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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
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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-
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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
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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
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