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`ThematerialonthispagewascopiedfromthecollectionoftheNationalLibraryofMedicinebyathirdpartyandmaybeprotectedlb‘yUZS.CooYiighllaW.
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`
`
`Parenteral Medications
`Volume1
`Setond Edition, Revised and Expanded
`
`Edited by
`
`Kenneth E. Avis
`The University of Tennessee
`Memphis, Tennessee
`
`Herbert A. liebermun
`H.H. Lieberman Associates, Inc.
`Consultant Services
`
`Livingston, New Jersey
`
`leon Imhmun
`Lachman Consultant Services
`
`Westbury, New York
`
`
`
`'
`
`15.
`
`-——-A
`
`1
`
`Marcel Dekker, Inc.
`
`New York s Basel 0 Hong Kong
`
`=
`
`ALKERMES Exh. 2048
`
`Luye V. Alkermes
`IPR2016-1096
`
`ALKERMES Exh. 2048
`Luye v. Alkermes
`IPR2016-1096
`
`
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`:hl'Mellufiufidéo'S'llKQ‘PGlOSIOJC-isqflewpuelinedpull;aliqeulogpawi0MalquieuoueNout;ououoenooan)tum;paidoo$9M969dsunuomusicalaqi
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`Library of Congress Cataloging — in— Publication Data
`
`Pharmaceutical dosage forms , parenteral medications / edited by
`Kenneth E. Avis, Herbert A. Lieberman, and Leon Lachman. " 2nd ed. ,
`rev . and expanded.
`p .
`cm.
`Includes bibliographical references and index.
`ISBN 0—8247-8576-2 (v. 1 : alk.' paper)
`1. Parenteral solutions.
`2‘. Pharmaceutical technology.
`Kenneth E.
`II. Lieberman, Herbert A.
`III. Lachman, Leon.
`[DNLM: 1. Infusions, Parenteral.
`WB 354 P536]
`R8201.P37P48 1992
`615'. 19--dc20
`DNLM IDLC
`for Library of Congress
`
`_
`I. AVIS,
`
`.
`2. Technology, Pharmaceutmal.
`
`91 -38063CIP
`
`9720“2?:2
`
`it)!i“fiat/(.9.
`
`This book is printed” acid-free paper.
`
`Copyright © 1992 MARCEL DEKKER, INC. All Rights Reserved
`/
`.
`Neither this boaé'hor any part may be reproduced or transmitted in any form
`or by any means, electronic or mechanical, including photocopying, micro-
`filming, and recording, or by any information storage and retrieval system,
`without permission in writing from the publisher.
`
`MARCEL DEKKER,-INC.
`270 Madison Avenue, New York, New York 10016
`
`Current printing (last digit):
`10 9 8 7 6 5 4 3 2 1
`
`PRINTED IN THE UNITED STATES OF AMERICA
`
`
`
`
`
`
`
`0.3.Copyrightlaw)“
`
`
`
`
`
`
`
`ThematerialonthispagewascopiedfromthecollectionoftheNationalLibraryofMedicinebyathirdpartyandmaybeprotectedby
`
`
`
`
`
`
`
`Contents
`
`
`
`..4“
`
`
`
`,
`
`Preface
`Contributors
`Contents of Pharmaceutical Dosage Forms: Parenteral Medications,
`Second Edition, Revised and Expanded, Volumes 2 and 3
`Contents of Pharmaceutical Dosage Forms: Tablets, Second Edition,
`Revised and Expanded, Volumes 1—3
`Contents of Pharmaceutical Dosage Forms: Disperse Systems,
`Volumes 1 and 2
`
`x1
`
`xiii
`
`xv
`
`XVii
`
`Chapter 1 The Parenteral Dosage Form and Its Historical Development
`Kenneth E. Avis
`
`I. The Dosage Form
`11. History of Parenteral Medications
`Appendix A: Glossary of Terms
`Appendix B: Highlights in the History of
`Parenteral Medications
`References
`
`'
`
`Chapter 2
`
`Parenteral Drug Administration: Routes, Precautions,
`Problems, Complications, and Drug Delivery Systems
`
`Richard J. Duma, Michael J. Akers, and
`Salvatore J. Turco
`
`Introduction ‘
`I. General Indications for Parenteral
`
`II.
`
`Administration of Drugs
`Pharmaceutical Factors Affecting Parenteral
`Administration
`
`Specific Routes of Administration
`III.
`IV. Distribution of Parenterally Administered Agents
`
`1
`
`1
`4
`12
`
`14
`15
`
`17
`
`17
`
`18
`V
`19
`
`21
`39
`
`vii .
`
`
`
`
`
`viii
`
`Contents
`
`V.
`
`Precautions, Problems, Hazards, and
`Complications Associated with Parenteral Drug
`Administration
`VI. Methods and Devices for Drug Delivery Systems
`VII.
`Summary
`References
`
`Chapter 3
`
`Biopharmaceutics of Injectable Medications
`
`Sol Motola
`
`I.
