Impact of sodium butyrate and mild hypothermia on metabolic and physiological behaviour of CHO TF 70R cells

Background: To reduce costs associated with productivity of recombinant proteins in the biopharmaceutical industry, research has been focused on regulatory principals of growth and survival during the production phases of the cell culture. The main strategies involve the regulation of cell proliferation by the modulation of cell cycle control points (G1/S or G2/M) with mild hypothermia and the addition of sodium butyrate (NaBu). In this study, batch culture strategies were evaluated using CHO TF 70R cells producing the recombinant human tissue plasminogen activator (rh-tPA), to observe their individual and combined effect on the cellular physiological state and relevant kinetic parameters. Results: NaBu addition has a negative effect on the mitochondrial membrane potential (ΔΨm), the values of which are remarkably diminished in cultures exposed to this cytotoxic compound. This effect was not reflected in a loss of cell viability. NaBu and mild hypothermic conditions increased the doubling time in the cell cultures, suggesting that these strategies triggered a general slowing of each cell cycle phase in a different way. Finally, the individual and combined effect of NaBu and mild hypothermia produced an increase in the specific rh-tPA productivity in comparison to the control at 37°C without NaBu. Nevertheless, both strategies did not have a synergistic effect on the specific productivity. Conclusions: The combination of NaBu addition and mild hypothermic condition causes an impact on physiological and metabolic state of CHO TF 70R cells, decreasing cell growth rate and improving glucose consumption efficiency. These results therefore provide a promising strategy to increase specific productivity of rh-tPA.

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

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 percent and in t-PA production around 80 percent, 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 effect on the metabolism of the main carbon and energy sources (glucose and glutamine) and the level of by-product formation (lactate and ammonia).