Effects of environmental factors on recombinant protein production in Chinese Hamster Ovary cells

Abstract

For the optimization of recombinant protein production in Chinese hamster ovary (CHO) cells, the effects of various environmental factors such as temperature, pH on cell growth and protein quality were systematically investigated using CHO cell lines producing erythropoietin (EPO), humanized anti-4-1BB antibody, therapeutic antibody and follicle stimulating factor (FSH). To determine the effect of low culture temperature on erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells, rCHO cells producing EPO (LGE10-9-27) were cultivated at 30, 33, and 37℃, respectively. At a culture temperature lower than 37℃, cell growth was suppressed, but cell viability remained high for a longer culture period. When the culture temperature was lowered from 37℃ to 33℃, more than 2.5-fold increase in the maximum EPO concentration was achieved. This enhanced EPO production at 33℃ was not just because of the extended culture longevity with the decreased release of proteolytic enzymes from dead cells, but mainly because of enhanced specific EPO productivity ($q_{EPO}$). The $q_{EPO}$ at 33℃ was $0.35 ± 0.08 μg/10^6$ cells/hr, which was approximately 4-fold higher than that at $37℃. Although the highest $q_{EPO}$ of $0.49 ± 0.14 μg/10^6$ cells/hr was obtained at 30℃, the maximum EPO concentration was lowest because the detrimental effect of lowering culture temperature on cell growth outweighed its beneficial effect on $q_{EPO}$. Like $q_{EPO}$, the relative EPO mRNA content increased by lowering culture temperature, indicating that the increased transcription level of EPO was responsible in part for the enhanced $q_{EPO}$ at low culture temperature. The quality of EPO produced at 33℃ in regard to isoform pattern, sialic acid content and in vivo biological activity was comparable to or even better than that produced at 37℃. Taken together, the results obtained here demonstrate the potential of the application of the low culture temperature to the commerc...