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Small-molecule-based affinity chromatography method for antibody purification via nucleotide binding site targeting.
- Source :
-
Analytical chemistry [Anal Chem] 2012 Sep 18; Vol. 84 (18), pp. 7721-8. Date of Electronic Publication: 2012 Sep 07. - Publication Year :
- 2012
-
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.
- Subjects :
- Animals
Antibodies, Monoclonal, Murine-Derived isolation & purification
Binding Sites
Cattle
Indoles chemistry
Mice
Rituximab
Serum Albumin, Bovine chemistry
Serum Albumin, Bovine metabolism
Sodium Chloride chemistry
Antibodies isolation & purification
Chromatography, Affinity
Nucleotides metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1520-6882
- Volume :
- 84
- Issue :
- 18
- Database :
- MEDLINE
- Journal :
- Analytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 22928545
- Full Text :
- https://doi.org/10.1021/ac300952r