`
`II.
`
`Introduction
`
`Physicochemical and Physiological Factors
`Affecting Drug Absorption by Injection: An
`Overview
`
`III. Application of Pharmacokinetics to Biopharma—
`ceutic Investigations: Pharmacokinetic Models
`IV. Examples of Biopharmaceutic IPharmacokinetic
`Principles
`V. Regulatory Considerations for Bioequivalence
`Studies
`
`VI. Bioequivalence Study of Two Injectable Forms
`of the Same Drug
`Summary
`References
`
`VII.
`
`Chapter 4
`
`Preformulation Research of Parenteral Medications
`
`Sol Motola and Shreeram N. Agharkar
`
`I .
`
`Introduction
`
`11. Drug Substance Physicochemical Properties
`III. Accelerated Stability Evaluation '
`IV. General Modes of Drug Degradation
`V.
`Preformulation Studies for Proteins and Peptides
`VI.
`Preformulation Screening of Parenteral
`Packaging Components
`Summary
`Preformulation Worksheet
`References
`
`VII.
`VIII.
`
`Chapter 5
`
`Formulation of Small Volume Parenterals
`
`Patrick P. DeLuca and James C. Boylan
`
`I .
`
`Introduction
`
`Formulation Principles
`II.
`III. Container Effects on Formulation
`
`IV. Stability Evaluation
`V. Process Effects
`References
`
`41
`49
`56
`57
`
`59
`
`59
`
`60
`
`77
`
`98
`
`108
`
`109
`111
`112
`
`115
`
`115
`
`116
`140
`150
`154
`
`158
`163
`163
`169
`
`173
`
`173
`174
`227
`234
`244
`245
`
`
`
`
`
`
`
`g'MBIlu5!lfid03's'nKqpapadeaqflewpueAuadpull;aAqeu!o!paw10£12anlauoueNall],10uouaanooau;wan;peydooSBM959dsunuo"2113121119141
`
`
`
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`
`ThematerialonthispagewascopiedfromthecollectionoftheNationalLibraryofMedicinebyathirdpartyandmaybeprotectedbyUS.Copyrightlaw.
`
`
`
`
`
`
`
`Contents
`
`Chapter 6
`
`Formulation of Large Volume Parenterals
`
`Levit J. Demorest and Jeffrey G. Hamilton
`
`I.
`II.
`III.
`IV.
`V.
`
`Introduction ,
`
`Concepts of Formulation
`Formulation Development
`Solution Quality
`Summary
`References
`
`Chapter 7 Parenteral Products of Peptides and Proteins
`
`Yu‘Chang John Wang
`
`I.
`II.
`III.
`IV.
`
`V.
`
`Introduction
`Characteristics of Proteins and Peptides
`Formulation Principles
`Compatibility'with Packaging Components and
`Infusion Sets
`Formulation of Market Products
`References
`
`Chapter 8
`
`Sterile
`
`Leif E.
`
`I.
`II.
`III.
`IV.
`V.
`VI.
`VII.
`
`Diagnostics
`Olsen
`
`Introduction
`
`Diagnostic Products Defined
`Sterile Diagnostics
`Definitions
`
`Aseptic Manufacturing Considerations
`Validation Program
`Conclusion
`References
`
`Chapter 9 Glass Containers for Parenterals
`
`R. Paul Abendroth and Robert N. Clark
`
`I.
`II.
`III.
`
`IV.
`V.
`VI.
`VII.
`VIII.
`
`Introduction
`
`‘
`The Nature of Glass
`United StatesPharmacopeia Glassware
`Classifications
`The Manufacture of Glass Containers
`Chemical Performance
`
`Mechanical Performance
`The Container and C10sure as a System
`Quality Assurance
`References
`
`249
`
`249
`250
`273
`280
`281
`281
`
`283
`
`283
`284
`302
`
`310
`312
`317
`
`321
`
`321
`321
`322
`325
`330
`351
`359
`359
`
`361
`
`361
`361
`
`362
`369
`375
`380
`380
`382
`384
`
`
`
`
`
`Contents
`
`387
`
`387
`389
`398
`407
`422
`439
`443
`
`445
`
`445
`450
`451
`462
`463
`470
`477
`494
`503
`
`505
`507
`508
`
`513
`
`513
`
`513
`524
`
`532
`547
`552
`562
`
`563
`566
`
`569
`
`x C
`
`hapter 10 Use of Plastics for Parenteral Packaging
`John M. Anes, Robert S. Nase, and
`Charles H. White
`
`I.
`II.
`III.
`IV.
`V.
`VI.
`
`Introduction
`Fundamentals
`Fabrication Processes
`Important Criteria for Selection of Plastics
`Plastics Used in Parenteral Packaging
`Quality Assurance of Parenteral Containers
`References
`‘
`
`Chapter 11 Elastomeric Closures for Parenterals
`
`Edward J. Smith and Robert J. Nash
`
`I.
