(219e) Insights on Ligand-Protein Interactions in Affinity Membranes for Antibody Purification

Recently, we designed and synthesized a series of small molecules acting as synthetic ligands for IgG. These ligands are the results of several years of study of the nature of interaction of monoclonal antibodies, and in particular IgGs, with their natural ligand, namely protein A and with the available synthetic ligands. An integrated approach that combines molecular dynamics with experimental activity and crystallographic studies was used to find of a new binding site of IgG, which lies in proximity of the main binding site of protein A, known as the consensus binding site, but is not overlapped with it. These small ligands were thus specifically computationally designed to bind to the new binding site of IgG that is, according to our simulations, more easily accessible than the consensus binding site.

Preliminary experiments with commercial cellulosic membranes decorated with these ligands, in particular with one of them called HPTA, showed promising properties in terms of binding capacity, selectivity and overall recovery of IgG from complex mixtures. Indeed, there are several issues related to residual non-specific binding and to the antibody recovery with the elution step, that appear to be associated to the membrane surface and to the spacer arm and not to the ligand itself. This was demonstrated in fundamental studies in which the influence of the endcapping protocol and of the spacer arm on the new ligand selectivity was studied, using pure solutions of IgG, BSA and lysozyme, as well as mixtures of IgG and BSA and lysozyme.

Current work is focused on the modification of the properties of the membrane surface in proximity of the affinity ligand by functionalizing part of the surface with histidines or similar residues containing amines, that able to change their protonation state as a function of pH. A modification of the charge of the surface is indeed expected to change drastically the energy of interaction of the adsorbed antibody with the surface, thus improving selectivity.