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Biotechnol Bioeng ; 116(1): 76-86, 2019 01.
Article in English | MEDLINE | ID: mdl-30252938

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Chromatography, Affinity/methods , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Staphylococcal Protein A/chemistry , Staphylococcal Protein A/metabolism , Immunoglobulin G/chemistry , Immunoglobulin G/isolation & purification , Protein Binding , Protein Conformation , Scattering, Small Angle
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