Hyperosmotic Stress and Elevated pCO2 Alter Monoclonal Antibody Charge Distribution and Monosaccharide Content

Abstract Medium osmolality increases with pCO 2 at constant pH. Elevated pCO 2 and osmolality inhibit hybridoma growth to similar extents in both serum‐containing and serum‐free media. The combination of osmolality and elevated pCO 2 synergizes to negatively impact cell growth. IgG 2a glycosylation by hybridoma cells was evaluated under elevated pCO 2 (to 250 mmHg pCO 2 ) and/or osmolality (to 476 mOsm/kg). IgG 2a site occupancy did not change significantly under any of the conditions studied, which is consistent with the robust glycosylation of other antibodies produced under various environmental stresses. However, changes were observed in the IgG 2a charge distribution. Changes in the isoelectric point (pI) were greater under hyperosmotic stress, increasing by 0.32 and 0.41 pH units at 435 mOsm/kg in serum‐containing and serum‐free medium, respectively. Hyperosmotic stress also resulted in a concomitant increase in the heterogeneity of the charge distribution. The mean pI in serum‐containing medium decreased by 0.16 pH units at 250 mmHg pCO 2 when osmolality was controlled at 320 mOsm/kg but increased by 0.20 pH units when the osmolality increased with pCO 2 (195 mmHg pCO 2 –435 mOsm/kg). In serum‐free medium, elevated pCO 2 did not alter pI, regardless of medium osmolality. In contrast to elevated osmolality at control pCO 2 , elevated pCO 2 did not significantly alter the IgG 2a charge heterogeneity under any of the conditions studied. The IgG 2a was not sialylated, so sialylation changes were not responsible for changes in the charge distribution. IgG 2a galactose content decreased with elevated osmolality, as a result of either elevated NaHCO 3 or NaCl. However, when osmolality was controlled at elevated pCO 2 , the galactose content tended to increase. The mannose content decreased with increasing stress, while the fucose content remained relatively unchanged. It is likely that the observed increases in the pI of murine IgG 2a were due to increased organellar pH, which is reflected by increased specific β‐galactosidase activity in the supernatant.