Analysis of CHO Cells Metabolic Redistribution in a Glutamate-Based Defined Medium in Continuous Culture

Altamirano, Claudia; Illanes, Andrés; Casablancas, Antoni; Gámez, X.; Cairó, Jordi J.; Gòdia, C.

doi:10.1021/bp0100981

Cited by 121 publications

(120 citation statements)

References 36 publications

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“…On the other hand, despite its positive effect on cell growth, the increase in vitamin concentration did not affect the t-PA specific production rate (qtPA) nor its specific activity (atPA), as shown in Table 2. The maximum levels of t-PA obtained in the different cultures (Table 2) were of the same order of magnitude of those reported for this cell line in the literature, that is, from 0.2 to 5 mg l-1 (Hansen and Emborg, 1994;Altamirano et al 2001;Altamirano et al 2004). …”

Section: Resultssupporting

confidence: 82%

Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production

Altamirano¹,

Illanes²,

Canessa³

et al. 2006

Electron. J. Biotechnol.

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Recombinant CHO TF70R cells are able to grow and produce t-PA on serum-free medium BIOPRO1 (BioWhitaker Europe, Belgium). The purpose of the present study was to determine the effect of medium supplementation with vitamins, lipids, and specific amino acids on cell growth, t-PA production and biological functionality. Among vitamins, only biotin, folic acid, cobalamine and benzoic acid were required for improving growth and t-PA production. Lipid supplement allowed a significant increase cell concentration and t-PA specific activity and concentration, though its specific production rate decreased slightly. Medium supplementation with proline, serine and asparagine had also positive effects on cell growth. Besides, the addition of asparagine (even in the presence of glutamine) was essential for the production and biological quality of the t-PA. This systematic approach for media supplementation produced an increase in cell concentration around 100% and in t-PA production around 80%, with no detrimental effect on its biological activity. The effect of asparagine on t-PA production was unexpected and needs to be further studied. The above modifications of the production medium did not produce a significant

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Section: Resultssupporting

confidence: 82%

Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production

Altamirano¹,

Illanes²,

Canessa³

et al. 2006

Electron. J. Biotechnol.

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“…These results are indicative of heightened TCA cycle energetics in cell lines expressing anti-apoptotic genes although an exact interpretation of the cellular reactions awaits more detailed metabolic flux analysis (Altamirano et al, 2001;Martinelle et al, 1998). Irrespective of the fate of the consumed lactate, this is the first time it has been demonstrated that apoptotic R cell lines consume accumulated lactate, which in turn, presumably aids in their increased longevity and IVCC.…”

Section: Fate Of the Consumed Lactate And Amino Acid Profile In Apoptmentioning

confidence: 83%

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

Dorai

Kyung

Ellis

et al. 2009

Biotech & Bioengineering

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ABSTRACT: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.

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“…The reduced waste production (such as lactate and ammonium) and osmolality, due to minimal nutrient consumption, also contributed to this (Altamirano et al 2000). The regulation of the glucose feed rate in culture also increased culture longevity and t-PA productivity in CHO without affecting cellular specific productivity suggesting that the combination of carbon and energy source availability and growth rate can be a determinant of t-PA production in continuous culture (Altamirano et al 2001a). This can be used as an alternative application of the biphasic culture method using nutrient shift (Altamirano et al 2001a, b).…”

Section: Media Formulation Approaches For Nutrient Based Cell Prolifementioning

confidence: 94%

Proliferation control strategies to improve productivity and survival during CHO based production culture

2007

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Chinese Hamster Ovary cells are the primary system for the production of recombinant proteins for therapeutic use. Protein productivity is directly proportional to viable biomass, viability and culture longevity of the producer cells and a number of approaches have been taken to optimise these parameters. Cell cycle arrest, particularly in G1 phase, typically using reduced temperature cultivation and nutritional control have been used to enhance productivity in production cultures by prolonging the production phase, but the mechanism by which these approaches work is still not fully understood. In this article, we analyse the public literature on proliferation control approaches as they apply to production cell lines with particular reference to what is known about the mechanisms behind each approach.

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Analysis of CHO Cells Metabolic Redistribution in a Glutamate-Based Defined Medium in Continuous Culture

Cited by 121 publications

References 36 publications

Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production

Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

Proliferation control strategies to improve productivity and survival during CHO based production culture

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