The optimization of a membrane purification step to remove unreacted polysaccharide from a protein-polysaccharide conjugate is described. A key component in this work was the introduction of membrane backpulsing to reduce membrane fouling. The backpulsing parameters of pulse duration, frequency and pressure were optimized to minimize cycle time and buffer usage and to maximize process capacity, while still achieving the required 50-fold purification and greater than 90% step yield. It was determined that optimal performance requires frequent pulses (approximately 1 min(-1)) and short durations (less than 1 s). After implementing the optimal backpulsing conditions, the gradual deterioration in membrane performance which had been observed previously was eliminated and acceptable polysaccharide sieving was maintained throughout the diafiltration. Depending on the polysaccharide serotype, a 3.5-10-fold reduction in buffer usage and a 3-8-fold reduction in cycle time were accomplished. Interestingly, with the introduction of membrane backpulsing, it was observed that decreasing the shear rate at the membrane significantly increased polysaccharide sieving for backpulsed diafiltrations.