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

Productivity in a CHO perfusion culture reactor was maximized when pCO2 was maintained in the range of 30–76 mm Hg. Higher levels of pCO2 (> 150 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 5.55×10-17 mol cell-1 sec-1 and 5.36×10-17 mol 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 (kLa+kAA) 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 2.1×107 cells/mL, the low CO2 removal efficiency would limit culture density to only 2.4×106 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 pCO2) for a culture at 107 cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.A = volumetric agitated gas-liquid interfacial area at the top of the liquid, 1/mB = cell broth bleeding rate from the vessel, L/minCER = carbon dioxide evolution rate in the bioreactor, mol/min[CO2] = dissolved CO2 concentration in liquid, M[CO2]* = CO2 concentration in equilibrium with sparger gas, M[CO2]** = CO2 concentration in equilibrium with headspace gas, MCO2(1) = dissolved carbon dioxide molecule in water[CT] = total carbonic species concentration in bioreactor medium, M[CT]F = total carbonic species concentration in feed medium, MD = bioreactor diameter, mDI = impeller diameter, mDb = the initial delivered bubble diameter, mF = fresh medium feeding rate, L/minHL = liquid height in the vessel, mkA = carbon dioxide transfer coefficient at liquid surface, m/minkinfAsupO= oxygen transfer coefficient at liquid surface, m/min