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The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z‐domain. Little is known...

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Autores principales: L. Silva, Goncalo, Plewka, Jacek, Lichtenegger, Helga, Dias‐Cabral, Ana C., Jungbauer, Alois, Tscheließnig, Rupert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587469/
https://www.ncbi.nlm.nih.gov/pubmed/30252938
http://dx.doi.org/10.1002/bit.26843
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author L. Silva, Goncalo
Plewka, Jacek
Lichtenegger, Helga
Dias‐Cabral, Ana C.
Jungbauer, Alois
Tscheließnig, Rupert
author_facet L. Silva, Goncalo
Plewka, Jacek
Lichtenegger, Helga
Dias‐Cabral, Ana C.
Jungbauer, Alois
Tscheließnig, Rupert
author_sort L. Silva, Goncalo
collection PubMed
description Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z‐domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small‐angle X‐ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three‐dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.
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spelling pubmed-65874692019-07-02 The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface L. Silva, Goncalo Plewka, Jacek Lichtenegger, Helga Dias‐Cabral, Ana C. Jungbauer, Alois Tscheließnig, Rupert Biotechnol Bioeng ARTICLES Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z‐domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small‐angle X‐ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three‐dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects. John Wiley and Sons Inc. 2018-10-25 2019-01 /pmc/articles/PMC6587469/ /pubmed/30252938 http://dx.doi.org/10.1002/bit.26843 Text en © 2018 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle ARTICLES
L. Silva, Goncalo
Plewka, Jacek
Lichtenegger, Helga
Dias‐Cabral, Ana C.
Jungbauer, Alois
Tscheließnig, Rupert
The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
title The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
title_full The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
title_fullStr The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
title_full_unstemmed The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
title_short The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
title_sort pearl necklace model in protein a chromatography: molecular mechanisms at the resin interface
topic ARTICLES
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587469/
https://www.ncbi.nlm.nih.gov/pubmed/30252938
http://dx.doi.org/10.1002/bit.26843
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