugation to occur with the protein. This was supported by the poor degree of modification in the control (non-oxidised CA) experiment whereby the degree of sialylation achieved was estimated at 0.39 ± 0.03 moles of non-oxidised CA per 1.0 mole of aprotinin. Evidence for the synthesis of polysialylated aprotinin was sought for by ammonium sulphate precipitation, dialysis and subsequent size exclusion chromatography (Fig. 3.21) and later SDS-PAGE (Chapter 4). Although, SEC could not confirm definitively a covalent linkage between oxidised CA and the protein, the emergence of a higher molecular weight entity was identified and the absence of un-reacted components shown. Figure 3.20 suggests that the degree of sialylation of aprotinin was complete over 48 hours and therefore a suitable time to terminate the reaction.

122 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

1.00n r0.15 I (A ° 0.75 O lO O' - 0.10 fi) 3 O cs 0.50- (D (0 cn L_ CO -0.05 cn 8 0.25- 3 € 3

0.00 -0.00 10 20 30 Elution volume (ml)

0.4-1 1-0.15 E (A g 0.3- O ir> O' - 0.10 fi) Q) 3 O c 0.2- CD co cn h. CO o -0.05 cn (A 3 3

0.0 4100 0 10 20 30 Elution voiume (ml)

î’igure 3.21 Size exclusion chromatography of colominic acid (A) and aprotinin (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: aprotinin was (50: 1). Samples were chromatographed on Sephadex G-50 column (35.0 x 1.0 cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^). Arrows indicate the co elution volume of the conjugate (Ve = 11ml) and the void volume (Vq = 4.0ml) of the column.

123 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.3.13 Factors affecting the degree of modification of polysialylated insulin

In an attempt to investigate the correlation between reaction time and molar yield of polysialylated insulin, periodate oxidised CA (average mol. wt lOKDa) was coupled to insulin (5.7KDa) as described in experiment 1 (section 3.2.2.7.3). This reaction involved an initial ratio of 50: 1 (CA: insulin), where CA was in excess. The control experiments involved using non-oxidised CA instead of oxidised CA or CA omitted altogether under the same experimental conditions.

A time study was performed over 48 hours, whereby aliquots (1.0ml) of the reaction mixture were removed at intervals of 0, 6 , 12, 24 and 48 hours respectively. The proposed polysialylated insulin conjugate was isolated from each aliquot by ammonium sulphate precipitation and the results expressed in terms of CA: insulin molar ratios found in the precipitates and represented in Fig. 3.22.

2.5

| 1 2 . 0 (50: 1) K -D 'c n,.1.5 0.5 (control) (50: 1)

0.0 0 6 12 18 24 30 36 42 48 Time (h)

Figure 3.22 Colominic acid (CA): insulin molar ratios in the conjugates of insulin with CA (average mol. wt lOKDa), estimated after ammonium sulphate precipitation as a function of time. (À) non-oxidised CA: insulin (50: 1) control, (■) oxidised CA: insulin (50: 1). Values are mean ± s.d of three experiments.

124 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

The rate of reaction for the 50: 1 ratio of CA to insulin shown in Fig. 3.22 revealed an initial rapid reaction, which peaked at 12 hours and formed a plateau over the period of 12-48 hours. Conversely, the 50:1 control (non-oxidised CA) reaction exhibited an initial slow reaction rate, which gradually peaked at 12 hours and formed a plateau over a period of 24-48 hours. This result was consistent with the control results for aprotinin (Fig. 3.20).

The degree of modification of the polysialylated insulin preparations (section 3.2.2.13) as estimated from the precipitates after 48 hours were expressed in terms of CA: insulin molar ratios and are reported in table 3.5.

Table 3.5 Degree of polysialylation of insulin under the reaction conditions described and expressed in terms of moles CA per one mole insulin (Insulin: CA molar ratio). Non-oxidised CA anc insulin alone were included as controls. Initial Time of Molarity of Molar ratio of Colominic acid molar ratio of reaction phosphate buffer CA: insulin (CA) CA: insulin (hours) at pH 6.4 in conjugate CA(lOKDa) 50: 1 48 0.75M 1.90 ±0.05: 1.0 (Oxidised) Non-oxidised CA 50: 1 48 0.75M 0.42 ±0.03: 1.0 (control)

Results (table 3.5) showed that the proposed covalent coupling of CA to insulin (50: 1) via reductive amination, yielded a molar conjugation ratio of approximately 1.90 ± 0.05 moles of CA to 1.0 mole insulin. In contrast, coupling of non-oxidised CA to insulin (control) by reductive amination in aqueous solution yielded a conjugate containing approximately 4.5-fold less CA. Thus, the molar ratio of insulin coupled to non-oxidised CA was estimated at 0.42 ± 0.03 moles of CA to 1.0 mole insulin. This result reaffirmed the need for oxidation of CA prior to coupling to insulin such that a higher molar conjugation yield is attained. The molar conjugation results achieved from the 50: 1 reaction of CA with insulin suggests that the N-terminal amino groups present in insulin might have contributed in the Schiff base reductive amination reaction as well as the e-lysine amino residues. This observation was derived from the fact that each bovine insulin molecule consists of an A chain of 21 residues and a B chain of 30 residues (totalling 51 amino acids) and has only one lysine residue

125 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

(LysB29) (Sanger and Thompson, 1953). Indeed, Glazer et al. (1976) proposed that the terminal amino groups of proteins also posses reactivity to undergo the Schiff base reductive amination reaction. For example mPEG (750 and 2000) was covalently attached to the N-terminal amino acid residue (PheBl) as well as to the amino acid residue LysB29 of insulin, (Hinds et al, 2000). The molar conjugation result achieved from the 50: 1 reaction of CA with insulin reflects the surface availability of reactive 8-lysine residues (LysB29) and the N-terminal amino acid residue (PheBl) within the bovine insulin molecule.

Several covalent conjugates have been synthesised utilising polysaccharide polymers of varying molecular weights attached to insulin. For example, Mehvar (1994) covalently coupled insulin to dextrans with molecular weights of 40KDa, 70KDa and 500KDa, and Torchilin et al. (1977) reported on the attachment of insulin to modified Sephadex with molecular weights of 10-150KDa. The insulin contents (-20% w/w) of the insulin-dextran conjugates were higher than those reported for the insulin- Sephadex conjugates (3-6.2%). Interestingly, these differences were attributed to solubility differences between the carriers used i.e. soluble dextran and insoluble Sephadex. Although direct comparisons could not be made between the degree of modification of insulin with CA and the alternative polymers employed in the studies above, insight into the effect of molecular weight of the polymer on the degree of modification of insulin was gained in each case.

3.3.14 Size exclusion chromatographic (SEC) characterisation of polysialylated insulin

Size exclusion chromatography (SEC) was employed in an attempt to establish that CA had covalently coupled to insulin, forming a neoglycoprotein conjugate in the reaction mixture. Samples from the zero and 48 hour aliquots obtained from the time study in experiment 1 (section 3.2.2.7.3), were subjected to ammonium sulphate precipitation to ‘salt-out’ the proposed polysialylated insulin conjugate. These precipitates were then re-dissolved in PBS (pH 6.4), dialysed extensively (3 x 2L; 24h) against the appropriate PBS buffer at 4°C and applied to a Sephadex G-50

126 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins column. The zero hour incubation sample was regarded as the control (Fig. 3.23A). The eluted fractions were assayed for protein (A595nm) and total sialic acid (A570nm) content. Fig. 3.23B represents the elution profile of the 48 hour sample after application to the column.

Results (Fig. 3.23A) showed complete resolution of CA (average mol. wt lOKDa) and insulin (6.0KDa) in the zero time sample as the two peaks eluted separately. This was expected due to their differences in molecular weight, thus suggesting the presence of unreacted CA and insulin at zero time. Evidence to suggest that insulin had successfully coupled to oxidised CA was sought for in the elution profile of Fig. 3.23B, whereby CA was found to co-elute with insulin in fraction 12ml of the column. Fig. 3.23B clearly shows a shift in the native protein peak to the left and noticeably no peak in the fraction known to be native insulin from Fig. 3.23A above. These results might suggest the emergence of a coupled product and the success of the Schiff base reductive amination reaction however, it is home in mind that further experimentation is required to rule out other types of bonding. Furthermore, the lack of any unreacted CA, due to the absence of an extra peak at the elution volume (Ve = 13) for ‘free’ CA suggests that the ammonium sulphate precipitation method was suitable to isolate the proposed conjugate from unreacted components.

To conclude, polysialylation of insulin with CA following periodate oxidation at the hypothesised non-reducing end (carbon 7) and subsequent coupling to the amino groups of insulin by reductive amination led to a degree of modification estimated at 1.90 ± 0.05 moles of CA to 1.0 mole insulin. These results suggest the involvement and reactivity of both the e-lysine residue (LysB29) and the N-terminal amino acid groups of insulin. This result was however in contrast to the poor degree of sialylation achieved with the control (non-oxidised CA) experiment where only 0.42 ± 0.03 moles of non-oxidised CA reacted with 1.0 mole of insulin (Fig. 3.22 and table 3.5), thus, reaffirming the need for periodate oxidation of CA prior to coupling. This trend was in agreement with the results obtained for the other proposed polysialylated peptides and proteins studied in this chapter, i.e. polysialylated IgG (table 3.3) and aprotinin (table 3.4).

127 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

1.00n r0.15

(/) K 0.75- o m O’ - 0.10 0) 3 C 0.50- s s -0.05 CJ1 % 0.25- 3 3

0.00 ■0.00 0 10 20 30 Elution volume (ml)

0.4-1 1-0.15 I (/) 0 R 0.3- “ t lO O' - 0.10 fi) 3 § 0.2 - S (0 n o -0.05 1O l W) 3 3

0.0

Figure 3.23 Size exclusion chromatography of colominic acid (A) and insulin (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: insulin was (50: 1). Samples were chromatographed on a Sephadex G-50 column (40.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin^). Arrows indicate the co elution volume of the conjugate (Ve = 12ml) and the void volume (Vo = 4.0ml) of the column.

128 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Definitive evidence for the formation of the sialylated insulin conjugate was not achieved by size exclusion chromatography (Fig. 3.23), however a ‘new’ molecular weight derivative was identified from the shift of native components into the same elution volume (Ve = 12ml) of the column. It was established that further experimentation is required to rule out forms of bonding other than a covalent link. The reaction kinetics observed in Fig. 3.22 also suggested that the reaction of insulin with CA was possibly complete after 48 hours reaction. This was consistent with the data reported for IgG and aprotinin.

3.3.15 Factors affecting the degree of modification of polysialylated somatostatin

An attempt was made to couple somatostatin (1.64KDa) to activated CA (average mol. wt lOKDa) via the method of Schiff base reductive amination in the presence of NaCNBHa at pH 9.0 as in the synthesis of polysialylated IgG, aprotinin and insulin. This reaction involved an initial starting ratio of 10: 1 (CA: somatostatin) as in experiment 1.0 (section 3.2.2.7.4), where CA was in excess. The control experiments were conducted under the same experimental conditions except that non-oxidised CA was included instead of oxidised CA, or CA omitted altogether. A time study was performed over 48 hours, with aliquots (1.0ml) of the reaction mixture removed at intervals of 0, 6, 12, 24 and 48 hours. The polysialylated conjugates were isolated from each aliquot by ammonium sulphate precipitation and the results expressed in terms of CA: somatostatin molar ratios found in the precipitates and represented in Fig. 3.24.

Initially, (Fig. 3.24) the rate of reaction for the 10: 1 ratio of oxidised CA to somatostatin was quite rapid resulting in the formation of increasing amounts of polysialylated somatostatin over 12 hours. The rate of reaction reached a plateau over a period of 12-48 hours and the reaction was terminated after 48 hours when it was thought to have undergone completion. The control 10:1 (non-oxidised CA) reaction exhibited a fairly rapid initial rate of reaction, however this reaction yielded a much smaller degree of conjugate.

129 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.5 i (10: 1) O £ 3.0 II 2.5 P 2.0 re E o (/> 1.5 3 Ü o 1.0 E (control) o O 0.5 ( 10: 1) 0.0 0 6 12 18 24 30 36 42 48 Time (h)

Figure 3.24 Colominic acid (CA): somatostatin (SS) molar ratios in the conjugates were estimated by ammonium sulphate precipitation as a function of time. (A) non- oxidised CA: somatostatin (10: 1) control, (■) oxidised CA: somatostatin (10: 1). Values are a mean ± s.d of three experiments.

Table 3.6 summarises the degree of modification of the polysialylated somatostatin preparations post 48 hours reaction. The results are expressed in terms of CA: somatostatin molar ratios found in the precipitates post ammonium sulphate precipitation. Under the conditions described, proposed covalent coupling of periodate oxidised CA to somatostatin (10: 1) via reductive amination, in aqueous solution yielded a molar conjugation ratio of approximately 3.00 ± 0.17 moles of CA to 1.0 mole somatostatin (SS).

Table 3.6 The degree of polysialylation of somatostatin (SS) under the reaction conditions described and expressed in terms of moles CA per one mole somatostatin, (somatostatin: CA molar ratio). Non-oxidised CA and somatostatin alone were included as controls. Initial Time of Molarity of Molar ratio of Colominic acid molar ratio of reaction phosphate buffer CA: SS (CA) CA: SS (hours) at pH 9.0 in conjugate CA(lOKDa) 10: 148 0.75M 3.00 ±0.17: 1.0 (Oxidised) Non-oxidised CA 10: 1 48 0.75M 0.61 ±0.01: 1.0 (control)

130 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

In contrast, the molar ratio of somatostatin coupled to non-oxidised CA (control experiment) via reductive amination in aqueous solution yielded a conjugate containing approximately 5-fold less CA coupled to somatostatin. Considering that each somatostatin (SS) molecule consists of 14 amino acids of which 2 are lysine residues (Lys4 and Lys9), (Brazeaus et al., 1983), this might correspond to the polysialylation of 100% of the available lysine groups and also the free terminal amino group of somatostatin (Alai). This outcome was hypothesised when we considered the reactivity of somatostatin in terms of the availability of the reactive s- lysine residues and N-terminal amino residue. Somatostatin is a single chain polypeptide that is folded and held by a single disulphide bond, it is thus possible that the reactive 8-lysine residues (Lys4 and Lys9) and the N-terminal amino residue (Alai) would be more accessible to react with CA.

The requirement for long acting somatostatin is recognised (Du et al, 2001) and several companies have produced long acting analogues of somatostatin of which the best known is octreotide (section 1.7.3.4). In terms of research into polymer conjugates of somatostatin, a recent report described the conjugation of somatostatin to periodate activated dextran (40 KDa) and histidine via the method of Schiff base reductive amination (Du et al, 2001). Results revealed approximately 1.0 mole of oxidised dextran reacted with 3.0 moles of somatostatin.

3.3.16 Size exclusion chromatographic (SEC) characterisation of polysialylated somatostatin

Size exclusion chromatography (SEC) was employed in an attempt to validate the synthesis of polysialylated somatostatin. This technique involved applying samples from the zero and 48 hour aliquots obtained from the time study in experiment 1 (section 3.2.2.7.4). Ammonium sulphate precipitation of the reaction mixtures was performed to isolate the proposed polysialylated somatostatin. The pellets formed were re-dissolved in PBS, dialysed extensively (3 x 2L; 24h) against the appropriate buffer at 4°C and applied to a Sephadex G-50 column. The eluted fractions were collected and assayed for protein (A595nm) and total sialic acid (A570nm) content.

131 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

0.15n rO.15 Ê c (0 o o Ü 0.10 h0.10 ^ 0) 3 o O c

0.00 0.00 10 20 Elution volume (ml)

B O.IOOn i 1-0.075 E c (A o 0.075- O in h0.050 §■ 0) 3 u c 0.050 S s 8 0 -0.025 cn 0.025 3 1 3 0.000 0.000 10 20 30 40 Elution voiume (mi)

Figure 3.25 Size exclusion chromatography of colominic acid (A) and somatostatin (SS) (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: SS was (10: 1). Samples were chromatographed on a Sephadex G-50 column (45.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'*). Arrows indicate co elution of the conjugate (Ve = 14ml) and the void volume (Vo = 5ml) of the column.

132 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Figure 3.25A represents the elution profile of the zero time sample (control) and Fig. 3.25B the elution profile of the 48 hour sample post column application. Results (Fig. 3.25A) indicated complete resolution of CA (average mol. wt lOKDa) and somatostatin (1.64KDa) fi*om the zero hour sample as the two peaks eluted separately. Indeed, this was expected due to the vast differences in their molecular weights, respectively, thus suggesting the presence of unreacted CA and somatostatin at zero time.

In contrast, the peaks observable from the elution profile of the 48 hour sample (Fig. 3.25B) overlap, and there is a definite shift in the native protein peak to the left which coincides with that of CA. The co-elution of CA with somatostatin in fraction 14ml of the column perhaps suggests the emergence of a covalently conjugated entity, in this case polysialylated somatostatin. It is however necessary to further justify this finding in order to rule out other types of bonding, which may have occurred. Interestingly, the peak corresponding to the elution volume (Ve) of ‘free’ CA is absent in the elution profile shown in Fig. 3.25B, which may also indicate that ammonium sulphate precipitation was a suitable method for the isolation of the neoglycoprotein from any unreacted CA.

To summarise, under the conditions described for the proposed polysialylation of somatostatin, the degree of modification achieved was 3.00 ± 0.17 moles of periodate oxidised CA to 1.0 mole somatostatin. This was in contrast with the poor degree of modification achieved with the control (non-oxidised CA) experiment, whereby 0.61 ± 0.01 moles of non-oxidised CA reacted with 1.0 mole of somatostatin (table 4.5). Indeed, these trends were reflected for all the proposed polysialylated peptides and proteins synthesised in this chapter, i.e. polysialylated IgG, aprotinin, and insulin, thus emphasising the importance of periodate oxidation of CA prior to coupling with any peptide or protein. The polysialylation process appeared to be complete after 48 hours as indicated in Fig. 3.24 and seen for polysialylated IgG, aprotinin and insulin. In terms of the molar yield of polysialylated somatostatin achieved with a 10: 1 ratio, the results appeared to reflect high reactivity of SS i.e. freely accessible reactive e-lysine residues (Lys4 and Lys9) and the N-terminal amino group reactivity (Alai).

133 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

Definitive evidence (Fig. 3.25B) to suggest that polysialylated somatostatin had been successfully synthesised using the technique of SEC was not conclusive, however the emergence of a higher molecular weight entity was seen with the absence of free starting materials. It is proposed that further evaluations of the proposed neoglycoprotein are performed to identify absolutely the formation of the covalent linkage.

3.3.17 Solubility of the native and polysialylated peptide and protein therapeutics

Bovine IgG, aprotinin and somatostatin were all readily soluble in water and (0.15M) PBS at physiological pH (7.4). Bovine insulin however was only partially soluble in

(0.15M) PBS at physiological pH (7.4) and in K 2HPO4 (0.75M, pH 9.0). Therefore, insulin was solubilised by titrating 0.75M K 2HPO4 to pH 6.4 with O.IM HCl. Although all the previous Schiff base reductive amination reactions had been performed at pH 9.0, we were confident that polysialylation of insulin could occur at pH 6.4. Literature reported on the stability of sodium cyanoborohydride down to around pH 3-4 (Borch et al., 1971) and showed broadly constant activity between pH 5 and 9.0.

Interestingly, when bovine IgG, aprotinin and somatostatin respectively were added to the coupling reaction mixture consisting of K2 HPO4 (0.75M, pH 9.0) a slightly hazy, colourless solution resulted. It was hypothesised that a few factors may have been responsible for these observations, one of which could be that proteins are known to exhibit decreased solubility in solutions of high ionic strength. This is analogous to protein precipitation from solution by concentrated salt solutions i.e. ammonium sulphate precipitation. Another factor accounting for the decreased solubility of these peptides and proteins was working at pH 9.0. Proteins are known to become insoluble in water close to their isoelectric point (pi). The isoelectric point is the pH at which peptides or proteins carry no net charge and become least soluble. A review of the pi values for bovine IgG (9.1; theoretical), aprotinin (9.24; theoretical) and somatostatin (8.91; theoretical) indicated that they were all close to the working pH (9.0), with proteins therefore exhibiting diminished solubility. CA and oxidised CA (50mg/ml)

134 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

were completely soluble in K2 HPO4 (0.75M, pH 9.0). On addition of CA (oxidised or intact) the peptide and protein solutions turned cloudy, perhaps suggesting partial aggregation due to the ionic interaction, even though the ionic strength of the solution was high. Indeed, pH changes owing to the addition of CA may have affected the solubility of the otherwise soluble peptides and proteins. It is also hypothesised that cross-linking effects albeit minimal, may have contributed to the cloudy appearance. Another plausible explanation for the cloudy appearance is that CA precipitates the protein out of solution. For instance, Harris (1989a) reported this observation with polymers such as PEG.

Interestingly, after 48 hours of reaction the turbidity of reactions involving these peptides and proteins with oxidised CA was much less pronounced in comparison with their respective controls (non oxidised CA or protein alone). Bovine insulin (pi 5.3) however was soluble in both instances at pH 6.4. These qualitative observations of increased solubility of peptides and proteins on conjugation to CA were recognised (although further investigation were warranted) as an important characteristic of CA thus offering a potentially important drug delivery system for the intravenous administration of therapeutic peptides and proteins. Mehvar (1994) reported similar findings, where insulin was partially soluble on addition to the coupling reaction mixture although complete solubility was observed within 12 hours of reaction with dextran aldehyde. The increased solubility of the peptides and proteins was not surprising due to the highly polar nature of the polysaccharide. It has been postulated that glycosylation of proteins significantly influences their solubility, stability and serum half-life (Weitzhandler, et al., 1994).

3.3.18 Properties of colominic acid and the polysialylated proteins

It is proposed that the synthesis and in vivo fate of the polysialylated peptides and proteins prepared in this chapter would be directly influenced by the properties of CA. Thus it was deemed important to investigate the in vitro properties of CA such as stability under different pH and biodegradability in mouse blood plasma. The effects of lyophilisation and storage conditions on the stability of the native and

135 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

polysialylated peptides and proteins prepared in this chapter were also investigated.

3.3.18.1 Stability of colominic acid under varying pH Colominic acid was incubated under a range of pH conditions (7.4, 6.4 and 9.0) at 37°C for 48 hours and then applied to a Sephadex G-25 (25.0 x 1.0cm) column (3.2.2.9.1). These conditions replicated the conditions to which the polysialylated peptides and proteins were exposed (3.2.2.7).

Results (Fig. 3.26) showed slightly altered elution profiles for CA post exposure to pH 7.4, 6.4 and 9.0, respectively, thus suggesting some degradation of CA albeit minor. Zhang (1999) reported similar results when CA was incubated under a range of pH conditions (7.4, 4.7 and 9.0) at 37°C for 24 hours. However, when CA was incubated for one week under the same pH conditions at 37°C it was found to be acid-labile on exposure to pH 4.7, yielding low molecular weight derivatives but was unaffected on exposure to pH 7.4 and 9.0. In fact, it was reported that CA could be stored in (0.15M) PBS (pH 7.4) at 4°C for three months without producing oligosaccharides (Zhang, 1999).

7500000-

Z* 5000000 >

2500000

* A * A 4 6 8 10 12 14 16 18 20 22 24 Elution volume (ml)

Figure 3.26 Stability of CA after incubation in: (■) PBS (pH 7.4), (A) 0.751V’ (K2 HPO4 ) dipotassium hydrogen phosphate (pH 6.4) and (À) 0.75M K2 HPO4 (pH 9.0), respectively. Each solution was incubated at 37°C for 48 hours and aliquots (500pl) were individually applied to a Sephadex G-25 (25.0 x 1.0cm) column. No significant differences between the samples were observed.

136 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.3.18.2 Stability of colominic acid in mouse plasma The stability of CA in mouse plasma is an important characteristic of CA if it is to be exploited for the potential of peptide and protein delivery. Due to the rapid blood plasma clearance of CA (Gregoriadis et al., 1999) and the anticipated equally rapid disappearance of CA catabolites (if any) it is difficult to monitor its stability in vivo. Thus, tritiated CA (Chapter 2) was treated with fresh mouse plasma at 37°C for 24 hours in vitro. Aliquots were then applied to a Sephadex G-25 (25.0 x 1.0cm) column and compared with CA incubated in PBS. Results (Fig. 3.27) indicate that the elution profile of CA was not significantly different from that of CA in PBS. It would appear that mouse plasma proteins had not caused diminution effects on CA. Zhang (1999) reported on the susceptibility of CA to the enzyme neuraminidase, which depolymerised CA into lower molecular weight fragments. It was concluded that mouse plasma was devoid of this enzyme.

E 7500000- Q. O >» îg 5000000

1o =5 2500000 &

6 8 10 12 14 16 18 20 22 24 Elution volume (ml)

Figure 3.27 Stability of CA after incubation in: (■) PBS (pH 7.4) and (A) fresh cintillation fluid and measured for radioactivity (tritium). No significant differences between mouse plasma at 37°C for 24 hours. Aliquots (500pi) were applied to a Sephadex G-25 (25.0 x 1.0cm) column. lOpl aliquots from each fraction were mixed with 4.0ml s the samples were observed.

137 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.3.18.3 Stability of native and polysialylated peptides and proteins upon lyophilisation and storage using size exclusion chromatography The preferred method of storage for the native and polysialylated peptides and proteins prepared here was lyophilisation and storage at 4°C. It is well documented that many freeze-dried proteins exhibit significant loss of activity and/or stability resulting from the stresses arising from freeze-drying and freeze thawing (Crowe et al., 1990). For example, Tanaka et al. (1991) reported on the dénaturation of the enzyme catalase, which occurred when ~95% of the water was lost. Thus, SEC was employed as outlined in section 3.2.2.9.B in an attempt to investigate the effects of lyophilisation and storage at 4°C on the stability of the native and polysialylated constructs reported in this chapter. Results of the elution profiles for the native and polysialylated peptides and proteins in solution were not significantly different from those stored in the freeze-dried form or those stored for 3 months at 4°C (results not shown). Moreover, no increased or reduced molecular weight derivatives were detected for the native peptides and proteins suggesting that no significant protein aggregation or biodégradation respectively had occurred.

138 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins

3.4 Conclusions

The main conclusions summarised here are derived from the four key objectives that were proposed in the introduction of this chapter (section 3.1). These challenges were considered pertinent to the synthesis and characterisation of the ‘new’ peptide and protein biotherapeutics polysialylated in this thesis and believed to lay the foundations upon which future chapters are built.

The present study shows that controlled periodate oxidation of CA (under the conditions described in section 3.2.2.1) employed in the first-step of the coupling strategy did not cause diminution of the CA molecule. These findings are based on the elution profiles of oxidised and intact CA whereby almost identical profiles resulted (Fig. 3.1 A). It would appear that the oxidative process did not affect the integrity of the internal a- (2—>8)-linked N-acetylneuraminic acid residues of CA. In addition, the oxidised derivative of CA was found to mimic the abnormal elution behaviour of intact CA in gel permeation studies and the broad elution profiles possibly suggest the presence of a highly hydrated molecule. Various spectroscopic methods including MS, FT-IR and ’H-NMR were employed in the identification of the ‘newly’ introduced aldehyde (CHO) functionality of CA. This was hypothesised to occur at the non reducing terminus (C7) of CA on account of the selectivity of sodium periodate for vicinal diols although other hydroxyl groups may also be prone to oxidation. Unfortunately, no definitive elucidation of the CHO moiety was possible. The failure of these methods to elucidate CHO functionality of oxidised CA may be reasoned by considering the structural conformation that CA adopts in solution. For instance, a reactive aldehyde introduced at the non-reducing end of CA is prone to attack by water thus forming a cyclic hemiacetal (Fig. 3.4). The spectral characteristics of this product would not be expected to show an aldehyde group. In contrast, comparative results of the 2,4-DNP colorimetric test with oxidised and intact CA (Fig. 3.6) do suggest that periodate oxidation had successfully introduced aldehyde functionality to CA. The 2,4-DNP method is a qualitative method, which is unable to quantify aldehyde content or determine absolutely the site of CHO introduction. As such, the site of CHO introduction remains hypothetical and investigations of the latter

139 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins properties are highly recommended for future studies. L-lysine was exploited as a protein model in an attempt to establish the second-step of the coupling strategy, namely Schiff base reductive amination. L-lysine offers the minimum number of available amino groups for coupling to CA. Whilst cross-linking was considered; it was deemed unlikely to occur on account of the properties of CA. The apparent degree of modification revealed a stoichiometric reaction between CA and L-Lysine (table 3.2) and the 3-fold improved coupling yield compared with the control suggests that periodate oxidation facilitates coupling. Similar findings were reflected in the molar conjugation ratios of oxidised CA coupled to all the peptides and proteins studied here compared with their respective controls. Evidence to prove the synthesis of polysialylated L-lysine was not definitively confirmed by employing SEC, however the emergence of a ‘new’ heavier entity was identified co-eluting in the same volume of the column (Fig. 3.1 IB). Further evaluations are warranted to confirm the presence of a covalent link and rule out other types of bonding.

This study followed with the attempted synthesis of CA modified IgG, aprotinin, insulin and somatostatin and a collective review of the factors that influenced the apparent degree of polysialylation. The reaction conditions found most favourable to produce optimal conjugation yields in the present work required temperatures of 35-

40°C in 0.75M K2HPO4 buffer at pH 6-9 for 48 hours in the presence of NaCNBHs. Evidence to suggest the formation of the covalently coupled peptide or protein conjugates is attempted by employing SEC. Whilst elucidation of covalent linkage can not be made from the chromatograms (Figs. 3.16B, 3.2IB, 3.23B, 3.25B) representing samples from the apparent polysialylated peptide or protein reaction mixtures; the chromatograms do show the emergence of ‘new’ heavier peaks of CA and protein co eluting in the same fraction of the column. Interestingly, the controls conducted with peptide or protein alone under the reaction conditions did not show the emergence of a heavier peak when applied to SEC; perhaps suggesting that the peptide or protein is not polymerised under Schiff base reductive amination conditions (results not shown). In the case of CA-modified IgG applied to SDS-PAGE, successive bands correlating to aliquots removed from the reaction mixture over 48 hours appear to migrate slower through the gels compared with native controls, which might suggest the synthesis of

140 Chapter Three: Synthesis & characterisation ofpolysialylated peptides and proteins the neoglycoprotein. Other process variables found to affect the yield of coupling include the starting molar ratio of reactants as shown by polysialylated IgG (table. 3.3) and aprotinin (table. 3.4). The extent of polysialylation of IgG appeared to be independent of the size of CA, however it was realised that a larger molecular weight range of CA should be employed to definitively prove this relationship. It is hypothesised that the level of incorporation of CA onto the peptides and proteins maybe further compromised by the negative charge encompassing the growing neoglycoprotein (Roy et al., 1994) and the steric effect of too many CA molecules. Perhaps it could be postulated that an optimum stoichiometry exists which is responsible for balancing these effects. By comparison of the different degrees of polysialylation achieved with the different peptides and proteins utilised in this chapter, there would appear to be a relationship between extent of sialylation and reactivity of the peptide or protein. For instance, the protein IgG contains the most number of reactive amino groups for coupling with CA compared with the peptides aprotinin, insulin and somatostatin, however its degree of modification based on the percentage of available a-amino and e -lysine residues was the lowest (table 6.2). The relationship between the availability of amino residues of proteins and coupling yield is investigated further in Chapter 4.

Results of the elution profiles for the native and polysialylated peptides and proteins in solution were not significantly different fi*om those stored in the lyophilised state at 4°C for up to 3 months (results not shown). This might suggest no protein aggregation or biodégradation had occurred. Plasma proteins are predominantly responsible for peptide and protein clearance from blood plasma via opsonisation or enzyme activity. Thus it is of considerable interest to find that CA stability is not affected in the presence of mouse plasma (in vitro) at 37°C (Fig. 3.27). These findings are encouraging in terms of employing CA as a means to augment the circulatory half- lives of some of the peptide and protein therapeutics considered here and render them more effective in vivo. Accounts of the effects of chemical modification by polysialylation on the concomitant loss in the biological activity of some of the peptides and proteins are discussed in Chapter 4 and the in vivo behaviour of polysialylated IgG, aprotinin and insulin are presented in Chapter 5.

141 Chapter Four

I n v i t r o bioactivity of polysialylated peptides and proteins

142 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.1 Introduction

At the outset of the work conducted here, the effects of polysialylation were considered to bear potentially adverse effects on the biological activity of the peptides and proteins modified by polysialylation in Chapter 3, thus limiting their effectiveness in vivo. To that end, catalase, IgG2a (Mab) and aprotinin were selected to investigate the effects of polysialylation on their stability and biological properties. Additionally, the latter properties were also investigated for the three proteins modified using a novel polysialylation process developed and implemented in this chapter. Interestingly, catalase and asparaginase modified by the established method of polysialylation resulted in conjugates with reduced enzyme activity (albeit minor), although they were compensated for with improved pharmacokinetic profiles (Fernandes and Gregoriadis, 1996).

The emphasis of the research conducted in Chapter 3 was to optimise the efficiency of the polysialylation process. Unfortunately, the degree of modification of IgG (7.2%), aprotinin (29.2%) and insulin (63.3%) were considered poor to moderate based on the percentage of available a-amino and e-lysine residues modified with CA. Only somatostatin attained a quantitative yield of polysialylation. Although our research identified certain variables that could be manipulated to ‘custom-design’ the final bioconjugate, it was realised that the structural conformation (folding) of the peptide or protein also determined the percentage of reactive amino residues available for modification and the position of specific amino acids involved in biological activity. Thus the inherent 3-dimensional complex characteristic of peptides and proteins was identified as a contributory factor in limiting the efficiency of polysialylation. On the basis of these findings, new challenges presented with respect to improving the efficiency of polysialylation by modifying the peptide and protein as well as derivatising CA prior to coupling. Studies conducted by Visser and Blout (1971) and Prakash and Nandi (1976) on surfactant-protein interactions and the critical observations made by Anfmsen et al. (1963) on the renaturation of fully denatured ribonuclease provided the impetus for devising the modified polysialylation procedure. Their studies showed that certain concentrations of surfactant such as

143 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

sodium dodecyl sulphate (SDS) could bind to globular proteins and induce reversible changes in their structural conformation on removal of the dénaturant. Other protein dénaturants found to have a similar effect include acids, alkalis, urea and guanidine hydrochloride (Anfinsen et al., 1973). In this study however, SDS was chosen in preference to the two latter dénaturants as their basic amino functional groups were considered likely to react with CA in the coupling process. Also the use of acid or alkali dénaturants was avoided on account of their likely interference with the coupling process, CA and colorimetric assays of CA and proteins. Consequently, the anionic surfactant, SDS was used in an attempt to reversibly unfold six therapeutic peptides and proteins and further expose the reactive a-amino and e-lysine residues for modification with CA. Essentially, the strategy required applying a suitable concentration of SDS that allowed reversible unfolding of the protein molecule whilst retaining modest biological activity.

This chapter is organised around the four peptides and proteins polysialylated in Chapter 3 and also introduces mouse anti-bovine serum albumin monoclonal antibody (IgG2a) and catalase. The synthesis and characterisation of polysialylated catalase and IgG2a are reported here, as they are not covered in Chapter 3. Preliminary research employed catalase to establish the optimal concentration of SDS that resulted in the apparent reversible inactivation of the enzyme on removal of the dénaturant. Catalase was chosen because it represented a biologically active protein template that could be conveniently and easily used to optimise the conditions of SDS required to reversibly unfold the other peptides and proteins in our study. In brief, enzyme activity studies were performed on native catalase alone and in the presence of varying concentrations of SDS. SDS-treated catalase samples were subsequently dialysed extensively to remove the dénaturant and the enzyme activity re-measured. The concentrations of SDS found to inactivate the enzyme reversibly and irreversibly were applied to IgG, aprotinin and BSA and SEC performed. SEC was employed in an attempt to observe the effects of these concentrations of SDS on peptide and protein conformation during their proposed conformational transition from folded, unfolded to re-folded states. IgG, IgG2a, catalase, aprotinin and somatostatin were subsequently SDS-modified using the concentration of SDS found to inactivate catalase reversibly, polysialylated

144 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins and the bioconjugate yields determined. A direct comparison was made between these results and those obtained by the method of polysialylation established in Chapter 3. The SDS-modified bioconjugates were isolated fi-om their un-reacted components via the method of ammonium sulphate precipitation, established in section 3.2.2.8.2 and SEC was employed in an attempt to characterise the polysialylated SDS-modified peptides and proteins. Polysialylated SDS-modified IgG and aprotinin were further characterised by employing the method of SDS polyacrylamide gel electrophoresis (SDS-PAGE).

The effects of polysialylation on the stability and in vitro biological activities of catalase, aprotinin and IgG2a modified via both methods of polysialylation were investigated. IgG2a was employed instead of the non-specific polyclonal antibody IgG for the purpose of the above study. CA modified somatostatin (with and without SDS- modification) was not pursued in this study as it was not the subject of intensive study with respect to in vitro biological activity. Unfortunately, the in vivo biological properties of CA modified insulin (with and without SDS-modification) were not determined in this study due to difficulties with our project licence and financial constraints at the time of study. However, external studies have since been performed (Jain et al., 2002) which reported on the effects of CA modification upon the biological activity of insulin synthesised under the conditions described in our study.

Finally, the in vitro biological properties of polysialylated and SDS-modified polysialylated catalase, IgG2a and aprotinin were determined after fi*eeze-drying and storage and compared with the native controls, respectively.

145 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.2 Materials and Methods

This section details the specific materials and methods required to pursue the challenges proposed in section 4.1.

4.2.1 Materials

Table 4.1 Materials used in Chapter 4

Material Source** Benzoylarginine ethyl ester hydrochloride Fisher Chemical Company, UK Bovine serum albumin (98-99%) Sigma Chemical Company, UK Foetal calf serum (PCS) Sera-lab Ltd, UK Horseradish peroxidase-labelled goat anti-mouse Sigma Chemical Company, UK anti-bovine serum albumin JgG2a Hydrogen peroxide 30% Sigma Chemical Company, UK o-Phenylenediamine Sigma Chemical Company, UK Polystyrene (96-well) microtiter plates Dynatech laboratories. Inc., UK (Immulon IB) Silica quartz cuvette Sigma Chemical Company, UK Sodium dodecyl sulphate (SDS) (-99%) Sigma Chemical Company, UK Trypsin, from bovine pancreas Sigma Chemical Company, UK ** Manufacturers and suppliers full addresses may be found in Appendix 2 All other reagents were of analytical grade and may be found in previous chapters. The composition of reagent solutions and buffers can be found in Appendix 1.

4.2.2 Methods

4.2.2.1 Synthesis and characterisation of polysialylated lgG2a and catalase Oxidised CA (average mol. wt 1 OKDa) was covalently coupled to mouse anti-bovine serum albumin monoclonal antibody (IgG2a, 15OKDa) and the enzyme catalase

146 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

(240KDa), respectively. Coupling was attempted by the method of Schiff base, reductive amination in the presence of sodium cyanoborohydride as described for the polysialylated peptides and proteins prepared in section 3.2.2.7. The reactions were carried out for 48 hours in sealed vessels at 35-40°C. Controls were treated under the same reaction conditions described and involved reacting the native proteins alone or with non-oxidised CA. Stirring was kept to a minimum to avoid concomitant dénaturation of the proteins. The neoglycoproteins were isolated from the un-reacted components via ammonium sulphate precipitation and characterised using size exclusion chromatography (SEC).

4.2.2.1.1 Polysialylated IgG2a Prior to its use, IgG2a was dialysed extensively against deionised water (3 x 2L; 24h) at 4°C to remove the phosphate buffer, NaCl and sodium azide constituents in which IgG2a was presented. A 50; 1 starting molar ratio of oxidised CA (1.7mg, 0.165pmol) was reacted with IgG2a (0.5mg, 0.0033pmol) in 0.75M dipotassium hydrogen phosphate (2.5ml) at pH 9.0 in the presence of sodium cyanoborohydride (NaBHgCN) (lOmg, 0.16mmol). CA was in excess. Controls included IgG2a (0.5mg, 0.0033pmol) in the presence of non-oxidised CA (1.7mg, 0.165pmol) and IgG2a alone under the same reaction conditions as above. Aliquots (0.5ml) of the reaction mixture were removed at time intervals of 0 and 48 hours only as we were limited with the starting material (IgG2a). The neoglycoproteins were isolated via ammonium sulphate precipitation (section 3.2.2.8.2) and dialysed to purify the IgG2a conjugate. The reaction mixture was centrifuged at 4500xg for 40 min where the pellet (re-suspended in PBS) and the supernatant were assayed for sialic acid (Svennerholm, 1957) and IgG2a protein (Bradford, 1976) content. The conjugation yields were expressed in terms of CA (sialic acid): IgG2a molar ratios found in the pellets (table 4.2) and as a percentage of modified s-lysine residues.

Size exclusion chromatography (SEC) was utilised to characterise the zero time (control) and 48 hour aliquots. A Sephadex G-lOO column (40.0 x 1.1cm) was prepared and the eluted fractions (1.0ml) were assayed for CA and IgG2a content as above (Fig. 4.1). An investigation into the antibody-antigen binding properties of

147 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins native and polysialylated IgG2a were pursued in further experiments (section 4.2.2.6.1) by employing an Enzyme-Linked Immuno-Sorbent Assay (ELISA).

4.2.2.1.2 Polysialylated catalase A 50: 1 starting molar ratio of oxidised CA (50mg, S.Opmol) was reacted with catalase (24.0mg, 0.1 pmol) in 0.75M dipotassium hydrogen phosphate (5.0ml) at pH 9.0 in the presence of sodium cyanoborohydride (NaBHgCN) (20mg, 0.32mmol). CA was in excess. Controls included catalase (24.0mg, 0.1 pmol) in the presence of non-oxidised CA (50mg, 5.0pmol) and catalase alone under the same reaction conditions as above. Aliquots (1.0ml) of the reaction mixture were removed at time intervals of 0, 6, 12, 24 and 48 hours. The neoglycoproteins were isolated via ammonium sulphate precipitation (section 3.2.2.8.2) and dialysed to purify the catalase conjugates. The reaction mixtures were centrifuged at 3000xg for 30 min where the pellets (re suspended in PBS) and the supernatants were assayed for CA (Svennerholm, 1957) and catalase (A405nm) content. The conjugation yields were expressed in terms of CA: catalase molar ratios found in the pellets (Fig. 4.2).

Size exclusion chromatography (SEC) was utilised to characterise the zero time (control) and 48 hour aliquots. A Sephadex G-lOO column (45.0 x 1.1cm) was prepared and the eluted fractions (1.0ml) were assayed for CA and catalase content as above (Fig. 4.3). Further studies investigated catalase activity, the stability of catalase during coupling and kinetic properties (section 4.3.10.1).

4.2.2.2 Catalase enzyme activity assay Catalase and polysialylated catalase activity was measured spectrophotometrically at 240 nm by the method of Beers and Sizer (1952). The method involved monitoring spectrophotometrically the disappearance of hydrogen peroxide (H 2 O2 ) at 23°C. In brief, 2.9ml of 17.6mM H 2 O2 substrate solution in 0.05M phosphate buffer, pH 7.0 was added to 0.1ml catalase enzyme suitably diluted (~50U/ml) in 0.05M phosphate buffer, pH 7.0 and mixed in a 3.0ml silica cuvette. A blank cuvette was prepared containing the substrate solution and 0.1ml of 0.05M phosphate buffer, pH 7.0 only. Spectrophotometric analysis was performed at 240 nm whereby the time (min)

148 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins required for the absorbance of the mixture to decrease (AA) from 0.450 to 0.400

(corresponding to the decomposition of 3.45pmoles of H 2 O2 in the 3.0ml assay) was noted. Consequently, the enzyme activity for native unmodified and polysialylated catalase could be calculated by dividing 3.45 (pmoles of H 2 O2 ) by the time (min) taken for AA to decrease from 0.450 to 0.400. Catalase activity was represented as pmolmin'\ In order to determine the percentage residual enzyme activity and make comparisons between the native and polysialylated catalase constructs, activity was often corrected to activity/mass to take into account the differences in amount of enzyme present in each case. One unit (U) of catalase is defined as the amount of enzyme required to catalyse the decomposition of 1.0 pmol hydrogen peroxide per min at 23°C. The methodology described here for the purpose of catalase activity determination is employed in the stability studies performed on catalase and polysialylated constructs during the coupling process in section 4.3.10.1 and also for Micbaelis Menten kinetic studies in section 4.3.10.2.

