CO2 in large-scale and high-density CHO cell perfusion culture

Productivity in a CHO perfusion culture reactor was maximized when pCO(2) was maintained in the range of 30-76 mm Hg. Higher levels of pCO(2) (>105 mm Hg) resulted in CHO cell growth inhibition and dramatic reduction in productivity. We measured the oxygen utilization and CO2 production rates for CHO cells in perfusion culture at 5.55 x 10(-17) mol cell(-1) sec(-1) and 5.36 x 10(-17) mel cell(-1) sec(-1) respectively. A simple method to directly measure the mass transfer coefficients for oxygen and carbon dioxide was also developed. For a 500 L bioreactor using pure oxygen sparge at 0.002 VVM from a microporous frit sparger, the overall apparent transfer rates (k(L)a+k(A)A) for oxygen and carbon dioxide were 0.07264 min(-1) and 0.002962 min(-1) respectively. Thus, while a very low flow rate of pure oxygen microbubbles would be adequate to meet oxygen supply requirements for up to 2.1 x 10(7) cells/mL, the low CO2 removal efficiency would limit culture density to only 2.4 x 10(6) cells/mL. An additional model was developed to predict the effect of bubble size on oxygen and CO2 transfer rates. If pure oxygen is used in both the headspace and sparge, then the sparging rate can be minimized by the use of bubbles in the size range of 2-3 mm. For bubbles in this size range, the ratio of oxygen supply to carbon dioxide removal rates is matched to the ratio of metabolic oxygen utilization and carbon dioxide generation rates. Using this strategy in the 500 L reactor, we predict that dissolved oxygen and CO2 levels can be maintained in the range to support maximum productivity (40% DO, 76 mm Hg pCO(2)) for a culture at 10(7) cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.