Stability andCharacterization ofProtein andPeptide Drugs

Case Histories

Pharmaceutical Biotechnology

Series Editor: Ronald T. BorchardtThe University ofKansasLawrence. Kansas

Volume I PROTEIN PHARMACOKINETICS AND METABOLISMEdited by Bobbe L. Ferraiolo, Marjorie A. Mohler,and Carol A. Gloff

Volume 2 STABILITY OF PROTEIN PHARMACEUTICALS,Part A: Chemical and Physical Pathways of ProteinDegradationEdited by Tim J. Ahern and Mark C. Manning

Volume 3 STABILITY OF PROTEIN PHARMACEUTICALS,Part B: In Vivo Pathways of Degradation and Strategiesfor Protein StabilizationEdited by Tim J. Ahern and Mark C. Manning

Volume 4 BIOLOGICAL BARRIERS TO PROTEIN DELIVERYEdited by Kenneth L. Audus and Thomas J. Raub

Volume 5 STABILITY AND CHARACTERIZATION OF PROTEINAND PEPTIDE DRUGS: Case HistoriesEdited by Y. John Wang and Rodney Pearlman

Stability andCharacterization ofProtein andPeptide DrugsCase Histories

Edited by

Y.John WangScios Nova. Inc.Mountain View. California

and

Rodney PearlmanGenentech, Inc.South San Francisco. California

Springer Science+Business Media, LLC

Llbrary of Congress Catalog1ng-ln-Publ1catlon Data

Stabl11ty and characterlzatlon of proteIn and peptIde drugs casehlstorles I edlted by Y. Hang and Rodney Pea rlMan.

p. CM . -- (PharNaceutlcal blotechnology : v.51Includes blbl10graphlcal references and Index.

1. ProteIn drugs. 2. Drug stabl11ty. 3. Drug Stabl1lty.1. Hang, Y. John. 11. PearlMan, Rodney. 111. Serles .

[DNLM , 1. Protelns- -pharNacoklnetlcs. 2. Protelns --cheNlstry .3 . Peptldes--pharMacoklnetlcs . 4 . Peptldes--cheNlstry . CU 55 S77519931RS431.P75S69 1993615' .3--dc20DNLM/DLCfor Llbrary of Congress 93-7490

CIP

1098765432

ISBN 978-1-4899-1238-1 ISBN 978-1-4899-1236-7 (eBook)DOI 10.1007/978-1-4899-1236-7

© 1993 Springer Science+Business Media New YorkOriginally published by Plenum Press, New York in 1993.Softcover reprint ofthe hardcover I st edition 1993

All rights reserved

No partof this bookmaybe reproduced, storedin a retricvai system, or transmittedin any formor by any mcans, clcctronic, mcchanica1. photoeopying, microfilming.rccording, or othcrwisc, withoutwrlttenpcrmission fromthcPublishcr

To Doris and Anne

Contributors

Akwete Lex Adjei • Product Development, Pharmaceutical Products Di­vision, Abbott Laboratories, North Chicago, Illinois 60064

Thomas A. Bewley • Department of Pharmaceutical Research and Devel­opment, Genentech, Inc., South San Francisco , California 94080

Jens Brange • Novo Research Institute, Novo Nordisk A/S, DK-2880Bagsvaerd, Denmark

Stephen R. Davio • Drug Delivery R&D-Pharmaceutics, Upjohn Labo­ratories, The Upjohn Company, Kalamazoo, Michigan 49007

Jodi Fausnaugh • Institute of Analytical Research, Syntex Research, PaloAlto, California 94304

John Geigert • Quality Control Department, Chiron Corporation, Emer­yville, California 94608. Present address: Immunex Corporation, Seat­tle, Washington 98101

Leo Gu • Institute of Pharmaceutical Sciences, Syntex Research, PaloAlto, California 94304

Michael J. Hageman • Drug Delivery R&D-Pharmaceutics, UpjohnLaboratories, The Upjohn Company, Kalamazoo, Michigan 49007

L. Hsu • Product Development Pharmaceutical Products Division, Ab­bott Laboratories, North Chicago, Illinois 60064

vii

viii Contributors

Daniel J. Kroon • The R. W. Johnson Pharmaceutical Research Insti­tute, Raritan, New Jersey 08869

