One of the key parameters in perfusion culture is the rate of medium replacement (D). Intensifying D results in enhanced provision of nutrients, which can lead to an increase in the viable cell density (Xv). The daily MAb production of hybridoma cells can thus be increased proportionally without modifying the bioreactor scale, provided that both viable cell yield per perfusion rate (YXv/D) and specific MAb productivity (qMAb) 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 operated 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™/vortex-flow filter system, significant reductions in YXv/D and qMAb resulting from the use of gas sparging were observed at D > 1.57 vvd (Xv > 15 × 106 cells/mL). Through glucose supplementation, we have shown that the decrease in YXv/D encountered in presence of sparging was not resulting from increased cellular destruction or reduced cell growth, but rather from glucose limitation. Thus, increases in hydrodynamic shear stress imparted to the culture via intensification of gas sparging resulted in a gradual increase in specific glucose consumption (qglc) and lactate production rates (qlac), while no variations were observed in glutamine-consumption rates. As a result, while glutamine was the sole limiting-nutrient under non-sparging conditions, both glutamine and glucose became limiting under sparging conditions. Although a reduction in qMAb was observed at high-sparging rates, inhibition of MAb synthesis did not result from direct impact of bubbles, but was rather associated with elevated lactate levels (25–30 mM), resulting from shear stress-induced increases in qlac, qglc, and Ylac/glc. Deleterious effects of sparging on YXv/D and qMAb encountered in the Celligen™/vortex-flow filter system were eliminated in the sparging-free low-shear environment of the Chemap-HRI/ultrasonic filter system, allowing for the maintenance of up to 37 × 106 viable cells/mL. A strategy aimed at reducing requirements for sparging in large-scale perfusion cultures by way of a reduction in the oxygen demand using cellular engineering is discussed. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 435–450, 2000.