Active hypothermic growth : a novel means for increasing total recombinant protein production by CHO cells

Abstract

Recombinant human glycoproteins produced by Chinese Hamster Ovary (CHO) cells are an important class of therapeutic molecules and investigating means of improving the production rate and product quality of these glycoproteins is therefore of great interest. Culturing CHO cells under mild hypothermia (30-33 ⁰C) leads to growth arrest in the G₀/G₁ phase of the cell cycle and, in some cases, causes an increase in specific productivity of recombinant protein, as was shown here for the model CHO cell line producing human interferon-gamma (IFN-[gamma]). Controlled proliferation, achieved by inducing growth arrest in the G₀/G₁ phase by chemical, environmental or genetic means, is commonly used to increase CHO specific productivity and thus there is speculation that enhanced hypothermic productivity is due to growth arrest. However, it was proven here that the positive effect of hypothermia on recombinant protein production is due to elevated IFN-[gamma] mRNA levels instead. At both 32 ⁰C and 37 ⁰C, specific productivity is growth-associated, increasing as the percentage of cells in the S phase increased, demonstrating that a cell line can be both a growth-associated producer and have enhanced productivity under hypothermic conditions. It was hypothesized that the best production platform would be cells actively growing at low temperature and this was proven to be the case using two different methods, namely growth factor supplementation and selection of cells capable of hypothermic growth. Both methods gave multi-fold increases in total IFN-y production compared to the 32 ⁰C and 37 ⁰C controls, thereby validating the novel culture strategy of active hypothermic growth.

(cont.) Cells capable of achieving significant hypothermic growth were also isolated for the non-recombinant CHO-KI cell line and are now available for the future production of any recombinant protein. Glycoprotein quality is partially assessed by the level of glycosylation and IFN-[gamma] contains two potential N-linked glycosylation sites. This thesis gives the first report of a detrimental effect of hypothermic culture on glycosylation, showing a 4-5% decrease in the end-of-batch percentage of 2-sites occupied glycoforms relative to the 37 ⁰C control. However, this negative effect is completely eliminated by culturing under perfusion conditions.