High-density mammalian cell cultures in stirred-tank bioreactor without external pH control

• Interactions between CO2, lactate, and pH were leveraged to maintain culture pH.
• No external pH control required in both fed-batch and perfusion cultures in stirred-tank bioreactors.
• Cell specific perfusion rate at as low as 15 ± 2 pL/cell/day was achieved in perfusion cultures.
• High bioreactor productivity at 1.9 ± 0.1 g/L/day was achieved in perfusion cultures.

Maintaining desired pH is a necessity for optimal cell growth and protein production. It is typically achieved through a two-sided pH control loop on the bioreactor controller. Here we investigated cell culture processes with minimum or no pH control and demonstrated that high-density mammalian cell cultures could be maintained for long-term protein production without pH control. The intrinsic interactions between pCO2, lactate, and pH were leveraged to maintain culture pH. Fed-batch cultures at the same lower pH limit of 6.75 but different upper pH limits (7.05, 7.30, 7.45, 7.65) were evaluated in the 3 L bioreactors and comparable results were obtained. Neither CO2 sparging nor base addition was required to control pH in the pH range of 6.75–7.65. The impact of sparger configurations (drilled hole sparger vs. frit sparger) and scales (3 L vs. 200 L) on CO2 accumulation and culture pH was also demonstrated. The same principle was applied in two perfusion cultures with steady state cell densities at 42.5 ± 3.3 or 68.3 ± 6.0 × 106 cells/mL with low cell specific perfusion rates (15 ± 2 to 23 ± 3 pL/cell/day), achieving up to 1.9 ± 0.1 g/L/day bioreactor productivity. Culture pH level in the 3 L perfusion bioreactors was steadily maintained by controlling the residual lactate and pCO2 levels without the requirement of external pH control for up to 40 days with consistent productivity and product quality. Furthermore, culture pH could be potentially modulated via adjusting residual glucose levels and CO2 stripping capability in perfusion cultures. To the best of our knowledge, this is the first time a systematic study was performed to evaluate the long-term cell cultivation and protein production in stirred-tank bioreactors without external pH control.