`
`II.
`III.
`IV.
`V.
`VI.
`VII.
`VIII.
`IX.
`
`XI.
`
`Elastomeric Parenteral Packaging Components:
`
`A Physical Description
`Physical Description of Rubber
`Types of Rubber Used in Parenteral Packaging
`Closure Design
`Rubber Compounding
`Vulcanization Process
`Closure Manufacture and Control
`
`Closure Design Qualification
`Regulatory Considerations
`Interaction of Drug Formulations with
`Rubber Closures
`
`Contemporary Closure-Related Issues
`References
`
`Chapter 12 Parenteral Products in Hospital and Home Care
`Pharmacy Practice
`
`John W. Levchuk
`
`I.
`II.
`
`III.
`IV.
`
`V.
`VI.
`VII.
`
`.
`Introduction
`The Preparation of Sterile Dosage Forms in the
`Hospital and in Home Care
`Dispensing and Compounding Processes
`Technology of Sterile Compounding in the
`Hospital Pharmacy
`Clinical Supply and Use of Sterile Products
`Quality Assurance
`Conclusion
`
`Appendix: Abbreviated Sequence for Preparing a
`Series of Extemporaneously Compounded I.V.
`Admixtures
`References
`
`Index
`
`
`
`
`
`M?!tufiufidoo's'nKqpapa);onaqAewpueAuedpull}9Aqeugogpaw;oMEJQH1euoueNeq;;0uouaanoosq;woupagdooSEM952dSm;uoleglamwaqi
`
`
`
`
`
`
`
`
`
`
`
`7
`Parenteral Products of Peptides
`and Proteins
`
`Yu-Chang John Wang
`
`California Biotechnology, Inc. , Mountain View, California
`
`I. INTRODUCTION
`
`Scientific advances in molecular and cell biology have resulted in the develop-
`ment of two new biotechnologies. The first utilizes recombinant DNA to pro-
`duce protein products. Given the amino acid sequence of a protein, the gene
`responsible for producing such protein can be isolated, modified, and recom-
`bined with a plasmid DNA (Fig. 1). The modified plasmid can then be im-
`planted into a host cell. The recombinant DNA will be replicated and tran-
`scribed by the host cell to produce the specific protein in large quantities.
`Escherichia coli have been modified to produce growth hormone and growth
`factors, yeast to produce hepatitis B vaccine, and mammalian cells to produce
`erythropoietin. The advantages of such production are practically unlimited
`supplies of protein of high purity, and consistent quality in lot-to-lot produc-
`tion
`The second technology involves fusion of antibody-producing B-lympho-
`cytes and myeloma (tumor) cells to form hybridomas (Fig. 2). Hybridomas
`grown in vivo in ascitic fluid or in vitro in tissue culture produce a single
`species of antibody, known as monoclonal antibody, which recognizes a spe-
`cific antigen. In contrast, conventional production methods in laboratory
`animals produce polyclonal antibodies that are comprised of a wide variety of
`antibodies with different affinity and antigenic specificities. Monoclonal anti-
`bodies, because of their unique properties of homogeneity, specificity, and
`affinity, can be used to: (1) isolate and purify proteins, e.g. , Monoclat Fac-
`tor VIII:C is produced in this fashion; (2) neutralize a specific group of cells,
`e.g., Orthoclone OKT-3 neutralizes T-cells; (3) target specific cells or organs,
`e.g., antibodies to tumor antigens allow for tumor imaging or therapy by link-
`ing imaging radioactive isotopes or cytotoxic drugs to the antibodies.
`
`
`
`284 (cid:9)
`
`Donor DNA
`
`0
`
`Restriction
`enzymes
`
`O
`
`Plasmid DNA
`
`Wang
`
`Replication
`
`Expression
`
`Protein
`
`Recombinant
`DNA
`
`Host cell
`
`Figure 1 Recombinant DNA for protein production. The technique of recom-
`bining genes from one species with those of another. Plasmid is an extrachro-
`mosomal, independently replicating small circular DNA molecule. (1) Restric-
`tion enzymes cut DNA at specific places. The donor DNA, represented by a
`heavy band, contains the information to produce the protein of interest. (2)
`Ligase fuses donor DNA with plasmid together. (3) Calcium is used to open
`cell wall or cell membrane to allow recombinant DNA to enter the host cell (bac-
`terium, yeast or mammalian cell). (4) Replication of the host cells and recom-
`binant DNA increase its number. Expression of the DNA produces desired
`protein.
`
`New products approved by the FDA between 1982 and 1989 derived from
`these two new biotechnologies are listed in Table 1.
