; VOLUME 9, NUMBER 11
`
`.
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`.
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`‘ 2‘ ~\
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`NOVEMBER 1992
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`PHREEB 9(11)1375_1524 (1992)
`
`ISSN 0724-8741
`
`Official Journal of the
`American Association of
`
`PHARMACEUTICAL
`RESEARCH "
`
`Pharmaceutical Scientists
`
`PLENUM PRESS 0 NEW YORK AND LONDON
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`PHARMACEUTICAL RESEARCH
`Official Journal of the American Association of Pharmaceutical Scientists
`
`Pharmaceutical Research publishes innovative basic research and technological advances in the pharmaceutical—biomedical sciences.
`Research areas covered in the journal include: pharmaceutics and drug delivery. pharmacokinetics and pharmacodynamics, drug metab-
`olism, pharmacology and toxicology, medicinal chemistry, natural products chemistry, analytical chemistry, chemical. kinetics and drug
`stability, biotechnology, pharmaceutical technology, and clinical investigations, as well as articles on the soctal, economic, or management
`aspects of the pharmaceutical sciences.
`
`.
`.
`EDITOR-IN-CHIEF
`Wolfgang Sadée, School of Pharmacy, University of Califorma, San
`Francisco, California
`EDITOR—EUROPE
`Douwe D. Breirner, University of Leiden, Leiden, The Netherlands
`EDITOR—JAPAN
`Yuichi Sugiyama, University of Tokyo. Tokyo, Japan
`ASSOCIATE EDITORS
`Analysis and Pharmaceutical Quality
`James T. Stewart, University of Georgia, Athens, Georgia
`Biotechnology
`Randy Mrsny, Genentech, Inc., South San Francisco, California
`Clinical Sciences
`Joachim Grevel, Biomedical and Agricultural Services of Texas,
`Inc., Houston, Texas
`Economics, Marketing, and Management Sciences
`Francis B. Palumbo, University of Maryland, Baltimore, Maryland
`Medicinal and Natural Products Chemistry
`Rao S. Rapaka, National Institute on Drug Abuse, Rockville,
`Maryland
`Pharmaceutics and Drug Delivery
`Gordon L. Amidon, University of Michigan, Ann Arbor, Michigan
`David Fleisher, University of Michigan, Ann Arbor, Michigan
`Gordon L. Flynn, University of Michigan, Ann Arbor, Michigan
`Pharmaceutical Technology
`Larry L. Augsburger, University of Maryland, Baltimore, Maryland
`Pharmacokinetics, Pharmacodynamics, and Drug Metabolism
`Robert J. Wills, R. W. Johnson Pharmaceutical Research Institute,
`Raritan, New Jersey
`Bobbe L. Ferraiolo, R. W. Johnson Pharmaceutical Research
`Institute, Raritan, New Jersey
`EDITORIAL ADVISORY BOARD
`Bradley D. Anderson, University of Utah, Salt Lake City, Utah
`Jessie Lai-Sim Au, Ohio State University, Columbus, Ohio
`Shoji Awazu, Tokyo College of Pharmacy, Tokyo, Japan
`Leslie Z. Benet, University of California, San Francisco, California
`Nicholas S. Bodor, University of Florida, Gainesville, Florida
`Ronald T. Borchardt, University of Kansas, Lawrence, Kansas
`Harry Brittain, Bristol-Myers Squibb Pharmaceutical Research
`Institute, New Brunswick, New Jersey
`Hans Bundgaard, Royal Danish School of Pharmacy, Copenhagen,
`Denmark
`
`Patrick Couvreur, Université de Paris-Sud, Chatenay-Malabry,
`France
`Dan J. A. Crommelin, University of Utrecht, Utrecht, The
`Netherlands
`Stanley S. Davis, The University of Nottingham, Nottingham,
`England
`Klaus Florey, Bristol-Myers Squibb Pharmaceutical Research
`Institute, New Brunswick, New Jersey
`Kathleen M. Giacomini, University of California, San Francisco,
`California
`
`Frank Goodhart, Wamer-Lambert Company, Morris Plains, New
`Jersey
`
`Johnathan Hadgraft, University of Wales, Cardiff, Wales
`Mitsuru Hashida, Kyoto University, Kyoto, Japan
`William Higuchi, University of Utah, Salt Lake City, Utah
`Ryohei l-Iori, Kyoto University, Kyoto, Japan
`Tetsuya Kamataki, Hokkaido University, Sapporo, Japan
`Vincent H. L. Lee, University of Southern California, Los
`Angeles, California
`Gerhard Levy, State University of New York, Buffalo, New York
`James D. McChesney, The University of Mississippi, University,
`Mississippi
