(570d) High Purification of Binary Protein Mixtures Having Close Molecular Weights By Ultrafiltration

Mixtures of proteins/biopharmaceuticals having close molecular weight ratios (MWRs) are generally separated by chromatography. Conventional ultrafiltration (UF) can separate binary protein mixtures if the MWR of the two proteins is at least ~7–10. For lower MWRs, novel countercurrent cascades of 3-5 stages can yield relatively pure proteins. To separate similarly sized proteins in one device, High Performance Tangential Flow Filtration (HPTFF) technique was developed. HPTFF achieves high selectivity but does not yield almost pure protein in the permeate. The Internally Staged Ultrafiltration (ISUF) technique was proposed to yield almost pure protein in the permeate. The ISUF technique uses a stack of 3 UF membranes without any gaskets/spacers in-between. Permeate from membrane 1 with selective skin facing the feed mixture becomes the feed to the next membrane 2 located below and so on. We have investigated separations of a number of binary protein mixtures using the ISUF configuration in a stirred cell: (1) hemoglobin (MW 64,677 Da) and BSA (MW 66,430 Da) having a MWR of 1.03; (2) α-lactalbumin (14,400, MW) and β-lactoglobulin (18,400, MW; present naturally as a dimer, 36,800) mixture having a MWR of 2.55 and encountered in whey protein concentrate separation. In each case, we have investigated the effect of pH, ionic strength and feed pressure using as required 100kDa or 30kDa MWCO Ultracel® and Omega® membranes having a particular charge. We have investigated the effect of diavolumes on the purity of the retained protein as well as other performance indicators. We have also investigated the purification of a protein from a virus using a stacked set of UF membranes. Virus filtration for validation of virus clearance is difficult when the size of the mAb approaches that of small virus particles.