Binary protein mixture separation and purification by a cassette having an internally staged ultrafiltration membrane stack

Recently, a mixture of hemoglobin (Hb) and bovine serum albumin (BSA) was separated into two highly purified fractions with high recovery via internally staged ultrafiltration (ISUF) having a stack of three identical flat 100 kDa ultrafiltration (UF) membranes in a stirred cell. Selectivities as high as 1000–4000+, and high recoveries of individual species were achieved. Successful separation and purification of IgG from a BSA-IgG mixture was also achieved in a stirred cell using a modified ISUF technique with two 100 kDa membranes on a 70 kDa membrane; here BSA was a model for host cell proteins (HCPs) in post-protein A eluate. We explored here separation and purification of the Hb-BSA system in a developmental cassette of 88 cm 2 membrane surface area, having a stack of three 100 kDa membranes on each side of the channel. The channel gap was smaller than that in regular cassettes resulting in higher flow pressure drops. The Hb-BSA separation was studied over a diavolume (DV) range of ∼0–6. The Hb-BSA selectivity was as high as 1600 and remained above 300 till 6 DV. Lower feed pressure and a particular permeate collection mode leading to reduced transmembrane pressure drop were crucial for higher performance. Species recoveries were lower for the DV range used especially compared to those in a stirred cell. Successful separation and purification of BSA-IgG system was also demonstrated with similar limitations. Increased flow pressure drop due to flow channel height reduction in existing cassette frames and lower flow rates leading to higher concentration polarization suggest modification of the cassette frame for high performance ISUF for difficult-to-separate protein mixtures in biopharmaceutical separation processes. • Internally staged ultrafiltration (ISUF) separates difficult binary protein mixtures. • Each channel side in a scaled-up ISUF cassette had a three 100 kDa membrane stack. • Cassette selectivity of Hb-BSA was a high of 1600 and remained >300 till 6 diavolume. • A modified ISUF stack can yield very high cassette performance for BSA-IgG system. • Larger channel gap to reduce flow pressure drop will enhance system performances.