The prominence of insect cell culture has grown rapidly due to its ability to produce baculovirus biopesticides and recombinant proteins using the Baculovirus Expression Vector System. A critical problem in the mass production of these products is CO₂ accumulation to inhibitory levels within the bioreactor. The current research investigated the effect of elevated CO₂ concentrations on insect cell growth and metabolism and the roles of oxidative stress and intracellular pH (pH_i) in CO₂ inhibition.

Spodoptera frugiperda Sf-9 insect cells were cultured in a 3 L bioreactor (1.2 L working volume) controlled at 20% air saturation, 27°C and a pH of 6.2. The cells were exposed to a constant CO₂ concentration by purging the medium with CO₂ and the headspace with air. The experiments were repeated for different CO₂ concentrations and samples were taken every 24 h to determine cell density, viability, metabolism and oxidative stress.

The population doubling time (PDT) of Sf-9 cells increased with increasing CO₂ concentration. Specifically, the PDT for 0-37, 73, 147, 183 and 220 mm Hg CO₂ concentrations were 23.2 ± 6.7, 32.4 ± 7.2, 38.1 ± 13.3, 42.9 ± 5.4 and 69.3 ± 35.9 h (n = 3 or 4; 95% confidence level), respectively. An 80 mL working volume shaker flask was maintained as a control and had an average PDT of 24.9 ± 3.1 h (n = 7; 95% confidence level). The viability of cells in all experiments was above 90%. The osmolality for all bioreactor experiments was observed to be 300 to 360 mOsm/kg, a range that is known to have a negligible effect on insect cell culture. Elevated CO₂ concentration did not alter the cell specific glucose consumption rate (2.5 to 3.2 x 10^-17 mol/cell-s), but slightly increased the specific lactate production rate from -3.0 x 10^-19 mol/cell-s to 10.2 x 10^-19 mol/cell-s. Oxidative stress did not contribute to CO₂ inhibition in uninfected Sf-9 cells as no significant increase in the levels of lipid hydroperoxide and protein carbonyl concentrations was discovered at elevated CO₂ concentration. The experimental method used to establish the calibration curve for determining pH_i was well documented. Further investigations are required to determine the role of pH_i in CO₂ inhibition.