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.

Advances in improving mammalian cells metabolism for recombinant protein production

Background: The production of recombinant proteins for therapeutic use represents a great impact on the biotechnology industry. In this context, established mammalian cell lines, especially CHO cells, have become a standard system for the production of such proteins. Their ability to properly configure and excrete proteins in functional form is an enormous advantage which should be contrasted with their inherent technological limitations. These cell systems exhibit a metabolic behaviour associated with elevated cell proliferation which involves a high consumption of glucose and glutamine, resulting in the rapid depletion of these nutrients in the medium and the accumulation of ammonium and lactate. Both phenomena contribute to the limitation of cell growth, the triggering of apoptotic processes and the loss of quality of the recombinant protein. Results: In this review, the use of alternative substrates and genetic modifications (host cell engineering) are analyzed as tools to overcome those limitations. In general, the results obtained are promising. However, metabolic and physiological phenomena involved in CHO cells are still barely understood. Thus, most of publications are focused on specific modifications rather than giving a systemic perspective. Conclusions: A deeper insight in the integrated understanding of metabolism and cell mechanisms is required in order to define complementary strategies at these two levels, so providing effective means to control nutrients consumption, reduce by-products and increase process productivity.

Simultaneous environmental manipulations in semi-perfusion cultures of CHO cells producing rh-tPA

We evaluated the combined effect of decreasing the temperature to a mild hypothermia range (34 and 31ºC) and switching to a slowly metabolizable carbon source (glucose substituted by galactose) on the growth and production of a recombinant human tissue plasminogen activator (rh-tPA) by Chinese hamster ovary cells in batch and semi-perfusion cultures. In batch cultures using glucose as a carbon source, decreasing the temperature caused a reduction in cell growth and an increase in specific productivity of rh-tPA of 32 percent at 34ºC and 55 percent at 31ºC, compared to cultures at 37ºC. Similar behaviour was observed in cultures at 34ºC using galactose as a carbon source. Nonetheless, at 31ºC, the specific productivity of rh-tPA strongly decreased (about 58 percent) compared to the culture at 37ºC. In semi-perfusion culture, the highest rh-tPA specific productivity was obtained at 34ºC. Similarly, whether a decrease in the temperature is accompanied of the replacement of glucose by galactose, the rh-tPA specific productivity improved about 112 percent over that obtained in semi-perfusion culture carried out at 37ºC with glucose as the carbon source. A semi-perfusion culture strategy was implemented based on the combined effect of the chosen carbon source and low temperatures, which was a useful approach for enhance the specific productivity of the recombinant protein.