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 degrees 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 degrees 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 degrees C.
Kaufmann
H
H
Institute of Biotechnology, ETH Zurich, CH-8093 Zurich, Switzerland.
Mazur
X
X
Marone
R
R
Bailey
J E
JE
Fussenegger
M
M
D000469:Alkaline Phosphatase / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D018797:Cell Cycle Proteins / Q000494:pharmacology; D002455:Cell Division / Q000502:physiology; D006224:Cricetinae; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D015870:Gene Expression / Q000187:drug effects; D011786:Quality Control; D012313:RNA / Q000378:metabolism; D013696:Temperature; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation
D018797:Cell Cycle Proteins; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D012313:RNA; D000469:Alkaline Phosphatase; D013752:Tetracycline