`Dirk K. F. Meijer, University of Groningen, Groningen, The
`Netherlands
`Hans P. Merkle, Swiss Federal Institute of Technology, Ztirich,
`Switzerland
`Bemd W. Miiller, Christian-Albrcchts-Universitat, Kiel, West
`Germany
`Shozo Muranishi, Kyoto Pharmaceutical University, Kyoto, Japan
`Ernst Mutschler, Johann Wolfgang Goethe Universitat, Frankfurt
`am Main, West Germany
`Tsuneji Nagai, Hoshi University, Tokyo, Japan
`Masahiro Nakano, Kumamoto University Hospital, Kumamoto,
`Japan
`Sidney D. Nelson, University of Washington, Seattle, Washington
`Tetsuo Osa, Tohoku University, Sendai, Japan
`Lennart Paalmw, University of Uppsala, Uppsala, Sweden
`Michael J. Pikal, Eli Lilly Company, Indianapolis, Indiana
`Edward G. Rippie, The University of Minnesota, Minneapolis,
`Minnesota
`Joseph R. Robinson; University of Wisconsin, Madison, Wisconsin
`Malcolm Rowland, University of Manchester, Manchester, England
`Karl Rozman, The University of Kansas, Kansas City, Kansas
`Jerome Schentag, SUNY at Buffalo, New York
`Paul L. Schiff, University of Pittsburgh, Pittsburgh, Pennsylvania
`Joseph Schwartz, Philadelphia College of Pharmacy and Science,
`Philadelphia, Pennsylvania
`Tomio Segawa, Hiroshima University School of Medicine,
`Hiroshima. Japan
`l-Iitoshi Sezaki, Setsunan University, Hirakata, Japan
`Anthony A. Sinkula, The Upjohn Company, Kalamazoo, Michigan
`Valentino J. Stella, The University of Kansas, Lawrence, Kansas
`Larry Stemson, Eastman Pharmaceuticals, Great Valley,
`Pennsylvania
`Heinz Sucker, University of Bern, Bern, Switzerland
`Akio Tsuji, School of Pharmaceutical Sciences, Tokyo, Japan
`Elizabeth B. Vadas, Merck Frosst Canada Inc., Pte. CIaire-Dorval.
`Canada
`
`George Zografi, University of Wisconsin, Madison, Wisconsin
`BOOK REVIEW SECTION
`John F. Fitzloff, University of Illinois at Chicago, Illinois
`Joyce Mordenti, Genentech, South San Francisco, California
`Praveen Tyle, Agouron Pharmaceuticals, La Jolla, California
`(Send books for review to the Editor—in-Chief, W. Sadée, sec
`Instructions for address)
`ASSISTANT EDITOR
`G. C. M. Beelen, San Francisco, California
`
`Pharmaceutical Research is published monthly by Plenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013. Pharmaceutical Research is
`abstracted or indexed in Bioscience Abstracts. Chemical Abstracts, Excerpta Medica, Gazette de l‘APGI, Index Medicus/MEDLARS, and International
`Pharmaceutical Abstracts. © 1992 Plenum Publishing Corporation. Ph
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`
`
`PHARMACEUTICAL RESEARCH
`Official Journal of the American Association of Pharmaceutical Scientists
`
`Volume 9 Number 11
`November 1992
`
`CONTENTS
`
`REVIEW
`
`Monoclonal Antibodies in Hapten Immunoassays
`Olivier N. Chappey, Pierre Sandouk, and Jean—Michel G. Scherrmann
`
`(Biotec)
`
`1375
`
`RESEARCH ARTICLES
`
`An Alternative Approach for Assessment of Rate of Absorption in Bioequivalence Studies
`Mei-Ling Chen
`
`(PPDM)
`
`1380
`
`Identification of Sites of Degradation in a Therapeutic Monoclonal Antibody by
`Peptide Mapping
`Daniel J. Kroon, Alysia Baldwin-Perm, and Praful Lalan
`
`(Biotec/APQ)
`
`1386
`
`REPORTS
`
`Localization of Binding Sites for Epidermal Growth Factor (EGF) in Rat Kidney: Evidence for the
`Existence of Low Affinity EGF Binding Sites on the Brush
`(Pharmacul/Biotec)
`Border Membrane
`Dong-Chool Kim, Manabu Hanano, Yasushi Kanai, Norio Ohnuma, and Ynichi Sugiyama
`
`Skin Alteration and Convective Solvent Flow Effects During Iontophoresis II. Monovalent Anion and
`Cation Transport Across Human Skin
`(PDD)
`Sandra M. Sims, William 1. Higuchi, and V. Srinivasan
`
`1394
`
`1402
`
`Noninvasive in Vivo Percutaneous Absorption Measurements Using
`X—Ray Fluorescence
`J. David Robertson, Elizabeth Ferguson, Michael Jay, and Dennis J. Stalker
`
`(PUD/APO)
`
`1410
`
`Lamellar Structures Formed by Stratum Corneum Lipids in Vitro: A Deuterium Nuclear Magnetic
`Resonance (NMR) Study
`{PDD}
`William Abraham and Donald T. Downing
`
`1415
`
`Local Enhanced Topical Delivery (LETD) of Drugs: Does It Truly Exist?