4.2.2.3 Micbaelis Menten: Efficacy of catalase and polysialylated constructs The efficacy of native, polysialylated and polysialylated SDS-modified catalase (~ 40pg enzyme) was derived from the rates of catalysis respectively, measured at different H2 O2 concentrations of 10, 20, 30 and 40mM. The apparent maximal velocity (Vmax) and Micbaelis constant (Km) were estimated from the Hanes Woolf plot (Palmer, 1985). The Hanes Woolf plot was achieved using the reaction kinetics software (Pharmacia LKB Biocbrom Ltd., Cambridge, UK, version 2.1, 1994) of the Wallac spectrophotometer.

4.2.2.4 Sodium dodecyl sulphate (SDS): an anionic surfactant used to reversibly unfold peptides and proteins In this study, the anionic detergent SDS was employed for the purpose of reversibly unfolding the six therapeutic peptides and proteins introduced in this thesis, in an attempt to further increase their number of available reactive amino residues for coupling to CA as described in section 4.1. To that end, catalase was employed initially as a biologically active template to establish the optimal concentration of SDS

149 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins that allowed complete reversible inactivation of the enzyme. The SDS concentration resulting in the complete irreversible dénaturation of the enzyme was also determined. These concentrations of SDS were subsequently applied to IgG, aprotinin and BSA, and SEC employed before and after dialysis to remove the dénaturant in an attempt to observe the conformational changes (folding) of the proteins. The rational for using SEC and the effects of SDS on the resorcinol method for sialic acid determination and changes in the conformation of the CA molecule are explored in section 4.2.2.4.2. The effects of SDS on the Bradford method for protein determination are explored in section 4.2.2.S. The unfolding of proteins gives rise to the loss of secondary and tertiary structures, and often results in changes to their spectroscopic properties. Thus, examples of other techniques used to analyse protein conformation often exploit the spectroscopic properties of peptides and proteins. Such examples include circular dichroism spectroscopy (CD), optical rotatory dispersion (ORD), differential scanning calorimetry (DSC), FT-IR, and NMR (Reubsaet et a/., 1998).

4.2.2.4.1 Optimisation of the sodium dodecyl sulphate (SDS) methodology Triplicate samples of catalase were dissolved (0.03mg/ml) in 0.05M phosphate buffer, pH 7.0 and left stirring gently for 1.0 hour at 20°C. Aliquots (0.1ml) were removed and catalase activity was determined as described in section 4.2.2.2 and expressed as pmolmin'^ (control). The subsequent experiments determined catalase activity in the presence of SDS. Solid SDS was added to each catalase solution to achieve SDS concentrations of 0.01, 0.02, 0.03, 0.06 and 0.09%. Each sample was left stirring gently for 1.0 hour at 20°C and the same procedure was conducted to measure catalase enzyme activity as for catalase in the absence of SDS. The percentage residual enzyme activity for each catalase sample in the presence of SDS was determined by comparison with catalase activity in the absence of SDS (control) and plotted against SDS concentration (Fig. 4.7). In continuation of these studies, all the SDS containing catalase solutions were dialysed extensively (3 x 2L; 24h) against distilled water at 20°C since a precipitate formed at 3°C in the presence of SDS detergent. Aliquots (0.1ml) were removed fi*om each sample and catalase activity was re-investigated in an attempt to investigate the reversibility as described in section 4.2.2.2 (Fig. 4.8). The optimal concentration of SDS was chosen as that which allowed almost 100%

150 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins reactivation of the catalase enzyme when SDS was removed. This concentration was then employed in an attempt to reversibly unfold the other peptides and proteins, studied in this chapter.

4.2.2.4 2 Analysis of the effect of sodium dodecyl sulphate (SDS) on peptide and protein conformation by size exclusion chromatography (SEC) Various techniques used to study peptide and protein conformation are outlined earlier in section 4.2.2.4. These techniques are often used collectively to identify changes in protein structure undergoing the transition fi*om denatured to the refolded state in the presence or absence of protein dénaturants. Unfortunately, many of the spectroscopic techniques such as CD and ORD were unavailable at the time of conducting our research and therefore size exclusion chromatography (SEC) was chosen in our study as a means to observe the effects of SDS on peptide and protein conformation. The principle involves changes in the hydrodynamic volume of the proteins between the native folded and unfolded states, which can be reflected in the different elution profiles obtained by SEC (Shalongo et al., 1989).

In brief, BSA, IgG and aprotinin were individually dissolved (1 mg/ml) into 5.0ml 0.15M PBS, pH 7.4 (control) and into solutions of 0.15M PBS containing l.OmM SDS (0.029%) and 0.15M PBS containing 3.12mM SDS (0.09%) respectively. The latter concentrations were established in section 4.2.2.4.1 as those that resulted in the reversible and irreversible inactivation of catalase respectively, after removal of the detergent. All the samples were left stirring gently for 1.0 hour at 20°C. Aliquots (0.5ml) removed from each solution containing BSA (Fig. 4.11) and IgG (Fig. 4.9) were characterised on a Sephadex G-lOO column (30.0 x 1.1cm) and those firom solutions containing aprotinin (Fig. 4.10), on a Sephadex G-50 column (30.0 x 1.1cm). The eluted fractions (1.0ml) were assayed for protein (Bradford, 1976) content. All the solutions containing dissolved proteins in the presence of SDS (l.OmM and 3.12mM) and in its absence were then dialysed extensively (3 x 2L; 24h) against distilled water at 20°C and aliquots (0.5ml) re-characterised as described above. It was hoped that SEC could characterise the transitional states of the proteins before, during treatment with SDS and after dialysis i.e. folded, unfolded and re-folded states.

151 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

CA dissolved (0.4mg/ml) into 5.0ml 0.15M PBS, pH 7.4 containing l.OmM SDS (0.029%) was left stirring overnight, dialysed extensively (3 x 2L; 24h) against 0.15M PBS and a calibration curve prepared employing the resorcinol method (Svennerholm, 1957). An aliquot (0.5ml) was removed from the CA containing SDS solution and characterised on a Sephadex G-75 column (45.0 x 1.1cm). The resultant chromatogram was compared with CA dissolved (0.4mg/ml) in 5.0ml 0.15M PBS alone (Fig. 4.12). These experiments were conducted in an attempt to investigate the effects of SDS upon the resorcinol method for sialic acid determination and changes in the conformation of the CA molecule.

4.2.2 5 Synthesis of SDS-modified peptide and protein conjugates with colominic acid In an attempt to further improve the efficiency of polysialylation of peptides and proteins, all six therapeutic peptides and proteins investigated in this thesis were modified with SDS prior to coupling. Peptide and protein calibration curves were repeated according to the dye-binding assay (Bradford, 1976) in section 2.2.22 to determine whether SDS used in the modification of the six peptides and proteins, even after extensive dialysis, interfered with protein determination. The concentration of SDS that resulted in the apparent reversible unfolding of IgG and aprotinin after removal of the detergent (4.2.2.4.2) was chosen to modify these peptides and proteins. The procedure was as follows:

In short, 15mg, (O.lpmol) IgG, 0.65mg, (O.lpmol) aprotinin, 0.60mg, (O.lpmol) insulin, 0.82mg, (0.5pmol) somatostatin, 24.0mg, (O.lpmol) catalase and 0.5mg, (0.0033pmol) IgG2a respectively, were dissolved in 5.0ml (IgG2a in 2.5ml) 0.75M

K2 HPO4 , pH 9.0 (insulin, pH 6.4) containing l.OmM (0.029%) SDS. The mixtures were warmed gently to dissolve the SDS completely and then left stirring gently for 1.0 hour at 20°C. IgG, aprotinin, insulin, somatostatin and catalase mixtures were all then reacted with oxidised CA (50mg, 5.0pmol) in the presence of NaBHgCN (20mg, 0.32mmol). IgG2a was reacted with oxidised CA (1.7mg, 0.165 pmol) in the presence of NaBHsCN (lOmg, 0.2mmol). All preparations were incubated for 48 hours at 35-

152 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

40°C. Stirring was kept to a minimum to avoid protein dénaturation and consequent loss in biological activity. CA was in excess in all the experiments. Controls included reacting the individual peptides and proteins in the presence of non-oxidised CA and alone under the reaction conditions above. The conjugates were isolated by ammonium sulphate precipitation (70% saturation) followed by centrifugation at 3000xg for 30 min (catalase) or 4500xg for 40 min (IgG and Ig02a) or 6000xg for 40 min (aprotinin, insulin and somatostatin). The supernatants were decanted and the pellets drained and re-dissolved into 5.0ml (Ig02a in 2.5ml) 0.15M PBS, pH 7.4. The re-dissolved pellets were extensively dialysed (3 x 2L; 24 h) at room temperature against 0.15M PBS, pH 7.4 to avoid precipitation of SDS. Protein concentration was estimated by the Bradford method (Bradford, 1976) and catalase by measuring the absorbance at 405nm. CA content was measured at 570 nm by the resorcinol method (Svennerholm, 1957). The conjugation yields were expressed in terms of CA: protein molar ratios found in the pellets (table. 4.5).

SEC was employed in an attempt to characterise all the SDS-modified peptide and protein-CA conjugates (Figs. 4.13-4.18). Polysialylated SDS-modified IgG and polysialylated SDS-modified aprotinin were further characterised by employing the method of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and reviewed in section 4.3.9. Evaluations of the in vitro biological properties of polysialylated SDS- modified IgG2a and aprotinin were performed in section 4.3.10 as well as stability and kinetic studies of polysialylated SDS-modified catalase during the coupling procedure.

4.2.2.6 In vitro stability and biological activity studies This section initially attempted to evaluate the stability of native unmodified, polysialylated and SDS-modified polysialylated catalase during the coupling process. In essence, aliquots (-10pi) containing ~25-35pg catalase were removed from the reaction mixtures of the three formulations at time intervals of 0, 6, 12, 24 and 48 hours and assayed for catalase enzyme activity by the method described in section 4.2.2.2. Blanks included CA to eliminate absorbance due to the presence of CA in the conjugated samples. Catalase content was determined spectrophotometrically (A405nm) for each sample and derived from the catalase standard curve (Fig. 4.4).

153 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Catalase activity was therefore represented for each time sample as pmolmin'Vg’^- The percentage residual enzyme activity was determined for the different time samples from the three formulations by comparison with their respective positive controls (tables 4.6-4.S). The positive controls for each of the three formulations included the catalase samples at zero time. The percentage residual enzyme activity determined for each time point of the three different formulations was then plotted against time (Fig. 4.24) to reveal the stability of the three formulations during the coupling process.

The biological properties of native IgG2a and aprotinin before and after chemical modification of the two proteins via polysialylation and SDS-modified polysialylation were also investigated here. A potential drawback in chemically modifying IgG2a via the two approaches mentioned above includes the loss of antibody-antigen recognition. Thus, an Enzyme-Linked Immuno-Sorbent Assay (ELISA) was employed in an attempt to compare the in vitro antigen binding ability of polysialylated and SDS-modified polysialylated IgG2a with native unmodified IgG2a. The ELISA methodology adopted from Catty and Raykundalia (1989) offered a convenient measure of the antigen binding properties of the native and polysialylated immunoglobulins (IgG2a). Aprotinin is a trypsin-kallikrein inhibitor, thus the in vitro biological activity of native, polysialylated and SDS-modified polysialylated aprotinin was determined by measuring their inhibitory action respectively upon a solution of trypsin of known activity. The inhibitory activity of native aprotinin and polysialylated aprotinin conjugates was calculated from the difference between the initial activity and the residual activity of the trypsin.

4.2.2.6.1 Enzyme-Linked Immuno-Sorbent Assay (ELISA) In short, microtiter plate wells were coated with an antigen, to which the primary antibody i.e. native or polysialylated IgG2a are introduced. This is followed by a thorough rinsing and the introduction of a secondary antibody. The secondary antibody was an enzyme-labelled anti-immunoglobulin raised against mouse IgG2a in a different animal species. Addition of the enzyme’s substrate solution to the wells

154 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins produces a colour change whose absorbance can be related to the primary antiboby- antigen binding properties. The procedures were as follows:

Antigen coating Polystyrene microtiter plates were coated with 60pL per well of bovine serum albumin (BSA) dissolved (4pg/ml) in coating 0.05M carbonate-bicarbonate buffer, pH 9.6. The plates were incubated at 37°C for Ih or overnight at 4°C.

Blocking Plates were washed three times with washing buffer consisting of PBS and 0.05% v/v Tween 20 (PBS-T) to remove unbound antigen and tapped dry. Then, 60pL of 1% BSA solution in PBS-T was added per well to avoid non-specific antibody binding. After incubation at 37°C for Ih the plates were washed three times with PBS-T and tapped dry.

Primary antibody application Appropriately diluted (10-lpg/ml) aliquots (60pL) of native or CA conjugated IgG2a in 0.05M phosphate buffer, pH 7.0 were loaded in doubles on the top wells. The concentrations were then diluted down the plate from lOpg/ml to l.Opg/ml. After incubation at 37°C for Ih or overnight at 4°C the plates were washed again three times with PBS-T and tapped dry.

Secondary antibody application 50pL of horseradish peroxidase conjugated goat anti-mouse IgG2a (diluted 1/4000 in washing buffer supplemented with 5% foetal calf serum and 1% BSA) was added to each well and incubated at 37°C for Ih. Plates were washed three times with PBS-T and tapped dry.

Substrate reaction 200pL of freshly prepared 0.3M citrate-phosphate buffer, pH 5.0, containing o- phenylenediamine (OPD) and 30% H2O2 as the substrates of the enzymatic reaction

155 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins were added to each well and incubated again at 20°C for 25-30 min. The reaction was stopped by the addition of 25 pL H 2 SO4 (1.5 M) solution.

Optical density: Antibody-antigen binding determination The absorbance was read for each sample at 492nm in an ELISA microplate reader (Titertek Multiskan® MCC/340). The concentration of antibody bound determined the strength of the optical signal. A concentration (pg/ml) versus optical density standard curve was prepared for serially diluted native, polysialylated and SDS- polysialylated IgG2a constructs (10-lpg/ml). The optimum optical signal achieved (-1.0) for native unmodified IgG2a (lOpg/ml) was chosen to correspond to 100% antibody-antigen binding ability and was used comparatively to determine the percentage residual antibody-antigen binding affinity for IgG2a under the reaction conditions and both polysialylated conjugates. Similar comparisons were made for data obtained fi-om solutions containing 6pg/ml and 2pg/ml IgG2a respectively. Each plate was run with a series of control wells. The controls included: coating buffer only, antigen only, blocking protein solution and CA only. For details on the preparation of the ELISA buffers and solutions see Appendix 1.

4.2.2.6.2 The trypsin-kallikrein inhibitory-activity assay of aprotinin The activity of aprotinin is determined by measuring its inhibitory action on a solution of trypsin of known activity. The inhibitory activity is calculated fi*om the difference between the initial and residual activity of trypsin. Essentially, two assays were performed as described in the British Pharmacopoeia (1999). In brief. Assay One: measured the residual activity of trypsin after its preincubation (10 min) with the inhibitor i.e. native aprotinin or aprotinin conjugates at pH 8.0 at 25°C. Assay Two: measured trypsin activity in the absence of aprotinin fi*om the hydrolysis rate of benzoyl-L-arginine ethyl ester hydrochloride by titration with O.IM sodium hydroxide (NaOH), on a pH-stat. Initially, inhibitory-activity assays were conducted for native unmodified aprotinin (positive control) and then compared with native aprotinin (CA absent), polysialylated and polysialylated SDS-modified aprotinin (all reacted for 48 hours under coupling conditions). The procedure in detail was as follows:

156 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Assays for trypsin activity and inhibition An atmosphere of nitrogen was maintained in the reaction vessel into which 0.0015M borate buffer solution (9.0ml) was reacted with 1.0ml of freshly prepared (6.9g/l) benzoyl-L-arginine ethyl ester hydrochloride. The mixture was stirred continuously and the pH adjusted to 8.0 by the addition of l.OM sodium hydroxide. When the temperature had reached equilibrium at 25°C, 1.0ml of 0.0015M borate buffer, pH 8.0 containing 0.08 microkatals (0.1 mg/ml) trypsin and 0.015mg/ml aprotinin (positive control) was added and a timer started. The mixture was maintained at pH 8.0 by the addition of O.IM sodium hydroxide using a 0.01 ml-graduated syringe and the volume required noted every 30s. The reaction was continued for 6 min and then terminated (Assay One). This assay procedure was repeated as above but instead of reacting the substrate with trypsin pre-incubated with aprotinin, 1.0ml of 0.0015M borate buffer, pH 8.0 containing trypsin diluted (0.05mg/0.04microkatals/ml) alone was used (Assay Two). Aprotinin (positive control) used in assay one was considered to reflect 100% inhibitory activity of native aprotinin and was substituted in turn with each of the other three aprotinin formulations containing ~ O.Smg aprotinin and their percentage inhibitory activity determined respectively.

Determination of the inhibitory activity of aprotinin Aprotinin inhibitory activity was calculated by measuring the volumes of O.IM sodium hydroxide required per second in both the trypsin enzyme activity and inhibition assays and employing the results into the equation below. The inhibitory activity of aprotinin was expressed in European Pharmacopoeia Units per mg. 1.0 Ph. Eur. U. inhibits 50% of the enzymatic activity of 2 microkatals of trypsin (British Pharmacopoeia, 1999). 4000 (2«2- «0 m

« 1 : Volume of O.IM NaOH required per second in the enzyme inhibition assay (involving trypsin-aprotinin solution).

« 2 : Volume of O.IM NaOH required per second in the enzyme activity assay (involving trypsin solution), m : mass of aprotinin

157 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Controls included reacting trypsin in the presence of oxidised CA alone. For details on the preparation of the buffers and solutions see Appendix 1.

4.2.2.7 Freeze-drying The effects of ffeeze-drying on the in vitro biological properties of native unmodified catalase, IgG2a, aprotinin and their respective conjugates prepared by two methods of polysialylation were determined after rehydration fi-om the dried state. Essentially, conjugates prepared as previously described i.e. polysialylated IgG2a (4.2.2.1.1), catalase (4.2.2.1.2), aprotinin (3.2.2.7.2) and the respective polysialylated SDS- modified counterparts (4.2.2.S) were frozen (- 40°C) and freeze dried (Edwards Modulyo, UK) overnight. Similarly, native unmodified IgG2a, catalase and aprotinin dissolved (0.5mg/ml) in 0.15M PBS were frozen and freeze-dried overnight. The individual samples were re-hydrated to their original volume with water, then appropriately diluted and assayed for their residual biological activity. Catalase enzyme activity was determined by the spectroscopic method of Beers and Sizer (1952) as described in section A.2.2.2. The Enzyme-Linked Immuno-Sorbent Assay (ELISA) described in section 4.2.2.6.I (Catty and Raykundalia, 1989) was employed to determine IgG2a antigen binding affinity and the trypsin activity-inhibition assay described in section 4.2.2.6.2 (B.P, 1999) was utilised to determine aprotinin inhibitory activity.

4.2.2.S Statistical analysis The student’s t-test was used for comparing between two groups and multiple comparisons were made using a one-way analysis of variance test (ANOVA) with equal variances at significant level: P < 0.05). ANOVA is based on the assumption that not only do populations have a normal distribution but also have the same variance. In practice, data often showed differences in variances but since the sample size in each group (n = 3) was consistent, the equality of variances is not considered crucial (Erickson and Nosanchuck, 1992; Ryan et al., 1985). All statistical analyses were performed using GraphPad InStat (GraphPad Software, version 2.01, 2001).

158 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3 Results and Discussion

Having applied various methods in section 4.2.2 to explore our research aims and hypotheses in section 4.1, this section reports the results and discusses the outcomes.

4.3.1 Preparation of polysialylated IgG2a

An attempt was made to couple mouse anti-BSA monoclonal antibody IgG2a (150KDa) to CA (average mol. wt lOKDa) via the method of reductive amination in the presence of NaCNBHg at a 50: 1 CA: IgG2a molar ratio as described in section 4.2.2.1.1. Controls included reacting IgG2a in the presence of non-oxidised CA, and IgG2a alone under the same reaction conditions as above. The reactions were performed over 48 hours where an aliquot (500pl) was removed at zero time only. The technique of ammonium sulphate precipitation was employed in an attempt to isolate polysialylated IgG2a and the results expressed in terms of CA: IgG2a molar ratio found in the pellets (table 4.2). Unfortunately, it was not possible to conduct a time study for polysialylated IgG2a or apply different starting molar ratios of CA and IgG2a due to the limited amount of starting material of IgG2a in our possession.

Table 4.2 Degree of polysialylation of IgG2a under the reaction conditions described and expressed in terms of moles CA per one mole IgG2a (CA: IgG2a molar ratio). Non-oxidised CA was included as the control. Values are mean ± s.d of three

Initial Time of Molarity of Molar ratio of Colominic acid molar ratio of reaction phosphate buffer CA: IgG2a (CA)/ IgG2a CA: IgG2a (hours) at pH 9.0 in conjugate CA (lOKDa) 50: 1 48 0.75M 4.04 ±0.22: 1.0 (oxidised)

Non-oxidised CA 50: 1 48 0.75M 1.22 ±0.16: 1.0 (control)

Table 4.2 summarises the apparent degree of polysialylation of IgG2a estimated after ammonium sulphate precipitation following a 48 hours reaction. The results show that coupling IgG2a to oxidised CA via reductive amination yields a conjugate containing

159 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.04 ± 0.22 moles of CA per mole of IgG2a. Interestingly, the apparent degree of modification of IgG with oxidised CA modified under the same reaction conditions (section 3.3.2) was found to be marginally higher (4.25 ± 0.20 moles of CA per mole of IgG). In comparison, the control experiment involving the conjugation of non- oxidised CA to IgG2ayielded an apparent molar ratio of 1.22 ±0.16: 1.0 (CA: IgG2a). Thus, direct coupling of non-oxidised CA to IgG2a, yielded a conjugate containing approximately 3-fold less CA than IgG2a coupled to oxidised CA. Similarly, IgG reacted with non-oxidised CA (control experiments) yielded a poor molar ratio of 1.25 ± 0.36: 1.0 (CA: IgG). These results suggest that periodate oxidation of CA prior to conjugation with IgG or IgG2a facilitate the coupling procedure. This phenomenon was observed for all the polysialylated peptides and proteins synthesised in this thesis. An explanation for the poor molar yields of conjugation observed with non-oxidised CA (control experiments) compared with oxidised CA was outlined earlier with respect to polysialylated IgG in terms of the structural conformation that oxidised CA and non-oxidised CA adopt in solution (section 3.3.2).

Although IgG2a is a subclass of IgG and is differentiated by small differences in the amino acid sequences in the constant region of the heavy chain, both antibodies possess similar structures and molecular weights. These similarities between IgG and IgG2a would suggest why similar degrees of modification were obtained under the same reaction conditions with oxidised and non-oxidised CA even though different species had been used.

160 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.2 Size exclusion chromatographic characterisation of polysialylated IgG2a

Size exclusion chromatography was employed in an attempt to provide evidence for the formation of polysialylated IgG2a. The reaction was conducted over 48 hours where a sample was removed at time zero and 48 hours. The samples were subjected to ammonium sulphate precipitation in an attempt to isolate the proposed neoglycoproteins. The precipitates were centrifuged, re-dissolved in PBS (pH 7.4) and dialysed extensively (3 x 2L; 24h) against PBS (pH 7.4) to purify the IgG2a conjugate. Aliquots from the zero time and 48 hours samples were individually applied to a Sephadex G-lOO (40.0 x 1.1cm) column and the eluted fractions assayed for IgG2a (A595nm) and total sialic acid (A570nm) content. The zero hour sample was taken immediately after all the reagents had been added and therefore regarded as the control (Fig. 4.1).

The elution profiles representing CA and IgG2a at zero time observed in Fig. 4.1 A were not completely resolved although the differences in their molecular weights should have resulted in complete separation by this technique. It is proposed that the incomplete resolution of IgG2a and CA may be due to the polydisperse nature of CA and the increased hydrodynamic volume it adopts in solution as previously described in section 3.3.3. Nevertheless, the elution profiles of IgG2a and CA at zero time (Fig. 4.1 A) do suggest independent elution of the reactants and suggest the presence of un reacted components. In contrast (Fig. 4. IB), the elution profiles of CA and IgG2a after 48hours reaction are completely overlapping. Both ‘new’ peaks representing CA and IgG2a after 48 hours reaction appear to have shifted to the left and co-elution occurs in the same volume (Ve =14ml) of the column. The emergence of a ‘new’ heavier entity might suggest the formation of a neoglycoprotein however; further investigations are warranted to definitively confirm covalent coupling and rule out other forms of bonding. The proposed polysialylated IgG2a conjugate fractions were pooled and used to investigate the effects of polysialylation on antibody-binding affinity (section 4.3.10.3) and later freeze-dried to investigate the effects of lyophilisation on antibody-binding affinity (section 4.3.11).

161 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

1.5-, 1-0.15

0) o O- 1.0 - - 0.10 0) 3 8 U1 0.5- -0.05 CJlCO 3 3

0.0 0.00 010 20 30 40 Elution volume (ml)

0.15-1 1- 0.20 E c 0) o -0.15 o O" 2 . 0.10- 0) Q) 3 o o c - 0.10 (D (Q SI *01 Q 0.05- COCJl (0 -0.05 3 5 3 0.00 0.00 0 10 20 40 Elution volume (ml)

Figure 4.1 Size exclusion chromatography of colominic acid (V) and IgG2a (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: IgG2a was (50: 1). Samples were chromatographed on a Sephadex G-lOO column (40.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^). Arrows indicate the co-elution volume of the conjugate (Ve = 14ml) and the void volume (Vo = 7ml) of the column.

162 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.3 Preparation of polysialylated catalase

In this study, an attempt was made to couple periodate activated CA (average mol. wt lOKDa) to catalase (240KDa) by reductive amination in the presence of NaCNBH] at a 50: 1 CA: enzyme molar ratio as described in section 4.2.2.1.2. Controls included reacting catalase in the presence of non-oxidised CA and catalase alone under the same reaction conditions. The reaction was followed over 48 hours where aliquots (1.0ml) of the reaction mixture were removed at intervals of 0, 6, 12, 24 and 48 hours. The technique of ammonium sulphate precipitation was employed in an attempt to isolate the proposed polysialylated catalase conjugate and the results expressed in terms of CA: catalase molar ratio found in the pellets and represented in Fig. 4.2. Bioconjugates prepared by employing differing starting molar ratios of catalase and CA were not pursued in this study as it was not the subject of intensive study with respect to the polysialylation of catalase.

4n 0

1 Q) z o rs s < 0) o E z O

0 6 12 18 24 30 36 42 48 Time (h)

Figure 4.2 Colominic acid (CA): catalase molar ratios in the conjugates of catalase with CA (average mol. wt 1 OKDa), estimated after ammonium sulphate precipitation as a function of time. (■) non-oxidised CA: catalse (50: 1) control, (T) oxidised CA: catalase (50: 1). Values are mean ± s.d of three experiments.

163 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Results (Fig. 4.2) show a relatively rapid initial rate of reaction for polysialylated catalase for the first few hours, with the CA: catalase molar ratio increasing gradually from 6 to 48 hours. The reaction was terminated after 48 hours. In contrast, the control (non-oxidised CA) exhibited a much slower initial rate of reaction for the first 12 hours, which only marginally increased over the 12-48 hour period.

Table 4.3 Degree of polysialylation of catalase under the reaction conditions described and expressed in terms of moles CA per one mole catalase (CA: catalase molar ratio). Non-oxidised CA was included as the control. Values are mean ± s.d of three experiments. Colominic acid Initial Time of Molarity of Molar ratio of (CA)Zcatalase molar ratio of reaction phosphate buffer CA: catalase in CA: catalase (hours) at pH 9.0 conjugate CA(lOKDa) 50: 1 48 0.75M 3.45 ±0.29: 1.0 (Oxidised)

Non-oxidised CA 50: 1 48 0.75M 0.64 ±0.10: 1.0 (control)

Table 4.3 summarises the apparent degree of modification of catalase with CA estimated after 48 hours reaction and ammonium sulphate precipitation. Results showed that the degree of modification of catalase with CA was marginally lower (3.45 ± 0.29 moles of CA per mole of catalase) than reported by Fernandes and Gregoriadis (1996) where 3.80 moles ± 0.35 moles of CA reacted per mole of catalase modified by the same method. In contrast, the molar ratio of catalase coupled to non- oxidised CA (control experiment) via reductive amination in aqueous solution, yielded a conjugate containing approximately 5-fold less CA coupled to catalase (0.64 ±0.10: 1.0). These results are in agreement with the values obtained for catalase modified under the same conditions by Fernandes and Gregoriadis (1996) and suggest that oxidation of CA facilitates coupling to catalase.

Catalase has a molecular weight of 242KDa and contains 108 lysine residues (Weissmann et al., 1975). Therefore, based on the number of available e-lysine amino residues per catalase molecule it is estimated that CA could couple to 3.19% of these residues when CA is reacted with catalase (50: 1). Interestingly, Fernandes and

164 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Gregoriadis (1996) reported that CA was attached to 0.7% e-amino lysine groups when reacted with catalase at a starting molar ratio of 10: 1 (CA: catalase). Other catalase conjugates coupled with dextran (Marshall and Humphreys, 1977) and mPEG (Abuchowski et al., 1977) successfully attained a high degree of coupling and are discussed in more detail in section 4.3.10.1.

4.3.4 Size exclusion chromatographic characterisation of polysialylated catalase

Evidence to show the presence of polysialylated catalase in the reaction mixture was attempted by performing size exclusion chromatography. Aliquots (500ul) from the reaction mixtures at time zero and 48 hours (section 4.2.2.1.2) were applied to a Sephadex G-lOO column (40.0 x 1.1cm) and the eluted fractions were measured for catalase (A405nm) and total sialic acid (A570nm) content. The zero time sample was taken immediately after all the reactants had been added to the reaction mixture. It also functioned as the control in this experiment (Fig. 4.3A). Figure 4.3B shows the elution profile of the 48 hours sample.

Under the SEC conditions described, the two peaks observed in Fig. 4.3A were suitably resolved (as expected) due to the difference in the molecular weights of catalase (240KDa) and CA (average mol. wt lOKDa). The independent peaks suggest the presence of un-reacted CA and catalase at zero time. The broad elution profile of CA observed is attributed to its polydisperse nature. In contrast, the peaks representing CA and catalase after 48 hours reaction (Fig. 4.3B) are shifted to the left and suggest co-elution of the two reactants. The emergence of a ‘new’ derivative with increased molecular weight in elution volume (Ve = 13ml) of the column suggests the presence of a neoglycoprotein. However, further investigations are warranted to confirm a covalent linkage has occurred and rule out the possibility of other forms of bonding.

Proposed polysialylated catalase fractions were pooled and employed in stability and enzyme kinetic studies (section 4.3.10) and later freeze-dried to investigate the effects of lyophilisation on enzyme activity and the cryoprotective effects of polysialylation (section 4.3.11).

165 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

1.5-1 r1.5 E c 0) o 0 h*- -T 0 “ 2 . 1.0 - - 1.0 Q) 3 8 8 g cn CO S 0.5- -0.5 01 (/) 3 3

0.0 0.0 0 10 20 30 40 Elution volume (ml)

1.5-, r1.5 E c cn o o O" S 1.0- - 1.0 0) 3 8 c 8 CO JQ cn CO 5 0.5- -0.5 cn U) 3 3

0.0 0.0 0 10 20 30 40 Elution volume (ml)

Figure 4.3 Size exclusion chromatography of colominic acid (V) and catalase (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: catalase was (50: 1). Samples were chromatographed on a Sephadex G-lOO column (45.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin"^). Arrows indicate the co elution volume of the conjugate (Ve = 13ml) and the void volume (Vo = 8ml) of the column.

166 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.5 Catalase: Spectrophotometric characterisation and quantification

In this study, catalase content was determined by employing the spectrophotometric method described in section 2.2.2.1. Essentially, the characteristic absorption maximum exhibited by catalase at 405nm (Soret band) in the near UV was used to determine its concentration. It was necessary to know the catalase content of the conjugates with a reasonable degree of accuracy in order to determine molar conjugation ratios and later when making comparisons between enzyme activity of the bioconjugates and native enzyme. To that end, a standard curve (Fig. 4.4) was prepared by measuring the absorbance of catalase samples of known concentration at 405nm.

2.5-1 I 2.0- o

0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Concentration (mg/ml)

Figure 4.4 Calibration curve obtained for catalase standard solutions. Catalase exhibits a characteristic absorption maximum (Soret band) at 405nm. Values are a mean ± s.d of 3 experiments, = 0.9988 represents linearity of 99.88%.

It was borne in mind that the extinction coefficient may have been affected by the chemistry of derivatisation i.e. polysialylation, leading to an error in the determination of concentration of catalase and retained activity. Therefore, the spectrophotometric properties of native, polysialylated and SDS-modified catalase were investigated. The results (Fig. 4.5A) show that the absorption features of native catalase in the near UV

167 Chapter Four: In vitro hioactivity of polysialylated peptides and proteins

3.5

2.5

1.5

0.5 0) O 0 c 200 (U 250 300 350 400 450 JQ o 3.5 (0 n < 3 2.5

1.5

0.5

0 200 250 300 350 400 450

Wavelength (nm)

Figure 4.5 UV-visible spectra of native, polysialylated and SDS-modified polysialylated catalase. A) native catalase; lOOpg/ml in PBS (black line) and 50pg/ml in 0.75M K2HPO4, pH 9.0 (blue line). B) zero time reaction mixture (thin black line), reaction mixture after 48 hours of catalase control (thick black line), polysialylated catalase (green line) and SDS polysialylated catalase (red line). The absorbance peaks at 405nm are similar for native and polysialylated catalase constructs.

168 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins are unaffected under the alkaline reductive amination conditions as its spectra is similar to native catalase in PBS, pH 7.4. The peak in the far UV however, was slightly shifted to a lower absorbance in alkaline buffer. It must be noted however that the change in absorbance observed in the far UV may not be indicative of specific changes of catalase absorption as certain buffers absorb within this region of the spectra. The spectra for the control sample (in the absence of reagents) reacted for 48 hours (Fig. 4.5B) shows a loss of the Soret band. It is hypothesised that this may have occurred owing to the turbidity of the sample, which is probably on account of protein aggregation.

Catalase at zero reaction time (in the presence of all reagents) and both conjugates (Fig. 4.5B) still exhibited the characteristic (Deisseroth and Dounce, 1970) Soret band at 405nm suggesting that no significant changes occur in the haem centre of catalase polysialylated by either method. Similar observations were made for native and polysialylated catalase conducted under the same conditions by Fernandes and Gregoriadis (1996). Interestingly, PEGylation was reported not to cause structural effects on the haem protein, horse cytochrome c (Mabrouk, 1994) as no major changes were detected in the spectrophotometric features of the enzyme on attachment of 6 PEG molecules. However, extensive modification of the enzyme (19 PEG molecules) did lead to changes in the near UV (Soret band). It was envisaged that SDS- polysialylated catalase might exhibit changes in the absorptivity in the near UV (405nm) owing to the 2-fold increase in amount of coupled CA (7.85 ± 0.45 moles) per mole catalase compared with polysialylated CA, however this was not evident by comparison of the spectra (Fig. 4.5B). It is realised that every protein is an entity in its own right and results cannot be extrapolated between one protein and another. Assuming therefore that the molar absorptivity of the Soret band is unaffected by polysialylation, it was proposed that we should be able to determine catalase content of the polysialylated constructs by the method suggested.

169 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.6 The influence of sodium dodecyl sulphate (SDS) on catalase inactivation and conformation of other peptides and proteins

The purpose of this study was two-fold; firstly it involved applying varying concentrations of SDS (0.35mM or 0.01% to 3.12mM or 0.09%) to catalase to determine which concentrations resulted in the optimal reversible and irreversible inactivation of the enzyme on removal of the dénaturant as described in section 4.2.2.4.I. Secondly, the concentrations of SDS found to optimally reversibly and irreversibly inactivate the model enzyme upon removal of the dénaturant were applied to IgG, aprotinin and BSA. It was hoped that changes in the hydrodynamic volume accompanying protein conformational changes (unfolding/folded states) on account of SDS treatment could be observed by employing the technique of size exclusion chromatography (SEC) as described in section 4.2.2.4.2.

4.3.6.1 Effects of varying concentrations of sodium dodecyl sulphate (SDS) on catalase activity Catalase enzyme activity in the absence (control) and presence of varying concentrations of SDS (0.01% or 0.35mM to 0.09% or 3.12mM) was investigated by employing the method described in section 4.2.2.4.2. Figure 4.6 shows the absorbance (A240nm) versus time spectrum obtained for native catalase dissolved (0.03mg/ml) in 0.05M phosphate buffer, pH 7.0 in the absence of SDS (control). Similar spectra were obtained for catalase exposed to an increasing concentration of SDS however, the gradient of the line became progressively less steep (spectra not shown). The activities determined from the spectra for the individual catalase samples were compared with the control (table 4.4) and the percentage residual activity plotted against SDS concentration (Fig. 4.7). Catalase catalyses the degradation of hydrogen peroxide to oxygen and water as represented in equation 1.0. The reaction is first-order and therefore the decomposition of the H 2O2 substrate is proportional to the concentration of the substrate and the enzyme.

catalase

2 HgOg 2 H2 O + O2 [equation 1.0]

170 Chapter Four: In vitro bioactivity’ of polysialylated peptides and proteins

Abs 3.00

2.50

2.00

1,50

1.00

0.50

0.00 0:00 5:00 10:00 Time (mm:ss)

Figure 4.6 Spectrum showing the decomposition of H 2O2 per min by native catalase (control) diluted (0.03mg/ml) in 0.05M phosphate buffer, pH 7.0. The decrease (AA) from 0.450 to 0.400 (at 240 nm) eorresponds to the decomposition of 3.45pmoles of H2O2 at 20°C per unit time and is a measure of enzyme activity (pmolmin.i). Slope of the graph is 0.015 A/min and the linearity is 99.90%.

Table 4.4 summarises the kinetie data obtained from the speetra derived for native catalase in the absenee (control) and presence of varying eoncentrations of SDS. All the spectra obtained exhibited a linear deeay of H 2O2 per min by catalase whereby the linearity was measured at > 99.90%

Table 4.4 Kinetic data obtained from the deeomposition of 30% H 2O2 per min at 20°C by native eatalase in the absence and presenee of different concentrations of SDS. Catalase in the absenee of SDS was included as the positive control (Pos. control) and used comparatively to determine pereentage residual activity. * Indicates values are the mean of 3 experiments and pereentage residual aetivity is represented as a mean ±

Catalase Time for decomposition of Enzyme activity Percentage residual

solutions 3.45pmoles H 2 O 2 (min)* (pmolmin’)* activity

Pos. control 3.43 1.01 100.00

0.35mM SDS 16.27 0.21 23.80 ± 1.34

0 . 6 9 m M S D S 22.05 0 . 1 6 18.73 ±2.83

l.OmM SDS 45.00 0.08 8.72 ± 1.98

2.08mM SDS 67.57 0.05 4.93 ± 1.98

3 . 1 2 m M S D S 300.30 0.01 l . l O i O . l l

171 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

100-f O (D > ,« 80- N (0 J ^ 60- S'S |: î 40-

20 -

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 SDS concentration (mM)

Figure 4.7 Catalase enzyme inhibition by different concentrations of SDS. Catalase samples dissolved (0.03mg/ml) in 0.05M phosphate buffer, pH 7.0 were incubated at 20°C for 1.0 hour alone (control) and in varying concentrations of SDS (0.35mM, 0.69mM, l.OmM, 2.08mM and 3.12mM). Catalase enzyme activity was measured in the presence and absence of SDS and expressed as a percentage residual enzyme activity of native catalase enzyme. Values are a mean ± s.d of 3 experiments.

The results (Fig. 4.7) show that increasing the concentration of SDS from 0.35mM to 3.12mM resulted in decreasing the percentage residual enzyme activity of catalase from 23.80 ± 1.34% to 1.1 ± 0.11%. Even at low concentrations of SDS (0.35mM), approximately 76% of the residual enzyme activity of catalase was lost. The fact that the low concentration (0.35mM) of SDS could bring about a large loss in the activity of catalase suggested that the binding affinity of catalase for SDS is very high. Complete inactivation of catalase enzyme was observed at > 3.12mM SDS concentration. The 99.55 ± 0.49% retention of enzyme activity for native catalase in the absence of SDS (control), treated under the same conditions as the catalase containing-SDS solutions indicated that the process of gentle stirring for 1.0 hour at room temperature was not detrimental to the residual activity of catalase.

The mechanism of action of SDS is unclear however, several authors e.g. Steinhardt and Reynolds (1969) and Ananthapadmanabhan (1993), have given an overview on

172 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins protein-SDS interactions. They suggest that due to the amphiphilic nature of SDS, the interaction with proteins is expected to be strongly dependant upon its concentration in solution. At low concentrations the protein is partially unfolded by the electrostatic interactions with SDS and at higher concentrations the interactions between SDS and proteins are primarily of hydrophobic character. The association between the hydrophobic side chains of the protein residue and the hydrophobic tails of SDS reduces the contact of the chains with water. Because of such interactions, high concentrations of SDS often result in considerable structural deformation/denaturation of the protein (Rao and Prakash, 1993). Such structural deformations of the protein would in turn influence the activity of the protein. The inactivation of catalase with SDS of varying concentrations positively demonstrated this correlation.

4.3.6.2 Determination of the optimal concentration of sodium dodecyl sulphate (SDS) resulting in the reversible inactivation of catalase In continuation from the studies in section 4.3.6.1 an attempt was made to study the reversibility of the inactivation effect of SDS upon catalase. Aliquots (0.1ml) from the SDS containing catalase solutions ranging from 0.35mM (0.01%) to 3.12mM (0.09%) and catalase solutions in the absence of SDS (control) were dialysed extensively (3 x 2L; 24 h) against distilled water and the catalase activity re-investigated. The percentage residual enzyme activity for the dialysed catalase solutions were compared with those obtained prior to dialysis and the results shown in Fig. 4.8.

The data for the SDS-catalase dialysed solutions in Fig. 4.8 showed that catalase inactivated by treatment with SDS at concentrations of 0.35mM (0.01%), 0.69mM (0.02%), and l.OmM (0.029%) regained almost complete activity (98.0%, 97.7% and 98.0%) after extensive dialysis. However, the residual enzyme activity (4.93 ± 1.98%) of catalase in solutions consisting of 2.08mM (0.06%) SDS was only marginally improved to 9.67 ± 1.53% after dialysis. Catalase was irreversibly inactivated when treated with 3.12mM (0.09%) SDS even after its apparent removal by dialysis as the activity determined before dialysis (1.10 ± 0.11%) was approximately the same as after dialysis (1.0%). The positive control in this study included catalase solutions in the absence of SDS, treated under the same conditions as the catalase containing-SDS

173 Chapter Four: In vitro hioactivity of polysialylated peptides and proteins

100 SD S- catalase 0) (D solutions before IS d ialysis N (D C SDS- catalase 0) o(Ü (Ü solutions after 3 d ialysis ■D 8 CO 20

0.000 0.350 0.690 1.000 2.080 3.120 SDS concentration (mM)

Figure 4.8 C om parison of the percentage of catalase en zy m e activity retention during exposure to different concentrations of S D S and after extensive dialysis to rem ove SD S. Catalase sam ples dissolved (0.03m g/m l) in 0.05M phosphate buffer, pH 7.0 w ere incubated at room tem perature for 1.0 hour alone and in varying concentrations of SD S, 0.35m M , 0.69m M , l.Om M , 2.08m M and 3.12m M . Catalase enzym e activity w as m easured in the absence (control) and presence of varying concentrations of S D S and after extensive dialysis against distilled w ater to rem ove S D S . T h e results are expressed as a percentage residual en zy m e activity of native catalase en zym e. V alues are a m ea n ± s.d of 3 experim ents.

solutions. T he retention (98.30 ± 0.58% ) of en zym e activity for native catalase

(control) suggested that gentle stirring for 1.0 hour at ro o m tem perature and extensive dialysis (3 x 2L; 24 h) against distilled w ater w ere not detrim ental to the residual activity o f catalase.

