Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO(2) or pH.
Accumulation of CO(2) 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 CO(2) partial pressure (pCO(2)) 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. Flow-cytometric analysis revealed that PSA levels on Chinese hamster ovary (CHO) cells decrease with increasing pCO(2) 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 pCO(2), we showed that decreases in PSA correlate well with bicarbonate concentration ([HCO(3)(-)]). In fact, it was possible to offset a 60% decrease in PSA content at 120 mm Hg pCO(2) by decreasing the pH from 7.3 to 6.9, such that [HCO(3)(-)] was lowered to that of control (38 mm Hg pCO(2)). When the increase in osmolality associated with elevated [HCO(3)(-)] was offset by decreasing the basal medium [NaCl], elevated [HCO(3)(-)] 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 [NaHCO(3)]. In conclusion, we demonstrate the importance of pH and pCO(2) interactions, and show that [HCO(3)(-)] and osmolality can account for the observed changes in PSA content over a wide range of pH and pCO(2) values.
Zanghi
J A
JA
Department of Chemical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3120, USA.
Schmelzer
A E
AE
Mendoza
T P
TP
Knop
R H
RH
Miller
W M
WM
D000818:Animals; D001639:Bicarbonates / Q000032:analysis*; D016466:CHO Cells; D002245:Carbon Dioxide / Q000032:analysis*; D006224:Cricetinae; D003470:Culture Media / Q000737:chemistry; D005434:Flow Cytometry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000737:chemistry*; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry*; D009994:Osmolar Concentration; D012794:Sialic Acids / Q000737:chemistry*
D001639:Bicarbonates; D003470:Culture Media; D008562:Membrane Glycoproteins; D019006:Neural Cell Adhesion Molecules; D012794:Sialic Acids; C021319:polysialic acid; D002245:Carbon Dioxide