`Stephen C. McNeil, Russell 0. Potts, and Michael L. Francoeur
`
`(PDD)
`
`1422
`
`Physicochemical Properties of Salts of p-Aminosalicylic Acid. 1: Correlation of Crystal Structure and
`Hydrate Stability
`(PDD)
`Robert T. Forbes, Peter York, Victor Fawcett, and Leonard Shields
`
`(PDD)
`In Vivo Evaluation of Enteric-Coated Naproxen Tablets Using Gamma Scintigraphy
`Ian R. Wilding, John G. Hardy, R. Andrew Sparrow, Stanley S. Davis, Paul B. Daly, and
`John R. English
`
`Distribution and Elimination of the Glycosidase Inhibitors l—Deoxymannojirimycin and
`(PPDM)
`N-Methyl-l-Deoxynojirimycin in the Rat in Vivo
`Esther D. Faber, Roelof Costing, Jacques J. Neejjes, Hidde L. Ploegh, and Dirk K. F. Meijer
`
`1428
`
`1436
`
`1442
`
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`

`

`CONTENTS (Continued)w
`
`Pharmacokinetic Disposition of Multiple Dose Transdcrmal Nicotine in Healthy
`Adult Smokers
`Gregory M. Kochak, Jim X. Sun, R. Leslie Choi, and Anthony J. Piraino
`
`Age and the Pharmacokinetic—Pharmacodynamic Relationship of Phenobarbital in Rats:
`“Pseudo"—Longitudinal vs Cross-Sectional Study Design
`Annemiek M. Stijnen, Meina'ert Danhof, and Cornelius F. A. van Bezooijen
`
`IPPDM)
`
`1451
`
`(PPDM)
`
`1456
`
`Glutathione S-Transferase-Mediated Metabolism of Glyceryl Trinitrate in Subcellular Fractions of
`Bovine Coronary Arteries
`(ppDM}
`David T.-W. Lau, Elaine K. Chan, and Leslie Z. Benet
`
`1460
`
`Kinetics and Mechanism of Degradation of Zileuton, a Potent 5-Lipoxygenase Inhibitor
`Francisco J. Alvarez and Russell T. Slade
`
`(APOJ
`
`1465
`
`Synthesis of 2-(3-Substituted-1,2,4-oxadiazol-5-yl)—8-methyl-8-azabicyclo[3.2.1]octanes and
`2a-(3-Substituted-1,2,4-oxadiazol-S—yl)-8-methyl-8-azabicyclo[3.2.ljoct—Z-enes as Potential
`Muscarinic Agonists
`(MNPC)
`David J. Triggle, Yong Wha Kwon. Philip Abraham, M. Abdur Rahman, and F. Ivy Carroll
`
`1474
`
`Relative Lipophilicities and Structural—Pharmacological Considerations of Various
`Angiotensin-Converting Enzyme (ACE) Inhibitors
`Sunanda A. Ranadive, Andrew X. Chen, and Abu T. M. Serajuddin
`
`{MNPC}
`
`1480
`
`The Development of a Microwave Fluid-Bed Processor. 1. Construction and Qualification of a
`Prototype Laboratory Unit
`Michael K. Doelling, David M. Jones, Richard A. Smith, and Robert A. .Nash
`
`(PT)
`
`1487
`
`The Development of a Microwave Fluid—Bed Processor. II. Drying Performance and Physical
`Characteristics of Typical Pharmaceutical Granulations
`Michael K. Doelling and Robert A. Nash
`
`(PT)
`
`1493
`
`TECHNICAL NOTES
`
`In Vivo Evaluation of Biodegradable Progesterone Microspheres in Mares
`Pramoa' K. Gupta, Rahal C. Mehta, Robert H. Douglas, and Patrick P. DeLuca
`
`{PDD/PPDM)
`
`1502
`
`Sustained Propranol Delivery and Increased Oral Bioavailability in Dogs Given a Propranolol
`Laurate Salt
`Bruce J. Aungst and Munir A. Hussain
`
`Relationship Between Polymer Viscosity and Drug Release from a Matrix System
`Lucy Wan Sai Cheong, Paul Wan Sia Heng, and Lee Fun Wong
`
`(PDD)
`
`1507
`
`(PDD)
`
`1510
`
`Effect of Isoflurane Anesthesia on Antipyrine Pharmacokinetics in the Rat
`Francis L. S. Tse, David F. Nickel-son, and Renee Aun
`
`(PPDM)
`
`1515
`
