(466b) Tuning Electrostatics to Separate Proteins of Similar Molecular Weight and Isoelectric Point

Membrane filtration is primarily a size-based method, thus the separation of proteins with similar molecular weight and charge remains difficult to accomplish and is often performed with chromatography.  In this study, we customized charged poly(ether sulfone) (PES) membranes and showed that tuning the electrostatic interactions involved we can optimize the separation of two pairs of such proteins:  (i) lysozyme (Lys) and RNase A (MW 14.3 and 13.7 kDa, pI 11.3 and 9.6, respectively) and (ii) bovine serum albumin (BSA) and hemoglobin (Hem) (MW 69 and 67 kDa, pI 4.8 and 6.8, respectively).  The charged coating was covalently grafted to PES membranes using our patented photo-induced graft polymerization method, with which the charge density was controlled.  Then, modified membranes were tested in filtration experiments using a scaled-down industrial buffer mixer (Asahi Kasai Bioprocessing, Glenview, IL).  The system permits operation under tightly controlled pH or conductivity (ionic strength) gradients, in order to screen for the optimal operating conditions that maximize proteins purity and yield.  The effect of TMP was also accounted for.  For the first system, 0.5 mg/ml Lys and 0.15 mg/ml RNase A, using a negative membrane and operating at pH 11, we were able to remove RNase A and collect Lys with a purity of ~96% and selectivity ~11.   Utilizing the same approach, we challenged a second mixture, 0.5 mg/ml BSA and 0.15 mg/ml Hem, with the more difficult task to collect Hem.  The sieving coefficient for BSA was kept ~0.1 and the selectivity was >6.5.  These two examples demonstrate how tight control of the electrostatic interactions (pH and ionic strength) can be used to perform precise membrane separations. It also suggests the possibility to apply membrane filtration, with its advantages of continuous processing, lower cost and shorter operating time over chromatography, in more interesting commercialized therapeutics, like IgG isoforms.