Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies

Authors

  • Lauren Feeney,

    1. Department of Late Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080-4990; telephone: 650-467-4596; fax: 650-225-2006
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  • Veronica Carvalhal,

    1. Department of Late Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080-4990; telephone: 650-467-4596; fax: 650-225-2006
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  • X. Christopher Yu,

    1. Department of Protein Analytical Chemistry, Genentech, Inc., South San Francisco, California
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  • Betty Chan,

    1. Department of Protein Analytical Chemistry, Genentech, Inc., South San Francisco, California
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  • David A. Michels,

    1. Department of Protein Analytical Chemistry, Genentech, Inc., South San Francisco, California
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  • Yajun Jennifer Wang,

    1. Department of Protein Analytical Chemistry, Genentech, Inc., South San Francisco, California
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  • Amy Shen,

    1. Department of Early Stage Cell Culture, Genentech, Inc., South San Francisco, California
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  • Jan Ressl,

    1. Department of Late Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080-4990; telephone: 650-467-4596; fax: 650-225-2006
    Current affiliation:
    1. Department of Cell Culture and Fermentation, Perseid Therapeutics, LLC, 515 Galveston Drive, Redwood City, CA 94063-4720.
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  • Brendon Dusel,

    1. Department of Late Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080-4990; telephone: 650-467-4596; fax: 650-225-2006
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  • Michael W. Laird

    Corresponding author
    1. Department of Late Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080-4990; telephone: 650-467-4596; fax: 650-225-2006
    • Department of Late Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080-4990; telephone: 650-467-4596; fax: 650-225-2006
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Abstract

Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNATyr mischarging due to the structural similarities between tyrosine and phenylalanine. Biotechnol. Bioeng. 2013; 110: 1087–1097. © 2012 Wiley Periodicals, Inc.

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