A n explanation for the reversible and irreversible inactivation effects of S D S upon catalase m a y be derived from the overview on protein-SD S interactions discussed by

Steinhardt and Reynolds (1969) and A nanthapadm anabhan (1993) outlined earlier

section 4.3.6.1. B y considering the am phiphilic structure of S D S and the ch an ge in the

nature of interaction occurring between SD S and catalase at low to high concentrations of SDS, i.e. w eak electrostatic interactions to predom inantly

hydrophobic ones, it follows that increasing the concentration of S D S results in the

inactivation of catalase. It is also plausible therefore that the transient w eak

electrostatic interactions betw een S D S and catalase at low concentrations of S D S i.e.

174 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

0.01, 0.02 and 0.029%) could be reversed by dialysis to remove the dénaturant. The resultant renaturation of inactivated catalase back to its original enzymic activity can be explained by the “thermodynamic hypothesis” established by Anfinsen et al. (1973) from the studies conducted on the renaturation of fully denatured ribonuclease. In brief, the hypothesis states that the 3-dimensional structure of a native protein in its normal physiological milieu is in a state in which the Gibbs free energy of the system is lowest. Consequently, the native protein form is thermodynamically more stable than any other. As the native conformation of a protein is determined by its amino acid sequence, removal of the dénaturant results in a conversion from the inactive (unfolded) state to the active (folded) driven by the decrease in Gibbs free energy. To summarise therefore, the percentage residual enzyme activity of catalase was found to decrease with increasing concentrations of SDS (Fig. 4.7) as per the theory on protein- SDS interactions proposed by Ananthapadmanabhan (1993). The concentration of SDS that led to the optimal reversible inactivation of catalase was observed at 1 .OmM SDS (0.029%) and irreversible inactivation was observed at > 3.12mM SDS. Prakash et al. (1980) observed similar effects with SDS upon the a-globulin protein (section 4.2.2.4). They reported that partial unfolding of the protein occurred at concentrations of 1.OmM SDS and dénaturation occurred at > 2.5mM.

4.3.6.3 Application of size exclusion chromatography for the study of peptide and protein conformation Various researchers (Shalongo et ah, 1989; Becktel and Lindorfer, 1990 and Reubsaet et ah, 1998) have described the use of SEC for the purpose of studying protein conformation (folding). In this study, the critical concentrations of SDS that brought about the reversible (l.OmM) and irreversible (3.12mM) inactivation of catalase upon removal of the dénaturant in section 4.3.6.2 were applied to IgG, aprotinin and BSA and analysed via size exclusion chromatography to study the corresponding effects of SDS on protein conformation. Based on the background information (Shalongo et ah, 1989; Becktel and Lindorfer, 1990), it was hypothesised that changes in the hydrodynamic volume of these proteins due to surfactant-protein interactions might be reflected in differing elution volumes obtained by employing SEC. Although more specific analytical techniques such as circular

175 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins dichroism (CD) would offer more in the way of definitively characterising the conformational changes of proteins in terms of secondary/tertiary changes, SEC was used simply as the most accessible method by which a comparison of the changes in conformational state could be observed. It was hoped that SEC would characterise transitional states fi"om the native folded to unfolded and refolded states of the proteins during incubation with SDS and after dialysis.

4.3.6.3.1 Analysis of the effect of sodium dodecyl sulphate (SDS) on the conformation of IgG by size exclusion chromatography IgG was dissolved (l.Omg/ml) in 0.15M PBS, pH 7.4 (control) and into solutions (5.0ml) of 0.15M PBS containing l.OmM SDS and 3.12mM SDS, respectively. Aliquots (0.5ml) from the control and the other two solutions were applied to a

E c tf) S LU Ü

ütLI 8 <

10 20 10 20 Elution volume Elution volume

Figure 4.9 Chromatographic elution profiles for native IgG (-), IgG in l.OmM SDS (À) and IgG in 3.12mM SDS (A), before dialysis (A and B) and after (A1 and Bl) after dialysis. All samples were chromatographed on a Sephadex G-lOO (30.0 x 1.1 cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin’^) column. Arrows indicate void volume (Vo = 6.0ml) of the column.

176 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Sephadex G-lOO (30.0 x 1.0cm) column and the eluted fractions (1.0ml) assayed (A595nm) for IgG content. Subsequently, the SDS-treated IgG solutions and the control were dialysed extensively against distilled water at room temperature to remove SDS and aliquots (0.5ml) re- characterised as detailed above. The resultant chromatograms are shown in Fig. 4.9. Results (Fig. 4.9A) show that IgG incubated in 1 .OmM SDS solution is eluted as a single almost symmetrical peak before the native (control) protein. A proposed explanation for the early elution of SDS treated-IgG is that unfolding increases the hydrodynamic volume of the protein and results in a molecule with a larger Stokes radius that is eluted faster (Light and Higaki, 1987). The absence of multiple elution peaks suggests that partial unfolding of the IgG molecule may have occurred as opposed to complete dissociation. The completely dissociated sub-units of IgG i.e. heavy and light chains were seen in the SDS-PAGE gel (Fig. 3.19 section 3.3.10). Studies (Shalongo et al., 1989; Becktel and Lindorfer, 1990) following the conformational changes of peptides and proteins using SEC have reported that protein chains refold on removal of the dénaturant to form the secondary structure, thus the molecule becomes more compact (globular) and the Stokes radius decreases. Interestingly, the elution profile for IgG incubated in l.OmM SDS solution post dialysis resumed that of the native protein (Fig. 4.9A1). It is therefore plausible to suggest that reversible changes in the hydrodynamic volume of IgG might suggest refolding of the molecule. Essentially the change in elution volume of SDS-treated- IgG from Fig. 4.9A to Fig. 4.9A1 might demonstrate the reversible conformational changes of IgG undergoing transition from a partially unfolded state during incubation in l.OmM SDS solution (Fig. 4.9A) to the refolded state after dialysis (Fig. 4.9A1). The reversible unfolding of SDS-treated IgG might be explained from the mechanism of action of SDS at low concentration suggested by Steinhardt and Reynolds (1969) and Ananthapadmanabhan (1993) and the “thermodynamic hypothesis” established by Anfinsen et al. (1973) discussed earlier in section 4.3.6.2.

In contrast to the single almost symmetrical peak observed for IgG incubated in l.OmM SDS solution (Fig. 4.9A), the elution profile of IgG incubated in 3.12mM SDS solution was very broad (Fig. 4.9B). This may reflect gross unfolding due to the increased concentration of SDS. Although the elution profile is very broad, multiple

177 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins peaks were not observed suggesting that complete dissociation of IgG into the sub units had not occurred. It is suggested that IgG experiences considerable structural deformation/dénaturation in 3.12mM SDS solution. Indeed even after extensive dialysis to remove SDS, IgG incubated in 3.12mM SDS solution retained the same hydrodynamic volume and was not accompanied by the proposed refolding of the molecule as its elution profile did not resume that of the native protein (Fig. 4.9B1). It is probable that irreversible conformational changes of IgG by 3.12mM SDS results due to the extensive interaction between the SDS tails and hydrophobic regions of the protein thus increasing the aqueous solubility of the hydrophobic portions of the protein (Creighton, 1979). To summarise, it is suggested that exposure of IgG to l.OmM SDS solution results in reversible conformational changes (unfolding) of the molecule after dialysis a seen by the reversible changes in hydrodynamic volume (Fig. 4.9). However in 3.12mM SDS solution, the IgG molecule experiences irreversible conformational changes even upon dialysis to remove the dénaturant. Interestingly, the effects of l.OmM and 3.12mM SDS solution respectively upon the reversible and irreversible unfolding of IgG were mirrored by the results for the reversible and irreversible inactivation of catalase (section 4.3.6.2).

4.3.6.3.2 Analysis of the effect of sodium dodecyl sulphate (SDS) on the conformation of aprotinin by size exclusion chromatography The methodology outlined in section 4.2.2.4.2 was employed in an attempt to study the reversible and irreversible effects of SDS on the conformation of aprotinin by employing SEC. Figure 4.10A shows that aprotinin dissolved in l.OmM SDS solution is eluted as single broad peak before the native protein. The early elution of SDS- treated aprotinin relative to the native protein may be explained by the increase in the hydrodynamic volume of the protein. The absence of multiple peaks suggests that l.OmM SDS may have caused partial unfolding of the highly folded single polypeptide chain that comprises the aprotinin molecule. After extensive dialysis to remove l.OmM SDS it was shown that aprotinin almost resumed its original hydrodynamic volume thus possible suggesting it reverts to its near normal native folded state (Fig. 4.10A1). The change in elution volume of SDS-treated aprotinin from Fig. 4.10A to Fig. 4.10A1 after dialysis suggests the reversibility of the

178 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

E c lO S UJ 0

1 A1 o Bl (0 CD <

^ 4 20 30 0 10 20 30 Elution volume Elution volume Figure 4.10 Chromatographic elution profiles for native aprotinin (-), aprotinin in l.OmM SDS (A) and aprotinin in 3.12mM SDS (A), before dialysis (A and B) and after (A1 and Bl) dialysis. All samples were chromatographed on a Sephadex G-50 (30.0 xl.lcm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^) column. Arrows indicate void volume (Vo = 3.0ml) of the column. conformational state (refolding behaviour) of aprotinin). The very broad elution profile observed for aprotinin incubated in 3.12mM SDS solution (Fig. 4.1 OB) compared with aprotinin dissolved in l.OmM SDS solution (Fig. 4.10A) might suggest that a greater conformational change (degree of unfolding) has occurred due to the higher concentration of SDS. This finding suggests that aprotinin dissolved in 3.12mM SDS solution may have experienced a much greater loss in its conformational state. Upon dialysis of aprotinin dissolved in 3.12mM SDS, the elution profile (Fig. 4.1 OBI) suggests that aprotinin is unable to resume its original hydrodynamic volume and regain its native conformation. Thus, it is proposed that aprotinin experiences an irreversible conformational change when dissolved in 3.12mM SDS solution. A comparison of the chromatograms of IgG in Fig. 4.9 with those of aprotinin in Fig. 4.10 provides a good illustration of the parallel nature of the

179 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins detergent-induced transformations in the two proteins. In order to establish whether this phenomenon is a general one, the investigation was extended to include bovine serum albumin (BSA). BSA has been employed in several studies due to its structurally robust nature and several investigators have elucidated its structure and properties (Hughes, 1954 & Squire et oA, 1968). Our investigation sought to include the effects of SDS upon BSA on the basis of its unique structural characteristics.

4.3.6.33 Analysis of the effect of sodium dodecyl sulphate (SDS) on the conformation of bovine serum albumin (BSA) by size exclusion chromatography The effects of SDS on the conformational characteristics of BSA were studied by employing the methodology established in 4.2.2.4.2. The resultant chromatograms are presented in Fig. 4.11.

E c to S UJ 0 t

1Û: 8 A1 Bl <

30 0 10 20 30 Elution volume Elution volume Figure 4.11 Chromatographic elution profiles for native BSA (-), BSA in l.OmM SDS (À) and BSA in 3.12mM SDS (A), before dialysis (A and B) and after (A1 and Bl) dialysis. All samples were chromatographed on a Sephadex G-lOO (25.0 xl.lcm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^) column. Arrows indicate void volume (Vo = 3.0ml) of the column.

180 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Results (Fig. 4.1 lA) show that BSA dissolved in l.OmM SDS solution is eluted in the same fractions as the native (control) protein. This is contrary to the elution profiles observed for IgG (Fig. 4.9A) and aprotinin (Fig. 4.10A) dissolved in l.OmM SDS solution respectively. This suggests that l.OmM SDS has negligible effect on the hydrodynamic volume and hence conformation of BSA. Upon dialysis of BSA dissolved in l.OmM SDS solution the elution profile (Fig. 4.11A1) indicated no change in the hydrodynamic volume of BSA as expected. A possible explanation of the negligible effect of l.OmM SDS on the conformation of BSA was sought for in the unique native structure of the protein. The BSA molecule is composed of three homologous domains, which are divided into nine loops by 17 disulphide bonds. The loops in each domain are made up of a sequence of large-small-large loops forming a triplet (Peters et al., 1985). Interestingly, Katchalski et al. (1957) reported on the accessibility of the disulphide bonds of BSA to reducing agents under differing conditions. They reported that the conformation and relative location of the disulphides within BSA protect the disulphide bonds from attack by reducing agents. Other studies (Visser and Blout, 1971; Vermeer and Norde, 2000) employed techniques such as differential scanning calorimetry (DSC), circular dichroism spectroscopy (CD), infrared (IR), and optical rotatory dispersion (ORD) spectroscopy to interpret the effects of detergent binding on the conformation of proteins at a sub- molecular level. Interestingly, the data from early ORD investigations of proteins led Jirgensons (1966) to postulate that the conformations of proteins with substantial a- helical content are not altered by surfactants. A number of other studies also identified this phenomenon (Pasta et al., 1990). Indeed, CD and IR spectra data from SDS- treated haemoglobin and lysozyme are in agreement with the postulate (Glazer and Simmons, 1965). BSA is predominantly (67%) a-helical in its native conformation with the remaining polypeptide occurring in turns and extended regions (Carter and Ho, 1994). It is arguable therefore that the unique folding and high a-helical content of BSA inhibits the access of low (l.OmM) concentrations of SDS to the polypeptide chains thereby protecting itself from unfolding.

In contrast, the elution profile of BSA dissolved in 3.12mM SDS solution shows a single, almost symmetrical broad peak that is eluted before the native protein (Fig.

181 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.1 IB). This characteristic was observed for both IgG and aprotinin albeit at the lower concentration of l.OmM SDS. Although the elution profile does reflect changes in the hydrodynamic volume of BSA they are not gross in comparison with IgG (Fig. 4.9B) or aprotinin (Fig. 4.1GB) dissolved in 3.12mM SDS solution. The lack of multiple peaks may suggest that BSA experiences partial unfolding and is not fi*agmented. In order to ascertain whether BSA had experienced irreversible conformational changes in the presence of 3.12mM SDS, a sample was extensively dialysed and SEC performed. The resultant chromatogram (Fig. 4.1 IB 1) revealed a similar elution profile to that of BSA dissolved in 3.12mM SDS solution (Fig. 4.1 IB). This perhaps suggests that increasing the concentration of the detergent facilitated the conformational change albeit irreversibly.

SEC analysis of CA dissolved in l.OmM SDS solution (Fig. 4.12) exhibited an elution profile that was almost identical with that of CA in the absence of SDS. This suggests that CA may be unaffected by this concentration of SDS. Furthermore, the calibration curve obtained with dialysed SDS-CA aliquots was similar to that prepared in the absence of SDS suggesting that SDS does not interfere in the resorcinol method for sialic acid determination (results not shown).

1.00n E c o N 0 .7 5 - LO 0 Ü C 0 .5 0 - CO

J 0.25 <

0.00 50 Elution volume (ml) Figure 4.12 Elution profile of native CA av. mol. wt. lOKDa dissolved (0.4mg/ml) in 0.15M PBS, pH 7.4 (■) and CA av. mol. wt. lOKDa dissolved in 0.15M PBS, pH 7.4 containing l.OmM SDS (A) on a Sephadex G-75 (40.0 x 1.0cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^) column.

182 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

In conclusion, our results showed that increasing the concentration of SDS above a critical concentration (3.12mM) does cause irreversible dénaturation of the catalase enzyme. At lower concentrations {i.e. l.OmM) however, the enzyme maybe reversibly inactivated. Furthermore, results (Figs. 4.9 - 4.11) also suggest that certain proteins e.g. IgG, aprotinin and BSA are susceptible to reversible (l.OmM) and irreversible (3.12mM) SDS-induced conformational changes, however the effects are not universally similar for every protein.

At the outset of this work it was considered that a manipulation of this kind might result in protein aggregation and dénaturation of the protein. Indeed, Reubsaet et al. (1998) reported that in the process of folding and unfolding partially unfolded intermediates exist. These intermediates may form large soluble or insoluble aggregates, which may be monitored by alteration in size and formation of precipitates (Reubsaet et a/., 1998). It was therefore proposed, that any increase in protein size due to aggregation of the proposed refolded protein (post SDS removal) should be reflected by an early elution profile during SEC compared with its normal elution volume. Interestingly, the elution profiles obtained for IgG and aprotinin (Figs. 4.9A1, 4.10A1) after removal of l.OmM SDS were both similar to their individual native counterparts thereby suggesting no aggregation had occurred in both cases. However, the elution profiles obtained for both proteins after removal of 3.12mM SDS (Figs. 4.9B1, 4.1 OBI) do suggest that aggregation may have irreversibly occurred. Furthermore, IgG and aprotinin samples remained slightly cloudy even after dialysis of 3.12mM SDS, whereas the same samples post l.OmM SDS treatment were clearer. It is plausible therefore to suggest that these findings indicate the absence of any major aggregation of IgG and aprotinin on refolding post dialysis of 1 .OmM SDS. It is proposed that other techniques are warranted to rule out completely the possibility of aggregation of the refolded peptides and protein after modification with l.OmM SDS. Many techniques have been reviewed by Reubsaet et al. (1998) for the purpose of protein aggregation analysis and they include turbidity measurements, scanning transmission electron microscopy (STEM), equilibrium dialysis, surface plasmon resonance (SPR) and SDS-PAGE.

183 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

The susceptibility of proteins to conformational changes due to SDS appears to be dependent upon factors such as the inherent 3-dimensional structure, a-helical content, number and location of disulphide bonds and the concentration of SDS applied. These results are consistent with the findings of Katchalski et al. (1957); Jirgensons (1966); Pasta et al. (1990) and Glazer and Simmons (1965) as described in section 4.3.6.3.3. Both IgG and aprotinin are low a-helical content proteins that contain relatively equal numbers of disulphide bonds (section 1.7.3), which are however linked in different quantities to a different number of chains within each molecule. Indeed, this phenomenon is true for IgG2a, catalase, insulin and somatostatin as they are all structurally different (section 1.7.3). BSA is conversely predominantly a-helical. It was however envisaged that IgG2a, catalase, insulin and somatostatin would be susceptible to conformational changes in the presence of l.OmM SDS. By virtue of our findings and substantial information on surfactant-protein interactions it was deemed justifiable to apply 1 .OmM SDS solution to the latter low a-helical content proteins to cause reversible unfolding of the proteins and thereby increase the number of available amino groups for coupling to CA. Finally, results indicated that both colorimetric assays for CA and proteins are unaffected by SDS after it has been dialysed. Additionally, the elution profiles of CA in the presence and absence of l.OmM SDS were almost identical, thereby suggesting that l.OmM SDS does not cause diminution of the CA molecule.

4.3.7 Degree of polysialylation of the SDS-treated peptides and proteins

In continuation from the previous study, 1 .OmM SDS was applied to the six different peptides and proteins introduced in this thesis in an attempt to increase the availability of their reactive a-amino and e-lysine residues and further facilitate the coupling of CA. It was also hoped that adequate biological activity was retained after this modification. Thus, in an attempt to further improve the efficiency of polysialylation of IgG, IgG2a, aprotinin, insulin, somatostatin and catalase, they were all modified with l.OmM SDS and polysialylated via reductive amination in the presence of NaBHsCN as described in section 4.2.2.5. The methods of sialic acid and protein determination were conducted as described in section 2.2, except calibration

184 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins curves were prepared using the six SDS-modified peptides, proteins and CA after dialysis. The molar yields of polysialylated SDS-modified peptides and proteins were expressed in terms of CA: Protein molar ratios found in the precipitates and are presented in table 4.5. For comparison, table 4.5 also incorporates data on the degree of modification of the six peptides and protein modified by the method of polysialylation established in Chapter 3 and their respective controls. These results were previously discussed in Chapter 3, except for polysialylated IgG2a and catalase, which are reported in section 4.3.1 and 4.3.3.

Table 4.5 Degree of modification of six therapeutic peptides and proteins by polysialylation and a modified polysialylation procedure. Reaction conditions and degree of modification of the six peptides and proteins are expressed in terms of moles CA per one mole of peptide or protein (or % of modified s-lysine and a-amino residues in parenthesis). Non-oxidised CA was reacted with the peptides and proteins

CA- Protein CA- (SDS) modified protein molar conjugation yields molar conjugation yields Preparation CONTROL CA-PROTEIN CONTROL CA-SDS PROTEIN CA: IgG 1.25 ±0.36: 1.0 4.25 ±0.20: 1.0 1.55 ±0.21: 1.0 12.27 ±0.3: 1.0 (50: 1) CA: Ig02a 1.22 ±0.16: 1.0 4.04 ±0.22: 1.0 1.48 ±0.11: 1.0 12.03 ±0.3: 1.0 (50: 1) CA: Catalase 0.64 ±0.10: 1.0 3.45 ±0.29: 1.0 0.98 ±0.10: 1.0 7.85 ±0.45: 1.0 (50: 1) CA: Aprotinin 0.39 ±0.03: 1.0 1.46 ±0.05: 1.0 0.45 ±0.02: 1.0 4.59 ±0.05: 1.0 (50: 1) CA: Insulin 0.42 ±0.03: 1.0 1.90 ±0.05: 1.0 0.61 ±0.03: 1.0 2.92 ±0.06: 1.0 (50: 1) CA: SS 0.61 ±0.01: 1.0 3.00 ±0.01; 1.0 0.55± 0.02: 1.0 3.00 ±0.02: 1.0 (10: 1)

Results (table 4.5) show that the apparent degree of polysialylation achieved by covalently coupling oxidised CA via reductive amination to the e-lysine and/or a- amino groups of all the SDS-modified peptides and protein (except somatostatin) were significantly improved in comparison with the conventional method of polysialylation established in chapter 3.

185 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Similar molar yields of conjugation were obtained for polysialylated SDS-modified IgG (12.27 ± 0.3 moles CA per mole IgG) and IgG2a (12.03 ± 0.3 moles CA per mole IgG2a). Since each molecule of IgG contains 92 s-lysine and 4 a-amino residues (Edelman et al, 1969), the molar ratio achieved for SDS-assisted polysialylation of IgG corresponds to an average of 12.78% of the total available amino groups. This is approximately 3-fold greater than by the established method of polysialylation (4.43%). In contrast, chemical modification of SDS-treated IgG and IgG2a by direct coupling to non oxidised CA (control) by reductive amination in aqueous solution yielded proposed conjugates showing an approximate 8 -fold decrease in the amount of coupled CA. Similarly, poor molar yields of conjugation were reported for the control experiments for polysialylayted IgG and IgG2a. The similarity in values obtained for IgG and IgG2a were anticipated on account that both immunoglobulins possess similar structures and molecular weights.

SDS-treated catalase modified by conjugation to oxidised CA via reductive amination in aqueous solution, yielded a proposed conjugate containing 2 -fold the amount of coupled CA (7.85 ± 0.45 moles CA per mole catalase) compared with the conventional method of polysialylation (3.45 ± 0.29 moles CA per mole catalase). On the basis that each molecule of catalase contains 108 8 -lysine and 1 a-amino residue (Weissmann et al, 1975), this ratio corresponds to the polysialylation of an average 7.20% of the total available amino groups. SDS-treated catalase coupled directly to non oxidised CA (control) by reductive amination, in aqueous solution led to a conjugate showing an 8 -fold decrease in the amount of coupled CA (0.98 ±0.10 moles of CA per mole of SDS-modified catalase) compared with oxidised CA coupled to SDS-treated catalase. Fernandes and Gregoriadis (1996) attempted to further increase the molar yield of polysialylated catalase by decreasing the size of CA reacted with catalase; however they were unable to significantly improve the molar yield of conjugation as compared with polysialylated SDS-modified catalase yields obtained in this study.

SDS-modified aprotinin coupled to oxidised CA via reductive amination in aqueous solution yielded an apparent conjugate containing 3-fold more coupled CA (4.59 ±

186 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

0.05 moles CA per mole aprotinin) than by the conventional method of polysialylation 1.46 ± 0.05 moles CA per mole aprotinin). Each aprotinin molecule consists of 4 e- lysine and 1 terminal a-amino residue (Roberts et al., 1996). Therefore, it was calculated that this ratio corresponds to the polysialylation of an average 91.8% of the total available amino groups of aprotinin. SDS-treated aprotinin modified by the procedure above without first incorporating an aldehyde group into CA (control) led to a very poor yield of conjugation where only 0.45 ± 0.02 moles of oxidised CA reacted per one mole of SDS-treated aprotinin. Thus, the extent of polysialylation achieved by direct coupling was 10-fold less than when oxidised CA had been employed in the conjugation reaction with SDS-treated aprotinin.

SDS-treated insulin coupled to periodate oxidised CA by reductive amination in aqueous solution, led to a proposed conjugate containing 1.5-fold the amount of coupled CA (2.92 ± 0.06 moles CA per mole insulin) compared with the conventional method of polysialylation (1.90 ± 0.05 moles CA per mole insulin). On the basis that each molecule of insulin comprises 1 e-lysine and 2 N-terminal reactive a-amino groups, it was estimated that an average of 97.3% of the total available amino groups were modified. Chemical modification of SDS-treated insulin by direct coupling of non oxidised CA (control) via reductive amination, in aqueous solution led to a conjugate with a very poor degree of conjugation (0.61 ± 0.05 moles of oxidised CA per one mole of SDS-treated insulin). Thus, prior incorporation of the aldehyde group in CA followed by coupling to the available amino residues of SDS-treated insulin resulted in a conjugate showing a 5-fold increase in the amount of coupled CA.

Finally, somatostatin (SS) was the only peptide to produce similar molar yields of conjugation when modified by both methods of polysialylation which were quantitative in comparison to the other proteins. Somatostatin contains 2 e-lysine and 1 N-terminal reactive a-amino groups (Brazeaus et ai, 1983), thus this corresponds to polysialylation of an average of 100% of the available amino groups. Direct coupling of SDS-treated somatostatin to non oxidised CA (control) by reductive amination, in aqueous solution led to a conjugate showing a 5-fold decrease in the amount of coupled CA (0.55 ± 0.02 moles of CA per mole of SDS-modified SS). Similar poor

187 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins molar yields of conjugation were achieved in the control experiment with non oxidised CA reacting with intact SS 0.61 ± 0.01 moles of CA reacted per mole of SS (table 4.5).

In conclusion, modification of the polysialylation process by the use of SDS resulted in peptide and protein conjugates with 1.5-3 fold more coupled CA in comparison with those prepared by the standard polysialylation technique. These results suggest that prior treatment of the peptides and proteins investigated here with l.OmM SDS may have increased the availability of the reactive e-lysine and a-amino groups thus facilitating coupling of CA and leading to greater polysialylation yields. It should be noted however, that as CA is polydisperse, values of degree of polysialylation would be average.

Our findings also suggest that periodate oxidation appears to facilitate coupling as seen from the poor molar yields of conjugation obtained by directly coupling the SDS- treated peptides and proteins to non-oxidised CA (control). The poor molar yields of conjugation observed with all the control experiments {i.e. SDS-modified or unmodified peptides and proteins reacted with non oxidised CA) might be explained by considering the structural conformation that CA adopts in solution as previously explained in section 3.3.2. Essentially, the reactive C-2 (keto) group at the reducing end of CA exists only in small amounts and may therefore limit the efficiency of direct coupling.

To date, there have been no reports with respect to such a modification with other chemically modified peptides and proteins. It is hoped that SDS-modified polysialylated peptides and proteins will be therapeutically useful on account of the expected increase in circulatory half-live of the constructs; however, we needed to consider the effects of these manipulations on the resultant biological properties of these bioconjugates. To that end, the latter properties are investigated in section 4.3.10 and an in vivo account of the effects of CA-modification on SDS-treated peptides and proteins is given in Chapter 5.

188 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.8 Size exclusion chromatographic characterisation of polysialylated SDS- modified peptides and proteins

Size exclusion chromatography was employed in an attempt to prove that CA had covalently coupled to the six SDS modified peptides and proteins under investigation here. Samples taken at zero time reaction and after 48 hours were removed from the six different peptide and protein reaction mixtures, dialysed extensively and applied to Sephadex gel columns as outlined in Chapter 3 for IgG (3.3.8), aprotinin (3.3.12), insulin (3.3.14), somatostatin (3.3.16) and Chapter 4 for Ig02a (4.3.2) and catalase (4.3.4). The 48 hour reaction mixtures containing the proposed conjugates were subjected to ammonium sulphate precipitation in an attempt to isolate the neoglycoprotein prior to column application. Size exclusion chromatographic characterisation of the six CA modified SDS-treated peptides and proteins and their native components at zero reaction time can be seen for IgG (Fig. 4.13), IgG2a (Fig. 4.14), catalase (Fig. 4.15), aprotinin (Fig. 4.16), insulin (Fig. 4.17) and somatostatin (Fig. 4.18).

As expected, the elution profiles obtained for the individual peptides and proteins at zero time were different fi*om their 48 hour counterparts. All the zero hour profiles (Figs. 4.13A-4.18A) showed resolved peaks, which suggested the presence of unreacted components. Some overlapping was observed for IgG (Fig. 4.13A), which may be due to the polydisperse nature of CA however; the peaks do suggest independent elution of the reactants. In contrast, the elution profiles of the reactants after 48 hours reaction (Figs. 4.13B-4.18B) are completely overlapping. The ‘new’ peaks representing CA and the peptide or protein co-elute in the same elution volume (Ve) of the column. The shift in peaks to the left suggests faster co-elution perhaps due to the emergence of a ‘new’ heavier entity. These observations have been previously interpreted as suggesting the presence of the neoglycoprotein and indeed here under the new modified conditions. Further experimentation is however warranted to definitively confirm covalent linkage has occurred and rule out other forms of bonding.

189 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

r1.5 E c (/) o 0 N "T c S 1.0- - 1.0 fi) 3 8 8 g 01 CO 0.5- -0.5 cn 3 3

0.0 0.0 0 1 0 2 0 30

Ve i B I.OOn r 2 . 0 E c 0 ) o o N 0.75- lO o- fi) o 3 o o c 0.50- (D (0 n 'a! L. CO cn 8 0.25- 3 3

0.00 10 20 Elution volume (ml)

Figure 4.13 Size exclusion chromatography of colominic acid (À) and SDS-modifiec IgG (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: IgG, (50: 1). Samples were chromatographed on a Sephadex G-lOO (40.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'*) column. Arrows indicate the co elution volume of the conjugate (Ve = 12ml) and the void volume (Vo = 7.0ml) of the column.

190 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

r 0 . 2 0 E c 0 o ) -0.15 -no If) O" fi) Q) 3 O c - 0.10 8 (0 n “oi CD O 0 .5 - CM (/) -0.05 3 3

0.0 0.00 0 1 0 2 0 30 4 0

B Ve 0.3-1 1- 0.20 E c o -0.15 O" m 0 .2 - 0 ) o c -0 . 1 0 (D (0 .Q s cn 8 -0.05 2

0.0 0.00 0 1 0 2 0 30 40 Elution volume (ml)

Figure 4.14 Size exclusion chromatography of colominic acid (À) and SDS-modifiec IgG2a (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: IgG2a: (50: 1). Samples were chromatographed on a Sephadex G-lOO (40.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^) column. Arrows indicate the co-elution volume of the conjugate (Ve = 14ml) and the void volume (Vo= 7.0ml) of the column.

191 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

r1.5 E c 0 ) o o lO O" - 1.0 &) Q) 3 O c 8 03 L. g o 0.5- -0.5 ai (/) 3 3

0.0 €.0 0 1 0 2 0 30 40

2 .0-1 r1.5 I (/) M 1.5- to lO O" - 1.0 Q) 3 8 g o -0.5 Ul ^ 0.5- 3 3

0.0 0.0 0 10 20 30 40 Elution volume (ml)

Figure 4.15 Size exclusion chromatography of colominic acid (A) and SDS-modifiec catalase (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: catalase: (50: 1). Samples were chromatographed on a Sephadex G-lOO (45.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^) colunrn. Arrows indicate co elution volume of the conjugate (Ve = 12ml) and void volume (Vo = 7ml) of the column.

192 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

I.OOn r0.15 E c 0 ) o 0.75- o If) O" - 0.10 fi) 3 8 0.50- 0 5 cn -0.05 cnCO 0.25- 3 3

0.00 0.00 0

Ve B 0.4-1 1-0.15 I 0 ) R 0.3- 0 If) O" - 0.10 0 ) 3

§ 0 .2 - s 0 jQ L. s -0.05 01 3 I 3

0.0 "0.00 0 1 0 2 0 30 Elution volume (ml)

Figure 4.16 Size exclusion chromatography of colominic acid (A) and SDS-modifiec aprotinin (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: aprotinin, (50: 1). Samples were chromatographed on Sephadex G-50 (35.0 x 1.0 cm; sample volume 0.5ml; PBS eluent; fiow rate, l.Omlmin'^) colunrn. Arrows indicate co elution volume of the conjugate (Ve = 11ml) and the void volume (Vo = 4ml) of the column.

193 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

1 .00-1 r0 .1 5 E c (fi g 0 .7 5 - 0 m O" - 0.10 0 ) 3 8 0 .5 0 - 1 SX 'cK k. CO -0.05 cn J 0 .2 5 - 3 3

0.00 0.00 0 1 0 2 0 30

B Ve 0.75-1 r0 .1 5 E i c (fi o 0 m 0 “ ^ 0 .5 0 - - 0.10 fi) 3 8 C S (0 SX s o 0 .2 5 - -0.05 01 0 ) 3 < 3

0.00 30 Elution volume (ml)

î'igure 4.17 Size exclusion chromatography of colominic acid (A) and SDS-modifiec insulin (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: insulin, (50: 1). Samples were chromatographed on a Sephadex G-50 (40.0 x 1.1cm; sample volume 0.5ml; PBS eluent; fiow rate, l.Omlmin'^) column. Arrows indicate co elution volume of the conjugate (Ve = 12ml) and the void volume (Vo = 4.0ml) of the column.

194 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

0.15n 1-0.15 ? c Vi o o

E . 0 . 1 0 ho.io ^ 3 s c s cn 0 .0 5 - K).05 g 3 3

0.00 K).00 0

Ve B

O . I O O n 1 r0.075 E c o OI N 0 .0 7 5 - m c r -0.050 Û) 3 8 O c 0 .0 5 0 - (D (0 cn L_ (O o Vo -0.025 cn (/) 0 .0 2 5 - 3 1 3

0.000 ■0.000 0 10 20 30 40 Elution volume (ml)

Figure 4.18 Size exclusion chromatography of colominic acid (A) and SDS-modified somatostatin (SS) (■) after A) zero time and B) 48 hours of reaction. Starting molar ratio CA: SS, (10: 1). Samples were chromatographed on a Sephadex G-50 (45.0 x 1.1cm; sample volume 0.5ml; PBS eluent; fiow rate, l.Omlmin"') column. Arrows indicate co-elution volume of the conjugate (Ve = 14ml) and the void volume (Vo = 5ml) of the column.

195 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.9 Electrophoretic characterisation of IgG and aprotinin modified by two methods of polysialylation

Immunoglobulin G (IgG) and aprotinin modified by both methods of polysialylation were fiirther characterised by employing the method of SDS polyacrylamide gel electrophoresis (SDS-PAGE). In brief, samples from the two proteins modified by either polysialylation techniques were run on SDS-PAGE gels and compared with their respective controls. The controls consisted of the native proteins and the native proteins subjected to the reaction conditions in the absence of CA. IgG and aprotinin samples respectively were run utilising a discontinuous SDS buffer system developed for SDS-PAGE employing either 12.5% (IgG) or 17.5% (aprotinin) polyacyrlamide gels. Both the SDS-PAGE gels were run under denaturing conditions outlined in section 2.2.4.1 and both molecular weight markers used may be seen in table 2.4. Details regarding buffer composition and gel mixture preparations are presented in table 2.3.

4.3.9.1 SDS polyacrylamide gel electrophoresis of IgG modified by two methods of polysialylation The electrophoretic behaviour of polysialylated IgG has been previously shown in section 3.3.10 (Fig. 3.19). Here, SDS-PAGE was employed in an attempt to characterise the proposed conjugates prepared by both methods of polysialylation and show the changes in molecular weight on conjugation compared with the starting materials. The SDS-PAGE gel consisted of low molecular weight markers (lane M) run against two controls consisting of native IgG (lane 1) and IgG under reaction conditions without CA (lane 2). The other samples were believed to consist of polysialylated IgG (lane 3) and polysialylated SDS-modified IgG (lane 4) isolated after 48 hours reaction respectively. All samples were extensively dialysed prior to SDS-PAGE. Under the denaturing conditions described, it was anticipated that each IgG containing sample would dissociate into four peptide sub-units, as SDS reduced each disulphide (Edelman, 1960). Indeed, analysis of the SDS-PAGE gel (Fig. 4.19) revealed two clearly defined bands for both controls (lane 1 and 2 ) of dissociated protomers with molecular weights of ~ 25KDa and 50KDa respectively.

196 Chapter Four: In vitro hioactivity of polysialylated peptides and proteins

Figure 4.19 SDS-PAGE of native IgG and IgG modified by two methods o polysialylation against low molecular weight markers (BioRad). SDS-PAGE was conducted on a 12.5% polyacrylamide gel under reducing conditions. Lane M: low molecular weight markers comprising of, phosphorylase b (97.4KDa), albumin (66.2KDa), ovalbumin (45.0KDa), carbonic anhydrase (31.0KDa) and trypsin inhibitor (21.5KDa). Lane 1: native IgG (control), Lane 2: native IgG after 48 hours reaction without CA (control). Lane 3: IgG-CA conjugate after 48 hours reaction and Lane 4: SDS-modified-IgG-CA conjugate after 48 hours reaction. The gel was stained with Coomassie blue. For other details refer to Chapter two.

These bands are consistent with the molecular weights of the heavy and light chains of IgG (Edelman, 1969). Even after incubation in l.OmM SDS and under the same reaction conditions in the presence of reagents (control, lane 2) IgG retained the same bands in SDS-PAGE with no evidence of polymerisation or lower molecular weight degradation products. Lane 3 (Fig 4.19) consisted of proposed polysialylated IgG isolated from its reaction mixture by ammonium sulphate precipitation after 48 hours reaction. The pattern of dissociated protomers is repeated as for the controls however the bands are broader in shape and more diffuse. This might suggest microheterogeneity (Carlsson, 1993) of the protein sample as a result of coupled colomininc acid chains, thereby suggesting the presence of the proposed neoglycoprotein. The same observation was made previously in section 3.3.10 for polysialylated IgG (lane 4) in Fig. 3.19 and by Fernandes and Gregoriadis (1996) for catalase and asparaginase modified with CA. Similarly, the sample run in lane 4 (Fig

4.19) revealed two broad diffuse protein bands, perhaps suggesting

197 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins microheterogeneity of the protein on account of coupled CA. These bands seem more exaggerated, compared with the neighbouring bands in lane 3. Unfortunately, the intensity of the protein band is reduced possibly due to uneven loading of the gel. The position of the protein bands (Fig 4.19) in lane 4 representing polysialylated SDS- modified IgG seem to have migrated slower than proposed polysialylated IgG (lane 3) and perhaps suggest a slightly higher molecular weight of this construct due to its increased CA modification.

Segrest and Jackson (1972) established that heavily glycosylated proteins migrate at a slower rate in SDS-PAGE due to the reduced affinity of the carbohydrate chains to the anionic detergent SDS. Therefore, application of SDS-PAGE in molecular weight determinations of a polysaccharide-protein conjugate would result in erroneously high estimations. However, Leach et al (1980) proposed that the negative sialic acid residues would compensate for the reduced affinity of SDS and render the measurement of molecular weight more precise. Previously in section 3.3.10 the molecular weight of proposed polysialylated IgG was estimated to be in the region of 194.4KDa. Unfortunately, no attempts were made here to determine the molecular weights of either of the two proposed conjugates (lane 3 and 4) due to the differences in the intensities of the protein bands caused by the possible uneven loading onto the gel.

4.S.9.2 SDS polyacrylamide gel electrophoresis of aprotinin modified by two methods of polysialylation Aprotinin modified by both methods of polysialylation together with controls of native aprotinin and aprotinin under the reaction conditions in the absence of CA were subjected to SDS-PAGE (Fig 4.20). These samples were calibrated against a combination of low and ultra-low molecular weight markers (lane M). Electrophoresis of both controls (lane 1 and 2 ) revealed single distinct protein bands with an estimated molecular weight of 6.5KDa (Fig. 4.20) which was consistent with that reported by Roberts et al. (1996) for the single polypeptide chain. Aprotinin retained the same single band (lane 2) in SDS-PAGE even after incubation for 48 hours under coupling conditions. This suggests that polymerisation and lower molecular weight derivatives

198 Chapter Four: In vitro hioactivity of polysialylated peptides and proteins

Figure 4.20 SDS-PAGE of native aprotinin and aprotinin modified by two methods of polysialylation against a combination of low and ultra-low molecular weight markers (BioRad). SDS-PAGE was conducted on a 17.5% polyacrylamide gel under reducing conditions. Lane M: low and ultra-low molecular weight markers comprising of, albumin (66.2KDa), ovalbumin (45.0KDa), carbonic anhydrase (3 LOKDa), triosphosphate isomerase (26.6KDa), trypsin inhibitor (21.5KDa), lysozyme (14.4KDa) and aprotinin (6.5KDa). Lane 1: native aprotinin (control). Lane 2: native aprotinin after 48 hours reaction without CA (control). Lane 3: aprotinin-CA conjugate after 48 hours reaction and Lane 4: SDS-modified-aprotinin-CA conjugate after 48 hours reaction. The gel was stained with Coomassie blue. For other details refer to Chapter two. of aprotinin under these conditions were not evident. In parallel with IgG after modification by polysialylation, the proposed microheterogeneity of aprotinin on account of coupled CA was demonstrated in SDS-PAGE by broad diffuse bands in lanes 3 and 4. These samples represented the proposed aprotinin conjugates prepared by two methods of polysialylation. The broad diffuse protein band observed in lane 4

(representing polysialylated SDS-modified aprotinin) was more pronounced and showed reduced electrophoretic mobility compared with that in lane 3 (representing polysialylated aprotinin). This suggests that the proposed aprotinin conjugate in lane 4 exhibits a higher molecular weight perhaps due to increased CA addition.

Unfortunately, the molecular weights of the respective conjugates in lane 3 and 4 were not determined due to differences in the intensities of the protein bands arising from the possible uneven loading onto the gel. In an attempt to avoid the aprotinin samples from running close to the edge of the 17.5% polyacrylamide gel as above, the samples

199 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

were simultaneously run on a 2 0 % polyacrylamide gel (results not shown). Unfortunately, the thicker gel heated unevenly (hot middle, cool edges) and so the protein bands were distorted, known as the smiling effect. It was anticipated that polysialylated SDS-modified aprotinin would show significantly reduced electrophoretic mobility due to higher substitution of CA molecules compared with polysialylated aprotinin, however this was not apparent. Similarly, anomalous migration was reported for Peg coupled IL-2 where conjugates ran to the same place on SDS-PAGE even though their molecular weights were quite different (Kunitani et al, 1991). In this case, it was reasoned that the locations of PEG moieties could have different effects on the radius depending on for example whether they were on the same sides of the molecule and may have had different effects on overall SDS binding.

