Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Abstract

CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well-known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50-62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early-temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO(2) with this product showed that the Neu5Gc levels were 46% lower at high pCO(2) conditions (140 mmHg) compared to moderate pCO(2) levels (20-80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics.

The combined effect of sodium butyrate and low culture temperature on the production, sialylation, and biological activity of an antibody produced in CHO cells

“…Thus, understanding factors that affect Neu5Gc in manufacturing is of significant importance when assessing the equivalency of different cell culture parameters. Our results of lower Neu5Gc content in the culture with NaBu addition are similar to those of Li et al [37]. Furthermore, the simultaneous application of NaBu addition and low culture temperature further decreased the Neu5Gc level, which would not be expected to lead to an increase in immunogenicity.…”

Culture temperature modulates monoclonal antibody charge variation distribution in Chinese hamster ovary cell cultures

“…To achieve robust cell culture process for mAbs with high quantity and quality, the effects of various process parameters and media components on cell growth, mAb production and product quality have been extensively studied (Aghamohseni et al 2014;Borys et al 2010).…”

Glycosylation in Cell Culture

“…As our understanding of specific affects of culture parameters improves, manipulation of culture variables by the modification of several factors and media additives can be used to tailor the glycosylation of the product. A recent example of this is the use of several culture parameters (sodium butyrate addition, reduced culture temperature, high pCO2 and use of sodium carbonate instead of sodium hydroxide for pH control) to reduce the amount of Neu5Gc incorporated into a recombinant protein produced in CHO cells (Borys et al 2010). Cellular responses are often unique to the cell line and recombinant protein, and therefore culture conditions and parameters are not necessarily transferable for similar glycosylation patterns.…”