High-level production of human thrombopoietin by anti-apoptotic CHO cells in serum-free suspension culture

Abstract

For the high-level production of human thrombopoietin (hTPO) in serum-free suspension culture of recombinant Chinese hamster ovary (rCHO) cells, the medium was optimized by the use of medium additives and CHO cells were engineered by the regulation of gene expression. The performance of a serum-free medium was enhanced by yeast hydrolysate addition. Sodium butyrate (NaBu) addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced a(2→3) sialylation, and decreased in vivo biological activity. To overcome detrimental effects of NaBu on apoptotic cell death and hTPO quality, I overexpressed Bcl-2 protein or downregulated caspases in rCHO cells producing hTPO. Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bcl2-14 cells) and 3 mM NaBu addition.