(106b) Optimizing Protein Fractionation with Electrostatics and a pH Gradient

For industrial applications, membrane separations are primarily a sized-based method. To improve these separations and reduce fouling, especially for fluid containing biological molecules such as proteins, we have initiated a concerted effort to modify the surface of poly(ether sulfone) (PES) membranes. Using our patented photo-induced graft polymerization method, we have added a negatively charged graft to a PES membrane. This charged membrane has the ability to separate proteins based on electrostatic considerations, similar to ion exchange chromatography. Using a scaled-down industrial buffer mixer (Asahi Kasai, Glenview, IL), we have conducted membrane filtration experiments under a pH gradient in order to obtain the most effective fractionation for maximizing protein purity and yield. As a proof-of-concept, we have effectively separated lysozyme from RNase A. Both of these proteins have similar molecular weights (14.3 and 13.7 kDa, respectively) and are close in pI (11.3 and 9.6, respectively). This separation is difficult using a membrane purely based on size. However, we have demonstrated that separating the proteins at pH 10 using a negatively charged membrane, gives excellent fractionation performance. We believe that these results demonstrate the first use of a pH gradient for the optimization of a membrane separation process. It also demonstrates that charged membranes and precise pH control can be used to perform precise membrane separations. This has implications for the fast and cost effective separation of IgG isoforms in commercialized therapeutics using membranes and pH control. We plan to test this next.