4.3.10 In vitro stability and biological activity studies of catalase, IgG2a and aprotinin modified by two methods of polysialylation

The in vitro stability of catalase during the coupling process and biological properties of IgG2a and aprotinin all modified by two methods of polysialylation are explored here and compared with their respective native unmodified counterparts as described in section 4.2.2.6. Initially, the percentage residual enzyme activity of native catalase (under reaction conditions) and the two polysialylated constructs during the coupling process were compared with native unmodified catalase (positive control). Subsequently, a more in depth study was conducted by employing a Hanes Woolf plot to investigate catalase enzyme kinetics as described in section 4.2.2.3. Secondly, the antigen binding properties of IgG2a modified by both polysialylation techniques was investigated using an Enzyme-Linked Immuno-Sorbent Assay (ELISA) as described in section 4.2.2.6.1, as the affinity of the Mab for its target antigen is an essential therapeutic criterion. Finally, the biological properties of the aprotinin conjugates prepared by both methods of polysialylation were determined by measuring their inhibitory activity on a solution of trypsin of known activity. The percentage residual activity reported for all the protein bioconjugates explored here were determined with respect to their relative native unmodified (positive controls) counterparts.

2 0 0 Chapter Four: In vitro bioactivity of polysialylated peptides and proteins

4.3.10.1 Determination of catalase stability during the conjugation process

Catalase samples (~10pl) containing ~25-35pg catalase taken at zero time (positive controls) and intervals of 6, 12, 24 and 48 hours from the reaction mixtures of three catalase formulations were independently assayed for enzyme activity (pmolmin 'pg'^) by the method reported in section 4.2.2.2. The formulations include catalase under reaction conditions with no CA, polysialylated catalase and polysialylated SDS- treated catalase. Spectra showing the time (min) required for the change in AA from

0.450-0.400 (representing the decomposition of 3.45pmoles) of 30% H 2O2by catalase taken from the three formulations at different time intervals were obtained. Spectra obtained for the zero time (positive controls) catalase samples of the three formulations are shown compared with the respective 48 hours samples only (Figs.

4.21-4.23) as the latter time samples represent the end of the bioconjugation process as reported throughout this thesis. Data derived from the spectra of all three formulations including time (min) for the decomposition 30% H 2O2, enzyme activity and percentage residual activities are presented in tables 4.6-4.8.

1,50 A1 1.00

0,50

0.00 0.00 0:30 1:00 1.30 Time (mm:ss)

1.50 B1

1.00

0.50

0 00 0:00 1:00 2:00 3:00 4:00 Time (mm:ss) Figure 4.21 Spectra showing the decomposition of 30% H 2O2 per min by catalase in the absence of CA Al) zero time (control) (slope = 0.150A/min, linearity = 100%, catalase content ~ 35pg and Bl) after 48 hours reaction (slope = 0.041 A/min, linearity = 99.9%, catalase content ~ 31pg. The decrease (AA) from 0.450 to 0.400 (at 240 nm) corresponds to the decomposition of 3.45pmoles of H 2O2 at 20°C per unit time and is used to measure enzyme activity (pm olm in'V g ')-

201 Chapter Four: In vitro bioactivity’ ofpolysialylated peptides and proteins

1.50 A2 - -4 1.00

0 50

0.00 0:00 0:30 1:00 1:30 Time (mm:ss) 1.50 B2

1.00

0.50

0,00 0:00 0:30 1:00 Time (mm:ss) Figure 4.22 Spectra showing the decomposition of 30% H 2O2 per min by polysialylated catalase at A2) zero time (control) (slope = 0.136A/min, linearity = 99.9%, catalase content ~ 30pg and B2) after 48 hours reaction (slope = 0.091 A/min, linearity = 99.9%, catalase content ~ 30pg. The decrease (AA) from 0.450 to 0.400 (at 240 nm) corresponds to the decomposition of 3.45pmoles of H 2O2 at 20°C per unit time and is a measure of enzyme activity (pmolmin 'pg ').

1.50 A3

1.00

0.50

0.00 0:00 1:00 Time (mm:ss)

1.50 B3

1.00

0.50

0.00 0:00 2:00

"igure 4.23 Spectra showing the decomposition of 30% H 2O2 per min by polysialylated SDS-modified catalase at A3) zero time (control) (slope = 0.120A/min, linearity = 100%, catalase content ~ 26pg and B3) after 48 hours reaction (slope = 0.077A/min, linearity = 99.9%, catalase content ~ 26pg. The decrease (AA) from 0.450 to 0.400 (at 240 nm) corresponds to the decomposition of 3.45pmoles of H 2O2 at 20°C per unit time and is a measure of enzyme activity (pm olm in'V g ')-

202 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Table 4.6 Data derived from the decomposition of 30% H 2O2 per min at 20°C by catalase reacted with non-oxidised CA taken at time intervals of the reaction. Catalase at zero time was included as the positive control. Samples contained ~ 31-35pg catalase. * Indicates values are the mean of 3 experiments and percentage residual

Catalase: CA Time for decomposition of Enzyme activity Percentage

reaction (h) 3.45pmoles H 2O2 (min)* (pmoImin'Vg*^)* residual activity

0 (pos. control) 0.30 0.33 99.99 ±0.01

6 0.62 0.18 54.30 ±2.50

1 2 0.77 0.14 43.30 ± 2.52

24 1.16 0 .1 0 29.00 ± 1.00

48 1 .2 2 0.09 26.30 ± 1.20

Table 4.7 Data derived from the decomposition of 30% H 2O2 per min at 20°C by polysialylated catalase taken at time intervals of the reaction. Catalase at zero time was included as the positive control. Samples contained ~ 30pg catalase. * Indicates values are the mean of 3 experiments and percentage residual activity is represented as a mean ± s.d of 3 experiments. Catalase: CA Time for decomposition of Enzyme activity Percentage residual

reaction (h) 3.45pmoles H 2O2 (min)* (pmolmin'Vg’)* activity

0 (pos. control) 0.37 0.32 99.95 ± 0.05

6 0.39 0.31 94.70 ± 2.30

1 2 0.44 0.27 84.00 ± 3.00 24 0.51 0.23 73.30 ± 0.60

48 0.55 0 .2 1 66.70 ± 1.50

Table 4.8 Data derived from the decomposition of 30% H 2O2 per min at 20°C by polysialylated SDS modified catalase taken at time intervals of the reaction. Catalase at zero time was included as the positive control. Samples contained ~ 26pg catalase. * Indicates values are the mean of 3 experiments and percentage residual activity is represented as a mean ± s.d of 3 experiments. Catalase: CA Time for decomposition of Enzyme activity Percentage residual

reaction (h) 3.45pmoles H 2O2 (min)* (pmolmin *pg^)* activity

0 (pos. control) 0.42 0.32 99.99 ±0.01

6 0.49 0.27 84.30 ± 2.50

1 2 0.53 0.25 77.70 ±0.58

24 0.63 0 .2 2 69.30 ± 1.53

48 0.65 0 .2 0 63.70 ± 1.53

203 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

1 0 0

a 80- >

(0 60-

c 40-

20 -

0 6 12 18 24 30 36 42 48 Time (h) Figure 4.24 Loss of enzyme activity of catalase and catalase conjugates during the coupling process. Native catalase (■), polysialylated catalase ( o ) and polysialylated SDS-modified catalase (A). The polysialylated enzymes were prepared with a CA: catalase molar ratio of 50: 1. Results are mean ± s.d. of 3 separate experiments.

Figures 4.21-4.23 showed that catalase enzyme activity is linear. The percentage residual enzyme activity determined for the three catalase formulations during the coupling process were plotted against time and represented in Figure 4.24. Results (Fig. 4.24) suggest that native unmodified catalase is significantly (ANOVA, p = 0.0001, p< 0.05) less stable (retaining 26.30 ± 1.20% activity) than the polysialylated (retaining 66.70 ± 1.50% activity) and polysialylated SDS-modified (retaining 63.70 ± 1.53% activity; 48h) catalase conjugates respectively. At the end of the coupling process, catalase modified by either method of polysialylation respectively yielded bioconjugates with 2-fold retention of stability, compared to native catalase. Likewise, Fernandes and Gregoriadis (1996) demonstrated that covalent attachment of CA to catalase (70.80 ± 1.3% activity) conferred improved stability over the native enzyme (29.1 ± 0.9% activity) at the end of the coupling process. It was suggested that the coupling process (or perhaps the presence of CA in the reaction mixture) protects catalase from inactivation. Indeed, Combes et al (1990) reported on similar protective effects of other polyols. A comparison of the percentage residual enzyme activities of polysialylated (66.70 ± 1.50% and polysialylated SDS-modified (63.70 ± 1.53%) catalase revealed no significant (p = 0.05, P < 0.05) difference even though the latter

204 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

conjugate contained twice as much the amount of coupled CA (table 4.9). Similarly, Fernandes and Gregoriadis (1996 and 1997) reported that activity retention of conjugates of both polysialylated catalase and asparaginase respectively synthesised with differing amounts of oxidised CA (under the same conditions without prior SDS treatment) was independent of the amount of coupled CA. Although these results support our findings, the implications of partially unfolding catalase by modification with SDS prior to covalent coupling to CA led us to postulate that the conjugate would exhibit a significantly decreased activity at the end of the coupling procedure in comparison with CA coupled to native catalase. This was anticipated due to interference from the covalently attached bulky CA chains on the correct re-folding of the enzyme after removal of the dénaturant. However, on the contrary, the results obtained for polysialylated SDS-modified catalase were surprisingly encouraging in that stability at the end of the coupling procedure seemed only marginally reduced compared with polysialylated catalase (table 4.9).

Table 4.9 Comparison between degree of modification and residual enzymatic activity of catalase conjugates prepared by polysialylation and a modified polysialylation procedure with native catalase. Degree of modification is expressed as the % of modified amino groups of the protein and the control included catalase reacted with non oxidised (non ox) CA. The percentage residual enzyme activity of native catalase in the absence of CA and catalase conjugates was determined at the end of the coupling procedure by employing the method of Beers and Sizer, (1952). Results are mean ± s.d of three experiments. N.D = not de termined. Preparation Modification degree % initial activity % CA: SDS-mod. catalase 7.20 ±0.41 63.70 ± 1.53 (50:1) CA: catalase 3.17 ±0.27 66.70 ± 1.50 (50: 1) CA (non ox): catalase 0.59 ± 0.09 N.D (50: 1) Catalase (CA absent) - 26.3 ± 1.2

A possible explanation for the improved stability of both polysialylated conjugates over the native enzyme during the coupling procedure is based on the factors frequently responsible for enzyme dénaturation and the steric stabilisation effects conferred by coupling polymers to proteins (Abuchowski et al, 1997). It was

205 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins reasoned that covalent attachment of a linear, flexible, hydrophilic polymer to available but non-essential groups on an enzyme provides a protective shell around the enzyme during the coupling process. At the same time, the shell is permeable to the small substrates such that enzymatic activity can continue. Under the coupling conditions described, it is likely that native catalase experiences destabilising effects from stirring and chemical reagents leading to unfolding and often resulting in aggregation of the protein molecules. Aggregation is a common cause of protein dénaturation, which if avoided can result in the improved stability of an enzyme and an unfolded protein could refold effectively on return to a suitable environment (Perutz, 1980). Interestingly, Cleland et al. (1992) reported that the fully reduced bovine carbonic anhydrase b enzyme had a strong tendency to aggregate. However in the presence of polyethylene glycol (PEG), aggregation was inhibited and the refolding was enhanced. In addition, Buchner et al. (1991) found that PEG and the chaperonin, GroEL, were able to bind to partially unfolded protein structures and inhibit their aggregation without altering the rate of refolding. The methods employed for such investigations included techniques previously mentioned in section 4.3.6.3.

With regards to the stability of polysialylated catalase and polysialylated SDS- modified catalase during the coupling procedure it is conceivable that at the low (3 and 7%) degree of modification achieved for each conjugate respectively, coupling occurs mainly with non-essential amino groups of the catalase molecule. Thus, at the end of the coupling process, in contrast with native catalase, polysialylated catalase and SDS-modified catalase might benefit from the steric stabilising effects of coupled CA whilst allowing diffusion of the substrate to the active sites of the enzyme. The reason for the considerable preservation of activity of polysialylated SDS-modified catalase even after partial reversible unfolding of the catalase molecule by SDS modification is unclear. However, at the level of modification achieved with polysialylated SDS-modified catalase, it is plausible to suggest that CA behaves like PEG described by Buchner et al. (1991) above. Thus, re-folding of catalase on removal of the dénaturant might be enhanced in the presence of CA and the molecule re-assumes its correct conformation such that enzyme activity is unaffected. Further investigations into this subject are nevertheless necessary.

206 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Several catalase conjugates have been synthesised by various procedures with the aim of attaining a high degree of coupling of enzyme and polymer, whilst retaining good enzymatic activity. For example, Marshall and Humphreys (1977) conducted a systematic study to determine the conditions required for the synthesis of soluble catalase-dextran conjugates using the cyanogen bromide activation procedure. Results indicated that increasing the starting molar ratios of dextran (60-90KDa) and catalase in the coupling reaction from 6.25: 1 to 25: 1 (dextran: catalase) resulted in an increased modification of e-amino lysine residues of catalase from 76 to 92%, with a significant concomitant decrease in residual enzymic activity from 6 8 to 47%. Unfortunately, a direct comparison cannot be made between the degree of modification and resultant enzymatic activity of dextran-catalase with CA-catalase due to the differing coupling procedures involved. However, it was interesting to note that activity retention of dextran-conjugates synthesised with differing amounts of dextran was dependent on the amount of coupled dextran. These findings were contrary to ours and a plausible suggestion might be that catalase enzyme activity is dependant upon the degree of modification but at high levels of modification. For example, 76-92% of the e-lysine residues were modified by dextran (which probably includes modified e-lysine residues required for activity) in comparison with 3-7% for polysialylated catalase conjugates (table 4.9).

Interestingly, decreasing the molecular weight of dextran from 60-90KDa to 40KDa resulted in marginally lowering the modification of e-amino lysine residues of catalase by attachment of dextran from 8 8 to 83%, whilst the residual enzymic activity was increased from 62 to 77%. All other reaction conditions were consistent for these comparisons. Increased stability of enzymes was shown to result from the grafting of hydrophilic macromolecules such as mPEG of varying molecular weights (Nucci et al, 1991; Abuchowski et al, 1977), albumin (Poznansky, 1986), cellulose (Barker and Somers, 1968) and other polymers such as poly(N-acryloylmorpholine) (PacM) and poly(N-vinylpyrrolidone) PVP.

207 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.10.2 Michaelis Menten: Catalase enzyme kinetics In the previous section, (4.3.10.1) the catalytic properties of native, polysialylated and polysialylated SDS-modified catalase were evaluated and compared during the coupling process under fixed conditions. In this study, the efficacy of native catalase versus the polysialylated constructs is investigated by employing the Michaelis Menten model to account for their kinetic characteristics. In brief, the rates of catalysis (V) of native, polysialylated and SDS-modified polysialylated catalase (40pg appropriately diluted) were individually measured under varying substrate [S] concentrations of 10, 20, 30 and 40 mM H 2O2 as described in section 4.2.2.3 and presented in tables 4.10-4.12. The apparent Michaelis constant (Km) and the maximal rate (Vmax) were derived from the method of Hanes-Woolf.

Table 4.10 Kinetic data determined from the decomposition of varying substrate [S] concentrations of 10, 20, 30 and 40 mM H 2O2 per min at 20°C by catalase reacted with no CA after 48 hours. Samples contained 40pg catalase appropriately diluted. * Indicates values are the mean of 3 experiments and substrate [s]/ velocity V is represented as a mean ± s.d of 3 experiments. Substrate [S] AAmln’^ Time for decomposition of [S]/V

Concentration (mM) Velocity (V)* 3.45pmoles H 2O2 (min)*

1 0 0.044 1.14 224.3 ± 4.0

2 0 0.079 0.63 251.7 ±2.5 30 0.108 0.46 277.0 ± 3.6 40 0.130 0.38 308.0 ± 1.5

Table 4.11 Kinetic data determined from the decomposition of varying substrate [S] concentrations of 10, 20, 30 and 40 mM H 2O2 per min at 20°C by polysialylated catalase reacted after 48 hours. Samples contained 40pg catalase appropriately diluted. * Indicates values are the mean of 3 experiments and substrate [s]/ velocity V is represented as a mean ± s.d of 3 experiments. Substrate [S] AAmin ^ Time for decomposition of [S]/V

Concentration (mM) Velocity (V)* 3.45pmoles H 2O2 (min)*

1 0 0.043 1.16 232.0 ± 2.6

2 0 0.078 0.64 249.7 ±5.0

30 0 . 1 1 2 0.41 272.7 ± 6.4 40 0.137 0.36 292.7 ± 1.5

208 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Table 4.12 Kinetic data determined from the decomposition of varying substrate [S] concentrations of 10, 20, 30 and 40 mM H 2O2 per min at 20°C by polysialylated SDS- modified catalase reacted after 48 hours. Samples contained 40pg catalase appropriately diluted. * Indicates values are the mean of 3 experiments and substrate [s]/ velocity V is represented as a mean ± s.d of 3 experiments. Substrate [S] AAmin'^ Time for decomposition of [S]/V

Concentration (mM) Velocity (V)* 3.45pmoles H 2O2 (min)*

10 0.042 1.19 236.0 ± 4.6

2 0 0.080 0.63 250.1 ±4.5 30 0.113 0.44 265.7 ±1.5 40 0.143 0.35 280.3 ±1.5

A plot of the substrate concentration [S] divided by the reaction rate ([S]A/') versus substrate concentration [S] called the Hanes-Woolf plot, yields a straight line with a slope of 1/Vmax, a Km/Vmax (y) intercept and -Km (x) intercept (Fig. 4.25).

350n 300-

250-

100 -

50-

-150 -125 -100 -75 -50 -25 0 25 50

[H2 O 2 ] (mM)

Figure 4.25 Hanes-Woolf Plot for the H 2O2 reaction catalysed by (■) native catalase reacted for 48 hours (CA absent) (linearity 99.16%), (A) polysialylated catalase reacted for 48 hours (linearity 97.43%) and (T) polysialylated SDS-modified catalase reacted for 48 hours (linearity 97.27%) at 20°C in 0.05M sodium phosphate buffer, pH 7.0. The bioconjugates were prepared with a CA: catalase molar ratio of 50: 1. Results are mean ± s.d. of 3 independent experiments. Km values for the three preparations were obtained by extrapolation to the y-intercept and Vmax estimated from the x- intercept. The results were compared (p = 0.0001) using the t-test.

209 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

The Michaelis constant (Km) represents the substrate [S] concentration for which the (initial) rate of reaction reaches half of the maximum. Thus, the Km value is often determined to represent the enzyme-substrate affinity. Table 4.13 shows the Km values for native catalase (70.67), polysialylated catalase (102.70) and polysialylated

SDS-modified catalase (149.40) mmol T^ H 2O2. These results suggest a statistically

significant (p < 0 .0 0 0 1 ; t-test) reduction in catalase enzyme affinity for the substrate

(H2O2) upon increasing modification by polysialylation. Vmax values increased slightly with increasing percentage of amino groups modified, however it was not significant (p < 0.05).

Table 4.13 Comparison between degree of modification and kinetic properties such as Km and Vmax of catalase conjugates prepared by two methods of polysialylation and native catalase. Degree of modification is expressed as the % of modified amino groups of the protein and the control included catalase reacted with non oxidised (non ox) CA. The method for determining the kinetic data for native catalase in the absence of CA and catalase conjugates was derived fi*om a Hanes-Woolf plot according to the Michaelis Menton model. Results are mean ± s.d of three experiments. N.D = not determined. Preparation Modification Km Vmax degree % (mmol r* H 2 O2 ) ( A A 2 4 0 min *) CA: SDS-mod-catalase 7.20 ±0.41 149.40 0.67 (50:1) CA: catalase 3.17 ±0.27 102.70 0.48 (50: I) CA (non ox): catalase 0.59 ± 0.09 N.D N.D (50: 1) Catalase (CA absent) - 70.67 0.36

Fernandes and Gregoriadis (1996) conducted a similar study under the same conditions as our own where the kinetic data for native catalase (Km = 69.96, Vmax = 0.390) and polysialylated catalase (Km = 122.88, Vmax = 0.644) were investigated. In this study, it was anticipated that further modification of catalase by SDS modification prior to coupling with CA would decrease the enzyme-substrate affinity, as seen. It is arguable that the increasing partial loss in substrate affinity demonstrated by both catalase conjugates (on increasing percentage polysialylation) may be the result of steric interference from the covalently attached bulky CA strands with the diffusion of

2 1 0 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

substrate molecules into the enzymes active site. Fernandes and Gregoriadis (1996) proposed that repulsive forces generated between the negatively charged CA layer on the catalase surface and the electronegative oxygen atoms of H 2O2 hamper the access of hydrogen peroxide to the active site. The partial loss in substrate affinity of catalase by polysialylation agrees with the observation that chemical modification of enzymes can adversely limit their efficacy. For example, Kraehenbuhl et al (1974) found that conjugation of microperoxidase to a Mab caused an increase in Km from 0.2 to 0.4M and a reduction in Vmax fi-om 4 to 0.4mmol/ min per mg compared with the native enzyme. In contrast, CA coupled asparaginase exhibited the same affinity towards asparagine as the native enzyme (Fernandes and Gregoriadis, 1997). Wieder et al. (1979) observed a similar phenomenon with PEG-phenylalanine ammonia-lyase conjugates and concluded that this phenomenon was due to conformational changes of the enzyme on PEGylation, rather than the diffusional barrier of PEG.

4.3.10.3 Antibody binding properties of native IgGla and IgGla modified by two methods of polysialylation Antibody binding capacities of the native Mab IgG2a and both polysialylated IgG2a conjugates (reacted for 48 hours) were determined as a percentage of unmodified native IgG2a using a direct binding enzyme-linked immuno-sorbent assay (ELISA) as described in section 4.2.2.6.I. Figure 4.26 shows the titration curves obtained for all

four immunoglobulin-containing samples (diluted 1 0 -1 . 0 pg/ml) tested against the protein antigen bovine serum albumin, (BSA). The absorbance obtained at 492nm for native IgG2a (CA absent) and both IgG2a conjugates (reacted over 48hours) tested at equal concentrations (lOpg/ml) was compared with that of unmodified native IgG2a (positive control) to determine their percentage residual antibody binding affinities. These results are presented in table 4.14 and include the degree of modification achieved for the IgG2a bioconjugates.

Table 4.14 shows that while polysialylated (94.65 ± 1.8% residual antigen binding) and polysialylated SDS-modified (82.44 ± 1.3% residual antigen binding) IgG2a conjugates respectively retained substantial antibody-binding activity of the parent Mab IgG2a, the binding activity of IgG2a reacted for 48 hours in the absence of CA

2 1 1 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

10 11 Concentration (^ig/mi) Figure 4.26 Enzyme-linked immuno-sorbent assay (ELISA) titration curves of (o) native unmodified IgG2a (positive control) (linearity 99.81%% (A) IgG2a reacted for 48 hours in the absence of CA (linearity 99.08%), (T) polysialylated JgG2a reacted for 48 hours (linearity 99.94%) and (■) polysialylated SDS-modified IgG2a reacted for 48hours (linearity 99.86%). Each immunoglobulin sample was appropriately diluted 10.0 - 1 .Opg/ml and applied to the microtiter plate in duplicate. The absorbance was read at 492nm. Each plate was run with a series of controls as described in section 4.2.2.6.I. The polysialylated Mab constructs were prepared with a CA: IgG2a molar ratio of 50: 1. Results are mean ± s.d. of 2 independent experiments.

Table 4.14 Antibody binding data derived by employing a direct binding enzyme- linked immuno-sorbent assay (ELISA) for native IgG2a in the absence of CA and both IgG2a conjugates (all reacted for 48 hours) compared with native unmodified IgG2a (positive control). Samples were tested at IG.Opg/ml. Degree of modification of the bioconjugates is expressed in terms of moles CA per one mole of IgG2a. The percentage residual antibody binding activity for IgG2a in the absence of CA and both IgG2a conjugates (all reacted for 48 hours) were determined by comparison with the positive control. * Indicates values are the mean of 2 experiments or ** the mean ± s.d of 2 experiments otherwise results are mean ± s.d of three experiments. N.D = not determined. Degree of Absorbance % Residual antibody- Preparation polysialylation 492nm* antigen binding affinity** CA: SDS-mod IgG2a 12.03 ±0.3: 1.0 0.817 82.44 ± 1.3 (50:1) CA: IgG2a 4.04 ±0.22: 1.0 0.938 94.65 ± 1.8 (50: 1) CA (non ox): IgG2a 1.22 ±0.16: 1.0 N.D N.D (50: 1) IgG2a (CA absent) - 0.411 41.47 ±2.9 Native IgG2a - 0.991 100.00 (positive control)

2 1 2 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins was significantly (P < 0.05, t-test) reduced (41.47 ± 2.95 residual antigen binding). Thus, at the end of the coupling procedure, CA coupled to IgG2a via either method of polysialylation yielded bioconjugates with at least a 2-fold retention of antibody binding affinity compared to IgG2a reacted for 48 hours in the absence of CA. Interestingly, similar data was found for IgG2a formulations tested at other concentrations i.e. 6.0pg/ml and 2.0pg/ml (Fig. 4.26). The phenomenon reported here was demonstrated earlier with CA-catalase conjugates in section 4.3.10.1. It was anticipated that the higher degree of modification achieved with polysialylated SDS- modified IgG2a in comparison with polysialylated IgG2a would result in a conjugate with a significantly decreased antibody-binding capacity. In this instance, this was envisaged as a result of the derivatisation of an increased number of s-lysine residues perhaps on or near the antigenic binding sites of the antibody such that the attached bulky CA strands sterically hinder the antibody-antigen interaction. Furthermore, it was believed that reversibly unfolding the antibody by modification with SDS prior to coupling with CA would hamper the correct re-folding of the molecule on removal of the dénaturant due to interference from the attached CA strands. Thus, changes in the conformation of the antibody might influence the antibody binding capacity. On the contrary, a comparison of the percentage residual antibody binding activity of polysialylated IgG2a and polysialylated SDS-modified IgG2a revealed no significant (p = 0.05, p< 0.05) difference even though the later conjugate contained a 3-fold increase in the amount of coupled CA.

Examples of other Mabs reported to retain substantial antibody binding affinity after modification by coupling to polymers include PEGylated anti-CEA IgG antibody (Pedley et al., 1994), anti-hepatitis surface B antigen IgG antibody (Suzuki et al., 1984), IgG-Fab fragments (Chapman et al, 1999), IgG (Cunningham-Rundles et al., 1992) and murine monoclonal antibody (Mab A7) (Kitamura et al., 1991). Similar findings were reported pertaining to the increasing loss in antibody binding affinity upon increasing the degree of PEGylation. For instance, Mab A7 modified with varying molar ratios of PEG (5.0 KDa) led to conjugates consisting of 10 and 5 moles of PEG per mole Mab A l, retaining 80-90 % antigen binding activity respectively. Increasing the degree of modification further yielded a conjugate consisting of 15

213 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins moles of PEG per mole Mab A7 which retained only 7% antigen binding activity compared with the parent Mab A7. It was suggested that the antibody binding activity of PEG coupled Mab A7 decreased gradually with increased PEG content because of attachment of PEG to the antigenic-binding site of the Fab regions and/or on their neighbours (Kitamura et al., 1991). Since the antibody binding activity of IgGla chemically modified by either method of polysialylation was found to be independent of the amount of coupled CA (at least for the degree of coupling achieved here), it may be argued that polysialylation occurred mainly with the non-essential e-lysine and/or amino residues of the IgGla molecule. The latter molecule consists of three active fi*agments, two Fab and one Fc, which are joined by a hinge that allows flexibility of the molecule (Vandenbranden et al., 1981). It is plausible that this kind of mobility of IgGla may contribute towards the preservation of antibody-binding affinity upon modification by CA, as the antibody-antigen combining sites are unhindered. An explanation as to why SDS modification of IgGla did not seem to further disrupt it structural conformation on removal of the dénaturant has been proposed earlier in section 4.3.10.1 with regards to enhanced refolding of CA coupled to SDS-modified catalase.

4.3.10.4 Inhibitory activity of aprotinin and aprotinin modified by two methods of polysialylation The inhibitory activities of native, polysialylated and polysialylated SDS-modified aprotinin (~0.5mg) reacted for 48 hours under the coupling conditions were determined by employing the trypsin activity-inhibition assays as described in section 4.1.1.6.1. Aprotinin inhibitory activity (Ph. Eur. U. mg) was calculated for each preparation in table 4.15 by calculating the volumes of O.IM NaOH required per second in both the trypsin activity and inhibition assays and employing the results into the equation described in section 4.1.1.6.1. The inhibitory activity value (0.04 Ph. Eur. U. mg) determined for native unmodified aprotinin (O.OlOmg) used in this study (table 4.15) was calculated to be 100% the normal stated value (British Pharmacopoeia, 1999) and therefore was used as the positive control with which to determine the percentage inhibitory activity of the three other aprotinin formulations (table. 4.16).

114 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

Table 4.15 Trypsin activity data determined in the presence and absence of native unmodified aprotinin (0.01 mg), aprotinin reacted for 48 hours in the absence of CA and CA-modified aprotinin prepared by two different methods of polysialylation. Reaction conditions and degree of modification of the aprotinin conjugates are expressed in terms of moles CA per one mole of aprotinin (in parenthesis). The latter three preparations contained ~0.5mg aprotinin. The inhibitory activities (Ph. Eur. U. mg) of the four aprotinin containing preparations were calculated by measuring the volumes of O.IM NaOH required per second in both the trypsin activity and inhibition assays and employing the results into the equation described in section 4.2.2.6.2. Controls included reacting trypsin in the presence of CA only. * Indicates values are the mean of 3 experiments. Assay One: Assay Two: Aprotinin Preparation Trypsin inhibition (nj Trypsin activity (uz) Inhib. activity Vol. NaOH used per Vol. NaOH used per second (mls^ x 10'^* second (mls^ x 10^)* Ph. Eur. U. mg* CA: SDS- mod. aprotinin 5.28 3.06 0.068 (50:1) CA: aprotinin 5.56 3.06 0.046 (50: 1) Aprotinin 4.17 3.06 0.133 (CA absent) Aprotinin 6.11 3.06 0.040 (pos. control)

Table 4.16 Comparison between degree of modification and residual inhibitory activity of aprotinin conjugates prepared by two methods of polysialylation and native aprotinin in the absence of CA reacted for 48 hours under coupling conditions. Reaction conditions and degree of modification of the aprotinin conjugates are expressed in terms of moles CA per one mole of aprotinin (in parenthesis). The percentage residual inhibitory activity of native aprotinin and aprotinin conjugates were determined at the end of the coupling procedure by employing the trypsin activity-inhibition assay and comparison with the positive control. Results are mean ± s.d of three experiments. Non-oxidised CA denoted as (non-ox), N.D = not determined. Preparation Degree of polysialylation Inhibitory activity % CA: SDS-mod. aprotinin 4.59 ±0.05: 1.0 58.82 ±0.7 (50:1) CA: aprotinin 1.46 ±0.05: 1.0 86.96 ± 1.9 (50: 1) CA (non ox): aprotinin 0.39 ±0.03: 1.0 N.D (50: 1) Aprotinin (CA absent) - 30.08 ± 0.9

215 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

The results (table 4.16) show that whilst chemical modification of aprotinin by polysialylation resulted in a proposed conjugate retaining 86.96 ± 1.9% inhibitory activity, the inhibitory activity of polysialylated SDS-modified aprotinin was somewhat reduced to (58.82 ± 0.7%), compared with that of the parent serine protease inhibitor, aprotinin. A further reduction to (30.08 ± 0.9%) in the inhibitory activity was observed for native aprotinin (CA absent). Aprotinin modified by either method of polysialylation yielded proposed conjugates with low and high degrees of CA substitution, which benefited with a 2-3 fold improvement in inhibitory activity compared with native aprotinin at the end of the polysialylation process (table 4.16). The apparent improved stability conferred to aprotinin upon grafting with CA was consistent with results with catalase and JgG2a. SDS modification of aprotinin prior to conjugation with CA resulted in a proposed conjugate with a quantitative yield of polysialylation and a significantly (p = 0.0001, p < 0.05) reduced inhibitory activity compared with polysialylated aprotinin. Interestingly, extensive chemical modification of aprotinin by other methods led to conjugates exhibiting a severe loss in inhibitory activity, with initial inhibitory activity being completely abolished (Odya et al., 1978) for sodium meta-periodate activated dexran coupled aprotinin and 12% (Larionova et al., 1984) for D-lactose-modified aprotinin. It was suggested that during the preparation of these carbohydrate-containing derivatives of aprotinin, the

inhibitory activity was decreased due to modification of the 8 -NH2 group of lysine-15, which is the reactive site of aprotinin (Chauvet and Archer, 1967). In contrast, the polysialylation methods employed in the derivatisation of aprotinin here appear to be gentle since even polysialylated SDS-modified aprotinin retained a significant (59%) degree of inhibitory activity. The reduction in trypsin inhibition observed for polysialylated SDS-modified aprotinin could be due to steric hindrances against the formation of multiple contacts between the enzyme and inhibitor molecules. Similar reports were made for aprotinin coupled to (carboxymethyl) dextran derivatives of D- galactose where the inhibitory activity was measured to be 56% compared to free protein and for cyanogen bromide activated-dextran coupled to aprotinin (41% inhibitory activity) (Larionova et al., 1985).

216 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.11 Effect of freeze-drying and storage on the biological properties of polysialylated peptides and proteins Freeze-drying or lyophilisation was the most commonly used method for the storage of the native and polysialylated peptides and proteins investigated throughout this thesis. Carpenter et al. (1993) suggested that the freezing step (with its consequent freeze-concentrating effect) was the major cause of protein damage during freeze- drying. Prestrelski et al. (1993) reasoned that conformational changes {i.e. unfolding) in peptides and proteins during freeze-drying were responsible for their loss of biological activity. To that end, in continuation from previous studies on the in vitro biological properties of native and CA-modified conjugates prepared via two methods of catalase (4.3.10.1), IgG2a (4.3.10.3) and aprotinin (4.3.10.4), an attempt was made here to determine the biological potency of these three proteins and all conjugates after immediate re-hydration from the dried state. Activity assays were similarly conducted upon re-hydration after storage for 3months at 4°C. The activities observed for each of the dehydrated-re-hydrated protein samples and conjugates (after immediate re-hydration and after 3 month storage at 4°C) were compared with the activities reported for their respective protein counterparts in the initial hydrated state (4.3.10) to determine their percentage residual biological potency.

4.3.11.1 Catalase The catalytic activity of native unmodified catalase after immediate re-hydration from the lyophilised state was determined by the methodology reported in section 4.2.2.2 and compared with the activity of native catalase previously determined in the hydrated state (4.3.6.1). The results are reported as either the average or mean ± s.d of 2 experiments. Re-hydrated lyophilised native catalase exhibited an average catalytic activity of 0.059 pmolmin'^pg'\ which corresponds to 17.4 ± 1.22 % retention of its initial hydrated activity (table 4.4). It is well documented that catalase exhibits a significant loss of activity from the stresses arising from freeze-drying (Deisseroth and Dounce, 1970). In comparison, polysialylated catalase (0.096 pmolmin'Vg ^ 30.10 ± 1.5%) and polysialylated SDS-modified catalase (0.110 pmolmin'^pg'\ 34.33 ± 2.9%), conjugates exhibited a significant (p = 0.0001, p < 0.05) increase in the percentage residual activity after freeze-drying. These results suggest that polysialylation affords

217 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins some stabilisation to the native enzyme towards freeze-drying effects similar to cryoprotective agents. It was anticipated that the 2-fold higher degree of modification achieved with polysialylated SDS-modified catalase would result in a much greater hypothesised cryoprotective effect, however this was not seen. Unfortunately, the protective effects of polysialylation conferred to catalase via both methods of modification were not substantial and it is felt that further addition of stabilising agents would be required to protect catalase more efficiently during freeze-drying. Interestingly, Tanaka et a/. (1991) investigated the effects of saccharides (e.g. glucose, maltose, mannitol) as stabilising agents (lyoprotectors) on the dénaturation of catalase by freeze-drying and found that stabilisation of catalase via maltose was dependant on the weight ratio of saccharide to catalase and not the bulk concentration of maltose. It was reasoned that hydrogen bonding between the protein and the saccharide, serving as a water substitute when the hydration shell of the protein is removed was the mechanism behind the stabilisation of catalase. It is arguable therefore that the increased stability conferred to catalase on lyophilisation by grafting with CA may be due to the extensive hydrogen bonding established between CA and the protein. Consequently, the residual enzyme activities of native and CA modified catalase conjugates prepared by both methods of polysialylation were determined during freeze thawing.

Freeze-thawed native catalase exhibited an average catalytic activity of 0.33 pmolmin" Vg ^ which corresponds to 97.06 ± 1.77% of its initial hydrated value (table 4.4). In contrast polysialylated catalase exhibited an average catalytic activity of 0 . 2 2 pmolminVg’ (68.75 ± 1.66%) and polysialylated SDS-modified catalase 0.110 pmolmin'Vg’' (59.38 ± 2.1%). The latter three results were found not be significantly different from their initial hydrated activities respectively (4.3.10.1). This observation agrees with the findings that during the drying phase, the major stress that must be overcome is the removal of the protein’s hydration shell (~ 95% water loss), which can result in the denatuartion of catalase (Tanaka et al., 1991). Both polysialylated catalase conjugates were stored frozen at - 40°C for 3 months with no appreciable loss of enzyme activity upon thawing (results not shown).

218 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.3.11.2 IgG2a Freeze-dried native unmodified IgG2a was immediately re-hydrated and its antigen binding affinity determined by application of the direct binding Enzyme-Linked Immuno-sorbent assay (ELISA) previously described in section 4.2.2.6.L The average absorbance observed at 492nm for native Mab IgG2a (lOpg/ml) was 0.872, which corresponded with a loss of 12.0 ± 1.44% of its initial hydrated antibody-binding capacity (table 4.14). Similarly, Ressing et al. (1992) reported that fi*eeze-drying of the parent mouse monoclonal antibody (MN12) caused a 10% loss in the antigen- binding affinity compared with the hydrated form of the Mab. The average absorbance readings for re-hydrated lyophilised polysialylated (0.914) and polysialylated SDS- modified IgG2a (0.804) corresponded to 92.24 ± 1.5% and 81.10 ± 2.1% residual antibody binding affinities respectively. These results are only marginally reduced compared with their initial hydrated activities respectively (section 4.3.10.3, table 4.14). In contrast with native catalase and the polysialylated conjugates, native and polysialylated conjugates of IgG2a seemed to be more stable to the effects of lyophilisation. In this instance however, as only marginal losses in antibody-binding affinity were observed upon re-hydration of lyophilised IgG2a and IgG2a conjugates compared with their initial hydrated values, the hypothesised cryoprotective effect of polysialylation could not be evaluated. Interestingly, dehydrated-re-hydrated pure E. carotovora asparaginase enzyme was found to retain 1 0 0 % of the hydrated enzymes initial activity (Fernandes and Gregoriadis, 1996 and Heilman et al., 1983). Although it is unclear why the biological properties of IgG2a are only marginally affected after freeze-drying, it is plausible to suggest that IgG2a may be capable of maintaining its three-dimensional conformation during the drying process. Both polysialylated IgG2a conjugates were lyophilised and stored at 4°C for up to 3 months without significant loss of antibody-binding activity (results not shown).

4.3.11.3 Aprotinin The trypsin activity-inhibition assay previously described in section 4.2.2.6.2, was employed to determine the inhibitory activity of native aprotinin and both aprotinin conjugates after re-hydration from the lyophilised state. The inhibitory activity of reconstituted freeze-dried native aprotinin (O.Olmg) was calculated as 0.038 Ph. Eur.

219 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

U. mg, which by comparison with native aprotinin in its initial hydrated state (table 4.16) represents 94.0 ± 1.91% inhibitory activity retention. Similarly, no measurable loss in aprotinin inhibitory activity was observed for reconstituted freeze-dried polysialylated aprotinin (0.035 Ph. Eur. U. mg, 87.11 ± 1.42%) and polysialylated SDS-modified aprotinin (0.023 Ph. Eur. U. mg, 58.20 ± 1.70%) by comparison with their initial hydrated counterparts (section 4.3.10.4, table 4.16). Again, the hypothesised cryoprotective effect of polysialylation could not be determined in this instance. Although no reports were found with regards to the effects of lyophilisation on the dénaturation of aprotinin, it is possible that the three naturally occurring disulfide bonds that cross-link the molecule might contribute towards the apparent stability of aprotinin during freeze-drying. It has been proposed that proteins containing naturally occurring disulfide bonds generally increased their thermodynamic stability resulting in reduced configurational entropy (ASconf) of the unfolded state (Wang, 1999). The polysialylated aprotinin conjugates were stored in the lyophilised state for up to 3 months at 4°C with no appreciable loss observed in aprotinin inhibitory activity (results not shown).

2 2 0 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins

4.4 Conclusions

Two key objectives were proposed in the introduction of this chapter (section 4.1) and the main conclusions derived are summarised here. Firstly, the emphasis of the research conducted in this chapter was to evaluate the potential loss in biological potency experienced by catalase, IgG2a and aprotinin modified by polysialylation. Secondly, to develop and optimise a novel strategy to further improve the efficiency and therapeutic value of polysialylation by generating peptides and proteins with a greater degree of CA substitution.

The present study shows that controlled periodate oxidation of CA prior to coupling with IgG2a and catalase led to conjugates containing 3-5 fold the amount of coupled CA compared with the respective controls, thereby suggesting periodate oxidation facilitated coupling. These findings were mirrored in chapter 3 with respect to the synthesis of polysialylated IgG, aprotinin, insulin and somatostatin (section 3.4). The anionic detergent sodium dodecyl sulphate (SDS) was introduced in to the coupling reaction in an attempt to reversibly unfold the peptides and proteins introduced in this chapter and increase their number of free amine groups for coupling with CA. This technique was developed using catalase as a model protein and the optimal concentration of SDS (l.OmM) found to reversibly inactivate the enzyme appeared to cause reversible conformational changes of both IgG and aprotinin. These results were obtained by employing SEC to observe changes in the hydrodynamic volumes (elution profiles) of both proteins before and after dialysis to remove SDS (section 4.3.6.3). Bovine serum albumin (BSA) did not appear to be affected by l.OmM SDS in that no hydrodynamic changes were visible before and after dialysis to remove SDS. The observed reversible changes in the hydrodynamic volumes of IgG and aprotinin suggested that whilst aggregation of the refolded proteins may have occurred it did not appear be a gross in nature. In contrast, the hydrodynamic volumes of both proteins subjected to 3.12mM SDS were not reversed upon dialysis to remove the detergent. This suggests that the proteins may have experienced gross aggregation and therefore irreversibly denatured at this concentration. Moreover, IgG and aprotinin solutions containing l.OmM SDS obtained post dialysis appeared clear and colourless in

2 2 1 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins contrast with cloudy solutions obtained with exposure to 3.12mM SDS even after dialysis. Further experimentation is however warranted to rule out protein aggregation of the re-folded proteins. Although the reversible (l.OmM) and irreversible (3.12mM) SDS-induced conformational changes were found not to be universally similar for every protein (section 4.3.6.3.3); catalase, IgG, Ig02a, aprotinin, and insulin modified with l.OmM SDS prior to coupling with oxidised CA yielded conjugates containing 1.5-3 fold more coupled CA than by the standard polysialylation technique (table 4.5).

