Comparative analysis of two controlled proliferation strategies regarding product quality, influence on tetracycline-regulated gene expression, and productivity

Overexpression of the cyclin-dependent kinase inhibitor p27 and exposure to low temperature (30 degreesC) represent two strategies to establish contro lled proliferation processes for production of therapeutic proteins using C hinese hamster ovary (CHO) cells. Here we analyze the effect of growth inhi bition on the quality of the human model glycoprotein SEAP (secreted alkali ne phosphatase) for both strategies in monoclonal CHO-derived cell lines. Separation of purified SEAP samples using two-dimensional gel electrophores is showed that production by proliferation-controlled CHO cultures did not alter the overall integrity of the product. Further, oligosaccharide profil es were compared using HPEC-PAD analysis. No differences were detectable be tween SEAP profiles obtained from p27 growth-arrested and proliferating cul tures. However, production at 30 degreesC led to a significant increase in the degree of sialylation, an effect that is generally considered beneficia l 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 lo w temperature-induced upregulation of the tetracycline-dependent transactiv ator (tTA). This induction has been shown by Northern blot analysis to occu r at the mRNA level and is independent of the promoters driving the transac tivator. We also describe a novel bottleneck in productivity at low temperature foun d in p27 growth-arrested CHO cells cultivated at 30 degreesC. (C) 2001 John Wiley & Sons, Inc.