`Employing Ionization Behaviors to Resolve a Trace—Level Impurity: Determination of
`1,4-Dihydro-2,6-dimethyl-4—(3—nitrophenyl)-3,S-pyridinedicarboxylic Acid
`(APQ)
`Di-2—[methyl(phenylmethyl)amino]ethyl Ester in Nicardipine Drug Substance
`Michael B. Maurin, Rodney D. Viekery, Philip Ma, Joseph Manalo, and Munir A. Hassaln
`
`(Biotec)
`Degradation of Synthetic Salmon Calcitonin in Aqueous Solution
`Kang Choon Lee, Yoon Joong Lee, Hyun Myo Song. Chang Ju Chan, and Patrick P. DeLuca
`
`ASSOCIATION NEWS AND ANNOUNCEMENTS
`
`1518
`
`1521
`
`1524
`
`\—
`
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`Pharmaceutical Research, Vol. 9, No. 11, 1992
`
`Research Article
`
`Identification of Sites of Degradation in a Therapeutic
`Monoclonal Antibody by Peptide Mapping
`
`Daniel J. Kroon,"2 Alysia Baldwin-Ferro,l and Praful Lalanl
`
`Received April 1, 1992; accepted June 23, I992
`A peptide mapping procedure was developed to locate regions of a monoclonal antibody, OKT3, that
`undergo chemical modification as the molecule degrades upon storage. The structures of these peptide
`degradation products were investigated. Deamidation at specific asparagine residues and oxidation of
`a cysteine and several methionines were found to be major routes of OKT3 degradation. A unique
`chain cross-linked degradation product was also observed and characterized Changing the storage
`conditions of the antibody affected the relative distribution of degradation products. These results
`were useful in the development of more stable formulations for OKT3, and the methods can be used
`———-———_——
`in the characterization of other monoclonal antibodies intended for therapeutic use.
`
`KEY WORDS: OKT3, monoclonal antibody; peptide mapping; protein stability; degradation of pro-
`teins.
`
`INTRODUCTION
`
`Orthoclone OKT3 became the first monoclonal anti-
`body approved for therapeutic use in 1986. OKT3 is used
`clinically to reverse rejections of human kidney transplants
`and is being studied as an adjunct therapy in other organ
`transplants. This murine antibody derives its immunosup—
`pressive properties from being directed against a component
`of the human T-cell antigen receptor complex. When OKT3
`binds to the receptor, T-cell function is blocked and periph-
`eral T cells are removed from circulation, halting the pa-
`tients’ immune response to the transplanted tissue.
`The production of proteins for pharmaceutical purposes
`requires extensive characterization of the purity, biochemi-
`cal properties, and stability of the protein. Proteins have an
`inherent propensity to undergo a variety of chemical reac-
`tions that can lead to loss of structural integrity and bioac-
`tivity (for a review of stability of proteins, see Ref. 1). The
`effect of storage conditions and formulation on the protein
`drug candidate needs to be investigated to minimize forma-
`tion of damaged protein. As a marketed therapeutic, consid-
`erable effort has been extended in developing methods to
`analyze OKT3 and monitor the antibody’s stability. With a
`molecular weight of about 150,000, OKT3 is a relatively
`large protein and its size makes it extremely difficult to char-
`acterize fully its degradation products.
