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  • Poster Presentation
  • Open Access
  • Regulation of the secretion pathway of CHO cells for altered recombinant Mab production rates during the course of MTX amplification

    • 1,
    • 1,
    • 1,
    • 2 and
    • 1, 3
    Microbial Cell Factories20065 (Suppl 1) :P3

    https://doi.org/10.1186/1475-2859-5-S1-P3

    • Published:

    Keywords

    • Methotrexate
    • Gene Copy Number
    • Secretion Pathway
    • Stable Cell Line
    • Representative Gene

    Background

    Monoclonal antibody (Mab) production by mammalian cells is a complex, multiple-step process which is regulated at transcriptional, translational, and post-translational levels. A detailed understanding of how cells regulate this pathway is a prerequisite for designing genetic strategies for increasing antibody production [1]. Methotrexate (MTX), which is widely used in the creation of high-producing stable cell lines by amplification of gene copy number, provides an effective means to alter Mab production rates for mechanistic studies of the regulation of this pathway [2].

    Results

    In this work, stable CHO DG44 cell lines expressing a human anti-D Mab were created and single-cell clones were amplified to obtain a series of cultures with varying production rates. During the course of amplification, changes in the Mab gene copy numbers, transcriptional levels of Mab mRNAs, and accumulated intracellular Mab peptides were examined for each clone. In addition, changes in expression levels of representative genes with function in translation, folding, assembly, and degradation were determined. Gene copy number and transcription level were quantified by quantitative real time PCR, and the intracellular Mab peptides were quantified by western blotting and ELISA.

    Conclusion

    Results obtained in this work could help identify the rate-limiting steps and factors that are significant in limiting production rate for high-producing clones.

    Declarations

    Acknowledgements

    We thank Toh Poh Choo and Jessna Yeo who created the cell lines used in this work.

    Open AccessThis article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Authors’ Affiliations

    (1)
    Bioprocessing Technology Institute, Biomedical Sciences Institutes, 20 Biopolis Way, #06-01 Centros, Singapore, 138668
    (2)
    Division of Bioengineering, Nanyang Technological University, Nanyang Avenue, Singapore, 639798
    (3)
    Department of Chemical & Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119620

    References

    1. Gonzalez R, Andrews BA, Asenjo JA: Metabolic control analysis of monoclonal antibody synthesis. Biotechnol Prog. 2001, 17: 217-226. 10.1021/bp000165b.View ArticleGoogle Scholar
    2. Kim SJ, Kim NS, Ryu CJ, Hong HJ, Lee GM: Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol Bioeng. 1998, 58: 73-84. 10.1002/(SICI)1097-0290(19980405)58:1<73::AID-BIT8>3.0.CO;2-R.View ArticleGoogle Scholar

    Copyright

    © Yang et al; licensee BioMed Central Ltd. 2006

    This article is published under license to BioMed Central Ltd.

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