Strategies for enhancing therapeutic protein production and sialylation in recombinant CHO cells

Abstract

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cell line for the produc-tion of therapeutic proteins because of the ability of human like glycosylated protein production. Glycosyla-tion which is a critical factor of quality is known to affect the biological activity, efficacy, in vivo half-life, and solubility. In this study, we focused on the sialylation which is one of the N-glycosylation structure can increase the serum half-life by preventing the degradation of terminal glycans and affects thermal stability and solubility. Glycosylation including sialylation was affected by numerous culture parameters such as cell lines, culture modes, and environmental factors. Among them, accumulated ammonia caused by glutamine metabolism significantly reduced the terminal sialylation of therapeutic proteins. It is however well-known the negative effect of ammonia on glycosylation including sialylation, the exact mechanism is still poorly understood. In this study, in order to understand the effect of ammonia on sialylation, glycosylation related mRNA transcript levels were evaluated with NanoString nCounter analysis system. And for overcoming the negative effect of ammonia, TCA cycle intermediates were used as glutamine substitutes. To understand the effects of ammonia on therapeutic protein glycosylation patterns, recombinant CHO cells producing Fc-fusion protein were subjected to 10 mM ammonia. The addition of ammonia to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. Fifty-two N-glycosylation related gene expressions were also assessed by the NanoString nCounter system, which can provide digital readout using custom-designed color-coded probes. Among them, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5-fold compared to the control culture. Among them, expression level of neu1 and neu3 ...