Lotte Langkjrer • Novo Research Institute, Novo Nordisk A/S, DK-2880Bagsvaerd, Denmark

Mark C. Manning • School ofPharmacy, University ofColorado HealthSciences Center, Denver, Colorado 80262

James W. Mitchell • Oread Laboratories, Lawrence, Kansas 66044

Tue H. Nguyen • Department ofPharmaceutical Research and Develop­ment, Genentech, Inc., South San Francisco, California 94080

Kamlesh Patel • SmithKline Beecham Pharmaceuticals, King ofPrussia,Pennsylvania 19406

Rodney Pearlman • Department ofPharmaceutical Research and Devel­opment, Genentech, Inc., South San Francisco, California 94080

Denise Pretzer • Merck Research Laboratories, West Point, Pennsyl­vania 19486

Patricia E. Rao • The R. W. Johnson Pharmaceutical Research Institute,Raritan, New Jersey 08869 . Present address:Ortho Diagnostic Systems,Inc., Raritan, New Jersey 08869

Niek Roosdorp • Chiron Corporation, Emeryville, California 94608

Brenda S. Schulteis • Marion Merrell Dow Research Institute, KansasCity, Missouri 64134

Christopher D. Smith • Department of Pharmaceutical Chemistry, Uni­versity of Kansas, Lawrence, Kansas 66045

Nicholas Solli • Quality Control Department, Chiron Corporation,Emeryville, California 94608

David G. Vander Velde • NMR Laboratory, University of Kansas,Lawrence, Kansas 66045

Sriram Vemuri • Scios Nova Inc., Mountain View, California 94043

Contributors ix

Carole Ward • Department of Pharmaceutical Research and Develop­ment, Genentech, Inc., South San Francisco, California 94080

Peggy Woehleke • Quality Control Department, Chiron Corporation,Emeryville, California 94608

C. Tony Yu • Bristol-Myers Squibb, Pharmaceutical Research Institute,Buffalo, New York 14213

Preface to the Series

A major challenge confronting pharmaceutical scientists in the future will beto design successful dosage forms for the next generation of drugs. Many ofthese drugs will be complex polymers of amino acids (e.g., peptides, pro­teins), nucleosides (e.g., antisense molecules), carbohydrates (e.g., polysac­charides), or comple x lipids.

Through rational drug design, synthetic medicinal chem ists are prepar­ing very potent and very specific peptides and antisense drug candidates.These molecules are being developed with molecular characteristics thatpermit optimal interaction with the specific macromolecules (e.g., receptors ,enzymes, RNA, DNA) that mediate their therapeutic effects.However, ratio­nal drug design does not necessarily mean rational drug delivery, whichstrives to incorporate into a molecule the molecular properties necessary foropt imal transfer between the point of administration and the pharmacologi­cal target site in the body.

Like rational drug design, molecular biology is having a significant im­pact on the pharmaceutical industry. For the first time , it is possible toproduce large quantities of highly pure proteins, polysaccharides, and lipidsfor possible pharmaceutical applications. The design of successful dosageforms for these complex biotechnology products represents a major chal­lenge to pharmaceutical scientists.

Development of an acceptable drug dosage form is a complex processrequiring strong interactions between scientists from many different divi­sions in a pharmaceutical company, including discovery , development, andmanufacturing. The series editor, the editors of the individual volumes , andthe publisher hope that this new series will be particularly helpful to scientistsin the development areas ofa pharmaceutical company (e.g., drug metabo­lism, toxicology, pharmacokinetics and pharmacodynamics, drug delivery,preformulation, formulation, and physical and analytical chemistry). In ad-

xi

xii Preface to the Series

dition, we hope this series will help to build bridges between the developmentscientists and scientists in discovery (e.g., medicinal chemistry, pharmacol­ogy, immunology, cell biology, molecular biology) and in manufacturing(e.g., process chemistry, engineering). The design ofsuccessful dosage formsfor the next generation ofdrugs will require not only a high level ofexpertiseby individual scientists , but also a high degree of interaction between scien­tists in these different divisions of a pharmaceutical company.

