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The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
Author(s) -
L. Silva Goncalo,
Plewka Jacek,
Lichtenegger Helga,
DiasCabral Ana C.,
Jungbauer Alois,
Tscheließnig Rupert
Publication year - 2019
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26843
Subject(s) - protein data bank , chemistry , stoichiometry , protein g , monoclonal antibody , immunoglobulin light chain , protein structure , steric effects , recombinant dna , crystallography , chromatography , antibody , biology , biochemistry , stereochemistry , immunology , gene
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.