Solution to the high dissolved CO2 problem in high-density perfusion culture of mammalian cells

High dissolved CO2 concentration (DCO2) is a major problem causing growth inhibition, metabolic changes, and poor productivity in mammalian cell culture. DCO2 often accumulates up to 200-250 mmHg in perfusion bioreactors, while the optimal physiological range is typically 40-50 mmHg. Although the problem with CO2 inhibition is well recognized, this critical parameter is generally not systematically controlled in bioreactor systems. We present an efficient (yet simple) DCO2 control method independent of other bioreactor culture parameters, such as pH, dissolved oxygen, temperature, etc. The method involves two key elements: 1) a novel protein-free medium formulation, and 2) a specific reactor operation strategy. The technology was successfully implemented in the high-density perfusion cultures of BHK cells and tested at development and production scale with campaigns lasting 3-6 months. An optimal physiological level of DCO2 of 40-50 mmHg was consistently maintained even at high cell concentrations up to 20e06 cells/mL using either a membrane tubing or a direct gas sparging for oxygenation. Both oxygenation methods inadequately strip CO2 from the culture and result in accumulated DCO2 levels UP to 200 mmHg, but dropped to the targeted optimal level of 50 mmHg after implementation of the proposed technology. The major advantage of this DCO2 reduction control was the significant increase in cell growth rate (similar to30%) and specific production rate (similar to30%) observed in BHK cell line producing different recombinant proteins. This proposed DCO2 control method is applicable to a wide variety of cell culture processes, such as perfusion, fed-batch and batch, with a particular advantage in high cell density culture.