`In stability studies of OKT3, several analytical assays
`indicated that structural changes occurred in some batches
`of the product within 1 year of storage at 5°C and after a few
`months at 24 and 37°C. These assays include isoelectric fo-
`
`
`
`‘ The R. W. Johnson Pharmaceutical Research Institute, Route 202,
`PO. Box 300, Raritan, New Jersey 08869.
`2 To whom correspondence should be addressed.
`
`cusing, sodium dodecyl sulfate—polyacrylamide gel electro-
`phoresis (SDS/PAGE), ion-exchange HPLC, and gel perme-
`ation chromatography. These assays measure different phys-
`ical properties, and the observed changes are a reflection of
`the multiple modes of degradation that would be available to
`a protein molecule the size of OKT3. The results of addi-
`tional studies of the stability of the antibody suggested that
`three major mechanisms account for most of the OKT3 de—
`composition: oxidation of labile amino acids, deamidation of
`Asn or Gln residues, and fragmentation of peptide chains (2).
`This paper reports the use of a peptide mapping proce-
`dure to identify specific sites and chemical mechanisms of
`degradation in OKT3. The effect of different storage condi-
`tions on these degradative pathways was investigated. This
`information can be applied to optimizing production meth—
`ods and formulations for OKT3 to preserve molecular integ-
`rity. Also, since the major portion of the structure of anti-
`bodies is the constant region, in which the amino acid se—
`quence is conserved in antibodies of the same class.
`information on sites of OKT3 degradation will be useful in
`the development of other antibodies as therapeutic agents,
`as many of the same degradation pathways would be ex-
`pected to occur.
`
`MATERIALS AND METHODS
`
`OKT3 Antibody. Production lots of Orthoclone OKT3
`were used as a source of antibody for these studies. This
`material was formulated at 1 mg/ml sterile solution in pH 7.0
`phosphate-buffered saline containing polysorbate 80. Vials
`containing 5 ml of OKT3 solution were stored in a refriger-
`ator or in stability cabinets at elevated temperatures. For
`peptide mapping. the antibody was concentrated with a Cen-
`tricon 30 apparatus before use. The integrity of the antibody
`
`0724-8741/92/11001386w650/0 c 1992 Plenum Publishing Corporation
`
`1386
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`Sites of Degradation in a Therapeutic Monoclonal Antibody
`
`1387
`
`was checked on a Pharmacia PhastSystem electrophoresis
`unit by isoelectric focusing on pH 3-9 Phastgel IEF media
`and SDS/PAGE on 10—15% Phastgels, with and without re-
`duction of the sample by heating with 2-mercaptoethanol.
`The gels were stained with Coomassie Blue. Biological ac-
`tivity of the antibody was assessed by measuring binding to
`the antigen on T cells (the Potency Assay described in Ref.
`2); this assay was performed by the Ortho Biotech QA de-
`partment or the Bioanalytical Research group of R. W.
`Johnson Pharmaceutical Research Institute.
`Separation of 0KT3 H and L Chains. Disodium 2-ni-
`tro—5-thiosulfobenzoate (NTSB) was synthesized by the lit-
`erature procedure (3). The sulfitolysis reagent was prepared
`by adding 0.40 ml of NTSB solution to 4.6 m1 ofa solution of
`4.73 g of guanidine thiocyanate, 0.504 g of sodium sulfite, 20
`mg of EDTA, and 0.485 g ofTris hydrochloride in water. The
`pH of the solution was adjusted to 9.0 with KOH. To 1 m1 of
`OKT3 solution was added 1 ml of sulfitolysis reagent and the
`reaction allowed to proceed for 45 min. Portions of the re—
`action mixture were loaded onto a Pharmacia HR10/30 Su—
`perose 12 column and the column was eluted with phos-
`phate-buffered saline containing 6 M urea on a FPLC sys-
`tem. Fractions containing pure heavy (H) and light (L) chain,
`as determined by SDS/PAGE, were used for peptide map-
`ping. Material containing the 88—kD component was obtained
`as an incompletely separated small peak eluting in front of
`the H chain.