Finally, everyone involved with this series hopes that these volumes willalso be useful to the educators who are training the next generation of phar­maceutical scientists . In addition to having a high level of expertise in theirrespective disciplines, these young scientists will need to have the scientificskills necessary to communicate with their peers in other scientificdisciplines.

RONALD T. BORCHARDT

Series Editor

Preface

This volume attempts to provide the formulation scientist with case historiesinvolving the use of therapeutic proteins and peptides that have been mar­keted or are under clinical testing. In previous volumes of this series, funda­mental theories and principles of protein characterization and stability werepresented in depth by researchers in their fields of expertise. The way fromtheory to practice is not always obvious and straightforward. There is a needfor practical examples of how the principles and theories are put into use,specifically in the development of a pharmaceutical product. It is our hopethat this volume will fulfill such a need.

It is not a simple task to choose a panel of proteins and peptides from theover 200 agents in human clinical trials. We have tried to collect a widerepresentation of molecules of different sizes-from 10 amino acids (Leu­prolide) to 1020 amino acids (Muromonab CD3). The examples includeagents derived from various sources including monoclonal antibodies (Mur­omonab CD3), recombinant DNA (human and bovine growth hormones),natural source (fibrolase), and chemical synthesis (Leuprolide). Clearly thislist is not intended to be encyclopedic. It is the first time a collection of thissort has been made accessible to the formulation scientists involved in devel­oping protein and peptide products.

Although each chapter in this volume focuses primarily on the charac­terization and stability of a specific molecule, each has unique aspects.Chapter 1on human growth hormone provides detailed examples of charac­terization and stability of the protein from both biochemical and pharma­ceutical aspects. Mechanisms on protein aggregation and covalent bondmodifications are described. Chapter 2 presents spectral analysis on bovinegrowth hormone (somatotropin), a protein homologous to human growthhormone. Solubility, solution stability, and absorption of moisture by the

xiii

xiv Preface

freeze-dried solid are described, which will aid scientists facing similarchallenges.

Alteplase, tissue type plasminogen activator, is covered in Chapter 3.This monograph illustrates approaches to handling a heterogeneous prod­uct. Thermal analysis and stability-pH profile of multiple degradation path­ways are valuable information contained in this chapter. Literature on thestability of monoclonal antibodies is very sparse. Chapter 4 addresses anti­body stability ofMuromonab CD3 (Orthoclone OKT3). The sites and mech­anism of degradation were identified through isoelectric focusing and sup­ported by peptide mapping.

In Chapter 5, the specifications required of a peptide product are de­scribed. Also of interest in this chapter on Leuprolide is the discussion ofthebioavailability profile ofthe peptide when given by different routes ofadmin­istration. Through the discussion of ACTH stability and its active hexapep­tide segment, Chapter 6 presents a detailed mechanistic analysis ofdeamida­tion, which is a major route of degradation for all proteins and peptides.

Development of a reliable, quantitative bioassay is always a concern toformulation scientists. In Chapter 7, results from four different in vitro bioas­says for human interleukin-Iri are delineated. Extensive formulation studiesare also included. Chapter 8, on IL-2, presents a description ofmanufactur­ing procedures and the process of determining the shelf life of a proteinproduct.

The kinetics of aggregation, an obvious physical change which is thebane of many protein products, is discussed with regard to ai-antitrypsin inChapter 9. In Chapter lO, an extensi ve spectral analysis on the natural prod­uct fibrolase, by CD and NMR, is presented. Although insulin has beenavailable since the 1920s, the degradation pathways have not been com­pletely elucidated and presented in a systematic fashion . Chapter 11 consoli­dates all existing information in a concise monograph.

The editors wish to thank all of the contributors for their willingness toshare updated information that made this volume possible. It is hoped thatas more biotechnology products are approved, information related to charac­terization and stability can be made more accessible to the scientists in thisfield. Before that happens, this volume can at least provide a guide and serveas a handy reference to those in need of this kind of information.

