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

Nature Biotechnology volume 11, pages 12931297 (1993) | Download Citation

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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.

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References

  1. 1.

    and 1988. Growth factor receptor tyrosine kinases. Annu. Rev. Biochem. 57: 443–478.

  2. 2.

    , , , , , , and 1985. The neu gene: an erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science 229: 976–978.

  3. 3.

    , , , , , , and 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.

  4. 4.

    , , , , and 1987. Human breast cancer: correlation of relapse and survival with amplification of the HER2/neu oncogene. Science 235: 177–182.

  5. 5.

    , , , , , , , and 1989. Expression of c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer. Cancer Res. 49: 2087–2090.

  6. 6.

    , , , , , , , , , , , , , and 1992. Identification of heregulin, a specific inhibitor of p185erbB2 . Science 256: 1205–1210.

  7. 7.

    , , , , and 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.

  8. 8.

    1991. Monoclonal antibodies in diagnosis and therapy. Science 252: 1657–1662.

  9. 9.

    and 1992. Monoclonal and engineered antibodies for human parenteral clinical use: regulatory considerations. Trends. Biotech. 10: 305–309.

  10. 10.

    , , , , , , , , and 1992. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. USA 89: 4285–4289.

  11. 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. 12.

    , and 1982. The nucleotide sequence of a human immunoglobulin Cγ1 gene. Nucleic Acids Res. 10: 4071–4079.

  13. 13.

    , , , , , , , , and 1991. C-Terminal modification occurs in tissue culture produced OKT3. BioPharm 4: 38–43.

  14. 14.

    , and 1990. Structural characterization of a recombinant CD4-IgG molecule. Eur. J. Biochem. 194: 611–620.

  15. 15.

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

  16. 16.

    , , , and 1984. Transfected DNA is mutated in monkey, mouse and human cells. Mol. cell. Bio. 4: 1951–1960.

  17. 17.

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

  18. 18.

    , , and 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.

  19. 19.

    , , , , and 1989. Biosynthesis and incorporation into protein of norleucine by Escherichia coli . J. Biol. Chem. 264: 531–539.

  20. 20.

    , , and 1966. Formylation of amino acid analogues of methionine sRNA. Biochem. Biophys. Res. Commun. 24: 50–55.

  21. 21.

    , , , , , , , and 1988. Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli . Biochem Biophys. Res. Commun. 156: 733–739.

  22. 22.

    , , , , , , , , , , and 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.

  23. 23.

    , , , , , and 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

  24. 24.

    , and 1988. The role of quality control in biotechnology: an analytical perspective. Anal. Chem. 60: 2546–2557.

  25. 25.

    and 1990. Characterization of recombinant proteins. Meth. in Enzymoi. 193: 519–536.

  26. 26.

    , , , , , , and 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.

  27. 27.

    , , and 1990. Characterization of a soluble form of human CD4. Eur. J. Biochem. 188: 291–300.

  28. 28.

    , , , , and 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.

  29. 29.

    and 1991. Post-translational modifications of recombinant human tissue factor. Thromb. Res. 60: 367–376.

  30. 30.

    , , , , and 1993. Glycosylation pattern and disulfide bond assignments of recombinant human differentiation-stimulating factor. Arch. Biochem. Biophys. 302: 484–489.

  31. 31.

    , and 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, NY.

  32. 32.

    , and 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.

  33. 33.

    , , and 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego.

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Author information

Author notes

    • Reed J. Harris

    Corresponding author.

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

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DOI

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

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