Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity

Authors

  • Sharon X. Gao,

    Corresponding author
    1. Department of Analytical Biochemistry, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8265; fax: 858-401-5031
    • Department of Analytical Biochemistry, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8265; fax: 858-401-5031.
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  • Ying Zhang,

    1. Department of Analytical Biochemistry, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8265; fax: 858-401-5031
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  • Kensey Stansberry-Perkins,

    1. Department of Analytical Biochemistry, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8265; fax: 858-401-5031
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  • Alex Buko,

    1. Department of Analytical Biochemistry, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8265; fax: 858-401-5031
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  • Shujun Bai,

    1. Department of Protein Pharmaceutical Development, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8780; fax: 858-401-5484
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  • Vanessa Nguyen,

    1. Department of Protein Pharmaceutical Development, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8780; fax: 858-401-5484
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  • Mark L. Brader

    Corresponding author
    1. Department of Protein Pharmaceutical Development, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8780; fax: 858-401-5484
    • Department of Protein Pharmaceutical Development, Biogen Idec, 5200 Research Place, San Diego, California; telephone: +1-858-401-8780; fax: 858-401-5484.
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Abstract

Monoclonal antibody (mAb) fragmentation can be a widespread problem across the biotechnology industry and there is a current need to better understand the underlying principles. Here, we report an example of a high-purity human IgG1 mAb prepared from CHO cells exhibiting fragmentation that can be attributed to residual proteolytic enzyme activity. The concomitant occurrence of proteolytic and non-proteolytic peptide bond cleavage is shown and the respective fragmentation patterns characterized using high-resolution LC–MS. Fragmentation rates are monitored by SE-HPLC and SDS–PAGE over the pH range 4–6 and characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. After 20 days at 40°C, pH 4, ∼60% decrease in BIIB-mAb monomer peak occurred attributed to residual proteolytic activity. At pH 5, this value was ∼13%. These results have implications for formulation design studies and the interpretation of accelerated stability data. A simple method to screen for acidic protease activity using the proteolytic enzyme inhibitor pepstatin A is described. Biotechnol. Bioeng. 2011; 108:977–982. © 2010 Wiley Periodicals, Inc.

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