(156d) IgG-HCP Separation and Purification By Internally Staged Ultrafiltration

Purification of IgG from residual host cell proteins (HCPs) in post-Protein A chromatography is important since some HCPs bind with Protein A and elute with the monoclonal antibody (mAb), IgG; removal of HCPs from CHO cell lines is essential. To that end, an advanced separation and purification technique in biopharmaceutical manufacturing, namely, internally staged ultrafiltration (ISUF), is investigated here. This builds on successful results obtained in separating a binary protein mixture of hemoglobin and bovine serum albumin (BSA) into two highly purified fractions by using ISUF with a stack of three identical ultrafiltration (UF) membranes¹. Choosing BSA as a model for HCPs, separation of a binary mixture of IgG and BSA containing 1.0 mg/ml IgG and 0.1mg/ml BSA is successfully demonstrated here using a modified ISUF technique: two Omega 100 kDa membranes on top followed by one Omega 70 kDa membrane at the bottom. This modified configuration demonstrated exceptional performance with almost 100% rejection, 99% purity, and 99.5% retention of IgG, along with 96.5% recovery of BSA over 10 diavolume. This modified membrane stacking resulted from strategic considerations of membrane stacking and careful selection of molecular weight cutoffs and materials, and performance analysis of different membrane stacking configurations using rejection behaviors, purity levels, and recovery rates under varying diavolumes and pressure differentials. This approach enhances flexibility in membrane choices in ISUF and provides valuable insights for optimizing membrane-based biopharmaceutical separation techniques.

¹ Y. Song, L. Feng, S. Basuray, K. K. Sirkar*, S. R. Wickramasinghe, Hemoglobin-BSA Separation and Purification by Internally Staged Ultrafiltration, Separation and Purification Technology, 312, 123363 (2023).