Pharmaceutical Research

, Volume 9, Issue 11, pp 1386–1393 | Cite as

Identification of Sites of Degradation in a Therapeutic Monoclonal Antibody by Peptide Mapping

  • Daniel J. Kroon
  • Alysia Baldwin-Ferro
  • Praful Lalan


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.

OKT3, monoclonal antibody peptide mapping protein stability degradation of proteins 


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  1. 1.
    M. C. Manning, K. Patel, and R. T. Borchardt. Stability of protein pharmaceuticals. Pharm. Res. 6:903–918 (1989).Google Scholar
  2. 2.
    P. E. Rao and D. J. Kroon. Orthoclone OKT3; Chemical mechanisms and functional effects of degradation of a monoclonal antibody. In J. Wang and M. P. Born (eds.), Stability of Protein Pharmaceuticals—Case Histories Plenum, New York (1992).Google Scholar
  3. 3.
    T. W. Thannhauser, Y. Konishi, and H. A. Scheraga. Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins. Anal. Biochem. 138:181–188 (1984).PubMedGoogle Scholar
  4. 4.
    P. Matsudaira. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 263:10035–10038 (1987).Google Scholar
  5. 5.
    K. U. Yuksel and R. W. Gracy. In vitro deamidation of human triosephosphate isomerase. Arch. Biochem. Biophys. 248:452–459 (1986).Google Scholar
  6. 6.
    W. J. Chazin, J. Kadel, E. Thulin, T. Hofmann, T. Drakenberg, and S. Forsen. Identification of an isoaspartyl linkage formed upon deamidation of bovine calbindin D9k and structural characterization by 2D 1H NMR. Biochemistry 28:8646–8653 (1989).Google Scholar
  7. 7.
    W. Konigsberg. Subtractive Edman degradation. In C. H. W. Hirs and S. N. Timasheff (eds.), Methods in Enzymology, Vol. XXV Academic Press, New York, 1972, pp. 326–332.Google Scholar
  8. 8.
    T. Geiger and S. Clarke. Deamidation, isomerization and racemization at asparaginyl and aspartyl residues in peptides. J. Biol. Chem. 262:785–794 (1987).Google Scholar
  9. 9.
    S. Capasso, L. Mazzarella, F. Sica, and A. Zagari. Deamidation via cyclic imide in asparaginyl peptides. Peptide Res. 2:195–200 (1989).Google Scholar
  10. 10.
    B. A. Johnson, J. M. Shirokawa, W. S. Hancock, M. W. Spellman, L. J. Basa, and D. W. Aswad. Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone. J. Biol. Chem. 264:14262–14271 (1989).Google Scholar
  11. 11.
    B. N. Violand, M. R. Schlittler, P. C. Toren, and N. R. Siegel. Formation of isoaspartate 99 in bovine and porcine somatotropins. J. Protein Chem. 9:109–117 (1990).Google Scholar
  12. 12.
    S. Clarke. Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins. Int. J. Peptide Protein Res. 30:808–821 (1987).Google Scholar
  13. 13.
    W. Jiskoot, E. Coen Beuvery, A. A. M. de Koning, J. N. Herron, and D. J. A. Crommelin. Analytical approaches to the study of monoclonal antibody stability. Pharm. Res. 7:1234–1241 (1990).Google Scholar
  14. 14.
    E. A. Kabat, T. T. Wu, M. Reid-Miller, H. M. Perry, and K. S. Gottesman. Sequences of Proteins of Immunological Interest 4th ed., U.S. Department of Health and Human Services, 1987.Google Scholar
  15. 15.
    R. J. Harris, K. L. Wagner, and M. W. Spellman. Structural characterization of a recombinant CD4-IgG hybrid molecule. Eur. J. Biochem. 194:611–620 (1990).Google Scholar

Copyright information

© Plenum Publishing Corporation 1992

Authors and Affiliations

  • Daniel J. Kroon
    • 1
  • Alysia Baldwin-Ferro
    • 1
  • Praful Lalan
    • 1
  1. 1.The R. W. Johnson Pharmaceutical Research InstituteRaritan

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