Deciphering the inconsistent performance of Protein A membrane on host cell protein clearance

In downstream processing of antibodies and Fc-fusion proteins, Protein A affinity chromatography plays an important role in host cell protein (HCP) reduction. Recently, Protein A membrane has emerged as a promising alternative to resin-based Protein A columns. While Sartobind Rapid A Protein A membrane has been shown to possess stronger capability for removing product-related impurities than traditional Protein A columns, its capability for removing process-related impurities, particularly HCPs, in comparison to Protein A columns is not clear. Limited and conflicting information has been found in the literature in this regard. In the current study, we compared Sartobind Rapid A membrane with MabSelect SuRe LX resin on HCP removal using culture harvests from ten different projects. In all cases but one, the HCP levels of membrane eluates are lower than or comparable to those of column eluates. We speculated that the high flow rate adopted by Protein A membrane serves as both a favorable and an unfavorable factor for minimizing HCP copurification. On one hand, the short residence time reduces HCP leaching during elution; on the other hand, it hinders HCP-antibody disassociation during wash. Protein A membrane provides better and worse HCP clearance than Protein A column when the favorable factor and the unfavorable factor dominates, respectively. For Protein A membrane chromatography to achieve minimum HCP copurification, low and high flow rates should be applied for wash and other steps, respectively. Our work explained the discrepancy between previous studies and shed novel insights on HCP clearance by Protein A membrane.