Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37°C until viable cell concentration reached 1 × 107 cells/mL, and then culture temperature was shifted to 25°C, 28°C, 30°C, 32°C, 37°C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37°C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, qEPO, increased at low culture temperature and were the highest at 32°C and 30°C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32°C was not as high as the cumulative EPO production at 32°C although the qEPO at culture temperature below 32°C was comparable or even higher than the qEPO at 32°C. This implies that the beneficial effect of lowering culture temperature below 32°C on qEPO is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32°C in regard to acidic isoforms, antennary structures and sialylated N‐linked glycans was comparable to that at 37°C. However, at culture temperatures below 32°C, the proportions of acidic isoforms, tetra‐antennary structures and tetra‐sialylated N‐linked glycans were further reduced, suggesting that lowering culture temperature below 32°C negatively affect the quality of EPO. Thus, taken together, cell culture at 32°C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32°C was not deteriorated as obtained at 37°C. Biotechnol. Bioeng. 2008;101: 1234–1244. © 2008 Wiley Periodicals, Inc.