Catalase, Ig02a and aprotinin polysialylated by both methodologies benefited with improved stabilisation at the end of the coupling procedure relative to their native protein controls. Indeed, Fernandes and Gregoriadis (1996) observed this phenomenon for polysialylated catalase and asparaginase. The residual biological properties were mostly preserved for catalase (63-66%), IgG2a (82-94%) and aprotinin (59-87%) modified by both methods of polysialylation. Only, polysialylated SDS-modified aprotinin showed a significant decrease in potency compared with aprotinin polysialylated by the standard method. These results are testimony to the correlation that exits between the degree of modification of peptides and proteins and their residual biological activities. Further to our work, separate experiments were conducted regarding the in vivo pharmacodynamic properties of polysialylated insulin in mice (Jain et al., 2002). In this instance, insulin was coupled to oxidised CA as described in section 3.2.2.T.3 and resulted in a conjugate containing 1.8 moles of CA per mole insulin. These results were in agreement with ours where 1.9 ± 0.05 moles of CA reacted with one mole of insulin. The conjugation of insulin to CA led to a bioactive conjugate with a delayed subcutaneous pharmacodynamic profile (blood glucose lowering effect) showing an extended release profile. Similar findings were reported for carboxymethyl dextran coupled insulin (Baudys et al., 1998) and PEG coupled insulin (Uchio et al., 1999).

All the bioconjugates synthesised here (by either method of polysialylation) were characterised and evidence to prove the synthesis of polysialylated constructs was sought for by employing size exclusion chromatography. Covalent coupling of CA- modified or CA-modified SDS-treated peptide and protein constructs was not

2 2 2 Chapter Four: In vitro bioactivity ofpolysialylated peptides and proteins definitively confirmed by employing SEC, however the emergence of ‘new’ heavier entities were identified of CA and the respective peptide or protein co-eluting in the same volume of the column. Although further evaluations are warranted to confirm the presence of a covalent link and rule out other types of bonding these findings suggested the presence of the neoglycoprotein. Similar findings were reported for IgG and aprotinin constructs that were further characterised by employing SDS-PAGE (section 4.3.9). Finally, whilst the biological activities of native and polysialylated IgG2a and aprotinin were mostly preserved after fi-eeze-drying and storage at 4°C for up to 3 months, considerable diminished activity was observed with native and polysialylated catalase in the dried form. Fernandes and Gregoriadis (1996) and Deisseroth and Dounce (1970) reported similar observations. In this case, although polysialylation conferred a cryoprotective effect, catalase was found to be more stable to freeze thawing and subsequent storage at -40°C for up to 3 months (section 4.3.11.1). These results are encouraging in light of our objectives to synthesise bioactive polysialylated peptides and proteins, which exhibit improved therapeutic properties. Based on these findings, we proceed with accounts of the in vivo behaviour of polysialylated and polysialylated SDS-modified peptides and proteins in Chapter 5.

223 Chapter Five

Pharmacokinetic properties of polysialylated hioconjugates

224 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

5.1 Introduction

Efficacious delivery of peptide and protein drugs is dependent upon their prolonged plasma retention time and ability to reach target tissues in sufficient quantities to exert a pharmacological effect (Nucci et al., 1991). Owing to their poor oral bioavailability, the preferred route of administration is parenterally. Unfortunately, once in the blood circulation these molecules are rapidly cleared mainly due to renal ultrafiltration, inactivation by the immunosystem and plasma enzymes and thus can exhibit very short (~2-5min) half-lives (Chapter 1.0). The poor pharmacokinetic and pharmacodynamic properties of peptides and proteins means that repeated injections are necessitated for the maintenance of therapeutic efficacy, which can often provoke toxicity and increase the possibility of adverse immune responses. This is especially true for non-human derived proteins. These problems have been addressed with varying degrees of success by covalent conjugation of protein therapeutics to hydrophilic macromolecules such as albumin, dextran and mPEG (section 1.5). It was proposed that the hydrophilic shell that forms around the protein sterically hinders interactions with factors thought to be responsible for their clearance. Recently, low molecular weight CA (average molecular weight lOKDa) was used successfully as an alternative to non-biodegradable mPEG to augment the circulatory half-lives of enzymes (Gregoriadis et ah, 1999), other large biopolymers and liposomes (Zhang, 1999) and also small molecules (Gregoriadis et ah, 1993).

The purpose of the work described in this chapter was to demonstrate the effect of polysialylation on the pharmacokinetic properties of bovine IgG and two small peptides, aprotinin and insulin in experimental animals, compared with their unmodified counterparts. These peptides and proteins were also coupled to CA (average molecular weight lOKDa) by the alternative method of polysialylation established in section 4.2.2.S and their pharmacokinetic properties evaluated in vivo for comparison. These experiments were conducted with a view to determine whether CA modification of these biotherapeutics enhances their therapeutic potential. To that end, isotopic labelling (^^^I) methods were introduced in this chapter, for the purpose of peptide and protein quantification and hence derivation of pharmacokinetic

225 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

parameters such as circulatory half-life (ti/ 2 ), area under the curve (AUC), plasma clearance (Clp), volume of distribution (Vd), p elimination rate and mean residence time (MRT) (section 5.2.3.S). Both the Chloramine-T and lodo-Gen methods were initially assessed using IgG for their suitability with regards to radioiodination of proteins for in vivo plasma studies. The Chloramine-T method was chosen as a suitable radioiodination method for application to the three biotherapeutics. Consequently, trace amounts from the three biotherapeutics were ^^^I-labelled, purified and the labelling efficiency and specific activity determined as outlined in section 2.2.5.4.

Unlabelled bovine IgG, aprotinin and insulin containing trace amounts of highly radioiodinated molecules were then modified by both methods of polysialylation. The degree of modification achieved and specific activity was determined for each labelled polysialylated bioconjugate. Size exclusion chromatography (SEC) was employed to characterise and purify the radiolabelled bioconjugates. Finally, in vivo plasma clearance studies were performed using the ^^^I-labelled bioconjugates and their ^^^I-labelled native protein controls respectively.

226 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

5.2 Materials and Methods

This section details specific materials and describes methods that have been developed to address the challenges proposed in section 5.1.

5.2.1 Materials

Table 5.1 Materials used in Chapter 5

Material Source** Heparin solution Sigma Chemical Company, UK 50 pi microcapillary-tubes Supracaps Rat and mouse standard diet Bantin & Kingman Universal, UK ** Manufacturers and suppliers full addresses may be found in Appendix 2 All other reagents were of analytical grade and may be found in previous chapters. The composition of reagent solutions and buffers can be found in Appendix 1.

5.2.2 Animals

Male T/O outbred mice (25-30 g body weight) were purchased from Harlan-OLAC UK and acclimatised for a week prior to in vivo experimentation. A conventional diet and water were available ad libitum.

5.2.3 Methods

5.2.3.1 Radioiodination of peptides and proteins by the Chloramine-T method In an attempt to follow the fate of native, polysialylated and polysialylated SDS- modifred IgG, aprotinin and insulin in vivo, ‘trace’ amounts of the native peptide and protein moieties were radiolabelled with ^^^I and incorporated with the respective unlabelled moieties into the final constructs. For this purpose, the chloramine-T methodology was adopted from Greenwood and Hunter (1963) in section 2.2.5.2 of

227 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates this thesis. In brief, native IgG, aprotinin and insulin were individually dissolved in PBS (lOOpl, 0.5mg/ml) to which carrier free Na ^^^1 (50pCi, 2pl) mixed with phosphate buffer (lOpl, 0.25M, pH7.5) was added. Subsequently, chloramine-T dissolved in 0.05M phosphate buffer (lOpl, 2mg/ml, pH 7.5) was added to each peptide and protein mixture and incubated at 20°C for 10 min. Once ^^^1 had been incorporated into the tyrosine residues of the peptide or protein, the reactions were terminated by the addition of sodium metabisulphite (20pl, 1.2mg/ml in 0.05M phosphate buffer, pH 7.5). The final volume of each solution was made up to 0.5ml using potassium iodide (1 mg/ml in 0.05M phosphate buffer, pH 7.5). Subsequently, radiolabelled IgG, aprotinin and insulin were purified by application to PD-10 columns and the pooled fractions dialysed extensively (4 x 2L; 24) against 0.05M sodium phosphate, pH 7.5. The labelling efficiency and specific activity were determined for each peptide and protein-containing solution as outlined in section 2.2.5.4. The final radiolabelled peptide and protein tracer solutions were stored frozen at - 40°C in 100 pi aliquots in lead pots until further required.

5.2.3.2 Radioiodination of IgG by the lodo-Gen method The lodo-Gen method is an alternative to the chloramine-T method where iodination proceeds by a solid-phase mechanism that damages the protein less (Franker and Speck, 1978) whilst proteins are labelled to high specific activity. This method was employed comparatively with IgG only in an attempt to investigate whether iodination via the harsh oxidising agent, chloramine-T affects the clearance pattern of radiolabelled proteins. In brief, a solution of IgG dissolved in PBS (lOOpl, 0.5mg/ml) was added to the lODO-GEN® pre-coated iodination tube followed by the addition of sodium acetate (lOOpl, 0.35M, pH 4.0). Carrier free Na ^^^1 (lOOpCi, 2pl) was mixed with phosphate buffer (lOpl, 0.25M, Ph7.5) and also added into the tube. The mixture was incubated in a water bath at 40°C for 30 min with occasional agitation. The tube was then chilled on an ice-water bath whereby sodium hydroxide (lOOpl, l.OM) was added to quench the reaction. The final volume of the solution was made up to 0.5ml using PBS, pH 7.5. SEC was used to isolate radiolabelled IgG. The labelling efficiency, purification and the specific activity were determined as described in section 2.2.5.4.

228 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

S.2.3.3 Synthesis of radioiodinated polysialylated and polysialylated SDS- modified bioconjugates Aliquots containing radioiodinated tracers of IgG prepared by both radioiodination techniques and those of aprotinin and insulin prepared by the chloramine-T method of known specific activity (see section 5.2.3.1 and 5.2.3.2) were thawed and employed here in the synthesis of radiolabelled polysialylated and polysialylated SDS-modified peptide and protein conjugates. Essentially, oxidised CA (average mol. wt lOKDa) was coupled 50:1 (CA: protein) with IgG (150KDa), aprotinin (6.5KDa) and insulin (5.7KDa) via reductive amination established in section 3.2.2.2 and by the modified polysialylation technique described in section 4.2.2.5. Both polysialylation procedures involved coupling the radioiodinated peptide or protein ‘tracer’ molecules and unlabelled moieties with CA. Controls were conducted under the same reaction conditions except in the absence of oxidised CA. The final radiolabelled products were fi-eeze-dried until required for in vivo experiments. (50:1, CA: Protein)

5.2.3.3.1 ^^Î-labelled polysialylated peptide and protein conjugates ^^Î-labelled polysialylated IgG, aprotinin and insulin conjugates were synthesised as follows. Oxidised CA (25mg, 2.5pmol) was reacted with un-labelled IgG (7.5mg, 0.05pmol) as was oxidised CA (200mg, 20pmol) with aprotinin (2.6mg, 0.4pmol) and insulin (2.4mg, 0.4pmol) in 0.75M K 2 HPO 4 , (5.0ml) at pH 9.0 (insulin, pH 6.4) in the presence of NaBHgCN (lOmg, 0.16mmol for IgG, 80mg, 1.28mmol for aprotinin & insulin). To each reaction vessel, lOOpl (approximately ~l,000,000cpm or 0.5pCi) of the appropriate ^^Î-labelled peptide or protein tracer prepared by the chloramine-T method (section 5.2.3.1) was added. The IgG tracer prepared by the lodo-Gen method (section 5.2.3.2) was similarly employed in the synthesis of ^^Î-labelled polysialylated IgG. The reaction mixtures were incubated for 48 hours at 35-40°C. Controls were conducted for each peptide or protein under the same reaction conditions above but in the absence of oxidised CA.

The radiolabelled conjugates were isolated from reaction mixtures by ammonium sulphate precipitation (70% saturation) and centrifuged at 4500xg for 40 min (IgG) or 6000xg for 40 min (aprotinin and insulin). The respective native controls were

229 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates subjected to the same process. The supernatants were decanted and the pellets drained and re-dissolved into 5.0ml 0.15M PBS, pH 7.4. The conjugates and native protein controls were purified by extensive dialysis (3 x 2L; 24 h) at 4°C against 0.15M PBS, pH 7.4. Estimation of peptide or protein content was performed using the Bradford method (Bradford, 1976) and CA content determined by the resorcinol method (Svennerholm, 1957). The extent of radioactivity incorporated into each peptide or protein-containing solution was determined by transferring 5 pi aliquots to gamma vials and counted for 60 seconds in a Wallac, 1275 Minigamma y-counter. The results obtained were used to calculate specific activity (mCi/mg protein) of the final polysialylated bioconjugates and native peptide or protein controls. Finally, size exclusion chromatography was employed to characterise the radiolabelled polysialylated bioconjugates and native peptide and protein controls. Sephadex gel columns were prepared as outlined in Chapter 3 for IgG, aprotinin and insulin. The final products of known specific activity (mCi/mg protein) were fi-eeze-dried and stored at 4°C, until further required.

S.2.3.3.2 *^Î-labelled polysialylated SDS-modified peptide and protein conjugates The procedure for radiolabelling polysialylated SDS-modified IgG, aprotinin and insulin conjugates was similar to that used in the radioiodination of the polysialylated bioconjugates in section 5.2.3.3.I. However, in this instance the reactants were pre incubated for 1 hour in reaction mixtures containing l.OmM SDS before oxidised CA and NaBHsCN were added (4.2.2.5). In short, un-labelled IgG (7.5mg, 0.05pmol), aprotinin (2.6mg, 0.4pmol), insulin (2.4mg, 0.4pmol) and lOOpl of the appropriate ^^Î-labelled tracer molecules (containing ~l,000,000cpm or 0.5pCi) prepared by the chloramine-T method were dissolved in 0.75M K 2 HPO 4 , (5.0ml) at pH 9.0 (insulin, pH 6.4) in the presence of l.OmM SDS. The IgG tracer prepared by the lodo-Gen method (section 5.2.3.2) was similarly employed for the synthesis of ^^Î-labelled polysialylated SDS-modified IgG. The mixtures were warmed gently to dissolve the SDS completely and then lefi; stirring gently for 1 hour at room temperature. Subsequently, oxidised CA (25mg, 2.5pmol for IgG, 200mg, 20pmol for aprotinin & insulin) and NaBHgCN (lOmg, 0.16mmol for IgG, 80mg, 1.28mmol for aprotinin & insulin) were added to the reaction mixtures and the conjugation process allowed to

230 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates continue for 48 hours at 35-40°C. The procedures for isolation, purification, protein/CA determination and specific activity for the radiolabelled SDS-modified conjugates and controls were as described in section S.2.3.3.1.

5.2.3.4 In vivo plasma clearance study of *^^I-labelled native, polysialylated and polysialylated SDS-modified peptides and proteins The plasma clearance of ^^^I-labelled native, polysialylated and polysialylated SDS- modified IgG (prepared by both methods of iodination), aprotinin and insulin were determined using groups of four male T/O outbred mice (25-3Og body weight). The final radioiodinated native and polysialylated peptides and proteins prepared in section S.2.3.3 (including radioiodinated IgG constructs prepared using the lodo-gen method) were reconstituted with (0.2ml) filter-sterilized PBS prior to use. Each group of four mice was tail vein injected with ~2S0,000cpm (0.12SpCi/mg protein) for native, polysialylated and polysialylated SDS-modified IgG, aprotinin and insulin respectively. At time intervals after the injection, SOpl of heparinized blood was collected and diluted with 4S0pl PBS. All the diluted whole blood samples were transferred to y-vials and the radioactivity content measured for 60 seconds. The residual drug radioactivity in plasma was denoted in counts per minute (cpm) and expressed as a percentage of the initial dose. Assuming that the total mouse blood volume is 2.0ml i.e. 7% of the body weight (Senior et al., 1991), the percentage residual radioactivity in circulation for each construct was converted to concentration (mg/L) and plotted semi-logarithmically versus time. It is appreciated that other specific methods of determining blood volume exist involving weighing the individual animals that would have give more accurate results.

5.2.3.5 Estimation of pharmacokinetic parameters for native, polysialylated and polysialylated SDS-modified peptides and proteins after intravenous injection Following intravenous (i.v) administration, drug or other xenobiotic substances mix rapidly into plasma (central compartment) and then distribute into organs and tissues (tissue compartment). Distribution from the plasma may be faster or slow depending on the drug. Drugs exhibiting slow tissue distribution show curved semi-logarithmic concentration versus time plots. In general, in pharmacokinetic studies the

231 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates concentration versus time profile of a drug is studied in blood or blood plasma only. The results are then evaluated on the basis of a mathematical model and characterised in pharmacokinetic parameters. The primary pharmacokinetic parameters volume of distribution (L) and clearance (L/h) play a central role in pharmacokinetics from which all other pharmacokinetic parameters are derived i.e. ^-elimination rate constant (L/h), distribution, elimination half-life (h) and bioavailability (h.mg/L) from area under the curve (AUC).

Clearance (Cl) Clearance is probably the most important pharmacokinetic parameter, since it is the measure of the body’s ability to eliminate a drug from blood or plasma. It is defined as the ratio between the rate of elimination (dAb/dt) and the plasma concentration (C) or as the volume of body fluid that is ‘cleared’ from the drug per unit time. At its simplest, equation 1 defines this relationship:

Cl = Dose/AUC [Equation 1.0]

Volume of distribution (Vd) The second fundamental kinetic parameter useful in evaluating drug disposition is volume of distribution. The volume of distribution is defined as the ratio between the amount of drug in the body (Ab) to the concentration of drug in the blood (C). Equation 2 defines this relationship: V = Ab/C or V = D/AUC.k [Equation 2.0]

Elimination half-life (ti/2 p) The elimination half-life is the third fundamental kinetic parameter. It reflects the time taken for the plasma level of a drug to fall to half, once its clearance pattern is linear. It should be noted that the half-life of a drug reflects both its distribution and elimination (Gibaldi, 1991). It can be determined from the slope of the terminal phase of the concentration versus time profile on a semi-logarithmic scale: Equation 3 defines this relationship:

Ti/2 p = 0.693/p (h) [Equation 3.0]

232 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

Elimination rate constant (P) The elimination rate constant can be determined from the slope of the terminal phase of the concentration versus time profile on a semi-logarithmic scale: Equation 4 defines this relationship:

P = -slope X 2.303 (h-1) [Equation 4.0]

Bioavailability Bioavailability is defined as the fraction of unchanged drug reaching the site of drug action, or more usually the systemic circulation following administration by any route. It may be determined from the area under the curve of log concentration versus time curves from zero to 48 hours (AUCo^g) and 72 hours (AUC 0 .72) estimated by the linear trapezoidal rule. The extrapolated area under the plasma concentration time curves from 48 and 78 hours to infinity are estimated from C 4 g/p or C 72/P, where C72 and p are respectively the concentration at 48 hours, the concentration at 72 hours and the terminal elimination constant.

These pharmacokinetic parameters were determined for radiolabelled native, polysialylated and polysialylated SDS-modified IgG, aprotinin and insulin constructs respectively using the Dutch MW/Pharm pharmacokinetic software program. The program uses non-linear regression analysis to derive basic pharmacokinetic parameters for 1-3 compartmental models. The pharmacokinetic model that statistically suited the data generated with respect to the peptide and protein constructs studied here was a two-compartment model (single i.v. dosing).

S.2.3.6 Statistical analysis The student’s t-test was used to compare two groups and multiple comparisons were made using a one-way analysis of variance test (ANOVA) with equal variances at significance level: P < 0.05. When multiple comparisons were made for example, to investigate the correlation between degree of polysialylation and kinetics of the peptides and proteins, the results were analysed by ANOVA and significant P values were corrected by the Bonferroni method and are given in the text. ANOVA is based on the assumption that not only do populations have a normal distribution but also

233 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates have the same variance. In practice, data often showed differences in variances but since the sample size in each group (n = 4) was consistent, the equality of variances is not considered crucial (Erickson and Nosanchuck, 1992; Ryan et al., 1985). All statistical analyses were performed using GraphPad InStat (GraphPad Software, version 2.01, 2001).

234 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates

5.3 Results and Discussion

This section discusses the results obtained from the methodologies applied in section 5.2 with respect to our aims and objectives expressed in section 5.1.

5.3.1 Radioiodination of peptide and protein tracers for in vivo plasma clearance studies

Chloramine-T was chosen as the method for iodination and determining the in vivo plasma clearance of native and polysialylated peptides and proteins, by way of introducing the radioactive isotope into the tyrosine residues of the peptide or protein moieties. Based on the knowledge that radioiodination of proteins yields species with high specific activity (pCi/mg protein), ‘trace’ quantities of the peptides and protein were radioiodinated for incorporation into the polysialylated bioconjugates.

1000000n E Ü 750000-

> 500000-

250000-

0 10 15 205 Elution volume (ml)

Figure 5.1 Elution profiles of radioiodinated (□) IgG, ( o ) aprotinin and (À) insulin prepared by the chloramine-T method after application to a Sephadex-G-25, 1.0 x 10cm column (Pd-10) pre-equilibrated with 0.05M sodium phosphate buffer, pH 7.5. lOpl aliquots were transferred from each fraction to y-vials and counted for radioactivity per minute (cpm) in a Wallac, 1275 Minigamma y-counter. Arrow indicates elution of the radiolabelled proteins at fraction 4 (Ve, elution volume = 4.0ml).

235 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

To that end, the chloramine-T methodology adopted from Greenwood and Hunter (1963) was used to radiolabel IgG, aprotinin and insulin respectively as described in section 5.2.3.1. The radiolabelled peptides and proteins were separated from excess by application to a Sephadex-G-25, 1.0 x 10cm column (Pd-10) pre-equilibrated with 0.05M sodium phosphate buffer, pH 7.5 (Fig. 5.1). Aliquots (lO.Opl) were transferred from each fraction to y-vials and counted for 60 seconds in a Wallac, 1275 Minigamma y-counter. All three radiolabelled peptides and proteins were expected to elute in earlier fractions than the lower molecular weight ‘free’ molecules. The results (Fig. 5.1) show similar elution profiles for each peptide and protein where most of the eluted in fraction 4 (Ve = 4.0ml). The fact that and protein were found together in the pooled fractions from the first peak suggests it is protein-bound, whereas negligible amounts were measured in the pooled fractions of the second peak. This was typical for all three preparations and indeed for IgG radiolabelled by the lodo-Gen method outlined in section 5.2.3.2 (Fig. 5.2).

2000000n 1750000- Ü 1500000- > 1250000- - 1000000- Ü (Ü O 750000- ■o (Ü 500000- 250000-

0 10 15 205 Elution volume (ml) Figure 5.2 Elution profile of I-labelled IgG (■), prepared by the lodo-Gen method after application to a Sephadex-G-25, 1.0 x 10cm column (Pd-10) pre-equilibrated with 0.05M sodium phosphate buffer, pH 7.5. lOpl aliquots were transferred from each fraction to y-vials and counted for radioactivity per minute (cpm) in a Wallac, 1275 Minigamma y-counter. Arrow indicates elution of the radiolabelled protein at fraction 3. (Ve, elution volume = 3.0ml).

236 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

The labelling efficiency of the chloramine-T method i.e. extent of protein-bound radioactivity was estimated with respect to each iodinated peptide and protein as described in section 2.2.S.4 and represented as a percentage of the total radioactivity. Both aprotinin (82.18%) and insulin (84.51%) attained a high level of incorporation whereas IgG (90.08%) was even higher. This was anticipated due to the fact that by comparison the smaller peptides contain fewer tyrosine groups for incorporation. Indeed, aprotinin (Roberts et al, 1996) and insulin (Sanger and Thompson, 1953) contain 4 tyrosine residues each compared with IgG containing 48 tyrosine residues (Edelman et al, 1969). For in vivo work it is often important to use radiochemical tracers with a purity of at least 98%. Thus, the pooled fractions containing the isolated radiolabelled peptides and proteins respectively (1®^ peak. Fig. 5.1) were exhaustively dialysed and precipitated with TCA (section 2.2.5.4) to determine the purity of the radioactive tracers. Typically, all three radiolabelled compounds were purified to no less than 99.0% and the specific activity for the three radiolabelled tracers was determined at ~0.41-0.49pCi/pg protein.

One of the drawbacks reported by Franker & Speck (1978) with respect to using chloramine-T reagent pertained to the oxidative damage that peptides and proteins can sustain due to the harsh nature of this oxidising agent. Consequently, it was feared that peptide and protein tracers iodinated in this chapter via this reagent may affect their clearance pattern from the blood circulation. Thus, our attention was turned to the solid-phase reagent lodo-Gen as a milder alternative, where the initial investigation employed iodination of IgG for a comparison of techniques. Although the latter mechanism is reported to damage proteins less, the optimal reaction times were three times longer than using chloramine-T. In comparison with the chloramine-T method, labelling with lodo-Gen yielded an IgG tracer with high specific activity (~0.9)nCi/pg protein), comparable labelling efficiency (90.26%) and purity (> 90%). Interestingly, a comparison of the pharmacokinetic parameters derived from the plasma clearance profiles of the IgG constructs iodinated via chloramine-T (table 5.3, Fig. 5.6) and the IgG counterparts labelled via the lodo-Gen method (table 5.4, Fig. 5.7) revealed no significant difference. This suggested that iododerivatives of IgG (iodinated by either method) were equally acceptable for the use as tracers for in vivo

237 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates plasma clearance investigations. Ideally, comparisons between the pharmacokinetic parameters of labelled and unlabelled IgG constructs would have been beneficial however this could not be evaluated due to interference fi*om endogenous IgG antibodies in the blood. On the basis of these findings, the chloramine-T method was applied to aprotinin and insulin, respectively. It was however home in mind that whilst both radioiodination procedures have potential, it is necessary to establish the optimal reaction conditions and processes for each peptide and protein individually. For instance, Biscayart et al. (1989) concluded that the chloramine-T labelling procedure yielded ^^Î-labelled human growth hormone (hGH) tracers of high quality however; Wood et al (1981) found labelling with lodo-Gen produced a tracer (^^Î- labelled.hGH) of unsatisfactory quality. Several other studies have employed radioiodinated peptide and protein tracers prepared via the chloramine-T, lodo-Gen and other labelling procedures for the purpose of radioimmunoassay, pharmacokinetic and pharmacodynamic investigations. Mostly, these methods were found to be comparable producing equally acceptable iodinated products. Examples include: Trichosanthin (Ko et al, 1991), hGH (Biscayart et al, 1989) and insulin (Suzuki et al., 1972) which were iodinated via the chloramine-T process, Mab CC49 IgG (Pavlinkova et al, 1999) via lodo-Gen and IgG antibody fi*agment (Fab) (Chapman et al, 1999) via Bolton-Hunter reagent. In this study, the chloramine-T method yielded IgG, aprotinin and insulin tracers of high purity, labelling efficiency, and satisfactory specific activities suitable for plasma clearance studies. The isotope ^^Î has a half-life of 59.6 days, thus the iodinated peptide and protein compounds can be stored for acceptable lengths of time, the shelf life being only dependant upon their stability (4.3.11). The biological activities of the iododerivatives of IgG, aprotinin and insulin obtained by the chloramine-T method were not determined as the subject of intense review here was with respect to plasma clearance studies.

5.3.2 Radioiodination of polysialylated and polysialylated SDS-modified peptide and protein conjugates Polysialylated and polysialylated SDS-modified IgG, aprotinin and insulin conjugates were prepared under similar conditions to those used for the unlabelled conjugates except the reactions included iodinated peptide or protein tracers prepared by the

238 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates chloramine-T method for incorporation into the final product (section 5.2.3.3). Controls were conducted under the same coupling conditions but in the absence of oxidised CA. Iodinated peptide and protein conjugates and the controls were isolated from the respective reaction mixtures by ammonium sulphate precipitation and subsequently dialysed extensively to remove non protein-bound The molar yields of conjugation obtained for each of the radiolabelled conjugates was equivalent to their unlabelled counterparts (chapter 4, table 4.5).

The extent of protein-bound radioactivity incorporated into each polysialylated bioconjugate and control was determined and the results were used to calculate the specific activity (mCi/mg protein) of the final product. Table 5.2 shows the specific activities of radioiodinated IgG, aprotinin and insulin conjugates synthesised by two methods of polysialylation and their native controls respectively.

Table 5.2 Specific activities of iodinated polysialylated and polysialylated SDS- modified IgG, aprotinin and insulin conjugates determined by the amount of radioactivity measured per l.Omg of protein (mCi/mg protein) in the sample. Controls included iodinated native IgG, aprotinin and insulin without oxidised CA. Tracers were prepared by the chloramine-T method. Results shown are means of three jiirr X_____ X:______i r»9 j \ Specific activity (x 10"^) (mCi/mg protein) Preparation Native (control) Polysialylated SDS-polysialylated IgG 0.76 0.71 0.70 Aprotinin 0.68 0.61 0.63 Insulin 0.61 0.57 0.55

As expected, the extent of ^^^I incorporated into the native controls was slightly higher than with the CA modified peptide and protein conjugates respectively, which was possibly due to the competition between the labelled and unlabelled protein moieties for CA. Nevertheless, this method of radiolabelling produced bioconjugates with good specific activity (0.55-0.71 x 10‘^mCi/mg protein) and adequate labelling efficiency (63-90%) that were all independent of the extent of polysialylation. Introduction of a tritium (^H) second label into the double bond (C=N) of the polysialylated conjugates was considered however; Fernandes and Gregoriadis (1996) highlighted that this

239 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates technique had a poor labelling efficiency.

5.3.3 Size exclusion chromatographic (SEC) characterisation of radioiodinated polysialylated and polysialylated SDS-modified peptide and protein conjugates Size exclusion chromatography was employed in an attempt to establish that was bound with the native peptide and protein controls and indeed incorporated into the respective polysialylated and polysialylated SDS-modified peptide and protein conjugates. The radiolabelled polysialylated conjugates were characterised using SEC. Essentially, samples fi*om the radiolabelled native controls and the peptides and proteins modified by both methods of polysialylation (post 48 hours reaction) were applied to different Sephadex gel columns as described in Chapter 4 for IgG, aprotinin and insulin.

The chromatograms obtained for each iodinated IgG (Fig. 5.3), aprotinin (Fig. 5.4) and insulin (Fig. 5.5) product prepared using tracers fi-om the chloramine-T method (section 5.2.3.1) can be seen below. The elution profiles obtained for ^^Î-labelled native IgG and its polysialylated constructs prepared with tracers fi*om the lodo-Gen method (section 5.2.3.2) were not significantly different (not shown) fi*om IgG prepared with via the chloramine-T method. Typically, native IgG, aprotinin and insulin (Figs. 5.3A, 5.4A and 5.5A) controls eluted in the same fi*actions as their native unlabelled counterparts (Chapter 4) except that here, coincident ^^Î and peptide and protein peaks were identified. This suggested that the ^^Î-label was protein-bound in each case. Interestingly, no higher or lower molecular weight derivatives of any of the peptide or protein controls were evident even after incubation under the coupling conditions and in the presence of coupling and radiolabelling reagents. In contrast with the elution profiles of the radiolabelled native controls, IgG, aprotinin and insulin modified by both methods of polysialylation eluted more rapidly with co-elution of CA and the ^^Î-label in earlier fi*actions (Figs. 5.3B&C, 5.4B&C, 5.5B&C). These observations might suggest the emergence of the neoglycoprotein and coincident radioiodination of the peptides and proteins modified by both polysialylation, however further evaluations are warranted to rule out other forms of bonding.

240 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

Q) 1.5i r2000 O c g (0 a ■s -1500 5' D) 8 1.0 - O < 1000 O 0.5- ■o 500 3

0.0 0 10 20 30 Ve 2.01 r1500

a cs 1.5 (0 1000 2 ■s & 8 1.0 < .Q < 'o' 500 •a 0.5 Vo 3

0.0 0 10 20 30 Ve 2.01 r1500 g 8 Q. c 1.5 (0 1000 8 ■e a <

500 'cT Vo ■o 3

0.0 0 10 20 30 Elution volume (ml) Figure 5.3 Size exclusion chromatography of iodinated IgG compounds. A) native IgG (control), B) polysialylated IgG conjugate and C) polysialylated SDS-modified IgG conjugate after 48 hours of reaction. Samples were chromatographed on a Sephadex G-lOO column (40.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'’) and fractions (1.0ml) assayed for radioactivity (A), IgG (595nm) (■) and CA (570nm) (T). Arrows indicate elution (Ve) of the conjugate at fraction 12 (Vo, void volume = 7ml).

241 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates

o r15000.15i o c CO g € a -1000 8 & <

o 0.05- -500 ■O 3

0.00 0 10 20 30 Ve 0.41 r1500 g 8 Q. C 0.3- 5‘ -1000 D) I & < 8 0. 2 - < -500 Vo ■o 3

0.0 0 10 20 30 Ve 0.41 1000 g 8 a c 0.3 -750 5‘ &) eg o ■s A -500 <

Vo -250 ■o 0.1 - 3

0.0 0 10 20 30 Elution volume (ml) Figure 5.4 Size exclusion chromatography of iodinated aprotinin compounds. A) native aprotinin (control), B) polysialylated aprotinin conjugate and C) polysialylated SDS-modified aprotinin conjugate after 48 hours of reaction. Samples were chromatographed on a Sephadex G-50 column (35.0 x 1.0cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^) and fractions (1.0ml) assayed for radioactivity (□), aprotinin (595nm) (■) and CA (570nm) (À). Arrows indicate elution (Ve) of the conjugate at fraction 11. (Vo, void volume = 4.0ml).

242 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

o> 0.15n r1500 o c (Q g o. ■s 5" 1000 <

0.05 -500 'cT ■o 3

Q QQ rnfoM -m -efB-f m ë # » 0 10 20 30 Ve 0.41 r2000 g Q. s 0.3 1500 c fi)o ’ € o S 0.2 1000

< o T3 Vo 500 3

0.0 0 10 20 30 Ve 0.61 r2000 g 0.5 a s 1500 5’ c D) ■s(0 0.4 - 0.3- 1000 .QS ^ 0.2 O 73 Vo 500 3

0.0 0 10 20 30 Elution volume (ml) Figure 5.5 Size exclusion chromatography of iodinated insulin compounds. A) native insulin (control), B) polysialylated insulin conjugate and C) polysialylated SDS- modified insulin conjugate after 48 hours of reaction. Samples were chromatographed on a Sephadex G-50 column (40.0 x 1.1cm; sample volume 0.5ml; PBS eluent; flow rate, l.Omlmin'^) and fractions (1.0ml) assayed for radioactivity (□), insulin (595nm) (■) and CA 570nm) (À). Arrows indicate elution (Ve) of the conjugate at fraction 12. (Vo, void volume = 4.0ml).

243 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates

5.3.4 Pharmacokinetic analysis

The pharmacokinetic properties of ^^^I-labelled IgG (prepared via both iodination methods), aprotinin and insulin modified by both methods of polysialylation were evaluated after intravenous administration and compared with their respective native peptide or protein controls. In each case, the plasma clearance profiles were characteristic of an apparent two-compartment model, which was analysed using a biexponential decay curve of best fit. The pharmacokinetic parameters derived included: a) dose, b) area under the plasma concentration versus time curve (AUC), c) plasma clearance (Clp), d) volume of distribution (Vd), e) distribution half-life (ti/ 2 a), f) elimination half-life (ti/ 2 p), g) P elimination rate constant (Pel) and h) mean residence time (MRT).

5.3.4.1 Immunoglobulin G (IgG) 1000q

2 100 -

Time (h) Figure 5.6 Clearance of chloramine-T labelled IgG and polysialylated conjugates from circulation of intravenously injected mice. ^^^1-labelled native IgG (o), polysialylated lgG-^^^1 (A) and polysialylated SDS modified lgG-^^^1 (T) conjugates (~167,000cpm, 0.70mg each). Blood samples subjected to 0-counting for radioactivity and the data converted to concentration of IgG remaining after injection. Values denote means ± SD, n = 4 animals.

244 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

Figure 5.6 shows the semi-logarithmic plasma concentration-time profiles of intravenously injected ^^Î-labelled IgG compared with that of the similarly injected ^^Î-labelled polysialylated constructs. Radiolabelling was performed using the chloramine-T method. In each case, an initial decline was followed by a slower phase of decline. Much of the administered dose (-51% for native IgG and 48 to 41% of the two polysialylated constructs) was removed from the circulation of mice within minutes after injection, the remainder exhibiting slower linear clearance rates. The three clearance profiles suggested biphasic disposition kinetics. Indeed, the observed values closely (r^ = 0.98) fitted the computer-generated curve for a two-compartment model for 72 hours of observation using the MW/Pharm program. Figure 5.7 shows a very similar semi-logarithmic plasma concentration-time profile for intravenously injected *^Î-labelled IgG and the polysialylated constructs prepared using the lodo- Gen method (section 5.2.3.2).

1000q

s 100t

Time (h) Figure 5.7 Clearance of lodo-Gen labelled IgG and polysialylated conjugates fi*om circulation of intravenously injected mice. ^^Î-labelled native IgG ( o ) , polysialylated IgG-’^Î (À) and polysialylated SDS modified IgG-^^Î (T) conjugates (~300,000cpm, 0.60mg each). Blood samples subjected to 0-counting for radioactivity and the data converted to concentration of IgG remaining after injection. Values denote means ± SD, n = 4 animals.

245 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

The first phase of the plasma concentration-time plots is curved and known as the a- phase during which time the drug is distributed out of the plasma and into tissues or extravascularly. The second phase is straight and known as the ^-terminal phase, during which the complete volume of distribution has been attained and drug excretion proceeds. The transition between a and P phases occurred at about 4 hours after injection. The pharmacokinetic parameters were derived as described in section 5.2.3.5, compared statistically and presented in table 5.3 for chloramine-T labelled and table 5.4 for lodo-Gen labelled IgG and Polysialylated constructs.

Table 5.3 Pharmacokinetic parameters derived using the MW/Pharm program for chloramine-T labelled native and polysialylated IgG conjugates after intravenous administration in T/O outbred mice weighing 25-30 g. Multiple comparisons were made using a one-way analysis of variance test (ANOVA) and significant p values were corrected by the Bonferroni method and given in paranthesis. Results are mean ± S.D of four experiments. * not significantly different from native IgG at P > 0.05.

Polysialylated SDS- Formulation Native IgG Polysialylated IgG modified IgG

Dose (mg) 0.720 ± 0.045 0.734 ±0.027 0.726 ± 0.033

AUC X lO'* (h.mg/L) 0.8741 ±0.289 1.209 ±0.084 1.491 ±0.215

Clp X 10'^ (L/h) 8.237 ±0.031 6.069 ± 0.066 4.869 ± 0.024

VdxlQ-^ (L) 3.434 ±0.015 2.821 ±0.111 2.573 ± 0.039

T 1/2 a (h) 0.945 ±0.010 0.970 ±0.005* 1.030 ±0.080*

T i/2 P (h) 28.89 ± 1.038 32.21 ± 1.407* 36.63 ± 0.882

P(L/h) 0.0476 ± 0.002 0.0371 ±0.001 0.0322 ± 0.002

MRT (h) 40.35 ± 1.636 45.46 ± 1.145 51.81 ± 1.504

Although the distribution half-lives (ti /2 a) for polysialylated IgG (0.970 ± 0.005) and polysialylated SDS-modified IgG (1.030 ± 0.080 h) were slightly improved, unfortunately they were not statistically (P > 0.05) different from that of native IgG

(0.945 ± 0.010 h). Comparison of the terminal elimination half-lives (ti/ 2 P) of native IgG (28.89 ± 1.038 h) and polysialylated IgG (32.21 ± 1.407 h) revealed no significant difference (P > 0.05), however polysialylated SDS-modified IgG (36.63 ± 0.882 h) was significantly (P < 0.01) different from the native protein.

246 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates

Table 5.4 shows almost identical pharmacokinetic parameters and similar significance as determined in table 5.3. These results suggest that although chloramine-T is a harsh reagent, there was no significant difference between the two methods of iodination with respect to IgG.

Table 5.4 Pharmacokinetic parameters derived using the MW/Pharm program for lodo-Gen labelled native and polysialylated IgG conjugates after intravenous administration in T/O outbred mice weighing 25-30 g. Multiple comparisons were made using a one-way analysis of variance test (ANOVA) and significant p values were corrected by the Bonferroni method and given in paranthesis. Results are mean ± S.D of four experiments. * not significantly different from native IgG at P > 0.05. Polysialylated SDS- Formulation Native IgG Polysialylated IgG modified IgG Dose (mg) 0.61 ±0.010 0.64 ±0.010 0.63 ±0.015

AUC X 10“ (h.mg/L) 0.7593 ±0.071 1.098 ±0.179 1.306 ±0.161

Clp X 10'^ (L/h) 8.034 ±0.116 5.830 ±0.039 4.822 ± 0.047

Vdx 10'^ (L) 3.749 ±0.141 2.946 ±0.123 2.723 ±0.112

Ti/2 a (h) 1.061 ±0.015 1.325 ±0.034* 1.369 ±0.037*

T i /2 P ( h ) 32.35 ± 0.40 35.03 ± 0.60* 39.14 ±0.36

P(L/h) 0.0485 ± 0.001 0.0347 ±0.001 0.0328 ± 0.001

MRT(h) 44.16 ±0.969 49.09 ± 0.496 55.16 ± 1.056

Interestingly, Fernandes and Gregoriadis (1996) found that the of polysialylated catalase was not significantly improved compared with the native enzyme. On the basis of their plasma clearance work with polysialylated asparaginase, it was later suggested that measurement of enzyme activity rather than radioactivity and further increased enzyme modification with CA might have led to significant changes in half- life. Similarly, PEG-gulonolactone oxidase is another example where modification of the enzyme by polymer conjugation did not show a significantly different clearance pattern from that of the unmodified enzyme (Hadley et al., 1989). Even though the im p of the PEGylated enzyme was slightly greater than the native enzyme, their ti/ 2 a were the same. In this instance, the lack of improved pharmacokinetics was attributed

247 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates to either the small molecular weight (5Kda) of PEG used or the insufficient degree of protein modification. In contrast, Kitamura et al. (1991) reported that PEG modification enhanced the a- and |3- half-lives of the monoclonal antibody A7 (Mab

A7) in comparison with the parent antibody. Half-life values of 3.6 h (ti /2 a) and 33.6 h (ti/2 P) were reported for native ^^^I-labelled Mab A7 in intravenously injected mice in contrast with 5.4 h (ti /2 a) and 38.1 h (ti /2 p) observed for the PEG-Mab A7 (5.0:

1.0) conjugate. Although our elimination half-life (ti /2 P) values for native and polysialylated IgG conjugates (CA: IgG, 4.25: 1.0 and 12.27: 1.0, respectively) were similar to those of Kitamura et al. (1991), the distribution half-lives (ti /2 a ) did differ. Even though the same species were used in both investigations and the observation times were fairly similar, it is quite probable that differences in conjugation chemistry (N-hydroxysuccinimide activation of the hydroxy group of mPEG versus periodate activation of hydroxyl groups of CA) contributed to the differences.

With respect to polysialylated IgG, it seems reasonable to assume that the lack of improvement in a- and p- half-life compared with the native control was due to either the small molecular weight of CA (lOKDa) or the insufficient degree of protein modification. Indeed, Knauf et al. (1988) demonstrated how changes in the hydrodynamic radii and charge of rIL-2, an anti-cancer drug, upon PEGylation impacted on the systemic clearance of the protein. Thus, it was established that changes in clearance rate correlated with the degree of modification and size of the polymer used. Unfortunately, in the case of IgG, a further increase in the extent of polysialylation (2.9-fold) by employing the SDS-modification technique did not significantly (P > 0.05) improve the distribution half-life (ti /2 a) although the elimination half-life (ti/ 2 P) was statistically (P < 0.01) improved in comparison with the native protein. A comparison of the other pharmacokinetic parameters (table 5.3) revealed that the AUC (a measure of the amount of drug that passes through the plasma) was significantly increased (p < 0.001) with increasing degree of polysialylation whilst the apparent volume of distribution (Vd) was significantly reduced (P < 0.001). The increased AUC probably results from the significantly (P = 0.0002, ANOVA) slower terminal elimination rate (p) observed in the P-phase and significantly (P < 0.001) reduced plasma clearance with increasing degree of

248 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates polysialylation (table 5.3). Furthermore, the apparent volume of distribution (ratio of drug in the body/drug in circulation) for each conjugate was reduced after coupling with CA, probably reflecting the increased difficulty of the polysialylated constructs to extravasate to the extravascular space on account of the increased molecular weight of the complex. It is apparent fi-om pharmacokinetic parameters such as AUC, Clp, Vd and P elimination rate (table 5.3) that the increased systemic exposure of IgG is dependent upon the degree of modification by CA.