`Although these two new biotechnologies have greatly expanded the capa-
`bility for producing large amounts of high quality proteins, not all new pro-
`tein products are produced in this way. Small proteins and peptides, such
`as calcitonin, may be produced by chemical synthesis. Human serum albumin
`is sourced from human blood, urokinase from urine, and streptokinase from
`fungi. Most of these proteins and peptides are formulated as injectable prod-
`ucts, although there are some exceptions. Cyclosporin is given per os. Oxy-
`tocin and desmopresin are available as nasal preparations. A topical ointment,
`Elase, contains two lytic enzymes, fibrinolysin and desoxyribonuclease, in
`lyophilized form. Wound-healing growth factors are being developed for use
`as topicals.
`Among the parenteral formulations, most of them are in aqueous solution
`or in freeze-dried form for reconstitution. However, one of the potent luteiniz-
`ing hormone release hormone (LHRH) analogues, leuprolide, and bovine somato-
`tropin have been developed as erodable microspheres for depot injection.
`
`II. CHARACTERISTICS OF PROTEINS AND PEPTIDES
`
`A. Protein Structures
`
`Proteins and peptides are made of amino acids linked by peptide bonds. The
`sequence of these amino acids defines the protein's structure. The sequence
`of amino acids in human insulin is illustrated in Figure 3. A protein may also
`show a secondary structure, which is formed by either intrachain or inter-
`chain hydrogen bonds. These bonds may result in either an a-helix or 13-sheet
`
`
`
`Parenteral Products of Peptides and Proteins (cid:9)
`
`285
`
`Antigen
`
`t> 0 0
`0
`Clone
`sselectt
`
`B- lymphocytes 4 ° (cid:9)
`0
`_,.---"*"...
`I ®
` 2c
`C4 ----..."*.„46,
`
`0 (cid:9)
`
`0 (cid:9)
`Myelomo
`cells CAI'
`
`
`
`(a)©. 0
`
`*
`Hybridoma (cid:9)
`
`Ascitic fluid
`
` Cne„....`4 •
`
`Separation
`
`lY ii'
`
`
`Y
`Desired
`antibody
`
`Fermenter
`
`Figure 2 Monoclonal antibody production. (1) A mouse or rat is immunized
`with a specific antigen. The spleen is removed and antibody-producing cells
`(B-lymphocytes) are isolated. (2) Transformed myeloma cells from another
`animal are isolated. (3) B-lymphocytes are fused with myeloma cells deficient
`in a particular enzyme, to form hybridoma cells. (4) Only the fusion product,
`hybridoma cells, can grow indefinitely in culture. The unfused myeloma cells
`and lymphocytes die. The hybridoma cells are cloned and ones expressing
`the desired antibody are selected to culture in large amounts. The antibody
`can be produced by these hybridoma cells in either ascitic fluid or fermenta-
`tion tanks. The ascitic fluid or culture media is then processed to yield puri-
`fied monoclonal antibodies.
`
`structure. Figure 4 shows a peptide chain of a protein coiled to form an cx-
`helix.
`The tertiary structure or conformation of a protein refers to the spatial,
`three-dimensional structure of the polypeptide chain. Given the primary and
`secondary structure, there are four kinds of interactions cooperating which
`may contribute to the tertiary structure of a protein. They are: (1) hydro-
`gen bonds between residues in adjacent loops of the chain, (2) ionic interac-
`tions (salt bridges) between oppositely charged residues, (3) hydrophobic
`interactions between the aliphatic or aromatic residues, or (4) disulfide link-
`ages (Fig. 5). Protein, in its native state, exists as a tight, compact folded
`structure. The folded structure of insulin is shown in Figure 6. When the
`tertiary or secondary structure is destroyed, the protein unfolds and is in
`its denatured state.
`If dimers, trimers, or other oligomers are formed, the arrangement of
`these subunits is a quaternary structure. The best-known example of an
`oligomeric protein is hemoglobin that contains four chains and no disulfide
`linkages. The quaternary structure is essential to its oxygen-carrying activ-
`ity. Insulin also forms dimers and, in the presence of zinc, hexamers. The
`role of the zinc insulin hexamers is to provide a storage form that is thermo-
`dynamically stable and more resistant to enzymatic degradation than the un-
`associated monomer.
`
`(cid:9)
`
`
`286 (cid:9)
`
`Wang
`
`Table 1 (cid:9) Biotech Products Approved in the United States Between 1982
`and 1989
`
`Drug
`
`Company
`
`Date approved
`
`Indication
`
`Insulin
`Human growth
`hormonea (hGH)
`Interferon
`A2b (IFN)
`Muromonab-CD3
`
`Hepatitis B
`vaccine
`Tissue plasminogen
`activator (t-PA)
`Erythropoietin (EPO)
`Hepatitis B vaccine
`
`Lilly
`Genentech
`Lilly
`Schering
`Roche
`Ortho
`
`October 1982
`October 1985
`March 1987
`June 1986
`June 1986
`June 1986
`
`Merck
`
`July 1986
`
`Genentech
`
`October 1987
`
`Amgen
`SmithKline
`Biologics
`
`June 1989
`August 1989
`
`Diabetes
`Dwarfism
`
`Hairy cell
`leukemia
`Reverse kidney
`rejection
`Vaccine against
`hepatitis B
`Thrombolytic
`agent
`Anemia
`Vaccine against
`hepatitis B
`
`a
`Growth hormone from Genentech differs from Lilly's product by a single
`methionine at the N-terminus.