Y. JOHN WANG

RODNEY PEARLMAN

Contents

Chapter 1

Stability and Characterization of Human Growth Hormone

Rodney Pearlman and Thomas A. Bewley

1. Introduction. .... ... . . .... . . ... .... . . .... . . . . . ..... .... ... .. .. .. 11.1. History of hGH and hGH Preparations . . . . . . . . . . . . . . . . . . . . . 11.2. Clinical Use of hGH 31.3. Structure and Properties of Pituitary and Recombinant hGH 5

2. Analytical Characterization. . . . . 92.1. Spectroscopy 102.2. Electrophoresis 162.3. Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.4. Bioassays 192.5. Chromatographic Methods. ... . ... . . ... . . .. . . . . . . .. . . ... . . . 20

3. Degradation Pathways 263.1. Deamidation 273.2. Oxidation 293.3. Reduction/Interchange of Disulfide Bonds 313.4. Aggregation 363.5. Proteolysis/Hydrolysis 403.6. Shear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

4. Stability Profile of hGH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434.1. Solution Stability 434.2. Stability in the Solid State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

5. Formulations .. . . . . . . .... ... . . .. .. .. . . . . .. .... .. . .. . . .. . .. . .. . .. 476. Conclusions 48

References 50

xv

xvi Contents

Chapter 2

Characterization and Formulation Considerations for RecombinantlyDerived Bovine Somatotropin

Stephen R. Davio and Michael J. Hageman

1. Introduction .. . . . ... . . . ... .. . . .. .. . . . . . .. . . . . .. . . . .. . . . . . . . . . . . . 591.1. bSt Commercial Applications 591.2. bSt Primary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591.3. rbSt Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611.4. bSt Predicted Conformational Structure 61

2. rbSt Structural Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622.1. rbSt Primary Structure (Sequence) 622.2. rbSt Secondary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672.3. rbSt Tertiary Structure . . 692.4. rbSt Quaternary Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702.5. rbSt Bioassay Characterization 71

3. rbSt Conformational Stability .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723.1. The Effect of Solution pH on rbSt Conformation 723.2. The Effect of Denaturants on rbSt Conformation 73

4. rbSt Physical Properties . . . . . . 744.1. Solubil ity Properties 744.2. Dissolution Properties 77

5. Physical/Chemical Stability . . . . . . . . . 785.1. Solution Stability 785.2. Solid-State Stability 815.3. Absorption of Water in the Solid State 82

6. Formulations . .. . ... . . . .. ... . . .. .. . . . ... . . . . . . . . .. .. . . . . . .. . .... 82References 84

Chapter 3

Stability Characterization and Formulation Development of Alteplase,a Recombinant Tissue Plasminogen Activator

Tue H. Nguyen and Carole Ward

1. Introduction . . ... . .... ....... ... . .... . .. .. .. . .. . . . . . . . . . . . .. . . . .. 911.1. The Fibrinolytic System 921.2. Tissue Plasminogen Activator . . . . . . . . . . . . . . . . . . . . . . . . 921.3. Pharmacology and Clinical Use of Alteplase 93

2. Structure and Properties of Alteplase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942.1. Primary Structure 94

Contents xvii

2.2. Carbohydrate Composition 962.3. Solubility Behavior 97

3. Analytical Characterization . . 993.1. Spectroscopy 1003.2. Electrophoresis 1073.3. Chromogenic Enzymatic Assay 1093.4. Clot Lysis Assay 1113.5. HPLC 113

4. Stability of Alteplase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184.1. Differential Scanning Calorimetry Studies 1184.2. Stability in Solution 1204.3. Stability of Alteplase in the Lyophilized State (Activase) 126

5. Conclusions . ..... . ..... ... . ..... .. . .. .. .... ... .... . . ... . .. . .... 128References 129

Chapter 4

Orthoclone OKT3: Chemical Mechanisms and Functional Effectsof Degradation of a Therapeutic Monoclonal Antibody

Patricia E. Rao and Daniel J. Kroon

1. Background 1351.1. History of Clinical Use ofOKT3 1351.2. Basic Biochemical Structure of OKT3 1361.3. Discovery of OKT3 Hybridoma and in Vitro Functional

Effects 1382. Biological Effects of Degradation of OKT3 139

2.1. Effects on Binding Affinity 1392.2. Ability to Induce Mitogenesis 1402.3. Ability to Inhibit MLR 141

3. Biochemical Evidence of Degradation 1423.1. Shifts in IEF Pattern 1433.2. Alteration in HPLC-IEC Retention Times . . . . . . . . . . . . . . . 1443.3. Protein Chain Alterations Detected by SDS-PAGE 145

