TY - JOUR
T1 - Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO2 or pH
AU - Zanghi, James A.
AU - Schmelzer, Albert E.
AU - Mendoza, Thomas P.
AU - Knop, Richard H.
AU - Miller, William M
PY - 1999/10/20
Y1 - 1999/10/20
N2 - 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.
AB - 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.
KW - Bicarbonate
KW - CO
KW - Carbon dioxide
KW - Cell culture
KW - Chinese hamster ovary
KW - Neural cell adhesion molecule (NCAM)
KW - Osmolality
KW - Polysialylation
KW - Sialic acid
UR - http://www.scopus.com/inward/record.url?scp=0033589369&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033589369&partnerID=8YFLogxK
U2 - 10.1002/(SICI)1097-0290(19991020)65:2<182::AID-BIT8>3.0.CO;2-D
DO - 10.1002/(SICI)1097-0290(19991020)65:2<182::AID-BIT8>3.0.CO;2-D
M3 - Article
C2 - 10458739
AN - SCOPUS:0033589369
SN - 0006-3592
VL - 65
SP - 182
EP - 191
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
IS - 2
ER -