`bInterferons from Schering and Roche differ by one amino acid.
`
`In some proteins, there are carbohydrates attached to specific amino acid
`residues to form glycoproteins. For example, erythropoietin is a glycoprotein
`hormone with a molecular weight of 44 kilodaltons, having 40% of its weight
`accounted for by sialic acid, an amino sugar. Carbohydrate residues stabilize
`the tertiary structure and may influence receptor binding, bioactivity, and
`pharmacokinetics of the protein in vivo.
`
`B. (cid:9)
`I soelectric Point
`In the course of a potentiometric titration of a protein there is a pH at which
`the mean charge on the protein is zero. This pH is termed the isoelectric
`point, pI. Table 2 provides examples of pls for a number of therapeutic pro-
`teins and peptides. The pI may be approximately calculated from the amino
`acid composition data, i.e., pI = (pK1 + pK 2 + pK3 . . . + pKn)/n for n ioniz-
`able groups. However, because the dielectric constant in the immediate vicin-
`ity of an ionizable group depends on protein structure and, because hydrogen
`bonding may alter dissociation constants (Kas), the true p1 can differ signifi-
`cantly from the calculated one.
`Just like pKas of small organic compounds, the pI of a protein plays an
`important role in solubility. In general, near the pH of the isoelectric point,
`protein solubility is at its minimum. This trend is similar to the low solubility
`exhibited by a zwitterion compound.
`
`
`
`Parenteral Prod ucts of Peptid es and Proteins
`
`CO
`
`NH2Gly lle Val Glu Gln Cys Cys Thr Ser lle Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys AsnC0011
`
`S--S
`
`5
`
`S
`
`10 (cid:9)
`
`15
`
`S 21
`
`S
`
`NH2Phe Val Asn
`
`S
`1
`Gln His Leu Cys Gly Ser His Leu Val
`
`Glu Ala Leu Tyr Leu
`
`Val Cys Gly
`
`Glu
`
`Arg Gly Phe Phe Tyr Thr Pro LysiThrCOOR
`
`1 (cid:9)
`
`5
`
`10
`
`15
`
`20
`
`25 (cid:9)
`
`30
`
`Figure 3 Amino acid sequence of human insulin. Insulin has two chains, the A chain with 21 and the
`B chain with 30 amino acids. The two chains are connected by two disulfide cross-linkages, and one
`of the chains has an internal disulfide linkage. Although insulins isolated from the pancreas of a cow
`or a pig are used in treatment of diabetic patients, they are not identical with human insulin. The
`highly conserved amino acid residues, i.e., invariant among species, are boxed. Human insulin can
`be produced by E. colt or yeast with recombinant DNA or from pork insulin by an enzymatic trans-
`peptidation process which selectively substitutes a threonine for the alanine B30, thus forming the
`human-insulin molecules.
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`
`
`288
`
`Wang
`
`0
`
`ri (cid:9)
`
`t
`H
`i N
`
`C
`
`0 1 (cid:9)
`
`H (cid:9)
`
`Z1
`
`0
`
`Figure 4 Peptide chain of a protein coiled to form an a-helix. Configuration
`of the helix is maintained by hydrogen bonds shown as vertical dotted lines.
`
`C. Degradation Through Covalent Bonds
`The degradation of proteins and peptides can be divided into two main cate-
`gories: those that involve a covalent bond and those involving a conforma-
`tional change. The latter process is often referred to as denaturation.
`Numerous types of chemical reactions leading to modification of the co-
`valent structure are possible in a protein. The following describes only those
`that are likely to be seen in a protein or peptide formulation.
`
`Peptide Fragmentation
`The peptide bond (RNH—CO—R) is much more resistant to hydrolysis than is
`the ester linkage (R—O—CO—R) and peptide bonds are considered stable un-
`
`(cid:9)
`
`
`Parenteral Products of Peptides and Proteins (cid:9)
`
`289
`
`Disulfide
`Bond
`
`CH2
`
`Cys
`
`Cys
`
`C
`
`H
`
`CH2
`Hydrophobic N Tyr
`
`Interaction
`H3C ,CH3
`CH
`Leu
`CH
`
`Asp
`
`0 == (cid:9)
`H3y t (cid:9)
`(CH2)4
`
`Ionic
`Interaction
`
`Lys
`
`tH Phe
`
`Tyr
`
`CH2
`
`Hydrogen
`Bond
`
`(1;1N1 His
`
`OH
`CH2 Ser
`
`Figure 5 Interactions that stabilize folded conformation.
