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

Different ratio of antibody to Protein A domain of MabSelect SuRe are found depending on the isotherm region. In the linear range of the isotherme the 1:1 stoichiometry is the most probable configuration. In the non linear range we argue for a 2:1 stoichiometry. According to the stoichiometry antibodies adsorb in different orientations. The 3:1 stoichiometry may be found at higher bulk concentrations. AbstractStaphylococcal 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 scatterin g 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 th e 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 comp...
Source: Biotechnology and Bioengineering - Category: Biomedical Science Authors: Tags: ARTICLE Source Type: research