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

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.

Antibody Binding Heterogeneity of Protein A Resins

“…According to the shrinking core model, when the antibodies in a sample reach the adsorbent they bind to the first Protein A ligands at the surface of the beads with the subsequent molecules binding to the inner ligands until reaching the center of the particle. In a recent study we concluded that in the linear range of the isotherm, the antibodies would bind preferably to the outermost domains in an average stoichiometry of 1:1 or lower . Therefore, the reorientation/rearrangement of the molecules upon binding is more favorable than multilayer formation.…”

“…Also, as mentioned, having multiple binding sites per Protein A ligand is a source of heterogeneous binding, with high‐ and low‐energy sites. So, the binding of antibody is likely a stochastic phenomenon in which the distribution of the binding energy determines the probability of concomitant binding events …”

Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies

“…To overcome these drawbacks manufacturers have continuously engaged in the design of novel resins with improved characteristics. MabSelect SuRe™, used in the capture step of Reblozyl® (activin receptor type IIB to treat beta thalassemia) and Alprolix® (a coagulation factor IX for hemophilia B), is a resin functionalized with a tetrameric chains of alkali-stabilized protein A-derived ligand (of the Z-domain of staphycoccal protein A) produced in E. coli ( Silva et al, 2018 ). This ligand is NaOH tolerant, allowing a deep cleaning of the resin after each purification round, minimizing cross-contamination, and extending the lifetime of the resin.…”

In situ neutron scattering of antibody adsorption during protein A chromatography

“…Native Staphylococcus aureus protein A is a tetramer that, when stretched, has an approximate length of 10 nm, and a single domain length of ~3 nm (Protein Data Bank:1BDD [31]). Mazzer et al and Silva et al suggested the binding is usually at a 2:1 ratio [10,32] when used as an affinity chromatography ligand, whereas Plewka et al [17] estimated that antibodies bind to protein A molecules at a 1.2:1 ratio. They both agree however that the IgG molecules are bound to the protein A ligand at a tilted orientation near the resin surface [10,17].…”