Effects of elevated osmolality and carbon dioxide on the glycosylation of neural cell adhesion molecule and monoclonal antibody: Osmoprotectant compounds as mitigating agents

Mammalian cells are important for the production of complex, recombinant glycoproteins as diagnostic and therapeutic molecules. The protein of interest must be properly glycosylated, in addition to being produced at a high titer. Carbon dioxide (CO₂), a major metabolic waste product, can build up in high-density cultures with mass transfer limitations. Elevated bicarbonate concentration ([$HCO-3$]) under elevated pCO₂ (hypercapnia), as well as the increase in [Na+] due to base addition to control pH, causes a concomitant increase in medium osmolality. Using freezing point and vapor pressure osmometers, this increase in osmolality was found to be irreversible, even after extensive degassing.; The glycosylation of neural cell adhesion molecule (NCAM) and an IgG _2a antibody produced under hypercapnia (to 250 mm Hg pCO₂) and/or osmolality (to 530 mOsm/kg) was analyzed. CHO cell NCAM polysialylation (PSA), determined by flow cytometry, decreased with pCO₂. PSA content correlated well with [$HCO-3$], rather than pCO₂, per se—indicating a synergy between CO₂ and pH on PSA content. The role of the concurrent increase in osmolality was evaluated by (1) removal of NaCl from the basal medium to offset the increase osmolality at elevated pCO₂ and (2) supplementation of basal medium with NaCl at control PCO₂. Elevated [NaCl] decreased NCAM PSA in a dose-dependent manner, reaching 20% of control levels at 530 mOsm/kg. Osmotic compensation of hypercapnic medium mitigated most, but not all, of the decrease in PSA content, indicating that CO₂ per se may play a minor role in altering PSA content. Addition of 20 mM glycine betaine, an osmoprotectant compound, partially mitigated the decrease in PSA at >435 mOsm/kg or 250 mm Hg pCO₂.; IgG_2a glycosylation by hybridoma cells was robust under hypercapnia and/or hyperosmolality. Mean isoelectric point (pI) increased with osmolality and decreased with pCO₂, although there were medium-specific differences. Changes in pI were reflected in those of secreted β-galactosidase, an indicator of organellar pH changes. IgG_2a galactose content decreased with osmolality and increased with pCO₂, while mannose content decreased and fucose content remained unchanged for all conditions evaluated.