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 ex- amining 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 oligosac- charides on the neural cell adhesion molecule (NCAM), so that small changes in either core glycosylation or in polysialylation are amplified and easily measured. Flow- cytometric 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 concen- tration ((HCO3 ˛ )). In fact, it was possible to offset a 60% decrease in PSA content at 120 mm Hg pCO2 by decreas- ing 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 conclu- sion, 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. © 1999 John Wiley &