`
`less hydrolysis is assisted by a neighboring group. The formulation factor
`that most influences the hydrolytic rate is solution pH. The rate of hydroly-
`sis is in direct proportion to the activity of hydronium, or hydroxide ions,
`when in acidic or alkaline pHs, respectively. A rate-pH profile for hydrolysis
`of tetraglycine is shown in Figure 7. The reaction minimum is in the region of
`pH 3 to 5 [2]. Published examples of stability studies on therapeutic peptides
`are: degradation of nafarelin [3], thermal stability and degradation mecha-
`nism of secretin [4], acid hydrolysis of captopril, a quasidipeptide [5] , and
`heat stability of urokinase [6].
`Many peptides are stable enough to formulate as a ready-to-use parenteral
`formulation. For instance, D-Trp-LHRH was stable at 60°C for 5 days and
`seven cycles of freezing and thawing; gramicidin, although used only for derma-
`tological indications when dissolved in glycols, can be autoclaved. Information
`available on package inserts also shows it is possible to have good long-term
`stability of peptides. Leuprolide injection has a shelf-life of at least 2 years
`under refrigeration and 3 months at room temperature. LH-RH injection (Rele-
`fact) does not even require storage under refrigeration. Oxytocin injection
`was reported to be stable at room temperature for 5 years [7]. Protirelin, a
`tripeptide (PyrGlu-His-Pro), is stable for 20 hr at 80°C at both pH 3.3 and
`pH .6 [8] .
`Certain amino acids form the weak link of the chain. The bond between
`aspartic acid and proline or tyrosine is sensitive to acid hydrolysis [9]. The
`resultant products are peptides with aspartic acid at the C-terminus. The C-
`terminal peptide bond adjacent to serine is also a reactive one due to the neigh-
`boring group effect of the alcohol on serine [10].
`
`(cid:9)
`(cid:9)
`
`
`290
`
`Wang
`
`ct-helix
`
`I
`
`hydrophobic
`region
`
`A21
`
`Figure 6 Folded three-dimensional structure of insulin. The A-chain is ar-
`ranged between the two terminal arms of the B-chain and lies on top of the
`central a-helix (B9-B19). The hydrophobic regions are responsible for the
`(Adapted from Ref. 1.)
`association of monomer into dimers and hexamers. (cid:9)
`
`Table 2 (cid:9) pls of Selected Proteins
`
`Proteins
`
`Pepsin
`
`Erythropoietin
`
`Serum albumin
`
`Growth hormone
`
`Insulin
`
`Interleukin- 2
`
`Calcitonin (salmon)
`
`Chymotrypsin
`
`Basic fibroblast growth factor
`
`Interferon- y
`
`Lysozyme
`
`Nafarelin
`
`Protamines
`
`pI
`
`1.0
`
`3.5-4.0
`
`4.8
`
`5.0
`
`5.4
`
`7.8
`
`7.8
`
`9.1
`
`9.8
`
`10. 0
`
`11.0
`
`11.3
`
`12.0
`
`1
`
`
`
`Parenteral Products of Peptides and Proteins (cid:9)
`
`291
`
`pH
`
`Figure 7 Rate-pH profile for the hydrolysis of tetraglycine. Peptide solu-
`tions containing phosphate buffer, 0.03 M, and sodium chloride, 0.1 M, were
`incubated at 60°C. Samples were analyzed by HPLC assay. At pHs 1 to 3,
`the slope is -1, indicating an acid catalyzed reaction. In the pH region 4 to
`7, the reaction is hydroxide ion catalyzed. The plateau at pH 8 is caused by
`change in ionization state, zwitter ion to negatively charged ion, which is less
`susceptible to hydroxide-ion attack [2].
`
`Deamidation
`
`Deamidation refers to the removal of ammonia from the amide (RCONH 2) moiety,
`whether it is at the end of a C-terminus or at asparagine (Asn) or glutamine
`(Gin) residues. Stability studies of insulin [11], ACTH [12], human growth
`hormone [13] , and RS-26306 [10], an LH-RH analogue, have shown this route
`to be the major factor resulting in instability of these proteins and peptides
`in aqueous solution.
`In acidic media, the peptides deamidate by direct hydrolysis. Thus, an
`Asn or Gln residue yields an Asp or Glu peptide, respectively. The neighbor-
`ing amino acid does not affect the deamidation rate. Hydrolytic mechanisms
`in neutral or alkaline pHs are more complex, however. Under these condi-
`tions, the side chain carbonyl group on the Asn or Gln residue attaches to
`the nitrogen atom on the peptide backbone to form a cyclic imide intermediate.
`Depending on which bond in the cyclic imide breaks (Fig. 8), the reaction
`product can be: (1) the des-amido peptide, (2) the isopeptide, or (3) D-
`isomers. The formation of isopeptide is sometimes referred to as transpeptida-
`tion because an extra methylene group is inserted to the peptide backbone.
`At neutral to alkaline pHs, the rate of deamidation is significantly affected by
`the size of the amino acid on the C-terminal side of the Asn or Gln residue.
