Haimanti Dorai and Yun Seung Kyung contributed equally to this work.
Expression of anti-apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture†
Article first published online: 22 JAN 2009
Copyright © 2009 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 103, Issue 3, pages 592–608, 15 June 2009
How to Cite
Dorai, H., Kyung, Y. S., Ellis, D., Kinney, C., Lin, C., Jan, D., Moore, G. and Betenbaugh, M. J. (2009), Expression of anti-apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture. Biotechnol. Bioeng., 103: 592–608. doi: 10.1002/bit.22269
- Issue published online: 22 APR 2009
- Article first published online: 22 JAN 2009
- Accepted manuscript online: 22 JAN 2009 12:00AM EST
- Manuscript Accepted: 8 JAN 2009
- Manuscript Revised: 28 DEC 2008
- Manuscript Received: 21 OCT 2008
- CHO cells;
- mammalian cell culture;
- lactate consumption;
- mitochondrial membrane potential;
- caspase 3/7;
- amino acid;
In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity, and enhanced mitochondrial membrane potential (MMP). Two particular cell lines, one containing two anti-apoptotic genes, E1B-19K and Aven (EA167), and another containing three, E1B-19K, Aven, and a mutant of XIAP (EAX197), exhibited a reduction in caspase 3 activity of at least 60% and a 170% enhancement in mitochondrial membrane potential compared to controls when treated with staurosporine. In batch cell growth experiments, the peak viable cell densities and viabilities were higher resulting in a 186% increase in integrated viable cell densities. Analyses of metabolite utilization and formation of waste products indicated that the apoptotic resistant cell lines depleted all the lactate when grown in commercially available CD-CHO medium while significant levels (>1.8 g/L) accumulated in the host cell lines. When the lactate level was replenished daily in the apoptotic resistant cell lines, the cell lines consumed lactate and the culture longevity was extended up to four additional days compared to control cell lines. Furthermore, the anti-apoptosis cell lines also accumulated lower levels of ammonia. The ability of the apoptotic resistant cell lines to consume lactate was exploited by cultivating them in a “high” glucose medium containing 15 g/L (60 mM glucose) in which apoptotic resistant cell lines exhibited lower maximum lactate (1.8 g/L) compared to control cell lines which accumulated concentrations of lactate (2.2 g/L) that appeared to be deleterious for growth. The shaker flask titer of a therapeutic antibody product expressed in an apoptotic resistant cell line in “high” glucose medium reached 690 mg/L compared to 390 mg/L for a cell line derived from a control host cell line. These results represent to our knowledge the first example in the literature in which manipulation of the apoptosis pathway has altered the nutrient consumption profile of mammalian cells in culture; findings that underscore the interdependence of the apoptotic cellular machinery and metabolism and provide greater flexibility to mammalian bioreactor process development. Biotechnol. Bioeng. 2009;103: 592–608. © 2009 Wiley Periodicals, Inc.