Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium

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

“…Neither the apoptotic R nor the control cell lines exhibited sustained growth in the complete absence of glucose, that is, in the presence of lactate only (data not shown). Indeed, similar observations were made by Altamirano et al (2004Altamirano et al ( , 2006, where the authors showed that CHO cells were incapable of sustained growth in medium containing lactate as the sole carbon source. When both cell lines were cultured in the same custom-formulated medium containing ''high'' (60 mM) concentrations of glucose, EAX197 exhibited a higher VCD, viability and IVCC ( Figure 9A-C) than the control cell line especially at later times in the cell culture.…”

“…Numerous strategies have been devised to address the accumulation of excessive lactate build-up including (1) maintaining low medium glucose concentrations (Kurokawa et al, 1994;Xie and Wang, 1993;Zhang et al, 2004;Zhou et al, 1995), (2) feeding alternative sugars, including fructose (Martinelle et al, 1998, Altamirano et al, 2004Wlaschin and Hu, 2007), (3) partially knocking out lactate dehydrogenase (LDH) expression by homologous recombination or siRNA technology (Chen et al, 2001;Kim and Lee, 2007a); (4) over-expression of pyruvate carboxylase (Kim and Lee, 2007b); (5) use of dichloracetate (DCA), a pyruvate dehydrogenase (PDH) activator (via PDH kinase inhibition) (Stacpoole et al, 2003) and (6) oxamic acid, an LDH competitive inhibitor (Mothersill and Seymour, 1986). While the above strategies have been partially successful in reducing the lactate concentration, an alternative approach to consider is stimulating mitochondrial respiration in order to enhance culture performance.…”

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

“…The use of a nutritional shift from glucose during the growth phase to galactose for production phase after the third day of batch culture and 12th day of perfusion culture resulted in slow growth rate with enhanced viability and increased t-PA production when applied to CHO (Altamirano et al 2001b). Replacement of glutamine by glutamate and alternatively changing the carbon source from glucose to galactose in the production phase also resulted in an increase in culture longevity, viability and 1.3-fold improved volumetric t-PA production (Altamirano et al 2004). This improvement in volumetric productivity was not due to any changes in specific productivity (Altamirano et al 2004).…”

“…Replacement of glutamine by glutamate and alternatively changing the carbon source from glucose to galactose in the production phase also resulted in an increase in culture longevity, viability and 1.3-fold improved volumetric t-PA production (Altamirano et al 2004). This improvement in volumetric productivity was not due to any changes in specific productivity (Altamirano et al 2004). Recently improved growth and t-PA productivity has been achieved by growing CHO cells in the presence of glutamate (6 mM) with 5 mM glucose and 20 mM galactose compared to a standard culture containing 6 mM glutamate and 20 mM glucose (Altamirano et al 2006).…”

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

“…Such strategies have proven effective at reducing the overall yield of lactate on glucose, but require on-line monitoring of nutrient concentrations and automatic feed rate adjustment during the culture. Metabolic waste formation was also reduced through modifications of the medium composition, such as substituting glucose with galactose (Altamirano et al, 2000(Altamirano et al, , 2004o rp y r u v a t e ( Genzel et al, 2005), glutamate (Hassell and Butler, 1990)o ra s p a r a g i n e ( Kurano et al, 1990) for glutamine, or through the addition of TCA intermediates in the culture medium (Omasa et al, 2009 lactate or ammonia accumulation, these changes may also negatively impact on the growth and/or productivity of the cells. Finally, various separation techniques have also been explored for the in-situ selective removal of lactate and ammonia from the culture broth (Brose and van Eikeren, 1990;Chang et al, 1995;Nayve et al, 1991), but have not yet found widespread application.…”