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Abstract: This paper discusses the use of a novel tangential-flow, three-stage, countercurrent cascade ultrafiltration configuration for continuous purification of monoclonal antibody Campath-1H from simulated mammalian cell culture supernatant. The main limitation of conventional ultrafiltration based bioseparation, particularly when using a single-stage system is the trade-off between recovery and purity. The aim of this study was to try to improve both purity and recovery of the monoclonal antibody by using the novel cascade ultrafiltration system. Purification experiments were carried out with the three-stage cascade system as well as with single-stage and two-stage versions of the proposed system for comparison. The recovery and purification factor were both significantly higher with the three-stage system. The highest purity obtained using the three-stage cascade system was 85%. The experimental purity and recovery were compared with corresponding values obtained by simulation and found to be in reasonably good agreement. [Copyright &y& Elsevier]

Abstract: Composite ultrafiltration membranes were prepared by applying thin coating of polyvinyl acetate (PVAc) over ceramic support by using a modified dip-coating method. Well-known dip coating method was not found suitable for PVAc, as the polymer solution (N,N-di-methyl acetamide solvent), was not viscous enough for uniform coating. In the modified technique, a secondary solvent (water) was used for the removal of DMAc rather than drying it from the solution. For studying the effect of polymer concentration and dipping time over membrane parameters, twelve different membranes were prepared using four different concentrations (10%, 20%, 30% and 40%) and three dipping time (30s, 60s and 120s). Membranes were characterized with TGA, SEM, air and hydraulic permeability and acid/base treatment. From gas permeability study, the pore sizes were found between 150 and 9nm. The lowest pore size membrane (9nm) was applied for the purification of lysozyme from chicken egg white (CEW) using three different concentrations. pH 11.0 was found as most suitable for the purification using the prepared membrane and over 98% of transmission of LS were found. [Copyright &y& Elsevier]

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]