The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process

Authors

  • Catherine E.M. Hogwood,

    1. Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK; telephone: +44-1227-823746; fax: +44-1227-763912
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  • Andrew S. Tait,

    1. Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK; telephone: +44-2076792374; fax: +44-2079160703
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  • Nadejda Koloteva-Levine,

    1. Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK; telephone: +44-1227-823746; fax: +44-1227-763912
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  • Daniel G. Bracewell,

    Corresponding author
    1. Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK; telephone: +44-2076792374; fax: +44-2079160703
    • Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK; telephone: +44-2076792374; fax: +44-2079160703.
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  • C. Mark Smales

    Corresponding author
    1. Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK; telephone: +44-1227-823746; fax: +44-1227-763912
    • Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK; telephone: +44-1227-823746; fax: +44-1227-763912.
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  • Catherine E.M. Hogwood and Andrew S. Tait contributed equally towards this publication.

Abstract

Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D-PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post-protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D-PAGE can be used for monitoring and identification of HCPs post-protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell-engineering approaches can be applied to reduced, or eliminate, such HCPs. Biotechnol. Bioeng. 2013; 110: 240–251. © 2012 Wiley Periodicals, Inc.

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