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Microbial Cell Factories

, 5:P3 | Cite as

Regulation of the secretion pathway of CHO cells for altered recombinant Mab production rates during the course of MTX amplification

  • Yuan Sheng Yang
  • Janet Chusainow
  • Yan Ying Mao
  • Steven CL Ho
  • Miranda GS Yap
Open Access
Poster Presentation
  • 2.9k Downloads

Keywords

Methotrexate Gene Copy Number Secretion Pathway Stable Cell Line Representative Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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.

Notes

Acknowledgements

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

References

  1. 1.
    Gonzalez R, Andrews BA, Asenjo JA: Metabolic control analysis of monoclonal antibody synthesis. Biotechnol Prog. 2001, 17: 217-226. 10.1021/bp000165b.CrossRefGoogle Scholar
  2. 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.CrossRefGoogle Scholar

Copyright information

© Yang et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

Authors and Affiliations

  • Yuan Sheng Yang
    • 1
  • Janet Chusainow
    • 1
  • Yan Ying Mao
    • 1
  • Steven CL Ho
    • 2
  • Miranda GS Yap
    • 1
    • 3
  1. 1.Bioprocessing Technology Institute, Biomedical Sciences InstitutesSingapore138668
  2. 2.Division of BioengineeringNanyang Technological UniversitySingapore
  3. 3.Department of Chemical & Biomolecular EngineeringNational University of SingaporeSingapore

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