Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates

One of the key parameters in perfusion culture is the rate of medium replac ement (D). Intensifying D results in enhanced provision of nutrients, which can lead to an increase in the viable cell density (X-v). The daily MAb pr oduction of hybridoma cells can thus be increased proportionally without mo difying the bioreactor scale, provided that both viable cell yield per perf usion rate (Y-Xv/D) and specific MAb productivity (q(MAb)) remain constant at higher D. To identify factors prone to limit productivity in perfusion, a detailed kinetic analysis was carried out on a series of cultures operate d within a D range of 0.48/4.34 vvd (volumes of medium/reactor volume/day) in two different suspension-based systems. In the Celligen(TM)/vortex-flow filter system, significant reductions in Y-Xv/D and q(MAb) resulting from t he use of gas sparging were observed at D > 1.57 vvd (X-v > 15 x 10(6) cell s/mL). Through glucose supplementation, we have shown that the decrease in Y-Xv/D encountered in presence of sparging was not resulting from increased cellular destruction or reduced cell growth, but rather from glucose limit ation. Thus, increases in hydrodynamic shear stress imparted to the culture via intensification of gas sparging resulted in a gradual increase in spec ific glucose consumption (q(glc)) and lactate production rates (q(lac)), wh ile no variations were observed in glutamine-consumption rates. As a result , while glutamine was the sole limiting-nutrient under non-sparging conditi ons, both glutamine and glucose became limiting under sparging conditions. Although a reduction in q(MAb) was observed at high-sparging rates, inhibit ion of MAb synthesis did not result from direct impact of bubbles, but was rather associated with elevated lactate levels (25-30 mM), resulting from s hear stress-induced increases in q(lac), q(glc), and Y-lac/glc. Deleterious effects of sparging on Y-Xv/D and q(MAb) encountered in the Celligen(TM)/v ortex-flow filter system were eliminated in the sparging-free low-shear env ironment of the Chemap-HRI/ultrasonic filter system, allowing for the maint enance of up to 37 x 10(6) viable cells/mL. A strategy aimed at reducing re quirements for sparging in large-scale perfusion cultures by way of a reduc tion in the oxygen demand using cellular engineering is discussed. (C) 2000 John Wiley & Sons, Inc.