4. Preliminary Studies of Degradation Mechanisms... . .... .... .. . . 1475. Identification of Degradation Sites by Peptide Mapping..... .. . . . 148

5.1. Peptide Mapping Method ........ ...... .. ...... .. ........ .. 1485.2. Sites of Deamidation 1505.3. Potential Sites of Oxidative Degradation. . . . . . . . . . . . . . . . . . . . 1525.4. Formation ofInterchain Cross-links. . . . . . . . . . . . . . . . . . . . . . . . 153

6. Conclusion 155References 156

xviii

Chapter 5

Leuprolide and Other LH-RH Analogues

Akwete Lex Adjei and L. Hsu

Contents

1. Introduction .. .. . . . .. . . . . . . . . . . .. . . . . . .. . . . . .. . . . . .. . .. . .. . . ... . 1591.1. Pharmacology ofLH-RH 1591.2. Leuprolide Acetate and Other LH-RH Analogues . . . . . . . . .. . 1601.3. Chemistry and Structural Properties of LH-RH Analogues . . 161104. Analytical Characterization 1691.5. Physical Pharmacy of Leuprolide Acetate. . . . . . . . . . . . . . . . . . . 1771.6. Bioanalysis 184

2. Formulations and Dosimetry :..... ..... . .... 1852.1. Parenteral Product Forms . .. . .. .. .. . .. .. .. .. . .. .. .. . .. . .. . . 1862.2. Nasal Dosage Forms 1862.3. Inhalation Dosage Forms 187

3. Preclinical and Clinical Studies with Leuprolide Acetate 1883.1. Pharmacokinetics following Acute Administration .. . . . . . . . . 1883.2. Pharmacodynamics following Chronic Administration . . . . . . 190

4. Stability Studies and Degradation Kinetics 1914.1. Drug Substance Stability 1924.2. Formulation Stability 1934.3. Shelf-Life Projections 193

5. Conclusions 1946. Appendix: Schematic Representation of Ion-Pair Partition Model 195

References 196

Chapter 6

Stability of Adrenocorticotropic Hormone (ACfH) and Pathways ofDeamidation of Asparaginyl Residue in Hexapeptide Segments

Kamlesh Patel

1. Introduction . . .. . .. .... . .. . .. . ... . . . .. . . . . . .. . . . . .. . . . . . . . . . . . . . 2011.1. Pharmacology 2011.2. Clinical Use 2021.3. Structure and Physicochemical Properties 202104. Preparations and Routes of Administration. . . . . . . . . . . . . . . . . 203

2. Chemical Stability 2032.1. Deamidation of ACTH 2032.2. Deamidation of ACTH22

-27

• • •• • • • • • • • ••• • • • • •••• • • • • • • • • • • 207

Contents xix

3. Conclusions 218References 218

Chapter 7

Stability and Characterization of Human Interleukin-Iji

Leo Gu and Jodi Fausnaugh

I. Introduction . . . .. . . . . . . . . . . . . . . .. . . ... . . ...... . .... .... ...... ... 2211.1. Preparation of Recombinant hIL-I,B . . . . . . . . . . . . . . . . . . . . . . . . 2221.2. Pharmacology Evaluation and Clinical Applications 224

2. Structural Characterization and Analytical Methods 2252.1. Spectroscopy 2252.2. Amino Acid Analysis 2252.3. Sequence Analysis 2262.4. Secondary and Tertiary Structural Analysis. .. . . . . . . .. . . . 2282.5. Chromatographic and Electrophoretic Methods... ....... . .. 2302.6. Bioassays 2322.7. lmmunoassays .. . . ....... ..... .. . . ... .. .. . .. . . ... . . .... .. . . 234

3. Physicochemical Stability 2353.1. Unfolding and Refolding ofrhIL-I,B 2353.2. Freeze-Thaw Effect 2353.3. Aggregation and Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2373.4. Stability at or below 30°C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

4. rhIL-I ,B Formulation and Its Stability ..... .. .. ...... .. .... .. .. .. 241References 245

Chapter 8

Development and Shelf-Life Determination of RecombinantInterleukin-2 (Proleukin)