`Peptide Mapping. The H and L chain fractions were
`concentrated to 0.2 vol with a Centricon microconcentrator,
`then diluted with an equivalent volume of 50 mM Tris, pH
`8.5, buffer for trypsin digestion or 100 mM dibasic sodium
`phosphate for Glu-C endoproteinase digestion. To the sub-
`strate solution was added sequence grade enzyme (Boeh-
`ringer-Mannheim) in an enzyme to substrate ratio of approx-
`imately 1:50; the enzyme was added in two portions 4~5 hr
`apart. The digestion mixtures were incubated at 25°C for 20
`hr, then quenched by the addition of tritluoroacetic acid to
`10% volume. The digests were analyzed by reversed—phase
`HPLC on a 4.6 X 250-mm, 5-p.m Rainin Dynamax C18, 300A
`column eluted with gradients of increasing acetonitrile in
`0.1% trifluoroacetic acid (TFA) on a Perkin—Elmer gradient
`HPLC system. The eluate was monitored at 215 and 280 nm
`with a diode array detector. Data collection and processing
`were performed with a Nelson Chromatography Data Sys—
`tem on an IBM P52 computer. Sample chromatograms are
`shown in Fig. 2.
`Characterization of Peptides. Peptide components of
`the HPLC maps were manually collected and the fractions
`dried using a Savant Speedvac. The amino acid sequence of
`the peptides was determined on a Porton 2090B Integrated
`Micro-Sequencing System or an ABI 477A Protein Sequena—
`tor. Sequencing of OKT3 fragments directly from PVDF
`electroblots of SDS/PAGE gels was accomplished following
`the procedure described by Matsudaira (4) and loading the
`blots as instructed in a Porton user bulletin. The molecular
`weights of the peptides were determined by FAB mass spec-
`trometry on a Finnigan-MAT 8230 mass spectrometer with
`8-keV xenon atoms. The identity of some OKT3 peptide
`fragments was established by running the peptide map on an
`HPLC with a solvent stream splitter interfaced via a pneu—
`matically assisted electrospray source to a Sciex 'l'AGA
`
`6000B triple-quadruple mass spectrometer at Cornell Uni—
`versity. The calculated masses of the peptides were com-
`pared with the expected molecular weights of OKT3 tryptic
`fragments and the peptides were located on the map by si-
`multaneous detection by UV.
`Peptide Synthesis. To confirm the identity of several
`OKT3 peptide fragments, the HPLC retention times were
`compared with authentic peptides obtained by chemical syn-
`thesis. Peptides were synthesized on an ABI 430A Peptide
`Synthesizer using standard protocols for t-BOC amino acid
`derivatives. BOG—methionine sulfoxide and BOC-aspartic
`acid u-benzyl ester were purchased from Bachem Bio—
`science, Inc. The peptides were cleaved from the resin with
`HF containing 10% p-cresol for 1 hr at 0°C. After removal of
`the HF with vacuum and washing the residue with ethyl
`acetate, the peptide was extracted into TFA. The mixture
`was filtered and the filtrate reduced to small volume by ro-
`tary evaporation. The addition of ether to the residue pre-
`cipitated the crude peptides. The peptides were purified by
`prep reversed-phase HPLC and identity confirmed by amino
`acid analysis and FAB mass spectrometry.
`Preparation of Ftab’); Fragments. OKT3, 5 mg/ml,
`was dialyzed against 0.9% sodium chloride. The antibody
`was transferred to a polypropylene tube and diluted with 1.5
`ml 0.20 N, pH 4.1, citrate buffer. To this solution was added
`300 pl pepsin solution. 1.0 mg/ml. in 0.1 N citrate, pH 4.1.
`The mixture was incubated at 37°C for 2 hr, then another 150
`pl of pepsin solution added and the incubation continued.
`After an additional 1.5 hr, the digestion was stopped with
`0.50 ml
`1 M Tris. The solution was filtered through a 0.45-
`pm membrane filter, then concentrated with a Centricon 30
`apparatus to less than 2 ml. The protein was loaded onto a
`16/50 Pharmacia column of Supcrose 12 prep grade and
`eluted with PBS. The center fraction cut of the major peak
`was concentrated with a Centricon 30 and treated with the
`sulfitolysis reagent. followed by size exclusion chromatog-
`raphy as described for intact OKT3 above. With the F(ab’)2
`prepared from reference standard OKT3, SEC gave a single
`protein peak which contained both the L chain and the H
`chain F(ab’)2 fragment, as shown by peptide mapping com-
`parison with standard L and H chain digests. F(ab’)2 from
`degraded OKT3 had a second. earlier—eluting peak in the
`SEC which had an apparent molecular weight of 50 kD on
`SDS/PAGE. This material was digested with trypsin and the
`peptide map compared with the map of standard OKT3
`Ftab’)2.