There are many mechanisms by which foreign peptides and proteins are cleared from the circulation, which have been previously discussed in Chapter 1. In brie^ they include: non-specific uptake by the reticuloendothelial system (RES) (Delgado et al., 1992), receptor-mediated endocytosis by cells in which protein degradation eventually occurs, and by protein molecular mass, shape and charge (Bocci, 1987; Benbough et al., 1979) which determine the extent of transcapillary passage or renal filtration (Bocci, 1987). Although the liver and kidney are major sites of protein catabolism (Bocci, 1990; Kompella and Lee, 1991), proteolysis in body fluids such as plasma or at the cell surface by membrane bound proteases contribute significantly to the short half-lives of peptides and proteins (Bocci, 1990; Breimer, 1991). In the case of IgG (sialoglycoprotein), its unusually long survival time may be associated with a very slow rate of catabolism due in part to its carbohydrate content with terminal sialic acid residues. Allen and Chonn (1987) and Schauer (1982a, 1982b) reported that enzymatic removal of sialic acid from circulatory cells or from monosialogangliosides resulted in their rapid uptake into parenchymal cells of the liver. It is known that the uptake of immunoglobulins occurs after sialic acid residues are removed by sialylases resulting in the internalisation of the terminal galactose residues by receptor-mediated endocytosis by the parenchymal cells in the liver. Indeed, biodistribution studies performed after intravenously injecting mice with iodinated IgG showed accumulation in the liver and spleen (Pedley et al., 1994). Gregoriadis, 1975 reported that antibodies are removed at random by the liver at rates that depend on the class of protein, its source and the host animal. At the same time a considerable portion of the immunoglobulin undergoes transcapillary passage to enter the extravascular space. Interestingly, Kitamura et a/. (1991) reported that chemical modification of the

249 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates antibody (Mab A7) by coupling with mPEG (mol. wt 5KDa) significantly reduced organ uptake in the liver and spleen whilst uptake in the lung and kidney remained constant in comparison with the parent Mab A7. The permeability threshold of the kidney glomerular filtration system is around 70KDa (Pedley et al., 1994), which is smaller than the size of the antibody before polymer attachment and probably explains why accumulation in the kidney was similar for both modified and unmodified antibody. Although it is unclear why PEG-modified antibodies accumulated less in the liver and spleen it was rationalised as either an inherent property of PEG or steric hindrance of non-specific uptake and reduced proteolysis due to the masking effect of PEG (Kitamura et al., 1991).

Lobuglio et al. (1989) reported that the a half-life represented the equilibrium of IgG between the intra- and extravascular spaces. Lisi et al. (1982) showed that PEG- modified proteins were more resistant to proteolysis than the corresponding unmodified protein, suggesting that the p half-life reflects the catabolism of the immunoglobulin. Gregoriadis et al. (1999) also reported on the increased resistance of polysialylated catalase and asparaginase to plasma proteases compared with the native enzyme. Likewise, Zhang (1999) demonstrated that CA-modified liposomes exhibited increased stability in protein solutions and mouse plasma and that adsorption of plasma proteins to such liposomes was significantly reduced. Consequently, the CA- modified liposomes exhibited a 2-fold increase in the plasma retention time in contrast with the unmodified liposome. Interestingly, Roerdink et al. (1986) observed the correlation between protein adsorption to unmodified liposomes and their resultant increased plasma clearance. Although the action of CA is not fully understood, it may be inferred that, at least in part, the CA chains shield the sites on the antibody that are subject to proteolysis and thereby curtail the catabolism of IgG thus increasing its plasma residence time. In addition, it is also conceivable that the altered pharmacokinetic characteristics of polysialylated IgG may be attributed to charge modifications of the antibody itself. Thus, as a result of the loss of positively charged 8-amino groups on coupling with negatively charged CA, the resulting protein is intrinsically more negative. The altered charge could reduce the interaction of the CA- antibody complex with negatively charged blood and tissue components (Tomlinson,

250 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates

1990). Further investigations are however necessary to support these hypotheses. Interestingly, several investigators have attempted to improve Mab clearance by using various methods including charge modifications of the Mab itself. For example, Sharifi et al. (1998) demonstrated how lowering the isoelectic point by conjugation with chemical moieties of positive, negative or neutral charge residues on the surface of Mabs correlated with their decreased clearance time and improved tumour targeting.

In summary, although the half-lives of both CA-modified IgG conjugates where not markedly augmented in comparison with the native protein, the other pharmacokinetic parameters such as AUG, Clp, Vd and P elimination rate (table 5.3) were influenced upon further modification of IgG with CA. Pedley et al. (1994) demonstrated that PEG modification of an intact anti-CEA antibody had little effect on its clearance although similar modification of the Fab-fi-agment dramatically increased the plasma half-life and increased tumour accumulation. Although intact immunoglubulin (IgG) molecules have several practical limitations of their pharmacology due to their large size (ISOKDa) leading to slow clearance from the circulation, limited tumour accumulation and increased organ uptake, their inherent specificity makes them ideal for antibody-directed therapy (Colcher et al., 1998). It is realised that further investigations are warranted into the immunological and residual antigen-binding properties of polysialylated IgG conjugates before their therapeutic potential may be established. However, these results and our earlier antibody-binding studies conducted with polysialylated IgG2a (Mab) (section 4.3.10.3) indicate the feasibility of custom- designing CA-antibody and Fab fragment molecules which may be used to enhance both the diagnostic and therapeutic potential of monoclonal antibodies used in radioimmunotherapy and immunochemotherapy (Pedley et al., 1994).

5.3.4.2 Aprotînin Figure 5.8A shows the plasma clearance profile of '^^I-labelled native aprotinin, and both polysialylated aprotinin conjugates after intravenous injection into mice. The study was conducted over 48 hours. The data for the first 5 hours has been expanded from Fig. 5.8A and shown in Fig. 5.8B to show more clearly the earlier time points.

251 Chapter Five : Pharmacokinetic properties of polysialylated bioconjugates

100^

24 30 54 Ü 1000

ÛL

Time (h)

Figure 5.8 Clearance of chloramine-T labelled aprotinin and polysialylated conjugates from circulation. (A) Plasma clearance profile over 48 hours. (B) Expanded plasma clearance profile for the first 5 hours only. -labelled native aprotinin ( o ) , polysialylated a p r o tin in - (À) and polysialylated SDS modified a p r o tin in - (T) conjugates (-80,000-150,OOOcpm, 0.70mg each) were intravenously injected into groups of four T/0 outhred mice. Blood samples taken from the tail vein were subjected to 8-counting for radioactivity and the data converted to concentration of aprotinin remaining after injection. The pharmacokinetic profiles demonstrate hiphasic patterns of clearance, which are consistent with a two-compartment model. Values denote means ± SD, n = 4 animals.

252 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates

As anticipated, both polysialylated aprotinin conjugates remained in the circulation of intravenously injected mice for longer periods than native aprotinin (Fig. 5.8A and B, table 5.5). Thus, 48 hours after injection, native aprotinin was undetectable whereas the concentration of the polysialylated aprotinin constructs was still measurable at 0.5- 1.0% of the injected dose depending on the degree of polysialylation. The plasma clearance profiles for each of the aprotinin preparations showed biexponential decay with the bulk of the dose being eliminated very rapidly (~ 60% and 42-37% of native and polysialylated aprotinin conjugates respectively) within minutes after injection. The remainder of the dose exhibited a slower, linear clearance rate (Fig. 5.8 A) similar to that observed for IgG. The pharmacokinetic parameters generated using the MW/Pharm program for each aprotinin preparation were compared statistically and presented in table 5.5. Comparisons of the distribution half-lives (ti /2 a) for native aprotinin (0.0396 ± 0.0043 h) and polysialylated aprotinin (0.0432 ± 0.0025 h) revealed no significant difference (P > 0.05), however polysialylated SDS-modified

Table 5.5 Pharmacokinetic parameters derived using the MW/Pharm program for native and polysialylated aprotinin conjugates after intravenous administration in T/O outhred mice weighing 25-30 g. Multiple comparisons were made using a one-way analysis of variance test (ANOVA) and significant p values were corrected by the Bonferroni method and given in parenthesis. Results are mean ± S.D of four experiments. * not significantly different from native aprotinin at P > 0.05.

Polysialylated Polysialylated SDS- Formulation Native aprotinin aprotinin modified aprotinin Dose (mg) 0.670 ±0.021 0.620 ± 0.045 0.770 ±0.010

AUC X 10^ (h.mg/L) 0.312 ±0.258 0.608 ± 0.250 1.321 ±0.379

Clp X 10'^ (L/h) 2.144±0.151 1.019 ±0.201 0.583 ± 0.069

VdxlO'^(L) 3.426 ± 0.203 2.400 ±0.152 1.498 ±0.100

Ti/2 a X 10'^ (h) 3.965 ± 0.430 4.328 ±0.251* 4.834 ±0.117

T i n P ( h ) 11.08 ± 1.002 16.33 ± 1.019 17.82 ±0.629

p(L/h) 1.648 ±0.255 0.946 ± 0.029 0.584 ±0.013

MRT(h) 14.53 ± 0.725 22.23 ±0.661 24.73 ± 1.090

253 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates aprotinin (0.0483 ± 0.0011 h) was significantly (P < 0.05) different from the control.

Comparisons of the terminal elimination half-lives (ti /2 P) of polysialylated (16.33 ± 1.02 h) and polysialylated SDS-modified aprotinin (17.82 ± 0.63 h) revealed both were significantly different (P = 0.0002, ANOVA) from the control (11.08 ± 1.00 h). Analysis of the other pharmacokinetic parameters (table 5.5) revealed that the AUC and MRT were increased 1.9 to 4.2-fold and 1.5 to 1.7-fold respectively with increasing degree of polysialylation of aprotinin whilst the apparent volume of distribution and plasma clearance were significantly (P > 0.001) reduced. The increase in AUC and MRT with increasing degree of polysialylation probably resulted due to the significantly (P = 0.0006, ANOVA) reduced p elimination rates seen for both aprotinin conjugates (table 5.5). The reduction in apparent volume of distribution seen with increasing degree of polysialylation of aprotinin probably arises due to the increased opposition of the polysialylated constructs to diffuse into the extracellular fluid on account of increasing molecular weight. It is apparent therefore that pharmacokinetic parameters such as AUC, Clp, Vd, P elimination rate, elimination half-life (ti/2 P) and MRT (table 5.5) support the idea that increased systemic circulation of aprotinin is dependent upon its degree of modification by CA.

Interestingly, Larionova et al. (1984) reported on the pharmacokinetics of native and lactose-modified aprotinin in mongrel rats weighing 200-250g. Disappointingly, the author did not report data with respect to a and p half-lives for unmodified or modified aprotinin constructs for comparison with our investigations. Nevertheless, they found practically the same quantitative elimination for both preparations from the blood stream. Thus, one hour after intravenously injecting rats, 2-3% of the initial dose of native and lactose-modified aprotinin was present in the blood. These results were in agreement with our values for native aprotinin (2%) and polysialylated aprotinin (4%) even though mice were used in our investigation instead of rats. However, the plasma concentration of polysialylated SDS-modified aprotinin (7%), one hour after administration, was marginally better (Fig. 5.8). It was worth noting that lactose-modified aprotinin contained 2.0 moles lactose per mole aprotinin, polysialylated aprotinin contained 1.46 ± 0.05 moles CA per mole aprotinin and polysialylated SDS-modified aprotinin contained 4.59 ± 0.05 moles CA per mole

254 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates aprotinin. Interestingly, mongrel rats intravenously injected with aprotinin coupled to (carboxymethyl) dextran derivatives of D-galactosamine (CMD-2) and D-lactose (CMD-1) led to higher circulatory concentrations of 25-55% respectively one hour after injection (Larionova et aL, 1985). The aprotinin-CMD-1 and CMD-2 conjugates contained 14 and 38 D-galactose residues and per mole aprotinin, respectively. Whilst our results differed from those reported by Larionova et ah (1985), it should be home in mind that different conjugation chemistries and also different species were employed thus different basal metabolic rates existed between the two species (Spector, 1956). Although direct comparisons could not be made for the above reasons, the collective results are interesting as they demonstrate how increasing the degree of modification of aprotinin and the use of different carrier systems can prolong the systemic clearance of aprotinin. Other authors have reported values of

0.32-0.50 h and 5.25-8.28 h for the distribution (ti /2 a) and elimination (ti /2 P) half-life values for the two phases of native aprotinin injected intravenously in human subjects (Davis and Whittington, 1995).

With respect to the clearance mechanisms of aprotinin, it is known that following intravenous administration of radiolabelled aprotinin, rapid distribution occurs throughout the extracellular compartment (Kaller et al, 1978). This is due to aprotinins small molecular dimensions of 2.9nm x 1.9nm (Larionova et al, 1985) which enable it to overcome the tissue-blood barrier, resulting in its rapid decrease in serum concentration and accumulation in tissue compartments (Fritz et al, 1969). Animal studies have shown that accumulation of aprotinin occurs within the proximal tubular epithelial cells of the kidneys, after which aprotinin is actively reabsorbed by the proximal tubules and stored in phagolysosomes (Trautschold et al, 1967). Indeed, biodistribution studies performed by Larionova et al (1984; 1985) revealed that native aprotinin accrues in the kidneys. Interestingly, fixation of lactose and CMD-1 modified-aprotinin decreased 2-fold in the kidneys and increased 2-fold in the liver during the 2 hour observation period compared with native aprotinin. CMD-2 modified-aprotinin exhibited a similar distribution profile except it accumulated 10- fold in the liver compared to native aprotinin. It was reasoned that CMD-2 concentrated in the liver to a greater extent on account of its increased number of

255 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

D-galactose residues. With respect to the diminished accumulation of the CMD- aprotinin conjugates in the kidneys, it was assumed (Fritz et al, 1969) that the decrease in basicity of aprotinin after modification with the carboxyl-containing polysaccharides was responsible for this phenomenon. As stated earlier, the action of CA on the clearance mechanism of CA-modified peptides and proteins is unclear. However, it is possible that the improved persistence of CA-modified aprotinin conjugates in plasma may be attributed partly to decreased glomerular filtration on account of size and charge modifications due to CA, slower catabolism in the liver due CA shielding and reduced proteolysis. Further investigations are however warranted to fully substantiate these hypotheses.

In conclusion, this study showed that both CA-modified aprotinin conjugates were characterised by a change in their in vivo behaviour manifested by their increased plasma retention in comparison with native aprotinin. A comparison of the pharmacokinetic parameters such as AUC, Clp, Vd, p elimination rate, elimination half-life (ti/2 P) and MRT (table 5.5) between aprotinin conjugates revealed that increasing the degree of modification resulted in increasing the longevity of CA- modified aprotinin in the circulation. The improved pharmacokinetics observed for both aprotinin conjugates may serve as a prerequisite for a more effective therapeutic use of the inhibitor in diseases caused by proteinases such as acute pancreatitis and septicaemia (Larinova et aL, 1985). Interestingly, Larinova et al. (1984a) coupled the protease inhibitor gordox to the carboxymethyl ester of dextran (CM-D) and compared its therapeutic effect with the native peptide. They reported a 50 % increase in the survival of animals with acute pancreatitis treated with the long-acting conjugate in comparison with the native form, thereby doubling the lifetime of animals that died.

S.3.4.3 Insulin Intravenous administration of ‘^^I-labelled native and polysialylated insulin conjugates resulted in plasma concentration-time profiles (Fig. 5.9A) that were characteristic of an apparent two-compartment model, best described by the biexponential decay curve of best fit as seen with IgG and aprotinin.

256 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

24 36 42 48 54

0 1 2 3 4 5 Tim e (h) Figure 5.9 Clearance of chloramine-T radiolabelled insulin and polysialylated conjugates from circulation. (A) Plasma clearance profile over 48 hours. (B) Expanded plasma clearance profile for the first 5 hours only. ^^^I-labelled native insulin (o), polysialylated insulin-(A) and polysialylated SDS modified insulin- (▼) conjugates (-134,000, 0.4mg each) were intravenously injected into groups of four T/O outhred mice. Blood samples taken from the tail vein were subjected to 6- counting for radioactivity and the data converted to concentration of insulin remaining after injection. The pharmacokinetic profiles demonstrate hiphasic patterns of clearance, which are consistent with a two-compartment model. Values denote means ± SD, n = 4 animals.

257 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates

The study was conducted over 48 hours as with aprotinin and similarly, the data for the first 5 hours has been expanded from Fig. 5.9A and shown in Fig. 5.9B to show more clearly the earlier time points. As anticipated, both polysialylated insulin conjugates remained in the circulation of intravenously injected mice for longer periods than native insulin (Fig. 5.9A and B, table 5.6). Thus, 24 hours after injection, native insulin was undetectable whereas the concentration of the polysialylated insulin conjugates was still detectable at 1.6-2.6% of the injected dose depending on the degree of polysialylation. Within minutes after injection, up to 58% of native insulin had been eliminated in contrast with 42 to 36% of the polysialylated insulin conjugates respectively. The remaining dose followed a slower, linear clearance pattern (Fig. 5.9A) similar to IgG and aprotinin. Table 5.6 shows the pharmacokinetic data generated using the MW/Pharm program for each insulin preparation.

Table 5.6 Pharmacokinetic parameters derived using the MW/Pharm program for native and polysialylated insulin conjugates afl:er intravenous administration in T/O outhred mice weighing 25-30 g. Multiple comparisons were made using a one-way analysis of variance test (ANOVA) and significant p values were corrected by the Bonferroni method and given in parenthesis. Results are mean ± S.D of four experiments. * not significantly different from native insulin at P > 0.05.

Polysialylated Polysialylated Formulation Native insulin SDS- modified insulin insulin Dose (mg) 0.400 ±0.015 0.396 ±0.018 0.4640 ±0.010

AUC X 10^ (h.mg/L) 0.135 ±0.017 0.447 ±0.199 0.939 ± 0.238

Clp X 10'^ (L/h) 2.956 ± 0.240 0.884 ±0.170 0.493 ± 0.027

Vdx lO'^(L) 4.879 ± 0.222 2.318 ±0.072 1.241 ±0.112

Ti/2 a (h) 0.1123 ±0.006 0.1146 ±0.003* 0.1271 ±0.003*

T,/2 P (h) 11.44 ±0.106 17.41 ±0.343 18.16 ± 1.255

p(L/h) 2.258 ± 0.082 0.745 ±0.101 0.489 ± 0.077

MRT(h) 10.63 ±0.214 22.81 ±0.382 23.26 ± 0.278

258 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

The distribution half-life (ti /2 a) of native insulin (0.1123 ± 0.006 h) was not significantly (P > 0.05) different from polysialylated insulin (0.1146 ± 0.003b), or polysialylated SDS-modified insulin (0.1271 ± 0.003b). Comparisons of the terminal elimination half-lives (ti /2 P ) of polysialylated (17.41 ± 0.343b) and polysialylated SDS-modified insulin (18.16 ± 1.255b) revealed both were significantly different (P < 0.001) from the control (11.44 ± 0.106b). Comparisons of the other pharmacokinetic data (table 5.6) revealed that AUC and MRT were increased 3.3 to 6.9-fold and 2.1- fold respectively with increasing degree of polysialylation of insulin whilst the apparent volume of distribution, plasma clearance and p elimination rates were significantly (P > 0.001) reduced. It is quite probable therefore that the increased molecular weight of the polysialylated insulin conjugates contributed significantly to the difficulty of these molecules to extravasate into the extracellular fluids in comparison with native insulin. It is apparent that the hormones elimination half-life

(ti/2 P ) , P elimination rate, AUC, Clp, Vd and AUC are dependent upon its degree of polysialylation, i.e. the amount of CA attached to the surface of the peptide.

Interestingly, Baudys et al. (1998) intravenously injected rats with native and CMD modified-insulin and found that the CMD-insulin conjugate exhibited a significantly prolonged plasma profile. They reported a distribution half-life (ti /2 a) of 1.03 ± 0.22 min and elimination half-life (ti /2 p) of 12.4 ±3.4 min for native insulin. By contrast,

CMD-modified-insulin resulted in a distribution half-life (ti /2 a) of 4.94 ± 1.06 min and elimination half-life (ti /2 p) of 114.1 ± 28.6 min. Although the distribution half- life obtained for CMD-modified insulin was in agreement with the values obtained for both polysialylated insulin conjugates (~ 6.79 and 6.95 min respectively), the value obtained for native insulin injected into rats (1.03 ± 0.22 min) was not in agreement with our value (6.58 min). The differences were probably attributable to the different basal metabolic rates between species, or due to the fact that Baudys et al. (1998) injected much lower doses of insulin (between 0.004 and 0.006mg) per animal, which over extended periods of time could lead to underestimations of half-life. It is conceivable therefore, that the different observation times also contributed to the different elimination half-life (ti /2 P) values determined for native and CMD modified insulin (study period 250 min) compared with our study of native and polysialylated

259 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates insulin conjugates (study period 48 h). Numerous other studies have been conducted on the intravenous administration of native insulin, for example Gray et al 1985 reported on very short distribution half-lives (ti /2 a) of about 1.6 to 3.4 minutes in healthy human subjects. Similarly to endogenous insulin, exogenous insulin exhibits a very short half-life of ~ 4 minutes which can be attributed to its rapid distribution throughout the extracellular fluids following intravenous, subcutaneous or intramuscular administration. Insulin is rapidly metabolised, mainly in the liver, by the enzyme glutathione insulin dehdrogenase (Jones et al, 1982) and to a lesser extent in the kidneys and muscle tissue (Hoffman and Ziv, 1997). Interpretation of the pharmacokinetic parameters derived for both polysialylated insulin conjugates in the context of clearance mechanisms is complex. However it is possible that the improved persistence of the CA-modified insulin conjugates may be partly due to reduced hepatic and renal clearance and partly due to reduced susceptibility of the conjugates to systemic pro teases.

To summarise, this pharmacokinetic study in mice revealed that insulin modified by both polysialylation techniques resulted in conjugates that were retained in the plasma longer than the native hormone after intravenous injection. Furthermore, comparisons of pharmacokinetic parameters such as AUC, Clp, Vd, elimination half-life (ti /2 p), p elimination rate and MRT suggest that a correlation exists between degree of modification and systemic plasma clearance. Indeed these findings are consistent with those of Knauf et al (1988). Unfortunately, the impracticality of intravenous administration of insulin precludes this approach thus; subcutaneous injections are currently the predominant mode of insulin delivery (Hoffinan and Ziv, 1997). Although, further work must be done to fully characterise these conjugates in terms of immunological properties and pharmacodynamic profiles before their clinical relevance may be established, the in vivo work conducted to date on polysialylated insulin is nevertheless encouraging. When also taking into account the in vivo pharmacodynamic properties of polysialylated insulin reported by Jain et al (2002) (section 6.3) it is felt that CA-modified insulin may represent a new potential candidate for use as a soluble and fully bioactive insulin replacement. Recently, other insulin conjugates have also been reported as resembling intermediate acting insulin

260 Chapter Five: Pharmacokinetic properties o f polysialylated bioconjugates preparations for example, carboxymethyl dextran (CMD) coupled insulin (Baudys et aL, 1998) and mPEG coupled insulin (Hinds et aL, 2000). It is however realised that although many formulations exist, there is much work still to be done to further facilitate and improve insulin therapy.

261 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates

5.4 Conclusions

In this study, the radioiodination method of choice was the chloramine-T method adopted from Greenwood and Hunter (1963). However owing to its harsh oxidative nature (Franker & Speck, 1978) it was feared that this approach might influence the clearance pattern of radiolabelled proteins. Thus the milder lodo-Gen method was considered as an alternative. To that end, preliminary investigation involved radiolabelling IgG with ^^^I via both methodologies for comparison. Results showed that iodinated IgG tracers prepared by either method were of equally high specific activity (-0.49-0.9pCi/pg protein), comparable labelling efficiency (-90.0%) and purity (> 90.0%). Also, no significant differences were observed between pharmacokinetic parameters derived for native and polysialylated IgG conjugates prepared by both radioiodination techniques. Consequently, the chloramine-T method was applied to aprotinin and insulin respectively and resulted in iododerivatives with > 80.0% incorporation of ^^^I, no less than 99.0% purity and specific activity of -0.5pCi/pg protein. The radiolabelled IgG, aprotinin and insulin tracers were used to produce polysialylated peptide and protein conjugates with good specific activity (0.55-0.71 mCi/mg protein) and adequate labelling efficiency (63-90%) that were all independent of the extent of polysialylation. These results justified the suitability of the chloramine-T method for preparing radiolabelled tracers for incorporation into native and CA-modified peptides and proteins for the purpose of studying their in vivo plasma clearance.

Prior to the in vivo investigations, the iodinated native (controls), polysialylated and polysialylated SDS-modified IgG, aprotinin and insulin conjugates were successfully characterised by SEC (Figs. 5.3, 5.4, 5.5). The plasma clearance profiles obtained after intravenously injecting mice with native and polysialylated IgG, aprotinin and insulin conjugates were all characteristic of an apparent two-compartment model and analysed using a biexponential decay curve of best fit. Analysis of the pharmacokinetic data revealed that although the distribution half-lives (ti/iu) of polysialylated IgG, aprotinin and insulin conjugates were improved, unfortunately they were not significantly different from the native peptide or protein. Interestingly,

262 Chapter Five: Pharmacokinetic properties of polysialylated bioconjugates upon further modification only aprotinin was significantly improved. In contrast, only the elimination half-life (ti/iP) of polysialylated IgG was not significantly improved in comparison with the native protein. However, upon further modification, all three peptides showed a significant increase in their elimination half-life. A comparison of the other pharmacokinetic parameters for native, polysialylated and polysialylated SDS-modified IgG, aprotinin and insulin revealed that whilst AUC and MRT were increased with increasing degree of modification, the apparent volume of distribution (Vd), plasma clearance (Clp) and P elimination rate were all significantly reduced. The only exception was the non-significant increase in MRT of polysialylated IgG in comparison with the native protein. The lack in significant improvement of specific pharmacokinetic parameters observed for certain polysialylated peptides and proteins might be attributed to either the insufficient degree of protein modification at that level or the relatively small molecular weight (average mol. wt lOKDa) of CA used. Similar findings were reported for PEG-gulonolactone oxidase (Hadley et al., 1989). Despite these findings, the results collectively suggest that a correlation exists between degree of modification and systemic plasma clearance. Indeed, Knauf et al. (1988) reported similar findings for interleukin-2 chemically modified with water- soluble polymers. Interpretation of the pharmacokinetic data derived for each peptide and protein construct in the context of clearance mechanisms is complex. However, it is arguable that the increased plasma residence of CA-modified peptide and protein conjugates may be due to circumvention of hepatic and renal clearance and avoidance of plasma proteases. Although the immunological properties remain to be explored, these results are encouraging as they highlight the potential pharmaceutical relevance of polysialylation as an alternative means of improving the therapeutic efficacy of peptides and proteins.

263 Chapter Six

Discussion and Conclusions

264 Chapter Six: Discussion and Conclusions

6.1 General discussion

Polymer conjugation is of increasing interest for the delivery of (poly) peptides, enzymes and other proteins. These biotherapeutics have been coupled to natural and synthetic polymers to ameliorate their inherent unfavourable properties such as short plasma half-life, immunogenicity and instability, thus facilitating their therapeutic and diagnostic potential. Among the many successful conjugates are those produced from mainly water soluble or amphiphilic polymers such as dexrans, styrenemaleic acid anhydride or monomethoxypoly(ethylene glycol) (mPEG). PEGNOLOGis the most acclaimed, however because of the non-biodegradable nature of PEG and accumulation in tissues on chronic use, research has been directed into finding alternative macromolecules to enhance the efficacy of peptide and protein therapeutics.

Recently, naturally occurring biodegradable, hydrophilic polymers of N-acetyl neuraminic acid (PSAs) have emerged as a promising alternative to mPEG. Polysialic acid, namely low molecular weight colominic acid (CA) has been used to augment the half-life of proteins (Fernandes and Gregoriadis, 1997), small molecules such as conventional drugs and peptides (Gregoriadis et aL, 1993) and other large biopolymers and microparticles, such as liposomes (Zhang, 1999). On the basis of these findings, this thesis attempted to covalently conjugate a number of smaller peptides and other proteins to polysialic acid, (polysialylation), as a means to improve their therapeutic efficacy. In pursuit of developing polysialylated peptide and protein conjugates a number of novel methodologies were applied and validated. There were four main research areas studied within the framework of this thesis. The synthesis and characterisation of polysialylated peptides and proteins, the optimisation of efficiency of the polysialylation process, the investigation into the effects of polysialylation on the stability and biological properties of peptides and proteins, and the in vivo pharmacokinetic characterisation of native and polysialylated constructs. A summary of the main findings, correspesponding conclusions, implications/ recommendations for further work and future prospects are covered in this chapter.

265 Chapter Six: Discussion and Conclusion

As reviewed in Chapter 1, a variety of peptide and protein therapeutics have been conjugated to various natural and synthetic hydrophilic polymers such as albumin, dextran, mPEG, and SMA to achieve efficacious delivery of peptide or protein pharmaceuticals. Although these polymers have been successfully exploited with peptides and proteins, their application is sometimes limited due to their immunological or non-biodegradable characteristics. Thus, the low molecular weight and poorly immunogenic derivative of polysialic acid, known as colominic acid has been proposed, as an alternative to these polymers for the delivery of short-lived drugs and small peptides.

In Chapter 2, we have described various methodologies required for the characterisation and elucidation of peptide and protein conjugates, that were synthesised using the novel patented approach to peptide and protein delivery, known as polysialylation. Preliminary research involving the characterisation of colominic acid (CA), periodate oxidised CA and the synthesis of CA-modified L-lysine are explored in Chapter 3. Various spectroscopic and chemical techniques such as mass spectrometry (MS), nuclear magnetic resonance (NMR), Fourier transfbrm-infi-ared spectroscopy (FT-IR) and the 2,4-dinitrophenylhydrazine (DNP) test were applied in an attempt to identify the aldehyde functionality of periodate oxidised CA. Results showed that only the latter colorimetric test was able to indicate periodate oxidation had successfully introduced aldehyde functionality to CA. The 2,4-DNP method is a qualitative method, which was unable to quantify aldehyde content or determine absolutely the site of CHO introduction. Conjugation of oxidised CA to L-lysine, offered a convenient and simple model for the polysialylation of proteins. The apparent degree of modification revealed a stoichiometric reaction between CA and L- Lysine and the 3-fold improved coupling yield compared with the control suggests that periodate oxidation facilitated coupling. Having demonstrated the potential to polysialylate L-lysine by the reductive amination reaction, four broadly categorised macromolecules, namely IgG (a polyclonal antibody) the peptide hormones insulin and somatostatin and the serine protease inhibitor aprotinin were similarly modified. The yield of conjugation was expressed as a molar ratio of CA to protein and also as the percentage of available amino residues of the protein modified with CA. The

266 Chapter Six: Discussion and Conclusion conjugation ratios were determined by chemical methods and by spectrophotometric analysis as described in Chapter 2. Size exclusion chromatography (SEC) was employed in the purification and characterisation of the polysialylated constructs. Evidence to prove the synthesis of the polysialylated peptides and proteins was not definitively confirmed by employing SEC, however the emergence of ‘new’ heavier entities were identified with both CA and the protein co-eluting in the same volume of the column. Further evaluations are suggested to confirm the presence of a covalent link and rule out other types of bonding. IgG and aprotinin (chapter 4) conjugates were further characterised by employing SDS-PAGE, which similarly suggested the presence of the neoglycoproteins respectively. Interestingly, covalent conjugation of colominic acid to poorly water soluble proteins i.e. insulin enhanced their solubility at physiological pH and permitted the design of stable formulations for intravenous use. Table 6.1 summarises the physicochemical properties of the 4 peptides and proteins introduced in Chapter 3 and includes catalase introduced in Chapter 4.

Table 6.1 Physicochemical properties of the peptides and proteins used in this thesis. The isoelectric point is represented by pi, which is the pH at which the protein carries no charge. Mouse anti-bovine serum albumin monoclonal antibody (IgG2a) is not included as it is differentiated from IgG by only small differences in its amino acid sequences in the constant region of the heavy chain. Number of e-lysiue Molecular Number of disulphide Protein pi & (N-termiual NHz) weight (Da) bonds (S-S bonds) residues IgG 150, 000 9.1 4 92(4) Aprotinin 6,512 9.24 3 4(1) Insulin 5,733.5 5.3 3 1(2) Somatostatin 1638 8.91 1 2(1) Catalase 24,000 5.4 4 108(1)

It was demonstrated that the degree of polysialylation of the peptides and proteins used was directly related to the conditions of polysialylation and reactivity’s of constituent components. Even after considerable manipulation of the process variables, the degree of modification achieved with the polysialylated peptides and proteins was considered poor to moderate, based on the percentage of available N-

267 Chapter Six: Discussion and Conclusion terminal and 8-lysine residues of the protein modified with CA, the only exception being somatostatin (table 6.2). Interestingly, it emerged that the secondary structural conformation of peptides and proteins was a contributory factor in limiting the conjugation yield. Thus, in an attempt to further improve the conjugation yield and therapeutic value of polysialylation in vivo, the next challenge pertained to develop a novel strategy to expose a greater number of free amine groups of the protein and facilitate increased coupling with CA (Chapter 4). To that end, the anionic detergent sodium dodecyl sulphate (SDS) was introduced into the coupling reaction. It was anticipated that chemical modification by polysialylation and indeed SDS could potentially result in the concomitant loss of biological activity of the peptides and proteins, as has previously been reported by Fernandes and Gregoriadis (1996). Thus, the novel polysialylation process involved applying a suitable concentration of SDS that allowed reversible unfolding of the protein molecule such that biological activity was preserved. The initial investigation utilised catalase as a model protein to develop the technique. The optimal concentration of SDS found to completely reversibly inactivate the enzyme resulted in partially reversibly unfolding of both IgG and aprotinin (section 4.3.6.3). Consequently, the four peptides and proteins mentioned previously and catalase and IgG2a newly introduced in Chapter 4, were all subjected to SDS treatment and polysialylated. The benefit of SDS-modification was seen when the conjugates were found to contain up to three fold more coupled CA than by the standard polysialylation process. These results are encouraging as they suggest that SDS may have increased the availability of reactive amine groups for modification with CA and lead to greater polysialylation yields (table 6.2).

Biological potency and stability studies conducted in Chapter 4 offered an opportunity to assess the effect of both methods of polysialylation on the stability (under coupling conditions) and activity of the monoclonal antibody IgG2a, aprotinin and catalase. Polysialylation via either method conferred increased stability to all three molecules relative to their native counterparts at the end of the coupling reaction. Indeed, steric stabilisation of proteins after coupling with polymers has been commonly observed (Fernandes and Gregoriadis, 1996; Abuchowski ct ai, 1977). It was noteworthy that the residual biological activities of IgG2a and catalase were only

268 Chapter Six: Discussion and Conclusion marginally reduced with increasing degree of polysialylation. Polysialylated SDS- modified aprotinin however, showed a significant decrease in potency compared with aprotinin polysialylated by the standard method. Although SDS-modified proteins were expected to suffer severe loss of activity upon polysialylation, the results suggest that significant biological activity was largely maintained for all the polysialylated constructs (table 6.2). This suggested that the polysialylation procedure (or perhaps the presence of CA in the mixture) protects proteins fi*om inactivation. The reason for the considerable preservation of activity of polysialylated SDS-modified proteins even after partial reversible unfolding of the protein molecules by SDS is unclear. However, at the level of modification achieved with SDS-modified conjugates, it is hypothesised that CA behaves like PEG as described by Buchner et al. (1991). Thus, re-folding of the protein molecule on removal of the dénaturant is enhanced in the presence of CA and the molecule resumes its correct structure such that biological activity is unaffected. Further investigations into this subject are however warranted. Collectively, these results are testimony to the correlation that exists between the degree of modification of peptides and proteins and their resultant biological activity. They also suggest the gentle nature of the polysialylation process, which is in contrast to PEGylation known to often lead to drastic loss of activity.

Enzyme kinetic studies were performed using catalase to study the effects of polysialylation on the efficacy of the enzyme. The results showed significantly increased Km values with increasing degree of polysialylation, indicating a reduction in the catalase enzyme affinity for the substrate. It is arguable that the increasing partial loss in enzyme-substrate affinity observed with both polysialylated catalase conjugates arises as a result of changes in the microenviroment of the enzyme caused by the increasing hydrophilicity and negative charge of the coupled CA chains. It could also be explained by conformational changes of the enzyme upon polysialylation that affect the enzyme-substrate complex. Although it was considered that steric interference of the bulky CA chains could prohibit the substrate fi*om diffusing into the active site of the enzyme, this was thought unlikely on account of the low molecular weight of the substrate (H 2 O2 ).

269 Chapter Six: Discussion and Conclusion

The pharmcokinetic properties of radiolabelled IgG, aprotinin and insulin modified by both methods of polysialylation were evaluated after intravenous administration in mice in Chapter 5. In each case, plasma clearance profiles characteristic of an apparent two-compartment model were observed. Analysis of the pharmacokinetic parameters for each protein revealed that increasing the extent of polysialylation resulted in reducing clearance rates and increasing the longevity of the peptide or protein in circulation (table 6.2). The lack of significant improvement of the distribution half-life (ti /2 a) for the polysialylated peptides and proteins might be attributed to the insufficient degree of protein modification at that level or the relatively small molecular weight of CA used. The relationship between degree of modification and systemic exposure shown in these results was consistent with the findings for PEG-modified rIL-2 reported Knauf et al. (1988). Interpretation of the pharmacokinetic data derived for each peptide and protein with respect to clearance mechanism is complex. However, the increased systemic exposure of the polysialylated peptide and protein conjugates suggests that they are less susceptible to renal and hepatic clearance and plasma proteases that are often responsible for the rapid clearance of their native counterparts.

Table 6.2 summarises the main findings for each peptide and protein employed in this thesis pertaining to the degree of modification achieved via both methods of polysialylation, residual biological activity and in vivo pharmacokinetics. From the data represented here, it is possible to suggest that a correlation between parameters does exist as reported by Knauf et al. (1988). Although additional immunological evaluations are required, these results are nevertheless encouraging as they highlight the potential of polysialylation to overcome the limitations in using short-lived peptides and proteins in therapy whilst maintaining moderate biological potency.

270 Chapter Six: Discussion and Conclusion

Table 6.2 Comparison of the degree of modification, biological activity and pharmacokinetic evaluation for the six native and CA-modified peptides and proteins prepared by two methods of polysialylation studied in this thesis. The conjugates are all prepared using 50:1 (CA:Protein) starting molar ratios except somatostatin (SS) which involved 10:1 starting ratios. The degree of modification is expressed as moles of CA per mole protein or % of modified amine groups. Non-oxidised CA was reacted with the peptides and proteins as the control in the degree of modification experiments and peptides and proteins reacted for 48 hours in the absence of CA were included in the biological activity studies. * Indicates intravenous and subcutaneous bioactivity data reported by Jain et al (2002) and ** Not the subject of intensive study with respect to the peptide or protein in this thesis. Results are the mean ± s.d of three experiments. N/D= not determined.

Preparation Molar conjugation yields & Biological Pharmacokinetics (% modified amino residues) activity (%) Ti/2 P (h)

IgG (control) 1.25 ± 0.36: 1.0 (1.30 ± 0.38 %) - 28.89 ± 1.038

CA: IgG 4.25 ± 0.20: 1.0 (4.43 ± 0.21 %) - 32.21 ± 1.407

CA: SDS-IgG 12.27 ± 0.3: 1.0 (12.78 ± 0.34%) - 36.63 ± 0.882 IgG2a (control) 1.22 ±0.16: 1.0 N/D N/D

IgG2a (CA absent) - 41.47 ±2.9% N/D CA: IgG2a 4.04 ±0.22: 1.0 94.65 ± 1.8% N/D CA: SDS-IgG2a 12.03 ±0.3: 1.0 82.44 ± 1.3% N/D Aprotinin (control) 0.39 ± 0.03: 1.0 (7.80 ± 0.60%) N/D 11.08 ± 1.002

Aprotinin (CA absent) - 30.98 ± 0.9% N/D CA: Aprotinin 1.46 ± 0.05: 1.0 (29.20 ± 0.01%) 87.79 ± 1.9% 16.33 ± 1.019 CA: SDS-Aprotinin 4.59 ±0.05: 1.0 (91.80 ±0.01%) 59.02 ± 0.7% 17.82 ±0.629 Insulin (control) 0.42 ±0.03: 1.0 (14.0 ± 1.0%) N/D* 11.44 ±0.106 CA: Insulin 1.90 ± 0.05: 1.0 (63.33 ± 1.67%) N/D* 17.41 ±0.343 CA: SDS-Insulin 2.92 ± 0.06: 1.0 (97.30 ± 2.0%) N/D 18.16 ± 1.255 SS (control) 0.61 ± 0.01: 1.0 (20.33 ± 0.33%) N/D** N/D** CA: SS 3.00 ± 0.01: 1.0 (100 ± 0.33%) N/D** N/D** CA: SDS-SS 3.00 ± 0.02: 1.0 (100 ± 0.67%) N/D** N/D** Catalase (control) 0.64 ±0.10: 1.0 (0.54 ±0.09%) N/D N/D**

Catalase (CA absent) - 26.30 ± 1.2% N/D** CA: Catalase 3.45 ± 0.29: 1.0 (3.17 ± 0.27%) 66.70 ± 1.5% N/D** CA: SDS-Catalase 7.85 ± 0.45: 1.0 (7.20 ± 0.41%) 63.70 ± 1.5% N/D**

271 Chapter Six: Discussion and Conclusion

6.2 Conclusions

This thesis demonstrated the effectiveness of using the novel patented polysialylation technology in enhancing and prolonging plasma levels of a spectrum of diverse therapeutic peptides and proteins, which is a prerequisite for the optimal use of peptides and proteins in therapy. In light of the reduced clearance rates, improved stability and maintenance of potency of the polysialylated peptides and proteins, the role of polysialic acid as a means to overcoming the limitations of these molecules in therapy is clearly evident. In addition, polysialylation was also found to improve the solubility of poorly soluble insulin, which is important to maintain systemic injectability. Furthermore, advances in the development of the established method of polysialylation yielded superior bioconjugates, which contributes significantly to the potential use of polysialic acids in peptide and protein delivery.

The progress made with PSA and these new biotherapeutic conjugates presents a wide range of potential commercial opportunities. Indeed, with the ever-growing demand for protein and antibody-based pharmaceuticals for the treatment of immune disorders, cancer, diabetes, growth disorders and haematological disorders, the search for new polymers is of paramount importance. Despite these promising results, further scientific evaluations of the present approach of peptide and protein drug delivery are nevertheless required to determine whether these novel bioconjugate drug candidates will translate into viable products with superior efficacy, safety and lower cost compared with alternative drug delivery systems. Suggestions for further work are detailed in section 6.3.

272 Chapter Six: Discussion and Conclusion

6.3 Further work

Within the scope of this thesis, many important goals such the effects of polysialylation on stability, activity and in vivo half-life of a variety of large and small peptides and proteins were reached providing testimony to the pharmaceutical relevance of polysialylation. However, on account of finite time and resources, many unexplored issues remain critical for the development of clinically feasible peptide and protein drug candidates in the form of polysialylated derivatives. Some of the key issues as well as further opportunities for study are suggested below in order of priority with respect to the investigations undertaken in this project.

In order to ascertain the full potential of polysialylated insulin conjugates as bioactive insulin substitutes it is necessary to investigate their in vivo pharmacokinetic and pharmacodynamic dispositions via both the intravenous and subcutaneous routes of administration. Jain et al (2002) have recently conducted investigations of this nature, although their investigations did not include polysialylated SDS-modified insulin conjugates. Interestingly, they reported that the polysialylated insulin conjugate was bioactive and exhibited a protracted pharmacodynamic profile (blood glucose lowering effect) after intravenous or subcutaneous injection, suggesting a prolonged circulating time. The effect of disease state on the efficacious performance of the insulin conjugates is also important and therefore it would be advantageous to conduct these studies in diabetic and normal animals for comparison.

Having established the in vitro biological activities of specific polysialylated peptide and protein conjugates, further investigations are required to determine their in vivo potencies. This is essential when gauging the potential of these polysialylated peptides and proteins for use in the treatment of disease, targeting strategies or as a diagnostic tool.

The effect of polysialylation on the stability of enzymes in the presence of proteolytic enzymes and blood plasma has been previously demonstrated (Fernandes and Gregoriadis, 1996). However, it is considered important to evaluate the effects of such

273 Chapter Six: Discussion and Conclusion agents on the clearance of the peptides and proteins under study in this project. Whilst polysialylation is a suitable method to increase systemic exposure of short-lived peptides and proteins, the long-term immunological effects from chronic administration of polysialylated peptide or protein conjugates may preclude their use in therapy (Fernandes, 1996). It is therefore crucial to evaluate the immunological properties of the polysialylated peptide and protein conjugates versus their native counterparts. Such an investigation should evaluate their immunogenicity (ability to induce the production of specific antibodies), antigenicity (ability to react with pre formed antibodies), and the role of specific antibodies in the clearance of native and polysialylated constructs.

Although polysialic acid is biodegradable, its biodegradability after coupling to peptides and proteins is unknown and therefore warrants further investigation. Exosialidases are not expected to act on polysialic acid-protein conjugates since the terminal non-reducing end of the saccharide where lytic activity is exerted (Saito and Yu, 1995) is involved in the covalent linkage. Endosialidase enzymes on the other hand, allow bacteriophage to infect bacteria with capsular polysialic acids. Owing to the acid sensitive nature of the internal a-(2-8) linkages of polysialic acid (McGuire and Binkley, 1964), it is possible that degradation may occur in intracellular compartments such as lysosomes where the pH is low. Indeed, this mechanism was reasoned to be responsible for the terminal degradation of N-CAM polysialic acids (Rougon, 1993). It is also plausible that after degradation of the protein moiety, the remnant polysialic acid chains might be susceptible to degradation by exosialidases.

Biodistribution studies are equally essential as they offer valuable insight into the clearance mechanism of the native and polysialylated constructs and indicate the possibility of their accumulation in tissues upon chronic use. They may also elucidate whether polysialylated constructs show organ specificity and thus the potential for organ targeting. Further evaluations pertaining to the ability/inability of polysialylated conjugates to cross anatomical barriers en route to target cells is required if the coupled protein needs to penetrate intracellular compartment or bind with specific membrane receptors to exert their therapeutic effect.

274 Chapter Six: Discussion and Conclusion

It appears that the degree of polysialylation of peptides and proteins was substantially improved when SDS was introduced into the process and resulted in improving their in vivo pharmacokinetics whilst retaining considerable biological activity. Although this novel strategy is recommended for the future development of polysialylated peptide and protein pharmaceuticals, further investigations into the interactions between SDS and CA-modified peptides and proteins are warranted.

Despite the advances made in the development of polysialylated peptide and protein pharmaceuticals for clinical use, their future approval by the USA Food and Drug Administration (FDA) is currently jeopardised. This is due to the fact that polysialylated peptides and proteins yield a complex and heterogeneous mixture of end products. The heterogeneity of polysialic acid makes it is difficult to fractionate the different molecular weight entities and purify the single species of conjugates. It is also difficult to accurately establish the molecular weight of the conjugates or characterise them by conventional methods such as SEC and SDS-PAGE. In terms of gaining regulatory approval by licensing authorities in the future, it is advisable that polysialylated conjugates are of predetermined molecular weight that are well characterised.

Further opportunities worth exploring involve using higher molecular weight poly-a- (2-8) polysialic acids to increase the systemic exposure of other smaller proteins for example superoxide dismutase which has been modified with mPEG and is currently in clinical trials (Enzon Inc.). Continued research with polysialylated somatostatin is recommended to determine the advantages of this construct versus the octapeptide analogue octreotide (Sandostatin). The broader applications of polysialylation could also be investigated with respect to allergens, interferons, interleukins and conventional drugs such as Paclitaxel. Against the background of the conventional pharmaceutical industry the development of polysialylated peptide and protein pharmaceuticals in this newly emerging arena presents a number of challenges that must be met in order to translate this innovative research into novel products which satisfy market need.

275 Chapter Six: Discussion and Conclusion

6.4 Future prospects

Polysialylation is a promising technology that is not only limited to enhance the delivery of protein pharmaceuticals, but presents exciting new avenues for its application in the future. A few of the possibilities envisaged are discussed below.

Research into polymer conjugation of labile oligonucleotides, including antisence drugs is in its infancy, yet preliminary results seem very promising. Polysialylation of these compounds could potentially improve their stabilisation towards nucleases and render them more effective in vivo.

Surface modification of other drug-carrier technologies such as nano-microparticles, and liposomes may also benefit fi*om polysialylation. Indeed, polysialylation of the latter system has already been shown to be fruitful with the production of ‘stealth’ liposomes (Zhang, 1999). Similarly, polysialic acid may be applied to other new structures that are currently under development such as niosomes and dendrimers to further their therapeutic applications. The latter structures have demonstrated great potential in drug delivery (Delong et a/., 1997), owing to the complex but extremely precise architecture of their huge synthetic structure. Polysialylated micelles are another interesting class of potentially useful drug carriers.

A further application of polysialic acid takes inspiration fi*om nature’s own strategy for trafficking molecules in the body. For example, antibodies and antibody fi'agments such as F(ab) and F(ab’)2 have been utilised for the purpose of directing drugs to specific tumour tissues (Colcher et al., 1998). Whilst antibody fragments are used in preference to intact monoclonal antibodies as their smaller size enables better tumour penetration, they exhibit very short half-lives in vivo. Thus, polysialylation of these fragments could potentially be of benefit to their diagnostic and drug targeting potential.

276 Chapter Six: Discussion and Conclusion

Finally, the combination of polysialylated bioactive molecules and their entrapment into particles could be developed to exploit the best achievements of both technologies.

Many of these innovative technologies are emerging as a major development in the field of novel therapeutics and drug delivery systems. Polymer therapeutics such as Oncospar® and Adagen® are already approved by regulatory authorities for routine clinical use and treatments for cancer, and a large number of constructs are currently under evaluation as novel treatments for AIDS and other diseases (section 1.5.2.2). The chemical orthogonality and advantageous biological characteristics of polysialic acid over other natural and synthetic polymers provides optimism and impetus for reaching the ambitious possibility of tailoring today’s drug delivery problem into tomorrow’s successful therapeutic.

277 Hpa

References

278 ______References

References

Abuchowski, A., McCoy, J.R., Palczuk, N.C., Es, T. V. & Davis, F.F. (1977) Effect of covalent attachment of polyethylene glycol on immunogenicity and circulation of hovine liver catalase, J. Biol Chem., 252, 3882-3886.

Abuchowski, A. & Davis, F.F. (1979) Preparation and properties of polyethylene glycol-trypsin adducts, Biochim. Biophys. Acta., 578, 41-46.

Abuchowski, A., Kazo, G.M., Verhoest, C.R., Jr., van Es, T., Kafkewitz, D., Nucci, M.L., Viau, A.T. & Davis, F.F. (1984) Cancer therapy with chemically modified enzymes. I. Antitumour properties of polyethylene glycol-asparaginase conjugates. Cancer Biochem. Biophys., 7, 175-186.

Abuchowski, A., Me Coy, J.R., Palczuck, N.C., Van Es, T. & Davis, F.F. (1997) Alteration of immulogical properties of bovine serum albumin by covalent attachment of poly(ethylene glycol), J. Biol Chem., 252, 3578-3581.

Aebi, H.E. (1983) Catalase, in H.U. Bergmeyer, J. Bergmeyer & M. Grasl (eds.). Methods of Enzymatic Analysis, pp. 273-286, Weinheim, Verlag Chemie.

Algranati, N.E., Sy, S. & Modi, M. (1999) A branched methoxy 40KDa polyethylene glycol (PEG) moiety optimises the pharmacokinetics (Pk) of peginterferon a-2a (PEG-IFN) and may explain its enhanced efficiency in chronic hepatitis C (CHC) [abstract], Hepatology., 30, 401-402.

Allen, T.M. & Chonn, A. (1987) Large unilamellar liposomes with low uptake by the reticuloendothelial system, FEBS Lett., 223, 42-46.

Ananthapadmanabhan, K.P. (1993) in K.P. Ananthapadmanabhan & E.D. Goddard (eds.). Interactions of Surfactants with Polymers and proteins, pp. 319, Boca Raton, FL, CRC Press.

279 ______References

Andrews, P. (1965) The gel filtration behaviour of proteins related to their molecular weights over a wide range, Biochem. J., 96, 595-606.

Anfinsen, C.B. (1973) Principles that govern the folding of protein chains. Science., 181, 223-230

Azanza Perea, J.R. (2001) (Review) Pegylated interferons: preliminary review of their pharmacokinetic characteristics. Revista. Clinica. Espanola., 201, 205-212.

Banting, F. G. & Best, C.H. (1922) Pancreatic extracts, J. Lab. Clin. Med., 7, 464-472.

Barker, S.A. & Somers, P.J. (1968) Preparation and properties of a-amylase chemically coupled to microcrystalline cellulose, Carbohyd. Res., 8, 491-497.

Battersby, J., Clark, R., Hancock, W., Puchulu-Campanella, E., Haggarty, N., Poll, D. & Harding, D. (1996) Sustained release of recombinant human growth hormone fi*om dextran via hydrolysis of an imine bond, J. Controlled Release., 42, 143-156.

Baudys, M., Letoumeur, D., Liu, P., Mix, D., Jozefonvicz, J. & Kim, S.W. (1998) Extending insulin action in vivo by conjugation to carboxymethyldextran, Bioconj. Chem., 9, 176-183.

Beauchamp, C.O., Gonias, S.L., Menapace, D.P. & Pizzo, S.V. (1983) A new procedure for the synthesis of polyethylene glycol-protein adducts: effects on function, receptor recognition and clearance of superoxide dismutase, lactoferrin and a2-macroglobulin, Biochem., 131, 25-33.

Beckman, J.S., Minor, R.L., White, C.W., Repine, I.E., Rosen, G.M. & Freeman, B.A (1988) Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance, J. Biol. Chem., 263, 6884-6892.

280 ______References

Beers, R.F. & Sizer, I.W. (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, J. Biol. Chem., 195, 133-140.

Benbough, J.E., Wilbin, C.N., Rafter, T.N.A. & Lee, J. (1979) The effect of chemical modification of L-asparaginase on its persistence in circulating blood of animals, Biochem. Pharmacol, 28, 833-839.

Bentley, M.D. & Harris, J.M. (1999) Polyethylene glycol aldehyde hydrates and related polymers and applications in modifying, US Patent 5., 990, 237.

Bentley, M.D., Harris, J.M. & Kozlowski, A. (2001) Heterobifunctional polyethylene glycol derivatives and methods for their preparation, WO, 126692A1.

Berard, J.L. (1999) A review of interleukin-2receptor antagonists in solid organ transplantations, 19, 1127-1137.

Besson, C., Favre-Bonvin, G., O’Fagain, C. & Wallach, J. (1995) Chemical derivatives of Pseudomonas aeruginosa elastase showing increased stability. Enzyme and Microbial Technology., 17, 877-881.

Bhattachaijee, A.K., Jennings, H.J., Kenny, C.P., Martin, A & Smith, I.C.P. (1975) Structural determination of the sialic acid polysaccharide antigens of Neisseria meningitidis serogroups B and C with carbon 13 nuclear magnetic resonance, J. Biol. Chem., 250, 1926-1932.

Biscayart, P.L., Paladini, A.C., Vita, N. & Roguin, L.P. (1989) Preparation of 125- labeled human growth hormone of high quality binding properties endowed with long term stability, J. Immuno., 10, 37-56.

Bloom, S.R. & Polak, J.M. (1987) Somatostatin, BMJ., 295, 288-290.

281 ______References

Blundell, T.L., Dodson, G.G., Hodgkin, D.C. & Mercola, D. (1972) Insulin: The structure in the crystal and its reflection in chemistry and biology. Adv. Protein Chem., 26, 279-402.

Boag, F., Chappell, M., Becket, A.G. & Dandona, P. (1985) Lipohypertrophy and glomerulonephritis after the use of aprotinin in an insulin-dependent diabetic. New Engl. J. Med., 312, 245-246.

Bocci, V. (1987) Metabolism of protein anticancer agents, Pharmac. Ther., 34, 1-49.

Bocci, V. (1990) catabolism of therapeutic proteins and peptides with implications for drug delivery. Adv. Drug. Del. Rev., 4, 149-169.

Bock, E., Yavin, Z., Jorgensen, O.S. & Yavin, E. (1980) Nervous system-specific proteins in developing rat cerebral cells in culture, J. Neurochem., 35, 1297-1306.

Bonneaux, P., Dellacherie, E., Labrude, P., Vigneron, C. (1996) Hemoglobin- dialdehyde dextran conjugates: Improvement of their oxygen-binding properties with anionic groups, J. Protein Chem., 15, 461-465.

Borch, R.F., Bernstein, M.D. & Durst, H.D. (1971) The cyanohydridoborate anion as a selective reducing agent, J. Am. Chem. Soc., 93, 2897-2904.

Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem., 72, 248-254.

Braeckman, R. (2000) Pharmacokinetics and pharmacodynamics of protein therapeutics, in R.E. Reid (ed.). Drugs and the pharmaceutical sciences. Peptide and Protein Drug Analysis, pp. 633- 669, Marcel Decker, New York.

282 ______References

Brazeaus, R., Vale, W. & Burgus, R. (1983) Hypothalamic peptide that inhibits the secretion of immunoactive pituitary growth hormone, Science., 179, 77-79.

Breimer, D.D. (1991) Pharmacokinetic and pharmacodynamic basis for peptide and protein drug delivery, in Peptides. Theoretical and Practical Approaches to their Delivery, pp. 107-111, Capsugel Library.

Brekke, O.K. & Sanlie, T. (2003) Therapeutic antibodies for human disease at the dawn of the twenty-first century, Nat. Rev. Drug. Discov., 2, 52-62.

Brenner, B.M., Hostetter, T.H.& Humes, H.D. (1978) Glomerular permselectivity: barrier function based on discrimination of molecular size and charge. Am. J. Physiol, 234, (455-460).

Brenner, B. & Rector Jr, F. (1996) The kidney, in B. Brenner & F. Rector Jr (eds.), 5^ ed. Philadelphia (PA), W.B. Saunders Company.

Breslow, D.S., Edwards, E.I. & Newborg, N.R. (1973) Divinyl ether maleic anhydride (pyran) copolymer used to demonstrate the effect of molecular weight on biological activity, 246, 160-162.

British Pharmacopoeia (1973) Appendix 28, HMSO, London.

British Pharmacopoeia (1999) Vol. 1. The Stationary Office, pp. 1320-1322, London

Brucato, F. H. & Pizzo, S. V. (1990) Catabolism of streptokinase and polyethylene glycol-streptokinase: evidence for transport of intact forms through the bilary system in the mouse. Blood., 76, 73-79.

Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X. & Kiefhaber, T. (1991) GroE facilitates refolding of citrate syntheatase by suppressing dig^Qgàiion, Biochemistry., 30, 1586-1591.

283 ______References

Bukowski, R., et ah (2002) Pegylated interferon a-2b treatment for patients with solid tumours: a phase I/II study, J. Clin. OncoL, 20, 3841-3849.

Caliceti, P., Morpurgo, M., Schiavon, O., Monfardini, C. & Veronese, P.M. (1994) Preservation of thrombolytic activity of urokinase modified with monomethoxy poly(ethylene glycol), J. Bioact. Compat. Polym., 9, 252-266.

Caliceti, P., Schiavon, O., Morpurgo, M. & Veronese, P.M. (1995) Physico-chemical and biological properties of monofunctional hydroxy terminating poly(N- vinylpyrrolidone) conjugated superoxide dismutase, J. Bioact. Compat. Polym., 10, 103-120.

Carlsson, S.R. (1993) Isolation and characterisation of glycoproteins, in M. Pukuda & A. Kobata (eds.) Glycobiology, pp. 1-26, Oxford University Press.

Caron, A., Menu, P., Paivre-Piorina, B., Labrude, P. & Vigneron, C. (1999) The effects of stroma-fi*ee and dextran-conjugated haemoglobin on haemodynamics and carotid blood flow in haemorrhaged guinea pigs. Artificial cells, Blood Substitutes, & Immobilization Biotechnology, 27, 49-64.

Carpenter, J.P. & Crowe, J.H. (1989) An infrared spectroscopic study of the interactions of carbohydrates with dried protein. Biochemistry., 28, 3916-3922.

Carpenter, J.P., Prestrelski, S.J. & Arakawa, T. (1993) Separation of freezing- and drying-induced dénaturation of lyophilised proteins using stress-specific stabilisation. I. Enzyme activity and calorimetric studies. Arch. Biochem. Biophys., 303, 456-464.

Carter, D. C. & Ho, J.X. (1994) Structure of serum albumin. Adv. Protein. Chem., 45, 153-203.

284 ______References

Catty, D. & Raykundalia, C. (1989) ELISA and related enzyme immunoassays, in Antibodies: A Practical Approach Vol. //, pp. 97-154, Oxford, Oxford University Press.

Chan, W.L., Shaw, P.C., Li, X.B., Xu, Q.F., He, X.H. & Tam, S.C. (1999) Lowering of Trichosanthin immunogenicity by site-specific coupling to dextran. Biochem. Pharmacol., 57, 927-934.

Chapman, A.P., Antoniw, P., Spitali, M., West, S., Stephens, S. & King, D. (1999) Therapeutic antibody fi'agments with prolonged in vivo half-lifes, Nat. BiotechnoL, 17, 780-783.

Chapman, A.P. (2002) PEGylated antibodies and antibody fi'agments for improved therapy: a review. Adv. Drug. Del. Rev., 54, 531-545.

Chatelut, E., Rostaing, L., Grégoire, N., et al. (1999) A pharmacokinetic model for alpha interferon administered cubcutaneously, Br. J. Clin. Pharmacol., 47, 365-371.

Chauvet, J. & Archer, R. (1967) The reactive site of the basic trypsin inhibitor of pancreas. Role of lysine 15, J. Biol. Chem., 242, 4274-4275.

Chen, R.H.L., Abuchowski, A., Van Es T, Palczuk, N.C. & Davis, F.F. (1981) Properties of two urate oxidase modified by the covalent attachment of poly(ethyleneglycol), Biochim. Biophys. Acta., 660, 293-298.

Cho, J.W. & Tory, F.A. (1994) Polysialic acid engineering: Synthesis of polysialylated neoglycosphingolipid by using the polysialtransferase from neuroinvasive Escherichia coli Kl, Proc. Natl. Acad. Sci. USA., 91, 11427-11431.

Chrubasik, J., Meinadier, J., Blond, J. et al., (1984) Somatostatin, a potent analgesic. Lancet., 2, 1208-1209.

285 ______References

Chytry, V., Letoumeur, D., Baudys, M. & Jozefonvicz, J. (1996) Conjugates of insulin with copolymers of N-(2-hydroxypropyl)methacrylamide: effects on smooth muscle cell proliferation, J. Biomed. Mater. Res., 31, 265-272.

Cleland J.L., Hedgepeth, C. & Wang, D.I.C. (1992) Polyethylene glycol enhanced refolding of bovine carbonic anhydrase B, J. Biol. Chem., 267, 13327-13334.

Colcher, D., Pavlinkova, G., Beresford, G., Booth, B.J.M., Choudhury, A. & Batra, S.A. (1998) Pharmacokinetics and biodistribution of genetically-engineered antibodies, Q. J. Nucl. Med., 42, 225-241.

Combes, D., Graber, M. & Ye, W.N. (1990) Stabilising effect of polyhydric alcohols, Ann. New York Acad. Set., 613, 559-563.

Compton, S.J. & Jones, C.G. (1985) Mechanism of dye response and interference in the Bradford protein assay. Anal. Biochem., 151, 369-374.

Creighton, T.E. (1979) Intermediates in the refolding of reduced ribonuclease A, J. Mol. Biol, 129, 235-264.

Cunningham-Rundles, C., Zhuo, Z., Griffith, B. & Keenan, J. (1992) Biological activities of polyethylene-glycol immunoglobulin conjugates. Resistance to enzymatic degradation,/. Immunol. Methods., 152, 177-190.

Davis, F.F., et al. (1978) Enzyme polyethylene glycol: modified enzymes with unique properties, Eng., 4, 169-173.

Davis, F.F., Kazo, G.M., Nucci, M.L. & Abuchowski, A. (1991) Reduction of immunogenicity and extension of circulating half-life of peptides and proteins, in V.H.L. Lee (ed.). Peptide and Protein Drug Delivery, pp. 831-864, New York, Marcel Dekker, Inc.

286 ______References

Davis, R. & Whittington, R. (1995) Aprotinin. A review of its pharmacology and therapeutic efficacy in reducing blood loss associated with cardiac surgery. Drugs., 49, 954-983.

Deisseroth, A. & Bounce, A.L. (1970) Catalase: physical and chemical properties, mechanism of catalysis, and physiological role. Physiol. Rev., 50, 319-375.

Delgado, C., Francis, G.E. & Fisher, D. (1992) The uses and properties of PEG-linked proteins, Crit. Rev. Therap. Drug Carrier Syst, 9, 249-304.

Du, J., Hiltunen, J., Marquez, M., Nilson, S. & Holmberg, A.R. (2001) Technetium- 99m labelling of glycosylated somatostatin-14, Applied Radiation and Isotopes. 181-187.

Duncan, R. & Spreafico, F. (1994) Polymer Conjugates. Pharmacokinetic considerations for design and development, Clin. Pharmacokinet., 27, 290-306.

Duncan, R. (2003) The dawning era of polymer therapeutics (review), Nat. Rev. Drug. Discov., 2, 347-360.

Dyer, J.R. (1956) Use of periodate oxidations in biochemical analysis. Methods Biochem. Anal., 3, 111-152.

Edelman, G.M., Cunningham, B.A., Einar Gall, W., Gottlieb, P.D., Rutishauser, U. & Waxdal, M.J (1969) The covalent structure of an entire yG immunoglobulin molecule, Proc. Natl. Acad. Sci. USA., 63, 78-85.

Eremin, A.N., Litvinchuk, A.V. & Metelitsa, D.I. (1996) Operational stability of catalase and its conjugates with aldehyde dextrans and superoxide dismutase, Biokhimiia, 61, 664-679

287 ______References

Erickson, B.H. & Nosanchuck, T.A. (1992) Understanding Data, 2nd ed., Buckingham, Open University Press.

Evans, W.S., Sollenberger, M.J. & Lee Vance, M. (1998) Hypothalamic Pituitary Hormones, in T.M Brody et al. (eds.). Human pharmacology: molecular to clinical, pp. 571-583, Missouri: Mosby.

Fagnani, R., Hagan, M.S. & Bartholomew, R. (1990) Reduction of immunogenicity by covalent modification of murine and rabbit immunoglobulins with oxidised dextrans of low molecular weight. Cancer Res., 50, 3638-3645.

Fagnani, R., Halper, S. & Hagan, M. (1995) Altered pharmacokinetic and tumour localisation properties of Fab’ fi'agments of a murine monoclonal anti-CEA antibody by covalent modification with low molecular weight dextran, Nucl. Med. Commun., 16, 362-369.

Farah, R.A., Clinchy, B., Herrera, L. & Vitella, E.S. (1998) The development of monoclonal antibodies for the therapy of cancer, Crit. Rev. Eukaryotic. Gene. Expr., 8, 321-356.

Fernandes, A. (1996) Polysialylation of proteins: an approach to improve their therapeutic efficacy, PhD Thesis., University of London, U.K.

Fernandes, A. & Gregoriadis, G. (1996) Synthesis, characterisation and properties of sialylated catalase, Biochim. Biophys. Acta, 1293, 92-96.

Fernandes, A. & Gregoriadis, G. (1997) Polysialylated asparaginase: preparation, activity and pharmacokinetics, Biochim. Biophys. Acta, 1341, 26-34.

Fiddler, M.B., Hudson, L.D.S. & Desnick, R.J. (1977) Immunological evaluation of repeated administration of erythrocyte-entrapped protein to C3H/HeJ mice, Biochem. J., 168, 141-145.

288 ______References

Firme, J. (1982) Occurrence of unique polysialosyl carbohydrate units in glycoproteins of developing brain, J. Biol Chem., 257, 11966-11967.

Firme, J., Leinonen, M. & Makela, P.H. (1983) Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet., 2, 355-357.

Flanagan, P.A., Kopeckova, P., Subr, V. & Kopecek, J. (1992) Evaluation of antibody-N-(2-hydroxypropyl)methacrylamide copolymer conjugates as targetable drug carrires. 2. Body distribution of anti Thy-1.2 antibody, anti-transferrin receptor antibody B3/25 and transferring conjugates in DBA2 mice and activity of conjugates containing daunomycin against L1210 leukaemia in vivo, J. Contr. Rel, 18, 25-38.

Fleury, P. & Lange, J. (1932) Sur l’oxydation des acides alcohols et des sucres par l’acide périodique, in M. Aslam & A. Dent (eds.), Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences, 1st éd., pp. 216-229, Bath Press.

Fritz, H., Oppitz, K.H., Meckl, D., Kemkes, B., Haendle, H., Schult, H. & Werle, H. (1969) Distribution & excretion of naturally occurring & chemically modified protease inhibitors following intravenous injection in rats, dogs (and man), Hoppe- Seyler’s Z. Physiol. Chem. 350, 1541-1550.

Franker, P.J. & Speck, J.C., Jr. (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, l,3,4,6-tetrachloro-3a,6a-diphenylglycouril, Biochem. Biophys. Res. Commun. 80(4), 849-857.

Fujita, T., Nishikawa, M., Tamaki, C., Takakura, Y., Sezaki, H. & Hashida, M. (1992) Targeted delivery of human recombinant superoxide dismutase by chemical modification with mono- and polysaccharide derivatives. J. Pharmacol. Exp. Ther. 263. 971-978.

289 ______References

Fuke, L, Hayashi, T., Tabata, Y. & Ikada, Y. (1994) Synthesis of poly(ethylene glycol) derivatives with different branchings and their use for protein modification, J. Controlled. Release.., 30, 27-34.

Fung, W. J., Porter, J.E. & Bailon, P. (1997) Strategies for the preparation and characterisation of polyethylene glycol (PEG) conjugated pharmaceutical proteins. Polymers Reprint., 38, 565-566.

Gewurz, A., Grammer, L.C., Shaughnessy, M.A. & Patterson, R. (1986) Soluble copolymer of wasp venom with human albumin for venom immunotherapy, J. Allergy Clin. Immunol., 77, 520-523.

Gibaldi, M. (1991) Compartmental and noncompartmental pharmacokinetics, in Biopharmaceutics and Clinical Pharmacokinetics, 4th ed., pp. 14-23, Philadelphia, Lea & Febiger.

Gladysheva, I.P., Moroz, N.A., Papisova, A.I. & Larionova, N.I. (2001) Soybean Bowman-Birk Inhibitor conjugates with chemical dextran: Synthesis and antiproteolytic activity. Biochemistry., 66, 384-389.

Glazer, A.N., DeLange, R.J., & Sigman, D.S. (1976) Modification of protein side- chains: group-specific reagents, in T.S. Work & E. Work (eds.). Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 4, Part I, Chemical Modification o f Proteins, pp. 68-120, New York, American Elsiever Publishing Co., Inc.

Glazer, A.N. & Simmons, N.S. (1965) The cotton effect associated with certain tyrosine residues in ribonuclease, J. Amer. Chem. Soc., 87, 3991-3993.

Goddard, P. (1991) Therapeutic proteins-a pharmaceutical perspective. Adv. Drug. Del. Rev., 6, 103-131.

290 ______References

Gotschlich, E.C., Fraser, B.A., Nishimura, O., Robbins, J.B. & Liu, T-Y. (1981) Lipid on capsular polysaccharides of gram-negative bacteria, J. Biol Chem., 256, 8915- 8921.

Goodson, R.J. & Katre, N.V. (1990) Site-directed pegylation of recombinant interleukin-2 at its gylcosylation site. Biotechnology., 8, 343-346.

Gray, G.R., Schwartz, B.A. & Kamicker, B.J. (1978) Proteins containing reductively aminated disaccharides: chemical and immunochemical characterisation, in V.T Marchesi et al. (eds.). Cell Surface Carbohydrates and Biological Recognition, pp. 583-594, New York, Alan R, Liss, Inc.

Gray, R.S., Cowan, P., di Mario, U, Elton, R.A. & Clarke, B.F. (1985) Influence of insulin antibodies on pharmacokinetics and bioavailability of recombinant human and highly purified beef insulins in insulin dependent diabetes, BMJ., 290, 1687.

Greenwald, R.A. (1990) Superoxide dismutase and catalase as therapeutic agents for human diseases. Free Radie. Biol. Med., 8, 201-209.

Greenwald, R.A. (1991) Therapeutic usages of oxygen radical scavengers in human diseases: myths and realities. Free Radie. Res. Commun., 12-13, 531-538.

Greenwood, F.C. & Hunter, W.M. (1963) The preparation of 131-1 labelled human growth hormone of high specific radioactivity, Biochem. J., 89, 114-123.

Gregoriadis, G. & Allison, A.C. (1974) Entrapment of proteins in liposomes prevents allergic reactions in pre-immunised mice, FEBS Lett., 45, 71-74.

Gregoriadis, G. & Neerunjun, D.E. (1975) Control of the rate of hepatic uptake and catabolism of liposome entrapped proteins injected into rats. Possible therapeutic applications, Eur. J. Biochem., 47, 179-185.

291 ______References

Gregoriadis, G. (1981) Targeting of drugs: implications in medicine, Lancet, 2, 241- 246.

Gregoriadis, G., McCormack, B., Wang, B. & Lifely, R. (1993) Polysialic acids: potential in drug delivery, FEBS Lett, 315, 271-276.

Gregoriadis, G. & McCormack, B. (1994) Polysialic acids: In vivo properties and possible uses in drug delivery, in G. Gregoriadis (ed.). Targeting o f drugs, vol. 4, pp. 139-145, Plenum Press, New York.

Gregoriadis, G., Fernandes, A. & McCormack, B. (1999) Polysialylated proteins: An approach to improving enzyme stability and half-life in the blood circulation, S.T.P. Pharma, Set, 9(1), 61-66.

Gregoriadis, G., Fernandes, A., Mital, M., and McCormack, B. (2000). Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics. Cellular and Molecular Life Sciences, 57, 1964-1969.

Grossberg, S.E. & Sedmark, J.J. (1977) A rapid, sensitive and versatile assay for protein using Coomassie brilliant blue G250, Anal Biochem., 79, 544-552.

Hadley, K.B. & sato, P.H. (1989) Catalytic activity of administered gulonolactone oxidase polyethylene glycol conjugates. Enzyme., 42, 225-234.

Hames, B.D. (1990) One-dimensional polyacrylamide gel electrophoresis, in B.D. Hames & D. Rickwood (eds.). Gel Electrophoresis o f Proteins’. A Practical Approach, 2nd ed., pp. 1-147, Oxford University Press.

Harris, E.L.V. (1989a) Concentration of the extract, in E.L.V. Harris & S. Angal (eds.). Protein Purification Methods: A Practical Approach, pp. 125-174, Oxford, Oxford University Press.

292 ______References

Harris, J.M., Martin, N.E. & Modi, M. (2001) Pegylation: A novel process for modifying pharmaceutics, Clin. Pharmacokinet., 40, 539-551.

Harris, J.M. & Chess, R.B. (2003) Effect of pegylation on pharmaceuticals, Nat. Rev. Drug Discov., 2, 214-221.

Heilman, K., Miller, D.S. & Cammak, K.A (1983) The effect of ffeeze-drying on the quaternary tructure of L-asparaginase from Erwinia carotovora, Biochim. Biophys. Acta., 749, 133-142.

Hennink, W.E., Ebert, C.D., Kim, S.W., Breemhaar, W., Bantjes, R. & Feijen, J. (1984) Interaction of anti throbin III with preadsorbed albumin-heparin conjugates. Biomaterials., 5, 264-268.

Hermanson, G.T. (1996), Bioconjugate Techniques, (eds.), San Diego Academic Press.

Hemaiz, M.J., Sanchez-Montero, M. & Sinisterra, J.V. (1999) Modification of purified lipases from Candida rugosa with polyethylene glycol: a systematic study. Enzyme and Microbial Technology., 24, 181-190.

Hershfield, M.S., Chaffee, S. Koro-Johnson, L., Mary, A. & Smith, A.A. (1991) Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol, Proc. Natl. Acad. Sci. USA., 88, 7185-7189.

Hershfield, M.S. (1997) Biochemistry and immunology of poly(ethylene glycol)- modified adenosine deaminase (PEG-AD A), in J.M. Harris & S. Zalipsky (eds.), Poly(ethylene glycol): chemistry and biological applications, pp. 145-154, Philadelphia (PA): American Chemical Society.

293 ______References

Hildebrand, G.E. & Tack, J.W. (2000) Microencapsulation of peptides and proteins. Int. J. Pharmaceutics., 196, 173-176.

Hinds, K., Koh, J.K., Joss, L., Liu, F., Baudys, M. & Kim, S.W. (2000) Synthesis and characterisation of poly(ethylene glycol)-insulin conjugates, Bioconj. Chem., 11, 195-

201.

Hinds, K.D. & Kim, S.W. (2002) Effects of PEG conjugation on insulin properties. Adv. Drug Del. Rev., 54, 505-530.

Hirano, T., Todoroki, T., Monta, R., Kato, S., Ito, Y., Kim, K.H., Shukla, P.G., Veronese, P., Maeda, H. & Ohashi, S. (1997) Anti-inflammatory effect of the conjugate of superoxide dismutase with the copolymer of divinyl ether and maleic anhydride against rat re-expansion pulmonary oedema, J. Controlled Release., 48, 131-139.

Hixson, H.F. (1973) Water-soluble enzyme-polymer grafts: Thermal stabilisation of glucose oxidase, BiotechnoL Bioeng., 15, 1011-1016.

Hoffinan, A. & Ziv, E. (1997) Pharmacokinetic considerations of new insulin formulations and routes of administration, Clin. Pharmacokinet., 33, 285-301.

Hofmeister, F. (1888) Zur Lehre von der Wirkung der Salze, in M. Aslam & A. Dent (eds.), Bioconjugation: Protein Coupling Techniques for the Biomedical 1st Sciences, ed., pp. 588-592, Bath Press.

Hughes, W.L. (1954), in H. Neurath and K. Biley (eds.). The Proteins, Vol. 2b, pp. 663-755, Academic Press, New York.

Humphreys, J.D., Abramson, S.L. & Marshall, J.J. (1977) Attachment of carbohydrate to enzymes increases their circulatory lifetimes, FEBS Lett., 83, 249-252.

294 ______References

Inada, Y., Furukawa, M., Sasaki, H., Kodera, Y., Hiroto, M., Nishimura, H. & Matsushima, A. (1995) Biomedical and biotechnological applications of PEG- and PM-modified proteins. Trends BiotechnoL, 13, 86-91.

Inoue, S. & Iwasaki, M. (1978) Isolation of a novel glycoprotein from the eggs of rainbow trout: Occurrence of disialosyl groups on all carbohydrate chains, Biochem. Biophys. Res. Commun., 83, 1018-1023.

Ito, H.O., So, T., Ueda, T., Imoto, T. & Koga, T. (1997) Prevention of collagen- induced arthritis (CIA) by treatment with polyethylene glycol-conjugated type II collagen; distinct tolerogenic property of the conjugated collagen from the native one. Clinical and Experimental immunology, 108, 213-219.

Jain, R.K. (1987) Transport of molecules across tumour vasculature. Cancer Metastasis Rev., 6, 559-594.

Jain, S., McCormack, B., Hirst, D. H. & Gregoriadis, G. (2002) Polysialylated insulin: synthesis, characterization and pharmacological behaviour in-vivo, 5th International symposium on polymer therapeutics: From laboratory to clinical practice, Cardiff (Abstract)

Jancik, J., Schauer, R. & Streicher, H.J. (1975) Influence of membrane-bound n- acetylneuraminic acid on the survival of erythrocytes in man, Hoppe-Seyler’s Z Physiol. Chem., 356, 1329-1331.

Jennings, H.J. & Lugowski, C. (1981) Immunochemistry of groups A, B, and C meningococal polysaccharide tetanus toxoid conjugates, J. Immunol., 127, 1011-1018.

Jirgensons, B. (1966) Optical rotatory dispersion of nonhelical proteins, J.Biol. Chem., 241, 147-152.

295 ______References

Kagedal, L. & Akerstrom, S. (1971) Binding of covalent proteins to polysaccharides I. Preparation of soluble glycin-insulin and ampicillin dextran, Acta. Chem. Scand., 25, 1855-1859.

Kaller, H., Patzschke, K. & Wegner, L.A. (1978) Pharmacokinetic observations following intravenous administration of radioactive labelled aprotinin in volunteers, Eur. J. Drug. Metab., 2, 79-85.

Kamisako, T., Miyawaki, S., Gabazza, E.C., Isbibara, T., Kamei, A., Kawamura, N. & Adacbi, Y. (1998) Polyethylene glycol-modified bilirubin oxidase improves hepatic energy charge and urinary prostaglandin levels in rats with obstructive jaundice, J. Hepatol., 29, 424-429.

Kaneo, Y., Ogawa, K., Tanaka, T., Fujibara, Y. & Igucbi, S. (1994) A protective effect of glutatbione-dextran macromolecular conjugates on acetominopben-induced bepatotoxicity dependant on molecular size, Biol. Pharm. Bull., 17, 1379-1384).

Kaneo, Y., Uemura, T., Tanaka, T., Kanob, S. & Matsuoka, A. (1995) Pharmacokinetics of glutatbione-dextran macromolecular conjugate in mice, Biol. Pharm. Bull., 18, 1544-1547.

Kassell, B. & Laskowski, M. (1965) The basic inhibitor of bovine pancreas. V. The di sulphide linkages, Biochem, Biophys. Res. Commun., 20, 463-468.

Katcbalski, E., Benjamin, O.S. & Gross, V. (1957) The availability of the disulpbide bonds of human and bovine serum albumin and bovine gamma globulin to reduction by Tbioglycolic acid, J. Am. Chem. Soc., 19,4096-4099.

Kaufinan, S.H.E., Schauer, R. & Hahn, H. (1981) Carbohydrate surface constituents of T-cell mediating delayed-type hypersensitivity that control entry into sites of contigen d e p o s itio n ,160, 184-195.

296 ______References

Kinstler, O., Molineux, G., Treuheit, M., Ladd, D. & Gregg, C. (2002) Mono-N- terminal poly(ethylene-glycol)-protein conjugates. Adv. Drug Del Rev., 54, 477-485.

Kitamura, K., Takahashi, T., Yamaguchi, T, Noguchi, A., Noguchi, A., Takashina, K., Tsurumi, H., Inagake, M., Toyokuni, T. & Hakomori, S. (1991) Chemical engineering of the monoclonal antibody A7 by polyethylene glycol for targeting cancer chemotherapy. Cancer. Res., 51, 4310-4315.

Knauf, M.J., Bell,D.P., Hirtzer, P., Luo, Z.P., Young, J.D. & Katre, N.V (1988) Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers, J. Biol Chem., 263, 15064-15070.

Knowles, D.M., Sullivan, T.J., Parker, C.W. & Williams, R.C. (1973) In vitro antibody-enzyme conjugates with specific bactericidal activity, J. Clin. Invest., 52, 1443-1452.

Ko, W.H., Wong, C.C., Yeung, H.W., Yung, M.H., Shaw, P C. & Tam, S.C. (1991) Increasing the plasma half-life of trichosanthin by coupling to dextran, Biochem. Pharmacol, 42, 1721-8.

Kodera, Y., Tanaka, H., Matsushima, A. & Inada, Y. (1992) Chemical modification of L-asparaginase with comb-shaped copolymer of polyethylene glycol derivative and maleic anhydride, Biochem. Biophys. Res. Commun., 184, 144-148.

Kompella, U.B. & Lee, V.H.L. (1991) Pharmacokinetics of peptide and protein drugs, in V.H.L. Lee (ed.). Peptide and protein Drug Delivery, pp. 391-484, New York, Marcel Dekker, Inc.

Kraehenbuhl, J.P. (1974) Preparation and characterisation of an immunoelectron microscope tracer consisting of a heme-octapeptide coupled to Fab, J. Exp. Med, 139, 208-223.

297 ______References

Kundig, F.D., Aminoff, D. & Roseman, S. (1971) The Sialic Acids XII. Synthesis of colominic acid by a sialyltransferase from Escherichia coli K-235, J. Biol Chem., 246, 2543-2550.

Kunitani, M., Dollinger, G., Johnson, D. & Kresin, L. (1991) On-line characterisation of polyethylene glycol-modified proteins,/. Chromatogr., 588, 125-137.

Laane, A., Haga, M., Aaviksaar, A., Kesvatera, T. & Tougu, V. (1983) Activation of poly (N-(2-hydroxypropyl)methacrylamide) for binding of bioactive molecules I. Activation with 4-nitrophenyl chloroformate. Die Makromolekulare Chemie., 184, 1339-1344.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T^^Nature., 227, 680-685.

Lamka, J., Schiavon, O., Laznicek, M., Caliceti, P. & Veronese, P.M. (1995) Distribution of catalase, ribonuclease and superoxide dismutase modified by monomethoxy (polyethylene glycol) into rat central lymph and lymphatic nodes. Physiol. Res., 44, 307-313.

Langer, J.A. & Pestka, S. (1988) Interferon receptors, Immunol. Today., 9, 393-400.

Larionova, N.I., Kazanskaya, N.F. & Sakharov, I.Y. (1978) (Abstract) Soluble high molecular weight derivatives of pancreatic trypsin inhibitor. Isolation and properties of the inhibitor bound to carboxymethyl cellulose and diethylaminoethyl dextran, Biokhimiia., 43, 880-886.

Larionova, N.I., Mityushina, G.V., Kazanskaya, N.F., Blidchenko, Y.A. & Berezin, I.V. (1984) Carbohydrate-containing derivatives of the trypsin-Kallikrein inhibitor aprotinin from bovine organs. I. Modification with lactose. Characterisation and behaviour of the preparation in vivo, Hoppe-Seyler’s Z. Physiol. Chem., 365, 791-797.

298 ______References

Larionova, N.I., Kazanskaya, N.F., Mityushina, G.V., Blidchenko, Y.A. & Vladimirov, V.G. (1984a) Pharmacokinetics of the main polyvalent protease inhibitor bound to the carboxymethyl ester of dextran, Chim. Pharm. Zhurn., 18, 1167-1172.

Larionova, N.I., Mityushina, G.V., Kazanskaya, N.F., Blidchenko, Y.A. & Berezin, I.V. (1985) Carbohydrate-containing derivatives of the trypsin-Kallikrein inhibitor aprotinin from bovine organs. II. Inhibitor coupled to the (carboxymethyl)dextran derivatives of D-Galactose, Hoppe-Seyler. Biol Chem., 366, 743-748.

Larsen, C. (1989) Dextran prodrugs-structure and stability in relation to therapeutic activity, Drug Del Rev., 3, 103-154.

Lasic, D.D. & Martin, F.J. (1995) (eds.) Stealth liposomes, Boca Raton, CRC Press.

Latham, P.W. (1999) Therapeutic peptides revisited, Nat. Biotechnol, 17, 755-757.

