Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research
  • Published:

Assessing Genetic Heterogeneity in Production Cell Lines: Detection by Peptide Mapping of a Low Level Tyr to Gln Sequence Variant in a Recombinant Antibody

Bio/Technology volume 11, pages 1293–1297 (1993)Cite this article

Abstract

A recombinant antibody directed against the human epidermal growth factor receptor-2 extracellular domain was subjected to detailed structural characterization. Heterogeneity in the heavy chain was demonstrated by recovery of two forms of a tryptic peptide, with either glutamine or the expected tyrosine at residue 376. Subsequent experiments indicated that the Y376Q variant developed during transfection of the antibody heavy and light chain genes into Chinese hamster ovary cells. Levels of the Y376Q variant (range: 27% to 1%) in the purified antibody were inversely proportional to cell age. The established cell line was subcloned and found to be heterogeneous by polymerase chain reaction analysis of cell extracts and protein analysis of the purified antibody. Ten percent of subclones produced high levels of the Y376Q variant while 90% of the subclones produced antibody with only the expected heavy chain sequence. This report demonstrates the utility of peptide mapping as a sensitive tool for assessing genetic heterogeneity of recombinant cell lines.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Yarden, Y. and Ullrich, A. 1988. Growth factor receptor tyrosine kinases. Annu. Rev. Biochem. 57: 443–478.

    Article  CAS  PubMed  Google Scholar 

  2. Schechter, A.L., Hung, M.-C., Vaidyanathan, L., Weinberg, R.A., Yang-Feng, T.L., Francke, U., Ullrich, A. and Coussens, L. 1985. The neu gene: an erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science 229: 976–978.

    Article  CAS  PubMed  Google Scholar 

  3. Fukushige, S.-I., Matsubara, K.-I., Yoshida, M., Sasaki, M., Suzuki, T., Semba, K., Toyoshima, K. and Yamamoto, T. 1985. Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line. Mol. cell. Biol. 6: 955–958.

    Article  Google Scholar 

  4. Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A. and McGuire, W.L. 1987. Human breast cancer: correlation of relapse and survival with amplification of the HER2/neu oncogene. Science 235: 177–182.

    Article  CAS  PubMed  Google Scholar 

  5. Wright, C., Angus, B., Nicholson, S., Sainsbury, S.R.C., Cairns, J., Gullick, W.J., Kelly, P., Harris, A.L. and Horne, C.H.W. 1989. Expression of c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer. Cancer Res. 49: 2087–2090.

    CAS  PubMed  Google Scholar 

  6. Holmes, W.E., Sliwkowski, M.X., Akita, R.W., Henzel, W.J., Lee, J., Park, J.W., Yansura, D., Abadi, N., Raab, H., Lewis, G.D., Shepard, H.M., Kuang, W.-J., Wood, W.I., Goeddel, D.V. and Vandlen, R.L. 1992. Identification of heregulin, a specific inhibitor of p185erbB2 . Science 256: 1205–1210.

    Article  CAS  PubMed  Google Scholar 

  7. Fendly, B.M., Winget, M., Hudziak, R.M., Lipari, M.T., Napier, M.A. and Ullrich, A. 1990. Characterization of marine monoclonal antibodies reactive to either the human epidermal growth factor receptor or HER2/neu gene product. Cancer Res. 50: 1550–1558.

    CAS  PubMed  Google Scholar 

  8. Waldmann, T.A. 1991. Monoclonal antibodies in diagnosis and therapy. Science 252: 1657–1662.

    Article  CAS  PubMed  Google Scholar 

  9. Manohar, V. and Hoffman, T. 1992. Monoclonal and engineered antibodies for human parenteral clinical use: regulatory considerations. Trends. Biotech. 10: 305–309.

    Article  CAS  Google Scholar 

  10. Carter, P., Presta, L., Gorman, C.M., Ridgway, J.B.B., Henner, D., Wong, W.L.T., Rowland, A.M., Kotts, C., Carver, M.E. and Shepard, H.M. 1992. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. USA 89: 4285–4289.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Office of Biologies Research and Review. 1985. Points to consider in the production and testing of new drugs and biologicals produced by recombinant DNA technology. Food and Drug Administration, Bethesda MD.

  12. Ellison, J.W., Berson, B.J. and Hood, L.E. 1982. The nucleotide sequence of a human immunoglobulin Cγ1 gene. Nucleic Acids Res. 10: 4071–4079.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rao, P., Williams, A., Baldwin-Ferro, A., Hanigan, E., Kroon, D., Makowskji, M., Meyer, E., Numsuwan, V., Rubin, E. and Tran, A. 1991. C-Terminal modification occurs in tissue culture produced OKT3. BioPharm 4: 38–43.

    CAS  Google Scholar 

  14. Harris, R.J., Wagner, K.L. and Spellman, M.W. 1990. Structural characterization of a recombinant CD4-IgG molecule. Eur. J. Biochem. 194: 611–620.

    Article  CAS  PubMed  Google Scholar 

  15. Ehlen, T. and Dubeau, L. 1989. Detection of RAS point mutations by polymerase chain reaction using mutation-specific, inosine-containing oligonucleotide primers. Biochem. Biophys. Res. Commun. 160: 441–447.

    Article  CAS  PubMed  Google Scholar 

  16. Lebkowski, J.S., DuBridge, R.B., Antell, E.A., Griesen, K.S. and Calos, M.P. 1984. Transfected DNA is mutated in monkey, mouse and human cells. Mol. cell. Bio. 4: 1951–1960.