`In general, Asn is more labile than Gln, and is most labile when adjacent to
`glycine, which is the least obstructive to the formation of a cyclic imide.
`Deamidation rate-pH profile is V-shaped [14], usually with a minimal rate
`at a pH of about 4 to 5 (Fig. 9). In a number of synthetic peptides the half-
`
`
`
`292 (cid:9)
`
`0
`
`C -NH2
`CH2 \ R 0
`1 (cid:9)
`I (cid:9)
`II
`Ire -NH -CH -C-NH -CH-C rs,
`NH
`0 (cid:9)
`3 0
`
`r w-C ciR 0
`
`N-CH-C ••••
`b
`
`R-NH (cid:9)
`
`ivc
`H (cid:9)
`
`II
`0
`
`/mide Formation
`
`Wang
`
`0
`II
`H2..-C -0 (cid:9)
`0 II
`,NH-CH-C
`le -NH -C—ci Deamidation
`for Asn 8 Gln
`011 (cid:9)
`
`0
`
`R 0
`
`C H2\
`NH -CH -C ••••
`R'-NH-C _C-0G
`
`g Ponspeptidotion
`
`R 0
`%A-1 2 \ 1 (cid:9)
`II
`H N-CH-C
`R'-NH-C- c/ Rocemization
`
`0
`Figure 8 Reaction pathways in deamidation of asparagine. Deamidation of
`asparagine or glutamine goes through an intermediate, a cyclic imide. Three
`routes of degradation can take place, which leads to products of deamidation
`(removal of the amino group), transpeptidation (insertion of an extra methy-
`lene group), and racemization (D aspartic or glutamic acids).
`
`.0
`0
`
`O
`
`0
`
`-2
`
`-3
`
`-4
`
`5
`0
`
`2 (cid:9)
`
`4
`
`8 (cid:9)
`
`10
`
`12
`
`6
`pH
`
`Figure 9 Deamidation rate-pH profile of an active segment of adrenocortico-
`tropic hormone, Val-Tyr-Pro-Asn-Gly-Ala, in aqueous solution at 37°C and
`ionic strength of 0.5. The apparent rate is followed by the disappearance of
`the parent peak in HPLC. (From Ref. 14.)
`
`
`
`Parenteral Products of Peptides and Proteins (cid:9)
`
`293
`
`lives of deamidation reactions of Asns at 37°C in pH 7.2 phosphate buffer
`range from 2 days to nine years [15]. Not all Asns are equally labile; those
`buried within the interior portion of a protein are inaccessible to water and
`thus less reactive. In a large protein, secondary and tertiary structures
`play an important role in determining the site and the rate of deamidation.
`There are three asparagines in insulin (Fig. 3), the monodesamido- (A21)-
`insulin is the prevailing degradant formed in acid solution. In neutral solu-
`tion, deamidation is slow at 5°C. However, when accelerated at higher stor-
`age temperatures, deamidation of Asn at the B3 position can be detected [11].
`Figure 10 shows deamidation of insulin in neutral formulations at three differ-
`ent temperatures. With growth hormone, there are nine asparagines among
`191 amino acids, and deamidation occurs primarily at the Asn- 149 position [13].
`
`Oxidation of Cysteine
`
`Under neutral or basic conditions, the free thiol (—SH) group of a cysteine
`is the most reactive moiety of all amino acid components. The disulfide
`(—S—S—) bond formed from the oxidation of two thiol groups results in sig-
`nificant changes in conformation both intramolecularly and intermolecularly.
`Oxidation of the thiol group is promoted at both neutral and basic pH.
`The rate-pH profile for captopril, a quasi dipeptide, showed an increase in
`oxidation rate, starting at pH 5 (Fig. 11). This reaction can be effectively
`retarded by the addition of a metal chelating agent such as EDTA [5].
`A strategy to stabilize proteins that have reactive thiol groups is to re-
`place the cysteine with serine. Serine, a stable amino acid with a hydroxy
`(—OH) group, mimicks the size and polarity of cysteine. In many cases,
`these serine mutants retain full biological activities.
`
`Oxidation of Methionine
`
`The methiol (—S—CH3) moiety on methionine is susceptible to oxidation to
`form methylsulfoxide (—SO—CH3) derivatives. Of the three methionines in
`human growth hormone, Met-125 is most reactive, Met-14 is less so and Met-
`170 is not reactive at all [16]. The reactive methionine is likely to be the
`one exposed on a protein surface, and the unreactive one buried within the
`core. Air in the headspace of formulated and freeze-dried growth hormone
`can cause 40% of the growth hormone molecules to be oxidized during a 6 month
`storage period [17].