John Geigert, Nicholas Solli, Peggy Woehleke, and Sriram Vemuri

I. The IL-2 Molecule 2492. The IL-2 Manufacturing Process 251

2.1. Synthesis 2512.2. Recovery 2512.3. Purification 2522.4. Formulating and Finishing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 253

3. Stability-Indicating Test Methods .. .... ......................... 2533.1. Methods for IL-2 2533.2. Methods for Proleukin 255

xx Contents

4. Proleukin Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2575. Stability upon Reconstitution 259

References 261

Chapter 9

Formulation and Stability of Recombinant at-Antitrypsin

Sriram Vemuri, C. Tony Yu, and Niek Roosdorp

1. Introduction. .. . ...... ... . ... .. . . . .. ... .... ....... . . .. . ..... . ... 2632. Overview of the Manufacturing Process 2653. Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

3.1. Determination of Monomeric rAAT Content . . . . . . . . . . . . . . . 2653.2. Determination of rAAT Activity 2663.3. Determination of Total rAAT Protein . . .. ..... ..... .... .... 2663.4. Determination of Total Nitrogen ... . . . . .. . .. . . . . . . .. . . ... .. 267

4. Preformulation Studies 2674.1. Effect of pH on rAAT Solution Stability . . . . . . . . . . . . . . . . . 2674.2. Effect of Potassium Chloride on rAAT Solution Stability.. .. 2684.3. Effect of Ionic Strength on rAAT Solution Stability 2694.4. Effect of Citrate on rAAT Solution Stability 2704.5. Physical Stability of rAAT Solution 271

5. Formulation Development .. . .. . .. . . .. . .. . . .. . .. . .. .. .. . . .. . . 2775.1. Solution Stability Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2775.2. Lyophilization 279

6. Conclusion 284References 285

Chapter 10

Fibrolase: A Fibrinolytic Protein from Snake Venom

Denise Pretzer, Brenda S. Schulteis, Christopher D. Smith,David G. Vander Velde, James W Mitchell, and Mark C. Manning

1. Introduction . ... . . . . . . ... . ... .... .. ..... ..... .... ...... . .... .... 2872. Physical Properties 2893. Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

3.1. Proteolytic Activity with Oxidized Insulin B Chain as aSubstrate 290

3.2. Proteolytic Activity Assay with Azocasein as a Substrate 2913.3. Circular Dichroism Measurements 291

Contents xxi

3.4. Nuclear Magnetic Resonance Measurements 2913.5. Electrophoresis and Protein Quantitation . . 292

4. Effect of pH on Fibrolase Stability 2925. Stability of Fibrolase and Temperature Effects . .. ... . .. . . ... .. . .. 2986. Effectsof Zinc Binding on the Stability and Structure of Fibrolase 305

6.1. Effectsof EDTA Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3056.2. Proposed Zinc Binding Site 309

7. Summary 311References 312

Chapter 11

Insulin Structure and Stability

lens Brange and Lotte Langkjar

1. Introduction . . .... ..... ..... . . .... ... . .... . ..... .. ... . ... . ...... 3152. Insulin Structure 316

2.1. Primary Structure 3162.2. Secondary and Tertiary Structure 3162.3. Quaternary Structure (Self-Association) 3172.4. Structure in Pharmaceutical Formulations. . . . . . . . . . . . . . . . . . 3202.5. Stability Overview 321

3. Physical Stability 3223.1. Isoelectric and Metal-Ion-Induced Precipitation 3223.2. Fibrillation 3223.3. Adsorption Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3243.4. Influence of Insulin Species and Purity 3243.5. Physical Stability of Pharmaceutical Formulations . . . . . . .. . . 325

4. Chemical Stability 3274.1. Introduction 3274.2. Formation of Hydrolysis Products. . . . . . . . . . . . . . . . . . . . . . . . . . 3284.3. Di- and Polymerization 3324.4. Influence of pH and Auxiliary Substances 3344.5. Effect of Temperature 3344.6. Kinetics and Mechanisms 3364.7. Properties of Transformation Products 3404.8. Influence on the Quality of Pharmaceutical Preparations . . . . 340

5. Summary and Conclusions 342References 344

Index 351