Characterization of hybridoma cell responses to elevated pCO₂ and osmolality: Intracellular pH, cell size, apoptosis, and metabolism

CO₂ partial pressure (pCO₂) in industrial cell culture reactors may reach 150-200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. The inhibitory effects of elevated pCO₂ at constant pH are due to a combination of the increases in pCO₂ and [HCO₃^-], per se, and the associated increase in osmolality. To decouple the effects of pCO₂ and osmolality, low-salt basal media have been used to compensate for this associated increase in osmolality. Under control conditions (40 mm Hg-320 mOsm/kg), hybridoma cell growth and metabolism was similar in DMEM:F12 with 2% fetal bovine serum and serum-free HB GRO. In both media, pCO₂ and osmolality made dose-dependent contributions to the inhibition of hybridoma cell growth and synergized to more extensively inhibit growth when combined. Elevated osmolality was associated with increased apoptosis. In contrast, elevated pCO₂ did not increase apoptotic cell death. Specific antibody production also increased with osmolality although not with pCO₂. In an effort to understand the mechanisms through which elevated pCO₂ and osmolality affect hybridoma cells, glucose metabolism, glutamine metabolism, intracellular pH (pHi), and cell size were monitored in batch cultures. Elevated pCO₂ (with or without osmolality compensation) inhibited glycolysis in a dose-dependent fashion in both media. Osmolality had little effect on glycolysis. On the other hand, elevated pCO₂ alone had no effect on glutamine metabolism, whereas elevated osmolality increased glutamine uptake. Hybridoma mean pHi was ∼0.2 pH units lower than control at 140 mm Hg pCO₂ (with or without osmolality compensation) but further increases in pCO₂ did not further decrease pHi. Osmolality had little effect on pHi. Cell size was smaller than control at elevated pCO₂ at 320 mOsm/kg, and greater than control in hyperosmotic conditions at 40 mm Hg.