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.
Highlights
Staphylococcal protein A chromatography is the capture step of choice in the manufacturing of monoclonal antibodies because of its high selectivity and robustness (Hahn et al, 2005; Hahn, Shimahara, Steindl, & Jungbauer, 2006; Shukla, Hubbard, Tressel, Guhan, &Low, 2007)
MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z‐domain
We designed three‐dimensional (3D) models with protein data bank crystallographic structures of an IgG1 and the staphylococcal protein A B domain
Summary
Staphylococcal protein A chromatography is the capture step of choice in the manufacturing of monoclonal antibodies because of its high selectivity and robustness We compared the antibody–protein A complex radial densities provided by SAXS with theoretical configurations (protein AB domain from the crystal structure 1BDD and the antibody from the crystal structure 1HZH from Protein Data Bank [PDB]) and spatial rearrangement of antibodies and staphylococcal Protein A ligands using a molecular model approach We implemented this model to simulate different binding orientations of a crystallographic structure of an IgG1 to a tetrameric B‐domain protein A chain attached to an agarose structure to mimic the experimental system of a monoclonal antibody to MabSelect SuRe. In the current study, the methodology is explored on this very specific system of high industrial relevance, but it is applicable to a broad range of protein‐surface adsorption systems and can improve the understanding of protein binding in those systems
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