Temperature Reduction in Cultures of hGM‐CSF‐expressing CHO Cells: Effect on Productivity and Product Quality

We have demonstrated that temperature reduction from 37 to 33 °C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO‐K1‐hGM‐CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO‐K1‐hGM‐CSF cells were cultured in a biphasic mode: first, a 37 °C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 °C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 °C. Furthermore, the total rhGM‐CSF production increased 6 times in the culture shifted to 33 °C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N‐ and O‐glycosylation of the protein produced under both cell culture conditions using high‐pH anion‐exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM‐CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO‐K1‐hGM‐CSF cells without affecting product quality, representing a novel procedure for the rhGM‐CSF production process.