Biomimetic affinity chromatography for antibody purification: Host cell protein binding and impurity removal

Abstract

Peptide affinity chromatography has received increasing attention as an alternative to protein A chromatography in antibody purification. However, its lower selectivity than protein A chromatography has impeded its success in practical applications. In particular, efficient removal of contaminants, including host cell proteins (HCPs) and DNA, is a great challenge for peptide affinity chromatography in monoclonal antibody (mAb) manufacturing. In this work, a biomimetic peptide ligand (bPL), FYWHCLDE, was coupled onto Sepharose 6 Fast Flow (SepFF) to synthesize a peptide affinity gel, SepFF-bPL, for the investigation of the binding mechanism of HCP as well as the feasibility of antibody capture. The results showed that the SepFF-bPL column exhibited effective removal of mAb aggregates as well as mAb capture from feedstocks of various origins, whereas poor removal of HCP and DNA was found. Mechanistic studies of HCP binding indicated that electrostatic interactions dominated HCP binding on the SepFF-bPL gel and that ionic conductivity had a significant influence on HCP binding at low salt concentrations. Thus, combined chromatin extraction and anion exchange adsorption were introduced prior to SepFF-bPL chromatography for initial contaminant removal to reduce mAb aggregation induced by HCP and the loading burden of contaminants in SepFF-bPL chromatography. A proof-of-concept study of the purification train demonstrated a high recovery of mAb (68.7%) and low levels of HCP (23 ppm) and DNA (below the limit of detection) in the final product, which were acceptable for the mandatory requirements in clinical applications. This research provided a deep understanding of HCP binding on the peptide affinity column and led to the development of an effective purification train.