`
`RESULTS AND DISCUSSION
`
`Peptide Mapping. A peptide mapping procedure was
`developed to locate regions of OKT3 that were chemically
`altered as the antibody degraded upon storage. Mechanisms
`of OKT3 degradation were deduced by determining the
`structures of peptide fragments isolated from the enzyme
`digests produced in the mapping procedure. OKT3 samples
`stored under different conditions for various lengths of time
`were analyzed by this peptide mapping procedure.
`Native antibodies are not very susceptible to enzymatic
`digestion. Denaturation exposes sites for attack by enzymes
`but has a tendency to make the antibody insoluble. In our
`method, OKT3 was denatured and the disulfide bonds
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`1388
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`cleaved along with Cys S-sulfonation following the proce-
`dure of Thannhauser et a1. (3). Cys-sulfonation improves the
`solubility of the protein and subsequent peptide fragments.
`This technique also enabled the separation of the H and L
`chains of OKT3 by size exclusion chromatography. Inclu—
`sion of 6 M urea in the elution buffer was necessary to pre—
`vent the chains from aggregating. The H and L chains were
`then separately digested with enzymes, which greatly sim-
`plified the chromatography needed to separate the more than
`60 peptide fragments produced by trypsin digestion of the
`two chains. The digests were analyzed by reversed-phase
`HPLC to produce the peptide map. The peptide maps of
`degraded samples of OKT3 were compared to the maps of
`reference standard material to detect peptide fragments that
`had undergone structural changes.
`It should be noted that this method discriminated
`against detecting degradation due to hydrolysis of peptide
`bonds since the resulting chain fragments may be separated
`from the H and L chain by the size exclusion chromatogra-
`phy and not be included in the material digested with en—
`zymes for the peptide maps. Decomposition of 0KT3 due to
`chain fragmentation does occur over time, as evidenced by
`SDS/PAGE, and other methods are being used to study
`OKT3 degradation pathways of this type.
`Several enzymes were investigated as digesting agents
`for peptide mapping of OKT3; Glu-C and Lys—C endopro-
`teinases produced fewer peaks in the chromatogram than
`trypsin, but the large size of many of the fragments made
`complete chemical characterization of the peptides difficult
`and changes in the structure of one amino acid did not alter
`the retention times significantly. The more complex chro-
`matograms of the trypsin digests gave more useful informa-
`tion for localizing the sites of OKT3 degradation to specific
`residues in the amino acid sequence of the molecule. Glu-C
`and Lys-C digestions were useful in confirming the identity
`of certain peptides and amino acid modifications.
`Peptide fragments of OKT3 were identified by collecting
`the material eluting as peaks in the peptide map and deter-
`mining the structure of each peak component by amino acid
`sequencing, mass spectrometry, and, in some cases, coelu-
`tion with chemically synthesized peptides. Digestion of
`OKT3 chains with trypsin was found to result in several
`incomplete cleavages at Lys—Asp bonds, e.g., L182—183,
`and alternate cleavages at adjacent basic residues, e.g.,
`H362—363 (see OKT3 amino acid sequence; Fig. 1). Exam-
`ples of HPLC tryptic peptide maps of OKT3 L and H chains
`are shown in Fig. 2.
`Degradation upon Storage at Elevated Temperature;
`Asparagine Deamidation. At 37°C, the earliest detectable
`change in OKTS is a shift in the pattern of isoelectric focus-
`ing (IEF) gel bands toward a more acidic pl; observable
`changes occur in as little as 1 week (2). Though less sensitive
`than IEF, ion-exchange HPLC changes in retention time,
`indicating a more acidic species, are also observed at later
`time points. Samples held at 5°C, however, continue to ex-
`hibit IEF patterns with minimal change from control for 9
`months. The change in IEF pattern is not correlated with
`loss of antigen binding potency of OKT3. The rate of this
`IEF shift is not affected by the addition of antioxidants or
`filling the sample vials with an overlay of an inert gas such as
`nitrogen or argon. It had been proposed that the IEF acidic
`
`Kroon, Baldwin-Ferro, and lalan
`
`LIGHT CHAIN
`
`1 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YHNWYQQKSG TSPKRWIYDT
