title: 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 K(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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Notre Dame, Indiana 46556, United States.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Alves
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Stimple
authors.forename: Michael W
authors.initials: MW
authors.lastname: Handlogten
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Ashley
authors.forename: Tanyel
authors.initials: T
authors.lastname: Kiziltepe
authors.forename: Basar
authors.initials: B
authors.lastname: Bilgicer
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000302:isolation & purification; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D007211:Indoles / Q000737:chemistry; D051379:Mice; D009711:Nucleotides / Q000378:metabolism*; D000069283:Rituximab; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry
chemical_list: D000906:Antibodies; D058846:Antibodies, Monoclonal, Murine-Derived; D007211:Indoles; D009711:Nucleotides; C014612:indolebutyric acid; D012710:Serum Albumin, Bovine; D012965:Sodium Chloride; D000069283:Rituximab