`
`Disulfide Exchange
`
`Disulfide exchange takes place when a cystine (disulfide) bond is reduced
`to two cysteines; one of them then reacts with another cysteine to form a new
`disulfide (Fig. 12). The reaction is base (hydroxide ion) catalyzed and pro-
`moted by mercaptoethanol, which is sometimes used as an antioxidant. The
`reaction is concentration-dependent, and oligomers are frequently formed as
`a result of disulfide scrambling of bonds between peptide chains. Improper
`linkages of disulfide bonds were responsible for a reduction in biological ac-
`tivity of interleukin-2 (IL-2) (Fig. 12). There are three cysteines in IL-2
`at positions 58, 105, and 125. The native protein forms a disulfide linkage
`between the two cysteines at 58 and 105. Cleavage of this disulfide in IL- 2
`and the subsequent formation of two less-active isomers with disulfide bonds
`
`
`
`294
`
`Wang
`
`Figure 10 Deamidation of insulin during storage of Actrapid MC (BP-formula-
`tion) at different temperatures. Each point represents the mean of analyses
`of 4-6 different batches. The desamido insulin content was determined by
`basic disc electrophoresis followed by densitometric scanning of the stained
`gels. (From Ref. 11.)
`
`at incorrect positions are promoted by high pH and copper ions [18]. The
`number of disulfide bonds in therapeutic proteins can be shown in the follow-
`ing examples: IL-2: 1; salmon calcitonin: 1; interferon-a: 2; human growth
`hormone: 2; insulin: 3; urokinase: 12; t-plasminogen activator: 17; albumin: 17.
`
`k (days-I) x 103
`
`s 0
`\
`N—C—CH—CH2— SH (cid:9)
`CH3
`COOK
`
`R—S—S —R
`
`4
`pH
`
`Figure 11 Oxidation of the thiol moiety in captopril. Reaction rates at 50°C
`is followed by a peak on the HPLC chromatogram. All solutions contain di-
`sodium edetate. Without the chelating agent, reaction rates are much faster.
`(From Ref. 5.)
`
`
`
`Parenteral Products of Peptides and Proteins (cid:9)
`
`295
`
`Native Interleukin - 2
`
`Denaturing
`Conditions
`
` 105 SH
`S
`
`125
`
`Alkaline pH
`
`125
`
`SH
`
`SS 125
`
`Less active forms
`
`Figure 12 Schematic representation of the various disulfide-linked isomers of
`interleukin-2 [18]. Under denaturing conditions, i.e., high temperature or
`alkaline pH, a free sulfhydryl group forms a disulfide bond with the sulfhy-
`dryl group from an existing disulfide. The results are proteins with incorrect
`disulfide linkages and "non-native" conformation.
`
`With N-disulfide bonds, there exist (2N)! /2N x N! possible isomers. Cau-
`tion must be exercised to preserve the correct disulfide linkage throughout
`the production process for therapeutic proteins.
`
`Racemization
`
`The racemization reaction is catalyzed by both acid and base. Racemization
`of peptides and proteins results in the formation of diastereomers. As an
`example, racemization of the serine residue in a gonadotropin-releasing hor-
`mone analogue was one of the main degradation reactions when treated with
`0.1 N NaOH at 5°C for 48 hr [19]. Racemization under basic conditions is
`thought to proceed by abstraction of the a-proton from an amino acid in a
`peptide to give a negatively charged planar carbanion. A proton can then be
`returned to this optically inactive intermediate, thus producing a mixture of
`D- and L-enantiomers for the individual amino acid (Fig. 13). Since a pep-
`tide is composed of multiple chiral centers, the product formed is a diastereo-
`mer. Racemization is biologically significant since a peptide comprised of D-
`amino acids is generally metabolized much slower than a naturally occurring
`peptide made of only L-amino acids. For this reason, many new synthetic
`peptides, agonists or antagonists, incorporate D-amino acids. A pH depend-
`ency on racemization was demonstrated in an aqueous degradation study of a
`decapeptide, RS-26306 [10]. At neutral and alkaline pHs, racemization con-
`tributed to more degradation than deamidation (Fig. 14).
`
`(cid:9)
`
`
`296 (cid:9)
`
`Wang
`
`CHR 0 (cid:9)
`I (cid:9)
`II (cid:9)
`^, NH C—C^' s (cid:9)
`I (cid:9)
`H (cid:9)
`
`CHR 0 (cid:9)
`I (cid:9)
`II (cid:9)
`is-\1H C—C- (cid:9)
`•°
`
`H 0
`I
`"-.N1H-C—C^1
`CHR
`
`0-
`OH
`Figure 13 Racemization of an amino acid resulting in a mixture of enantiomers.
`
`Maillard Reaction
`In the Maillard reaction, the carbonyl group (RCH=----0) from glucose can react
`with the free amino group (e.g., R'NH2 in lysine) in a peptide to form a
`Schiff base (RCH=NR'). This reaction is acid catalyzed. During pasteuriza-
`tion (60°C, 10 hr), the loss of antithrombin III biological activity seen in the
`presence of reducing sugars, especially glucose, was attributed to this reac-
`tion [20]. An alternative assessment of poor blood-glucose control in diabetic
`patients is the measurement of glycosylated hemoglobin, which is the pr