`F____
`—«
`51
`SKLASGVPAH FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG
`101
`TKLEINRADA APTVSIFPPS SEQLTSGGAS VVCFLNNFYP KDINVKNKID
`151
`GSERQNGVLN SWTDQDSKDS TYSMSSTLTL TKDEYERHNS YTCEATHKTS
`201
`TSPIVKSFNR NEC
`
`HEAVY CHAIN
`
`1
`
`W
`QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLE IGY
`
`INPSRGYTNY NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCAREX
`51
`DDHYCLDYWG QGTTLTVSSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG
`101
`YFFEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT
`151
`CNVAHFASST KVDKKIEPRG PTIKFCFPCK CPAPNLLGGP SVFIFPPKIK
`201
`251 DVLHISLSPI VTCVVVDVSE DDFDVQISWF VNNVEVHTAQ TQTHREDYNS
`301
`TLRVVSALPI QHQDWMSGKE FKCKVNNKDL PAPIERTISK PKGSVRAPQV
`351 YVLPPPEEEM TKKQVTLTCM VTDFMPEDIY VEWTNNGKTE LNYKNTEPVL
`F“"
`401
`DSDGSYFHYS KLRVEKKNWV ERNSYSCSVV HEGLHNHHTT KSFSRTPGK
`
`F————————————————————4
`
`Fig. 1. Amino acid sequence of OKTS. The amino acid sequence of
`the light and heavy chains of OKT3 in the single-letter code as
`derived from cDNA sequencing. The underlined portions indicate
`peptide fragments which we identified as containing sites of degra-
`dation by peptide mapping and are referred to in the text. In the
`mature protein the amino terminus of both chains is cyclized to the
`pyroglutamic acid derivative, the carboxy terminal Lys of the H
`chains is processed off, and a carbohydrate chain is attached to Asn
`H299. OKT3 is an IgGZa isotype.
`
`band shift is due to the deamidation of Asn or Gln residues,
`as has been observed previously for other proteins (5,6).
`OKT3 held at 37°C for 2 months showed one major
`change in the trypsin-generated peptide map of both the L
`and the H chain. As shown in the L chain map of degraded
`OKT3 in Fig. 3, compared to the control in Fig. 2, the peak
`with a retention time (rt) of 26.5 min (peak 1) diminished in
`size, and two peaks with rt of 27.1 and 27.4 min increased
`considerably in size (peaks 2 and 3). The peak area ratio of
`peak 3 to peak 2 was 3:1. Sequencing of these peaks estab-
`lished that the normal tryptic fragment, peak 1, is L155-l68
`and the degradation peak 2 is the Asp”6 analogue of this
`peptide, Asn being at this position in undegraded OKT3. The
`peak 3 component showed a Gln at the first cycle of the
`sequencing, then the Edman degradation terminated. This
`result is consistent with this peptide'being the isoaspartate-
`156 analogue of L155—168, since Edman sequencing fails at
`isoAsp residues (7) and the other expected product of Asn
`deamidation is isoAsp (8). Asparagine deamidation via a cy-
`clic succinimide derivative has been shown to give both
`types of aspartate derivatives at similar product ratios in
`other peptides and proteins (9—11).
`As in the L chain, one major change was observed in the
`H chain tryptic map of thermally stressed OKT3. A decrease
`in the size of a peak identified by sequencing as H364—388
`was observed and two new nonresolved peaks, eluting
`slightly earlier, appeared (Fig. 4). The degradation peptides
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`Sites of Degradation in a Therapeutic Monoclonal Antibody
`
`1389
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` ‘
`
`
`Am...
`
`
`
`N
`
`i
`
`lemma
`
`J
`
`sit.
`10
`20
`30
`40
`50
`
`Time nun.)
`Fig. 2. Peptide maps of OKT3 light and heavy chains. Separated L
`and H chains from 0KT3 reference standard were digested with
`trypsin and the digests analyzed by HPLC on a Rainin Dynamax
`C18, S-um 300A column eluted with a gradient of 5 to 45% aceto-
`nitrile in 0.1% trifluoroacetic acid over 60 min at 1.5 ml/min.
`
`isolated from the combined nonresolved peaks were related
`to H364—388 by the identical results obtained in sequencing
`through 22 cycles. This peptide contains two asparagines,
`which could potentially deamidate. Asnm was observed in
`the sequencing, but the status of Asn386 could not be estab-
`lished due to the low yields at this cycle of the sequencing
`and carryover from Asnm. This is particularly true since the
`expected major product of deamidation, the isoAsp peptide,
`would be diagnosed only as a termination in the sequencing.
`Asparagine deamidation at H386 in this sample of OKT3 was
`confirmed by digesting the heavy chain with Glu-C endopro-
`teinase, which gives a small H383—390 fragment. Asn38°—,
`Asp386—, and isoAsp386—H383—390 peptides were synthesized
`