The art & science of micro-sparging in high-density perfusion cultures of animal cells

Sparging micron-sized gas bubbles in animal cell cultures is an effective means of obtaining a large as-liquid interfacial area and thus enabling the high O-2 transfer rate necessary to target high cell densities of 40-60 x 10(6) cells/mL. While the method is widely used, the key characteristics of microsparging are not systematically quantified. These include die size distribution of microbubbles in bioreactors, the long-term effects of microsparging rates on cells, the rate of CO2 accumulation, the effect of process conditions k(L)a, and others. In this work, all these phenomena are analyzed and quantified. A novel in situ laser-imaging technique was employed to measure the distribution of microbubble size in a 15L stirred-tank bioreactor, The effect of spatger pore size, sparging rate, Pluronic F68. antifoam, cell density and culture age on bubble size was determined, Long-term correlation between microsparging rate and cell death was quantified for spargers with 0.5 and 15 mutm sintered steel pores. It was found that sparging above 0.025 vvm using the 0.5 mum sparger was detrimental, while 0.054 vvm was detrimental with the 15 mum sparger. The effect of sparging rate on cell death at the sparger surface proved insignificant. To avoid CO2, inhibition, a novel cascade method for dissolved CO2(DCO2) control was developed. Continuous control of DCO2, DO and pH was achieved by simultaneously adjusting the total microsparging rate and the O-2/N-2/CO2 ratio. Overall, the optimization of microsparging resulted in the development of efficient continuous perfusion processes combining high cell densities of 40-60 x 10(6) cells/ml with high viability.