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Titolo:
Comparative analysis of two controlled proliferation strategies regarding product quality, influence on tetracycline-regulated gene expression, and productivity
Autore:
Kaufmann, H; Mazur, X; Marone, R; Bailey, JE; Fussenegger, M;
Indirizzi:
ETH Zurich, Inst Biotechnol, CH-8093 Zurich, Switzerland ETH Zurich Zurich Switzerland CH-8093 chnol, CH-8093 Zurich, Switzerland
Titolo Testata:
BIOTECHNOLOGY AND BIOENGINEERING
fascicolo: 6, volume: 72, anno: 2001,
pagine: 592 - 602
SICI:
0006-3592(20010320)72:6<592:CAOTCP>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
HAMSTER OVARY CELLS; RECOMBINANT-HUMAN-ERYTHROPOIETIN; PLACENTAL ALKALINE-PHOSPHATASE; MESSENGER-RNA DEGRADATION; MAMMALIAN-CELLS; LOW-TEMPERATURE; 2-DIMENSIONAL ELECTROPHORESIS; CHO CELLS; GROWTH; GLYCOSYLATION;
Keywords:
controlled proliferation; low temperature; glycosylation;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
46
Recensione:
Indirizzi per estratti:
Indirizzo: Fussenegger, M ETH Zurich, Inst Biotechnol, CH-8093 Zurich, Switzerland ETH Zurich Zurich Switzerland CH-8093 Zurich, Switzerland
Citazione:
H. Kaufmann et al., "Comparative analysis of two controlled proliferation strategies regarding product quality, influence on tetracycline-regulated gene expression, and productivity", BIOTECH BIO, 72(6), 2001, pp. 592-602

Abstract

Overexpression of the cyclin-dependent kinase inhibitor p27 and exposure to low temperature (30 degreesC) 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 notalter 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 degreesC led to a significant increase inthe 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 bythe 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 degreesC. (C) 2001 John Wiley & Sons, Inc.

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Documento generato il 22/10/20 alle ore 03:39:21