Small-Molecule-Based Affinity Chromatography Method for Antibody Purification via Nucleotide Binding Site Targeting

Abstract

The conserved nucleotide binding site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and underutilized site. Here we describe a novel affinity chromatography method that utilizes the NBS as a target for selectively purifying antibodies from complex mixtures. The affinity column was prepared by coupling indole butyric acid (IBA), which has a monovalent affinity for the NBS with a <i>K</i>_d ranging between 1 and 8 μM, to ToyoPearl resin resulting in the NBS targeting affinity column (NBS^IBA). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBS^IBA column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBS^IBA column consistently yielded >95% antibody recovery with >98% purity, even when the antibody was purified from complex mixtures such as conditioned cell culture supernatant, hybridoma media, and mouse ascites fluid. The results presented in this study establish the NBS^IBA column as a viable small-molecule-based affinity chromatography method for antibody purification with significant implications in industrial antibody production. Potential advantages of the NBS^IBA platform are improved antibody batch quality, enhanced column durability, and reduced overall production cost