Lawson, E.Q., Hedlund, B.E., Ericson, M.E., Mood, D.A., Litman, G.W. & Middaugh, R. (1983) Effect of carbohydrate on protein solubility. Arch. Biochem. Biophys., 220, 572-575.

Leach, B.S., Collawn, J.F.J. & Fish, W.W. (1980) Behaviour of glycopolypeptides with empirical molecular weight estimation methods. I. In sodium dodecyl sulphate. Biochemistry., 19, 5734-5741.

Lee, V.H.L. (1991) Peptide and protein drug delivery-problems and some solutions, in S.R. Bloom & G. Bumstock (eds.). Peptides: a target for new drug development, pp. 120-134, London, IBC Technical Services Ltd.

Levy, Y., Hershfield, M., Femandez-Mejia, C., Polmar, S.H., Scudiery, D., Berger, M. & Sorensen, R.K. (1988) Adenosine deaminase deficiency with late onset of recurrent infections: Response to treatment with polyethylene glycol-modified adenosine deaminase,/. Pediatr., 113, 317-317.

299 ______References

Lifely, M.R., Nowicka, U.T. & Moreno, C. (1986) Analysis of the chain length of oligomers and polymers of sialic acid isolated from Neisseria meningitidis group B and C md Escherichia coli kl and K92, Carbohyd. Res., 156, 123-135.

Lifely, M.R., Nowicka, U.T., Krambovitis, E. & Esdaile, J. (1988) Antigenicity of meningococcal group B oligo- and polysaccharides of defined chain length in, J.T. Poolman (ed.). Gonococci and Meningococci, pp. 147-152, Dordrecht, Kluwer Academic.

Light, A. & Higaki, J.N. (1987) Detection of intermediate species in the refolding of bovine trypsinogen. Biochemistry., 26, 5556.

Lindorfer, M.A. & Becktel, W.J. (1990) Spectroscopic and chromatographic studies of native and denatured states of T4 lysozymes, in J.J Villafranca (ed.). Current research in protein chemistry, pp 309, San Diego, Academic Press. Lisi, P.L., van Es, T., Abuchowski, A., Palczuk, N.C. & Davis, F.F. (1982) Polyethylene glycol: p-glucuronidase conjugates as potential therapeutic agents in acid mucopolysaccharidosis, J. Appl. Biochem., 4, 19-33.

Lobuglio, A.F., Wheeler, R.H., Trang, J., Haynes, A., Rogers, K., Harvey, E.B., Sun,L., Ghrayeb, J., & Khazaeli, M.B. (1989) Mouse/ human chimeric monoclonal antibody in man: kinetics and immune response, Proc. Natl. Acad. Sci. USA., 86, 4220-4224.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265-275.

Lu, Z.R., Kopeckova, P., Wu, Z. & Kopecek, J. (1998) Functionalized semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] for protein modification, Bioconj. Chem., 9, 793-804.

300 ______References

Maeda, H. (2001) SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy, Adv. Drug Del Rev., 46, 169-185.

Magnani, M., Rossi, L., Fratemale, A., Bianchi, M., Antonelli, A., Crinelli, R. & Chiarantini, L. (2002) Erythrocyte-mediated delivery of drugs, peptides and modified oligonucleotides. Gene Therapy., 9, 749-751.

Maini, R. et al. (1999) Infliximab (chimeric antitumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. Lancet., 254, 1932-1939.

Mandrell, R.E. & Zollinger, W.D. (1982) Measurement of antibodies to meningococcal group B polysaccharide: low avidity binding and equilibrium binding constants,/. Immunol, 129, 2172-2178.

Marshall, J.J. (1978) Manipulation of the properties of enzymes by covalent attachment of carbohydrate, TIBS., 3, 79-83.

Marshall, J.J. & Humphreys, J.D. (1977) Synthesis of a soluble catalase-dextran Biotechnol. Bioeng., 19, 1739-1760.

Marshall, J.J., Humphreys, J.D. & Abramson, S.L. (1977) Attachment of carbohydrate to enzymes increases their circulatory lifetimes, FEBS Lett., 83, 249-252.

Mastrobattista, E., Crommelin, D.J.A., Wilschut, J. & Gert, S. (2002) Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumour cells, J. Liposome. Res., 12, 57-65.

Matousek, J., Pouckova, P., Soucek, J. & Skvor, J. (2002) PEG chains increase aspermatogenic and antitumour activity of RNase and BS-RNase enzymes, J. Controlled Release.., 82, 29-37.

301 ______References

McGuire, EJ. & Binkley, S.B. (1964) The structure and chemistry of colominic acid. Biochemistry., 3, 247-251.

Means, G.E. & Feeney, R.E. (1995) Reductive alkylation of proteins. Anal Biochem., 224, 1-16.

Mehvar, R. (1994) Preparation and analytical separation of insulin-dextran conjugates from native insulin: application to preparation and characterisation of insulin-dextran conjugates. Drug. Dev. Ind. Pharm., 20(3), 395-404.

Mehvar, R. (2000) Dextrans for targeted and sustained delivery of therapeutic and imaging agents, J. Controlled Release., 69, 1-25.

Meijer, D.K.F. & Ziegler, K. (1993) Mechanisms for the hepatic clearance of ologopeptides and proteins in, K.L. Audus and T.J. Raub, (eds.). Biological barriers to protein delivery, pp. 339-408, New York, Plenum Press.

Melton, R.G., Wiblin, C.N., Foster, R.L. & Sherwood, R.F. (1987) Covalent linkage of carboxypeptidase G2 to soluble dextrans I. Properties of conjugates and effects on plasma persistence in mice, Biochem. Pharmacol, 36, 105-112.

Michael, G., Youngster, S., Gitlin, G., Sydor, W., Xie, L., Westreich, L. et al. (2001) Structural and biological characterisation of pegylated recombinant IFN-a2b, J. Interferon and Cytokine Res., 21, 1103-1115.

Mikolajczyk, S.D., Meyer, D.L., Fagnani, R., Hagan, M.S., Law, K.L. & Starling, J.J. (1996) Dextran modification of a Fab-P-lactamase conjugate modulated by variable pre treatment of Fab with amine blocking agents, Bioconj. Chem., 7, 150-158.

Monfardini, C., et al. (1995) A branched monomethoxypolyethylene glycolfor protein modification, Bioconj. Chem., 6, 62-69.

302 ______References

Monfardini, C, & Veronese, F.M. (1998) Stabilisation of substances in circulation, Bioconj. Chem.., 9, 418-450.

Morell, A.G., Gregoriadis, G., Scheinberg, I.H., Hickman, J. & Ashwell, G. (1971) The role of sialic acid in determining the survival of glycoproteins in the circulation , J. Biol. Chem., 246, 1461-1467.

Moreno, C., Lifely, M.R. & Esdaile, J. (1985) Immunity and protection of mice against Neisseria meningitidis group B by vaccination using polysaccharides complexed with outer membrane proteins: a comparison with purified B polysaccharide. Infect. Immun., 47, 527-533.

Mukheijee, A., et al. (2002) How to optimise pegvisomant treatment of acromegaly safety. Endocrine Abstracts., 4, 0C8.

Muhlenhoff, M., Eckhardt, M. & Gerardy-Schahn, R. (1998) Polysialic acid: three- dimensional structure, biosynthesis and function. Current Opinion in Structural Biology., 8, 558-564.

Murray, M.C., Bhavanandan, V.P. & Davidson, E.A. (1989) Modification of sialyl residues of glycoconjugates by reductive amination. Characterisation of the modified sialic acids, Carbohyd. Res., 186, 255-265.

Nakadka, R., Tabata, Y., Yamaoka, T. & Ikada, Y. (1997) Prolongation of serum half- life period of superoxide dismutase by polyethylene glycol modification, J. Controlled Release., 46, 253-261.

Nakagomi, K. & Ajisaka, K. (1990) Chemical modification of alpha-thrombin and in vivo characterisation of its anticoagulant activity, Biochem. Invest., 22, 75-82.

303 ______References

Nishimura, H., Takahashi, K., Sakurai, K., Fujinuma, K., Imamura, Y., Ooba, M. & Inada, Y. (1985) Modification of batroxobin with activated polyethylene glycol: reduction of binding ability towards anti-batroxobin and retention of dogs, Life Set.,

3 3 , 1467-1473.

Norman, P.S., King, T.P., Alexander, J.F., Kagey-Sobotka, A. & Lichtenstein, L.M. (1984) Immunological response to conjugates of antigen E in patients with ragweed hay fever, J. Allergy Clin. Immunol., 7 3 , 782-789.

Nucci, M.L., Shorr, R. & Abuchowski, A. (1991) The therapeutic value of poly(ethylene glycol)-modified proteins, Drug Del. Rev., 6, 133-151.

Odya, C., Levin, Y., Erdos, E.G. & Robinson, C.J.G. (1978) Soluble dextran complexes of kallikrein, bradykinin and enzyme inhibitors, Biochem. Pharmacol., 2 7 , 173-179.

Ogino, T., Inoue, M., Ando, Y., Awai, M., Maeda, H. & Morino, Y. (1988) Chemical modification of superoxide dismutase. Extension of plasma half-life of the enzyme through its reversible binding to circulating albumin. Int. J. Pept. Protein Res., 3 2 , 153-159.

Olson, K., Gehant, R., Mukku, V., et al. (1997) Preparation and characterisation of poly(ethylene glycol)ylated human growth hormone antagonist in, J.M. Harris & S. Zalipsky (eds). Polyethylene glycol. Chemistry and biological applications, pp. 170- 181, ACS, San Francisco (CA).

Omstein, L. & Davis, B.J. (1964) Disc electrophoresis I. Background and theory, in B.D. Hames & D. Rickwood (eds.). Gel Electrophoresis of Proteins’. A Practical Approach, 2nd ed., pp. 1-92, Oxford University Press.

Palmer, T. (1985) Understanding enzymes, 2nd ed., Chichester, Ellis Horwood Ltd.

304 ______References

Park, T.G., Lee, H.Y. & Nam, Y.S. (1998) A new preparation method for protein loaded poly (D, L-lactic-co-glycolic acid) microspheres and protein release mechanism study, J. Controlled. Release., 55, 181-191.

Pasta, P., Carrea, G., Longhi, R. & Zetta, L. (1990) Circular dichroism and proteolysis of human beta-endorphin in surfactant and lipid solutions, Biochim. Biophys. Acta., 1039, 1.

Paul, R.W., Choi, A.H.C. & Lee, P.W. K. (1989) The a-anomeric form of sialic acid is the minimal receptor determinant recognised by reovirus. Virology, 172, 382-385.

Paulson, J.C. (1985) Interactions of animal viruses with cell surface receptors, in M. Conn (ed.). The Receptors, pp. 131-219, Academic Press. Pautov, V.D., Anufrieva, E.V., Anan'eva, T.D., Savel'eva, N.V., Taratina, T.M. & Krakoviak, M.G. (1990) Structural-dynamic and functional properties of native and modified streptokinase, Molekuliarnaia Biologiia., 24, 44-50.

Pavlinkova, G., Booth, B.J.M., Batra, S.K. Colcher, D. (1999) Radioimmunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody construct, Clin. Cancer. Res., 5, 2613-2619.

Pedley, R.B., Boden, J.A., Boden, R., Begent, R.H.J., Turner, A., Haines, A.M.R. & King, D.J. (1994) The potential for enhanced tumour localisation by poly(ethylene glycol) modification of anti-CEA antibody, Br. J. Cancer., 70, 1126-1130.

Perutz, M.F. (1980) The stabilisation of proteins and other biological materials, in M. Aslam & A. Dent (eds.). Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences, 1st ed., pp. 736-763, Bath Press.

Peters, T., Jr. (1985) Serum albumin, Protein Chem., 37, 161-245.

305 ______References

Pickup, J.C., Bilous, R.W. & Keen, H. (1980) Aprotinin and insulin resistance. Lancet., 2, 93-94.

Pizzo, S.V. (1991) Preparation, in vivo properties and proposed clinical use of polyoxyethylene-modified tissue plasminogen activator and streptokinase. Adv. Drug Del. Rev, 6, 153-166.

Poznansky, M.J. & Chang, T.M. (1974) Comparison of the enzyme kinetics and immunological properties of catalase immobilised by microencapsulation and catalase in free solution for enzyme replacement, Biochim. Biophys. Acta., 334, 103-115.

Poznansky, M.J. (1979) In vitro and in vivo activity of soluble cross-linked uricase- albumin polymers: a model for enzyme therapy. Life Sci., 24, 153-155.

Poznansky, M.J. & Bhardwaj, D. (1980) alpha 1,4-Glucosidase-albumin polymers: in vitro properties and advantages for enzyme replacement therapy. Can. J. Physiol. Pharmacol., 58, 322-355.

Poznansky, M.J., Shandling, M., Salkie, M.A., Elliott, J. & Lau, E. (1982) Advantages in the use of L-asparaginase-albumin polymer as an anti tumour agent. Cancer Res., 42, 1020-1025.

Poznansky, M.J., Singh, R. & Singh, B. & Fantus, G. (1984) Insulin: carrier potential for enzyme and drug therapy. Science, 223, 1304-1306.

Poznansky, M.J. (1986) Enzyme-protein conjugates: new possibilities for enzyme therapy, in G.M. Ihler (ed.). Methods of Drug Delivery, pp. 59-82, Oxford, Pergamon Press.

Poznansky, M.J. (1988) Soluble enzyme-albumin conjugates: new possibilities for enzyme replacement therapy. Methods. Enzymol. 137, 566-574.

306 ______References

Prakash, V. & Nandi, P.K. (1976) Dissociation and dénaturation behaviour of sesame a-Globulin in sodium dodecyl sulphate solution, Int. J. Peptide Protein. Res., 8, 385- 392.

Prakash, V., Nandi, P.K. & Jirgensons, B. (1980) Effect of sodium dodecyl sulfate, acid, alkali, urea and guanidine hydrochloride on the circular dichroism of a-Globulin of sesamum indicum /, Int. J. Peptide Protein. Res., 15, 305-313.

Prestrelski, S.J., Arakawa, T. & Carpenter, J.F. & (1993) Separation of freezing- and drying-induced dénaturation of lyophilised proteins using stress-specific stabilisation. II. Structural studies using infrared spectroscopy. Arch. Biochem. Biophys., 28, 101-

121.

Rao, K.S. & Prakash, V. (1993) Interaction of sodium dodecyl sulfate with multi- ubunit proteins. A case study with carmin, J. Biol. Chem., 268, 14769-14775.

Rajagopalan, S., Gonias, S.L. & Pizzo, S.V. (1985) A non antigenic covalent streptokinase polyethylene glycol complex with plasminogen activator function, J. Clin, Invest.., 75, 413-419.

Reddy, K.R., Modi, M.W. & Pedder, S. (2002) Use of peginterferon a-2a (40KDa) (Pegasys®) for the treatment of hepatitis C, Adv. Drug Del. Rev., 54, 571-586.

Remy, M.H. & Poznansky, M.J. (1978) Immunogenicity and antigenicity of soluble cross-linked enzyme/albumin polymers: advantages for enzyme therapy. Lancet., 2, 68-70.

Ressing, M.E., Fiskoot, W., Talsma, H., van Ingen, C.W., Bewery, E.G. & Crommelin, D.J.A. (1992) The influence of sucrose, dextran and hydroxypropyl-p- cyclodextrin as lyoprotectants for a freeze-dried mouse IgGi monoclonal antibody (MN12), Pharm. Res., 9, 266-270.

307 ______References

Reubsaet, J.L.E., Beijnen, J.H. Bult, A., van Maanen, R.J., Marchai, J.A.D. & Underberg, W.J.M. (1998) Analytical techniques used to study the degradation of proteins and peptides: physical instability, J. Pharm. Biomed. Anal., 17, 979-984.

Richter, A. W. & Akerblom, E. (1984) Polyethylene glycol reactive antibodies in man: titer distribution in allergic patients treated with monomethoxypolyethylene glycol modified allergens or placebo, and in healthy donors, Int Allergy Appl. Immunol., 74, 36-39.

Richter, W. & Kagedal, L. (1972) Preparation of dextran-protein conjugates and studies of their immunogenicity. Int. Arch. Allergy. Appl. Immunol., 42, 885-902.

Ringsdorf, H. (1975) Structure and properties of pharmacologically active polymers, J. Polym. Sci. Symp., 51, 135-153.

Roberts, S., Wagner, B.K.J., Boulanger, M. & Richer, M. (1996) Review. Aprotinin, The Annals of Pharmacotherapy., 30, 372-379.

Roberts, M.J. Bentley, M.D. & Harris, J.M. (2002) Chemistry for peptide and protein PEGylation, Drug Del. Rev., 54, 459-476.

Rocca, M., et al. (1996) Pathophysiological and histomorphological evaluation of polyacryloylmorpholine vs polyethylene glycol modified superoxide dismutase in a rat model of ischemia/reperfusion injury. Int. J. Artif. Organs., 19, 730-734.

Roerdink, F.H., Gegts, J. & Scherphof, G. (1986) Effect of lipid composition on the uptake and intracellular degradation of liposomes by kupffer cells. In A. Kiru., D.L. Knook. & E. Wisse, (eds.). Cell of the hepatic sinusoid, kupffer cell foundation, pp. 125, Rijswijk, Netherlands

Rohr, T.E. & Tory, E.A. (1980) Structure and biosynthesis of surface polymers containing polysialic acid in Escherichia coli. J. Biol. Chem., 255, 23332-2342.

308 References

Rougon, G. (1993) Structure, metabolism and cell biology of polysialic acids, Eur. J. Cell Biol, 61, 197-207.

Rowan, J.W. (1951) Spectrophotometric evidence for the absence of free aldehyde groups in periodate-oxidised cellulose, J. Am. Chem. Sac., 73, 4484-4487.

Roy, R., Laferriere, C.A., Pon, R.A. & Gamian, A. (1994) Induction of rabbit immunoglobulin G antibodies against synthetic sialylated neoglycoproteins. Methods Enzymol, 247, 351-361.

Rutishauser, U. (1989) Polysialic acids as regulator of cell interactions in, R.U. Margolis & R.K. Margolis (eds.). Neurobiology of Glycoconjugates, pp. 367-382, Plenum Press, New York.

Ryan, T.A., Joiner, B.L. & Ryan, B.F. (1985) Minitab Handbook, Boston, PWS-Kent Publishing Company, Inc.

Saez-Llorens, X. et al (1998) Safety and pharmacokinetics of an intramuscular humanised antibody monoclonal antibody to respiratory syncital virus in premature infants with bronchopulmonary dysplasia, Paediatr. Infect. Dis., 17, 787-791.

Sato, H. (2002) Enzymatic procedure for site-specific pegylation of proteins. Adv. Drug Del. Rev., 54, 487-504.

Saito, M. & Yu, R.K. (1995) Biochemistry and function of sialidases, in A. Rosenberg (ed.). Biology of the Sialic Acids, pp. 261-313, New York, Plenum Press.

Saito, T., Nishimura, H., Sekine, T., Urushibara, T., Rodera, Y., Hiroto, M., Matsushima, A. & Inada, Y. (1996) Tolerogenic capacity of poly(ethylene glycol) (PEG)-modified ovalbumins in relation to their immunoreactivity towards anti ovalbumin antibody, J. Biomaterials Science, Polymer edition, 8, 311-321.

309 ______References

Sandbom, W.J. & Hanauer, S.B. (1999) Antitumour necrosis factor therapy for inflammatory bowel disease: a review of agents, pharmacology, clinical results and safety, Inflamm. Bowel. Dis., 5, 119-133.

Sanger, F. and E.O.P, Thompson (1953) The amino acid sequence in the glycyl chain of insulin. II. The investigation of peptides from enzymic hydrolysates, Biochem. J., 53, 366-374.

Sarff, L.B., McCracken, G.H., Schiffer, M.S., Glode, M., Robbins, J.B., Orskov, I. & Orskov, F. (1975) Epidemiology of Escherichia coli in healthy and diseased newborn. Lancet., 1, 1099-1103.

Sasaki, H., Ohtake, Y., Matsushima, A., Hiroto, M., Kodera, Y. & Inada, Y. (1993) Reduction of immunogenicity of bovine serum albumin conjugated with comb-shaped polyethylene glycol derivatives, Biochem. Biophys. Res. Commun., 197, 287-291.

Savoca, K.V., Davis, F.F., van Es T., McCoy, J.R. & Palczuk, N.C. (1984) Cancer therapy with chemically modified enzymes. II. The therapeutic effectiveness of arginase and arginase modified by covalent attachment of polyethylene glycol on the Taper liver tumour and the L5178Y murine leukaemia. Cancer Biochem. Biophys., 1, 261-268.

Schally, A.V. (1988) Oncological application of somatostatin analogues. Cancer Res., 48, 6977-6985.

Schauer, R. (1982a) Sialic acids, Carbohydr. Chem. Biochem., 40, 131-234.

Schauer, R. (1982b) (ed.). Metabolism and Function in Sialic acids-chemistry. Springer, Veinna.

310 ______References

Schauer, R., Kelm, S., reuter, G., Roggentin, P. & Shaw, L. (1995) Biochemistry and role of sialic acids, in A. Rosenberg (ed.). Biology of the sialic acids, pp. 7-67, New York, Plenum Press.

Schiavon, O., Caliceti, P., Ferruti, P. & Veronese, F.M. (2000) Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine), II Farmaco., 55, 264-269.

Schilling, R.J. & Mitra, A.K. (1992) Pharmacodynamics of insulin following intravenous and enteral administrations of porcine zinc insulin in rats, Pharm. Res., 9, 1003-1009.

Scott, M.D., Lubin, B.H., Zuo, L. & Kuypers, F.A. (1991) Erythrocyte defense against hydrogen peroxide: prominent importance of catalase, J. Lab. Clin. Med., 118, 7-16.

Sebeni, J. (1998) The interaction of liposomes with the complement system. Grit. Rev. Ther. Drug Carrier Syst., 15, 57-88.

Segrest, J.P. & Jackson, R.L. (1972) Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Methods Enzymol., 28, 54-63.

Senior, J., Delgado, C., Fisher, D., Tilcock, C. & Gregoriadis, G. (1991) Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation, Biochim. Biophys. Acta., 1062, 77-82.

Seymour, L., Duncan, R., Strohalm, J. & Kopecek, J. (1987) Effect of molecular weight (Mw) of N-(2-hydroxypropyl)methacrylamide copolymers on body distributions and rate of excretion after subcutaneous intraperitoneal and intravenous administration to rats, J. Biol. Mater. Res., 21, 1341-1358.

311 ______References

Shalongo, W., Jagannadham, M.V., Flynn, C. & Stellwagen, E. (1989) Refolding of denatured ribonuclease observed by size exclusion chromatography, Biochemistry, 28, 4820.

Sharifi, J., Khawli, A., Homick, J.L. & Epstein, A.L. (1998) Improving monoclonal antibody pharmacokinetics via chemical modification, Q. J. Nucl. Med., 42, 242-249.

Sheffield, W.P. (2001) Modification of clearance of therapeutic and potentially therapeutic proteins. Current Drug Targets, Cardiovas. &Haemat. Dis., 1, 1-22.

Sindelar, R.D. (1997) Principles of Medicinal Chemistry in, D.J.A. Crommelin & R.D. Sindelar, (eds.). Pharmaceutical Biotechnology. An Introduction for Pharmacists

Smith, O.P., Delgado, C., Malik, F. et al., (1991) Receptor binding studies of PEG modified GM-CSF with dissociated biological activities, Br. J. Haematol., 1, 15.

Soares, A.L., Guimaraes, G.M., Polakiewicz, B., de Moraes Pitombo, R.N. & Abrahao-Neto, J. (2002) Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli L-asparaginase, Int. J. Pharm., 237, 163-170.

Spector, T. (1978) Refinement of the Coomassie blue method of protein quantitation. Anal. Biochem., 86, 142-146.

Squire, P. G., Moser, P., O’ Konski, C.T. (1968) The hydrodynamic properties of bovine serum albumin monomer and dimer. Biochemistry., 7, 4261-4272.

Steinhardt, J. & Reynolds, J.A. (1969) Multiple Equilibria in Proteins, pp. 234-300, Academic Press, New York.

312 ______References

Suzuki, F., Daikuhara, Y., Ono, M. & Takeda, Y. (1972) Studies on the mode of action of insulin: properties and biological activity of an insulin-dextran complex, Endocrin., 90, 1220-1230.

Suzuki, T., Kanbara, N., Tomono, T., Hayashi, N. & Shinohara, I. (1984) Physiochemical and biological properties of poly(ethylene glycol)-coupled immunoglobulin G, Biochim. Biophys. Acta., 788, 248-255.

Suzuki, Y. (1994) Gangliosides as influenza virus receptors. Variation of influenza viruses and their recognition of the receptor sialo-sugar chains. Pro. Lipid. Res., 33, 429-457.

Svennerholm, L. (1957) Quantitative estimation of sialic acid II. A colorimetric resorcinol-hydrochloride acid method, Biochim. Biophys. Acta, 24, 604-611.

Tabata, Y., Noda, Y., Matsui, Y. & Ikada, Y. (1999) Targeting of tumour necrosis factor to tumour by use of dextran and metal coordination, J. Controlled Release., 59, 187-196.

Takakura, Y. (1996) Development of drug delivery systems for macromolecular drugs, Yakugaku Zasshi., 7, 519-532

Tanaka, K., Takeda, T. & Miyajima, K. (1991) Cryoprotective effect of saccharides on dénaturation of catalase by freeze-drying, Chem. Pharm. Bull., 39, 1091-1094. Tanaka, H., Satake-Ishikawa, R., Ishikawa, M., Matsuki, S. & Asano, K. (1991) Pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol in rats. Cancer Res., 51, 3410-3714.

Thompson, R.C., Armes, L.G., Evans, R.J. et al., (1992) Pegylation of polypeptides. International Patent Application Number Wo92/16221.

313 ______References

Tomlinson, E. (1990) Control of the biological dispersion of therapeutic proteins, in J.B. Hook & G. Poste (eds.). Protein Design and the Development of New Therapeutics and Vaccines, pp. 331-357, New York, Plenum Press.

Tomlinson, E. (1991) Site-specific proteins, in R.C. Rider & D. Barlow (eds.). Polypeptide and Protein Drugs: Production, Characterisation and Formulation, pp. 251-264, Chichester, Ellis Horwood Ltd.

Torchilin, V.P., ITina, E.V., Mazaev, A.V., Lebedev, B.S., Smirnov, V.N. & Chazov, E.I. (1977) Study of modified Sephadex-bound insulin in animal experiments, J. Solid-Phase Biochem., 2, 187-193.

Torchilin, V.P., Voronkov, J.L & Mazoev, A.V. (1982) The use of immobilised streptokinase (Strptodekaza) for the therapy of thromboses, Ther. Arch., 54, 21-28.

Torres, E. & Vazquez-Duhalt, R. (2000) Chemical modification of haemoglobin improves biocatalytic oxidation of PAHs, Bioch. Biophys. Res. Commun., 273, 820- 823.

Tory, F.A. (1990) Polysialylation of neural cell adhesion molecules. Trends Glycosci. Glycotechnol., 2, 430-449.

Tory, F.A. (1992) Polysialylation: From bacteria to brains, Glycobiology., 2, 5-23.

Trautschold, I., Werle, E. & Zickgraf-Rudel, G. (1967) Trasoyl, Biochem. Pharmacol, 16, 59-72.

Troy II, F.A. (1995) Sialobiology and the polysialic acid glycotype, in A Rosenberg (ed.). Biology of the Sialic Acids, pp. 95-144, New York, Plenum Press.

314 ______References

Tsutsumi, Y., Kihira, T., Tsunoda, S., et al. (1995) Molecular design of hybrid tumour necrosis factor alpha with polyethylene glycol increases its anti tumour potency, Br. J. Cancer., 71, 936-968.

Tsutsumi, Y., Tsunoda, S., Kamada, H., et al. (1997) Pegylation of interleukin-6 effectively increases its thrombopoietic potecy, Thromb. Haemost., 77, 168-173.

Uchio, T., Baudys, M., Liu, F., Song, S.C. & Kim, S.W. (1999) Site-specific insulin conjugates with enhanced stability and extended action profile. Adv. Drug, Del. Rev., 35, 289-306.

Ulbrich, K., Subr, V., Strohalm, J., Plocova, D., Jelinkova, M. & Rihova, B. (2000) Polymeric drugs based on conjugates of synthetic and natural macromolecules: I. Synthesis and physico-chemical characterisation, J. Controlled. Release, 64, 63-79.

Uren, J.R. & Ragin, R.C. (1979) Improvement in the therapeutic, immunological, and clearance properties of Escherichia coli and Erwina carotovora L-asparaginase by attachment to poly-DL-alanyl peptides. Cancer Res., 39, 1927-1933.

Vandenbranden, M., De Coen, J.L., Jeener, R., Kanarek, L. & Ruyschaert, J.M. (1981) Interactions of gamma-immunoglobulin with lipid mono-or bilayers & liposomes. Existence of two conformations of gamma-immunoglobulins of different hydrophobicities. Mol. Immunol, 18, 621-631.

Vermeer, A. W.P. & Norde, W. (2000) The infiuence of the binding of low moleculare weight surfactants on the thermal stability and secondary structure of IgG, Colloids and Surfaces A: Physiochem. Eng. Aspects., 161, 139-150.

Veronese, P.M., Schiavon, O. & Caliceti, P. (1997) Protein delivery: PVP a new polymer for protein conjugation, Proc. Int. Symp. Controlled Release Bioact. Mater., 24th, 507-508.

315 ______References

Veronese, F.M. & Caliceti, P. (1997) Branched and linear polyethylene glycol influence of the polymer structure on enzymological, pharmacokinetic and immunological properties of protein conjugates, J. Bioact. Compat. Polym., 12, 196- 207.

Veronese, F.M. & Morpurgo, M. (1999) Review. Bioconjugation in pharmaceutical chemistry, II Farmaco., 54, 497-516.

Veronese, F.M. (2001) Peptide and protein PEGylation: a review of problems and solutions. Biomaterials., 22, 405-417.

Veronese, F.M., Caliceti, P., Schiavon, O. & Sergi, M. (2002) Polyethylene glycol- superoxide dismutase, a conjugate in search of exploitation. Adv. Drug Del. Rev., 54, 587-606.

Viser, L. & Blout, E.R. (1971) Elastase. II. Optical properties and the effects of sodium dodecyl sulfate. Biochemistry., 10, 743.

Wall, D.A. & Maack, T. (1985) Endocytic uptake, transport and catabolism of proteins by epithelial cells. Am. J. Physiol, 248, 12-20.

Wang, W. (1999) Instability, stabilisation and formulation of liquid protein pharmaceuticals. Int. J. Pharm., 185, 129-188.

Wang, Y.S., et al. (2002) Structural and biological characterisation of pegylated recombinant a-2b and its therapeutic implications. Adv. Drug Del. Rev., 54, 547-570.

Weider, K.J., Palczuk, N.C., van Es, T. & Davis, F.F. (1979) Some properties of polyethylene glycol: phenylalanine ammonia-lyase adducts, J. Biol. Chem., 254, 12579-12587.

316 ______References

Weider, KJ. & Davis, F.F. (1983) Enzyme therapy II. Effect of covalent attachment of polyethylene glycol on biochemical parameters and immunological determinants of beta-glucosidase and a-galactosidae, J. Appl Biochem., 5, 337-347.

Weigele, M., De Bernardo, S.L., Tengi, J.P. & Leimbruber, W. (1972) A novel reagent for the fluorometric assay of primary amines, J. Am. Sac., 94, 5927.

Weissmann, G., Bloomgarden, D., Kaplan, R., Cohen, C., Hoffstein, S., Collin, T., Gotlieb, A. & Nagle, D. (1975) A general method for the introduction of enzymes, by means of immunoglobulin-coated liposomes into lysosomes of deficient cells, Proc. Natl Acad. Sci U.S.A., 70, 88-92.

Weitzhandler, M., Hardy, M., Co, M.S. & Avdalovic, N. (1994) Analysis of carbohydrates on IgG preparations, J. Pharm. Sci, 83, 1670-1675.

Whitaker, J.R. (1963) Determination of molecular weights of proteins by gel filtration on Sephadex, yfwfl/. Chem., 35, 1950-1953.

Wie, S.L, Wie, C.W., Lee, W.Y., Filion, L.G., Sehon, A.H. & Akerblom, E. (1981) Suppression of reaginic antibodies with modified allergens. III. Preparation of tolerogenic conjugates of common allergens with monomethoxypolyethylene glycols of different molecular weights by the mixed anhydride method. Int. Arch. Appl Immunol, 64, 84-99.

Wileman, T.E., Foster, R.L. & Elliot, P.N.C. (1986) Soluble asparaginase-dextran conjugates show increased circulatory persistence and lowered antigen reactivity, J. Pharm. Pharmacol, 38, 264-271.

Wileman, T.E. (1991) Review: Properties of asparaginase-dextran conjugates. Adv. Drug Del Rev., 6, 167-180.

317 ______References

Wong, K., Cleland, L.G. & Poznansky, M.J. (1980) Enhanced anti-inflammatory effect and reduced immunogenicity of bovine liver superoxide dismutase by conjugation with homologous albumin. Agents Actions, 10, 231-239.

Wood, W.G., Wachter, C. & Scriba, P.C (1981) Experiences using chloramines-T and l,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (lodogen) for radioiodination of materials for radioimmunoassay,/. Clin. Chem. Biochem., 19, 1051-1056.

Wyle, F.A., Artenstein, M.S., Brandt, B.L., Tramont, E.G., Kasper, D.L., Altieri, P.L., Berman, S.L. & Lowenthal, J.P. (1972) Immunological response to man to group B meningococcal polysaccharide vaccines, J. Infect. Dis., 126, 514-522.

Yagura, T., Kamisaki, Y., Wada, H. & Yamamura, Y. (1981) Immunological studies on modified enzymes. I. Soluble L-asparaginase/mouse albumin copolymer activity and substantial loss of immunogenicity. Int. Arch. Allergy Appl. Immunol., 64, 11.

Yamamoto, H., Miki, T., Oda, T., Hirano, T., Sera, Y., Akagi, M. & Maeda, H. (1990) Reduced bone marrow toxicity of neocarzinostatin by conjugation with divinylethermaleic acid copolymer, Eur. J. Cancer., 26, 253-260. **

Yamaoka, T., Tabata, Y. & Ikada, Y. (1994) Distribution and tissue uptake of polyethylene glycol with different molecular weights after intravenous administration to mice, J. Pharm. Sci., 83, 601-606.

Yang, Z., Zhang, Y., Markland, P. & Yang, V.C. (2002) Poly (glutamic acid) poly(ethylene glycol) hydrogels prepared by photoinduced polymerisation: Synthesis, characterisation and preliminary release studies of protein drugs, J. Biomed. Mater. Res., 62, 14-21.

Yoshinaga, K., Shafer, S.G. & Harris, J.M. (1987) Effects of polyethylene glycol substitution on enzyme activity, J. Bioactive. Compatible Polymers., 2, 49-56.

318 ______References

Zalipsky, S. (1995) Chemistry of polyethylene-glycol conjugates with biologically- active molecules, Drug Del Rev., 16, 157-182.

Zalipsky, S. & Harris, J.M. (eds), (1997) in. Polyethylene glycol: Chemistry and biological applications, ACS Symposium series no. 680, American Chemical Society, Washington DC.

Zhang, X. (1999) Polysialylated liposomes: preparation, characterisation and stability studies in vitro and in vivo, PhD Thesis., University of London, U.K.

Zhao, Q., Tolmachev, V., Carlsson, J., Lundqvist, H., Sundin, J., Janson, J. C. & Sundin, A. (1999) Effects of dextranation on the pharmacokinetics of short peptides. A PET study on mEGF, Bioconj. Chem., 10, 938-946.

Zimmerman, J.J. (1991) Therapeutic application of oxygen radical scavengers. Chest., 100,189S-192S.

Zolle, I., Hosain, P., Rhodes, B.A. & Wagner, H.N. (1970) Human serum albumin millimicrospheres for studies of the reticuloendothelial system J. Nuc. Med., 11, 379.

319 Publications

320 Publications

Publications

Papers Gregoriadis, G., Fernandes, A., McCormack, B., Mital M. & Zhang, X. (1998) (Review) Polysialic acids: potential for long circulating drug, protein, liposome and other microparticle constructs, in G. Gregoriadis & B. McCormack (eds.). Targeting of Drugs. Strategies for Stealth Therapeutic Systems^ Vol 6. pp. 93-205, Plenum Press, New York,.

Gregoriadis, G., Fernandes, A., McCormack, B., Mitah M. & Zhang, X. (1999) Polysialic acids: potential role in therapeutic constructs. Biotechnology and Genetic Engineering Reviews., 16, 203.

Gregoriadis, G., Fernandes, A., Mitah M. & McCormack, B. (2000) Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics (Review), Cellular and Molecular Life Sciences., 57, 1964-1969.

Presentations Mitah M and Gregoriadis, G. (1999) Polysialylated IgG: Synthesis and Characterisation. Fourth ULLA Summer School, European Consortium, Copenhagen, Denmark (Poster presentation).

Mitah M and Gregoriadis, G. (2000) Polysialylation of peptide and protein therapeutics: an approach to improving their stability and half-life in the blood circulation. 8^^ Annual HR Pharmaceutical Conference. Protein and Peptide Drug Delivery, London, UK (Oral communication).

321 Appendices

322 Appendix One: Buffers and reagents

Appendix One: Buffers and reagents

All solutions were made with deionised water except in the case of the electrophoresis buffers were UHQ water was used. The asterisk (*) denotes that a fresh solution must be made fresh daily.

0.05M Phosphate buffer, pH 7.0

1. Dissolve 6 .8 g KH 2 PO4 in water and make up to IL.

II. Dissolve 17.9g Na 2 HP0 4 .1 2 H2 0 in water and make up to IL

Mix the solutions I and II until pH 7.0

0.15M Phosphate buffer, pH 7.4 (PBS) NaCl 8.0g Na2HP04 0.025g NaH2P04.2H20 0.05g KCl 0.20g

Dissolve in water, adjust the pH to 7.4 and make up to IL. The solution may be made up times 10 for storage and diluted as required.

0.25M Phosphate buffer, pH 7.5

1. Dissolve 34g KH2 PO4 in water and make up to IL.

II. Dissolve 35.5g Na 2 HP0 4 .2 H2 0 in water and make up to 1L.

Mix the solutions I and II until pH 7.5

0.0015M Borate buffer, pH 8.0 Boric acid 0.046g KCl 0.056g

Dissolve in water, adjust the pH to 8.0 with NaOH and make up to IL.

323 Appendix One: Buffers and reagents

Electrophoresis stock buffers All the buffer solutions are filtered through Whatman No. 1 filter paper and stored at 4°C.

Acrylamide-bisacrylamide (30:0.8) stock solution Dissolve 30g of acrylamide and 0.8g bisacrylamide in a total volume of 100ml of water.

Hydrolysis of acrylamide monomer to yield acrylic acid and ammonia will occur upon prolonged storage. Prepare stock solution to last for 1-2 months, to ensure reproducible results.

Stacking gel buffer stock: 0.5M Tris-HCl, pH 6.8 Dissolve 6.0g Tris base in 40ml water. Titrate to pH 6.8 with IM HCL (-48ml), and make up to 100ml final volume with water.

Resolving gel buffer stock: 3.0M Tris-HCl, pH 8.8 Mix 36.6g of Tris base with 48.0ml IM HCl and make up to 100ml final volume with water.

Reservoir buffer stock: 0.25M Tris, 1.92M glycine, 1% SDS, pH 8.3 30.3g Tris, 144.0g glycine, and lO.Og SDS were dissolved in and made up to IL with water.

Sample solubilization buffer Water 4.0ml 0.5M Tris-HCl 1.0ml Glycerol 0.8ml 10% (w/v) SDS 1.6ml P- mercaptoethanol 0.4ml 0.5% Bromophenol blue 0.2ml

324 Appendix One: Buffers and reagents

10% (w/v) SDS Dissolve lOg of SDS in water to 100ml. This solution is stable at 20°C (room temperature) for several weeks but precipitates in the cold.

1.5% Ammonium persulphate* 0.15g of ammonium persulphate was dissolved in 10ml of water. This solution is unstable and was prepared fresh just before use.

Coomassie blue staining solution Prepare a 0.2% (w/v) Coomassie brilliant blue R in a solution containing acetic acid: methanol: water (10: 35: 55). On the day of use, dilute 1:10 with the same solution and CUSO4 to reduce background staining.

ELISA reagents

Coating buffer: 0.05M carbonate-bicarbonate buffer, pH 9.6 NazCOs 0.318g NaHCOs 0.586g

Dissolve in 150ml, adjust pH to 9.6 (IM NaOH) and make up to final volume of

2 0 0 ml.

Washing buffer: PBS-Tween 20 buffer (PBS-T) (0.05% (v/v) Tween 20) NaCl 200g Na2HP04.2H20 36.5g

KH2 PO4 5g Tween 20 12.5ml

Make up to 2.5L with water, after adjusting the pH to 7.2. The buffer is lOx concentrated and therefore diluted 1:10 prior to use.

325 Appendix One: Buffers and reagents

Peroxidase conjugated seconadary antibody solution* Diluted 1/4000 in PBS-T supplemented with 5% foetal calf serum (PCS) and 1% ESA.

Substrate buffer, pH 5.0* O.IM citric acid 48.6ml

0.2M Na2 HP0 4 .2 H2 0 50.6ml water 100ml o-phenylenediamine SOmg

30% H2 O2 SOpl

Mix together and adjust to pH 5.0. Prepare just before use.

Resorcinol reagent Cone, hydrochloric acid 80ml 1 % (w/v) Resorcinol solution 20ml 0.1M Cupric sulphate 250pl

Keep in the dark at 40°C for not more than a month

Bradford reagent 1. Dissolve lOOmg of Coomassie brilliant blue G-250 in 50ml of ethanol and filter to remove and undissolved material. Dilute the solution to 500ml with water. II. 17% Phosphoric acid (commercial 85% phosphoric acid diluted 1: 5)

These solutions are stable for months at 20°C (room temperature)

Solutions I and II are mixed in a 1: 1 volume ratio to form the colour reagent and kept at 4°C for up to two weeks.

326 Appendix Two: Manufacturers and suppliers

Appendix Two: List of manufactures and suppliers

Amersham Pharmacia Biothechnology, Buckinghamshire, UK Aldrich Chemical Company, Gillingham, Dorset, UK. Bantin & Kingman Universal, North Humberside, Hull, UK. BDH Laboratory Supplies, Poole, Dorset, UK. BioRad Laboratories Ltd, Hemel Hempstead, Herts, UK. Du Pont (UK) Ltd, Stevenage, Herts, UK. Dynatech laboratories Inc., Billingshurst, Sussex, UK. Edwards High Vacuum, Crawley, Sussex, UK. Elga Ltd, High Wycombe, Bucks, UK. EY Laboratories, USA Fisher Chemical Company, Loughborough, Leics, UK. Fisons Chemicals, Loughborough, Leics, UK. Fluka BioChemica, Poole, Dorset, UK. Harlan-OLAC UK, Bicester, Oxon, UK. Heraeus Equipment Ltd, Brentwood, Essex, UK. Lancaster Chemical Company, Lancashire, UK Medicell International Ltd, London, UK. Nalge (Europe) Ltd, Hereford, UK. Pharmacia LKB Biotechnology, St. Albans, Herts, UK. Pierce Chemical Company, USA. Rathburn chemicals Ltd, Walkerbum, Scotland, UK. Sartorius Ltd, Epsom, Surrey, UK. Sera-lab, Crawley Down, Sussex, UK. Sigma Chemical Company, Poole, Dorset, UK. Sorensen Bioscience, Inc., USA. Wallac UK Ltd, Crownhill, Milton Keynes, UK. Watson Marlow, Smith & Nephew Pharmaceuticals Ltd, Falmouth, Cornwall, UK. Whatman Scientific Ltd, Maidstone, Kent, UK.

r 327