We have demonstrated that temperature reduction from 37 to 33degreesC in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO-Kl-hGM-CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO-Kl-hGM-CSF cells were cultured in a hiphasic mode: first, a 37degreesC growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33degreesC. 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 37degreesC. Furthermore, the total rhGM-CSF production increased 6 times in the culture shifted to 33degreesC. 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.