Understanding ligand-protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads is the current standard for the primary capture of monoclonal antibodies. The high cost of Protein A activated supports, ligand leakage and the limited production capabilities of the bead-based chromatographic process are at the origin of the current research efforts towards feasible alternatives. Synthetic ligands, that mimic the interaction between Protein A and the Fc portion of immunoglobulins, have been immobilized on membrane supports. In this work, regenerated cellulose membranes with immobilized synthetic A2P have been characterized and tested for binding and elution performance using pure polyclonal human IgG solutions as a feed and several buffers for the elution step. A2P is a synthetic ligand obtained from a triazine scaffold, which binds the Fc region of the antibody. The effects of the membrane support and of the spacer arm on the ligand-ligate interaction have been studied in detail. The experimental results have been interpreted on the basis of molecular dynamics simulation of the interaction between IgG and the supported ligand. Molecular models offer in fact the possibility to investigate the nature of the interaction between protein and ligand from an atomistic standpoint, thus supplying information that can be hardly accessed following experimental procedures. MD simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer considered were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the conjunct interactions of surface, spacer and ligand with IgG contribute to the formation of the bond between protein and affinity membrane.