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Comparative analysis of two controlled proliferation strategies regarding product quality, influence on tetracycline‐regulated gene expression, and productivity
Author(s) -
Kaufmann Hitto,
Mazur Xenia,
Marone Romina,
Bailey James E.,
Fussenegger Martin
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/1097-0290(20010320)72:6<592::aid-bit1024>3.0.co;2-j
Subject(s) - chinese hamster ovary cell , transactivation , biology , microbiology and biotechnology , cell growth , growth inhibition , cell culture , gene expression , gene , biochemistry , genetics
Overexpression of the cyclin‐dependent kinase inhibitor p27 and exposure to low temperature (30°C) represent two strategies to establish controlled proliferation processes for production of therapeutic proteins using Chinese hamster ovary (CHO) cells. Here we analyze the effect of growth inhibition on the quality of the human model glycoprotein SEAP (secreted alkaline phosphatase) for both strategies in monoclonal CHO‐derived cell lines. Separation of purified SEAP samples using two‐dimensional gel electrophoresis showed that production by proliferation‐controlled CHO cultures did not alter the overall integrity of the product. Further, oligosaccharide profiles were compared using HPEC‐PAD analysis. No differences were detectable between SEAP profiles obtained from p27 growth‐arrested and proliferating cultures. However, production at 30°C led to a significant increase in the degree of sialylation, an effect that is generally considered beneficial for the in vivo efficacy of protein therapeutics. In the production context presented here, SEAP expression is controlled by the tetracycline‐ (tet) repressible gene regulation system. Here we show low temperature‐induced upregulation of the tetracycline‐dependent transactivator (tTA). This induction has been shown by Northern blot analysis to occur at the mRNA level and is independent of the promoters driving the transactivator. We also describe a novel bottleneck in productivity at low temperature found in p27 growth‐arrested CHO cells cultivated at 30°C. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 592–602, 2001.

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