Hemoglobin-BSA separation and purification by internally staged ultrafiltration

Separation and purification of a binary protein mixture having close molecular weights was studied using ultrafiltration (UF) membranes. The binary system of hemoglobin (Hb; MW 64.7 kDa; isoelectric point (pI) 6.8) and bovine serum albumin (BSA; MW 66.4 kDa; pI 4.7) having a molecular weight ratio (MWR) of only 1.03 was studied. It has been demonstrated that the internally staged ultrafiltration (ISUF) technique, which used a stack of three UF membranes without any gaskets/spacers in-between, can yield nearly pure Hb in the permeate from its mixture with BSA in an Amicon® stirred cell. Further, the highest purity of BSA, 99 %, was achieved at a pH 7.8 at 10.66 diavolume. This study achieved these results by avoiding membrane fouling by adjusting the pH of the buffer/solution close to 7.8 and operating at a low applied pressure difference of 10.34 kPa. When pH is at around 7.8, BSA is much more negatively charged than Hb; so BSA was much more strongly repelled by the negatively charged UF membranes. A slight net negative charge on the Hb will affect the permeability of Hb to a small extent through the negatively charged UF membrane. However, this can considerably reduce the attraction between BSA and Hb, avoid higher protein agglomeration and alleviate membrane fouling. Low membrane fouling improved UF membrane performance; the recovery of Hb, retention of BSA, and purification of Hb and BSA show good results. Besides, the reusability of UF membranes by in situ cleaning continued to show good performance even after undergoing regeneration 7 times. The results suggest ISUF technique may be used in practical systems for separating and purifying protein mixtures.