Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO₂ or pH

Accumulation of CO₂ in animal cell cultures can be a significant problem during scale-up and production of recombinant glycoprotein biopharmaceuticals. By examining the cell-surface polysialic acid (PSA) content, we show that elevated CO₂ partial pressure (pCO₂) can alter protein glycosylation. PSA is a high-molecular-weight polymer attached to several complex N-linked oligosaccharides on the neural cell adhesion molecule (NCAM), so that small changes in either core glycosylation or in polysialylation are amplified and easily measured. Flowcytometric analysis revealed that PSA levels on Chinese hamster ovary (CHO) cells decrease with increasing pCO₂ in a dose-dependent manner, independent of any change in NCAM content. The results are highly pH-dependent, with a greater decrease in PSA at higher pH. By manipulating medium pH and pCO₂, we showed that decreases in PSA correlate well with bicarbonate concentration ([HCO₃/^-]). In fact, it was possible to offset a 60% decrease in PSA content at 120 mm Hg pCO₂ by decreasing the pH from 7.3 to 6.9, such that [HCO₃/^-] was lowered to that of control (38 mm Hg pCO₂). When the increase in osmolality associated with elevated [HCO₃/^-] was offset by decreasing the basal medium [NaCl], elevated [HCO₃/^-] still caused a decrease in PSA, although less extensive than without osmolality control. By increasing [NaCl], we show that hyperosmolality alone decreases PSA content, but to a lesser extent than for the same osmolality increase due to elevated [NaHCO₃]. In conclusion, we demonstrate the importance of pH and pCO₂ interactions, and show that [HCO₃/^-] and osmolality can account for the observed changes in PSA content over a wide range of pH and pCO₂ values.