    Article  CAS  Google Scholar 

  17. Wurm, F.J. 1990. Integration, amplification and stability of plasmid sequences in CHO cell cultures. Biologicals 18: 159–164.

    Article  CAS  PubMed  Google Scholar 

  18. Lu, H.S., Tsai, L.B., Kenney, W.C. and Lai, P.-H. 1988. Identification of unusual replacement of methionine residues by norleucine in recombinant interleukin-2 produced by E. coli . Biochem Biophys. Res. Commun. 156: 807–813.

    Article  CAS  PubMed  Google Scholar 

  19. Bogosian, G., Violand, B.N., Dorward-King, E.J., Workman, W.E., Jung, P.E. and Kane, J.F. 1989. Biosynthesis and incorporation into protein of norleucine by Escherichia coli . J. Biol. Chem. 264: 531–539.

    CAS  PubMed  Google Scholar 

  20. Trupin, J., Dickerman, H., Nirenberg, M. and Weissbach, H. 1966. Formylation of amino acid analogues of methionine sRNA. Biochem. Biophys. Res. Commun. 24: 50–55.

    Article  CAS  PubMed  Google Scholar 

  21. Tsai, L.B., Lu, H.S., Kenney, W.C., Curless, C.C., Klein, M.L., Lai, P.-H., Fenton, D.M., Altrock, B.W. and Mann, M.B. 1988. Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli . Biochem Biophys. Res. Commun. 156: 733–739.

    Article  CAS  PubMed  Google Scholar 

  22. Seetharam, R., Heeren, R.A., Wong, E.Y., Braford, S.R., Klein, B.K., Aykent, S., Kotts, C.E., Mathis, K.J., Bishop, B.F., Jennings, M.J., Smith, C.E. and Siegel, N.R. 1988. Mistranslation in IGF-1 during over-expression of the protein in Escherichia coli using a synthetic gene containing low frequency codons. Biochem. Biophys. Res. Commun. 155: 518–523.

    Article  CAS  PubMed  Google Scholar 

  23. Lu, H.S., Fausset, P.R., Sotos, L.S., Clogston, C.L., Rohde, M.F., Stoney, K.S. and Herman, A.C. 1993. Isolation and characterization of three recombinant human granulocyte colony stimulating factor His to Gln isoforms produced in Escherichia coli . Protein Expression and Purification. In press

    Google Scholar 

  24. Garnick, R.L., Solli, N.J. and Papa, P.A. 1988. The role of quality control in biotechnology: an analytical perspective. Anal. Chem. 60: 2546–2557.

    Article  CAS  PubMed  Google Scholar 

  25. Scoble, H.A. and Martin, S.A. 1990. Characterization of recombinant proteins. Meth. in Enzymoi. 193: 519–536.

    Article  CAS  Google Scholar 

  26. Chloupek, R.C., Harris, R.J., Leonard, C.K., Keck, R.G., Keyt, B.A., Spellman, M.W., Jones, A.J.S. and Hancock, W.S. 1989. Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping. J. Chromatog. 463: 375–396.

    Article  CAS  Google Scholar 

  27. Harris, R.J., Chamow, S.M., Gregory, T.G. and Spellman, M.W. 1990. Characterization of a soluble form of human CD4. Eur. J. Biochem. 188: 291–300.

    Article  CAS  PubMed  Google Scholar 

  28. Leonard, C.K., Spellman, M.W., Riddle, L., Harris, R.J., Thomas, J.N. and Gregory, T.J. 1990. Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type I recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J. Biol. Chem. 265: 10373–10382.

    CAS  PubMed  Google Scholar 

  29. Paborsky, L.R. and Harris, R.J. 1991. Post-translational modifications of recombinant human tissue factor. Thromb. Res. 60: 367–376.

    Article  Google Scholar 

  30. Schmelzer, C.H., Harris, R.J., Butler, D., Burton, L.E., Yedinak, C.M. and Wagner, K.L. 1993. Glycosylation pattern and disulfide bond assignments of recombinant human differentiation-stimulating factor. Arch. Biochem. Biophys. 302: 484–489.

    Article  CAS  PubMed  Google Scholar 

  31. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, NY.

    Google Scholar 

  32. Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Innis, M.A., Gelfend, D.H., Sninsky, J.J. and White, T.J. 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego.

    Google Scholar 

Download references

Author information

Author notes

  1. Reed J. Harris: Corresponding author.

Authors and Affiliations

  1. Department of Medicinal and Analytical Chemistry, Genentech, Inc., 460 Point San Bruno Blvd., So. San Francisco, CA, 94080

    Reed J. Harris & Karen L. Wagner

  2. Department of Cell Culture and Fermentation Research and Development, Genentech, Inc., 460 Point San Bruno Blvd., So. San Francisco, CA, 94080

    Amy A. Murnane, Gian D. Polastri & Mary B. Sliwkowski

  3. Department of Quality Control, Genentech, Inc., 460 Point San Bruno Blvd., So. San Francisco, CA, 94080

    Shannon L. Utter, Edward T. Cox & Judith C. Helder

Authors
  1. Reed J. Harris
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amy A. Murnane
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shannon L. Utter
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karen L. Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Edward T. Cox
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gian D. Polastri
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Judith C. Helder
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mary B. Sliwkowski
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Harris, R., Murnane, A., Utter, S. et al. Assessing Genetic Heterogeneity in Production Cell Lines: Detection by Peptide Mapping of a Low Level Tyr to Gln Sequence Variant in a Recombinant Antibody. Nat Biotechnol 11, 1293–1297 (1993). https://doi.org/10.1038/nbt1193-1293

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1193-1293

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing