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....
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| Veröffentlicht in: | Analytical chemistry (Washington) 2012-09, Vol.84 (18), p.7721-7728 |
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| creator | Alves, Nathan J Stimple, Samuel D Handlogten, Michael W Ashley, Jonathan D Kiziltepe, Tanyel Bilgicer, Basar |
| description | 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 (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. |
| doi_str_mv | 10.1021/ac300952r |
| format | Article |
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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 (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. 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Chem</addtitle><description>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 (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. 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Stimple, Samuel D ; Handlogten, Michael W ; Ashley, Jonathan D ; Kiziltepe, Tanyel ; Bilgicer, Basar</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a439t-3424e5590bb6d459739ee1a6d0a991daba1c86a403b67119f4f1374160f1715f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Analytical chemistry</topic><topic>Animals</topic><topic>Antibodies - isolation & purification</topic><topic>Antibodies, Monoclonal, Murine-Derived - isolation & purification</topic><topic>Binding Sites</topic><topic>Cattle</topic><topic>Cell culture</topic><topic>Chemistry</topic><topic>Chromatographic methods and physical methods associated with chromatography</topic><topic>Chromatography</topic><topic>Chromatography, Affinity</topic><topic>Exact sciences and technology</topic><topic>Indoles - chemistry</topic><topic>Mice</topic><topic>Molecules</topic><topic>Nucleotides - metabolism</topic><topic>Other chromatographic methods</topic><topic>Pharmaceuticals</topic><topic>Rituximab</topic><topic>Serum Albumin, Bovine - chemistry</topic><topic>Serum Albumin, Bovine - metabolism</topic><topic>Sodium Chloride - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Alves, Nathan J</creatorcontrib><creatorcontrib>Stimple, Samuel D</creatorcontrib><creatorcontrib>Handlogten, Michael W</creatorcontrib><creatorcontrib>Ashley, Jonathan D</creatorcontrib><creatorcontrib>Kiziltepe, Tanyel</creatorcontrib><creatorcontrib>Bilgicer, Basar</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical chemistry (Washington)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Alves, Nathan J</au><au>Stimple, Samuel D</au><au>Handlogten, Michael W</au><au>Ashley, Jonathan D</au><au>Kiziltepe, Tanyel</au><au>Bilgicer, Basar</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Small-Molecule-Based Affinity Chromatography Method for Antibody Purification via Nucleotide Binding Site Targeting</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. Chem</addtitle><date>2012-09-18</date><risdate>2012</risdate><volume>84</volume><issue>18</issue><spage>7721</spage><epage>7728</epage><pages>7721-7728</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><coden>ANCHAM</coden><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 (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.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>22928545</pmid><doi>10.1021/ac300952r</doi><tpages>8</tpages></addata></record> |
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| subjects | Analytical chemistry Animals Antibodies - isolation & purification Antibodies, Monoclonal, Murine-Derived - isolation & purification Binding Sites Cattle Cell culture Chemistry Chromatographic methods and physical methods associated with chromatography Chromatography Chromatography, Affinity Exact sciences and technology Indoles - chemistry Mice Molecules Nucleotides - metabolism Other chromatographic methods Pharmaceuticals Rituximab Serum Albumin, Bovine - chemistry Serum Albumin, Bovine - metabolism Sodium Chloride - chemistry |
| title | Small-Molecule-Based Affinity Chromatography Method for Antibody Purification via Nucleotide Binding Site Targeting |
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