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Abstract: We discuss a continuous two-stage cascade ultrafiltration bioreactor-separator system for fragmentation of immunoglobulin G (IgG) by pepsin and purification of F(ab′)2 fragment thus generated. F(ab′)2 is frequently preferred to intact IgG in therapeutic applications due to lower immunogenicity and faster onset of action. The 10kDa MWCO membrane of the first stage bioreactor retained pepsin, IgG and F(ab′)2 while allowing degraded Fc sub-fragments through and the 70kDa membrane of the second stage retained both IgG and F(ab′)2 while allowing pepsin through. Due to this differential retention behavior, the pepsin concentration within the first stage was significantly higher than that in the feed. The first bioreactor therefore primarily carried out IgG digestion while the second stage primarily served as a separator for pepsin and F(ab′)2. The two-stage system was first assessed using pure equine IgG as feed. Under optimized feed and sweep flow rates, 97% IgG conversion with 93% pure F(ab′)2 product were obtained. A mathematical model for the bioreactor system was developed and simulated and experimental data were found to be in good agreement. When the bioreactor system was operated with unpurified equine serum as feed, close to 95% IgG conversion was observed. The F(ab′)2 product obtained from serum showed a single band on SDS-PAGE. The results demonstrated the suitability of the two-step cascade membrane bioreactor for production of F(ab′)2 from both IgG and unpurified equine serum. [Copyright &y& Elsevier]