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

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 Kd ranging between 1 and 8 ?M, to ToyoPearl resin resulting in the NBS targeting affinity column (NBSIBA). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBSIBA column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBSIBA 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 NBSIBA column as a viable small-molecule-based affinity chromatography method for antibody purification with significant implications in industrial antibody production. Potential advantages of the NBSIBA platform are improved antibody batch quality, enhanced column durability, and reduced overall production cost.