Experimental and theoretical investigation of effect of spacer arm and support matrix of synthetic affinity chromatographic materials for the purification of monoclonal antibodies.

The aim of this study was to elucidate the influence of each material component-the support, the spacer, and the surface chemistry-on the overall material performance of an affinity type purification media for the capture of immunoglobulin G (IgG). Material properties were investigated in terms of an experimental evaluation using affinity chromatography as well as computer modeling. The biomimetic triazine-based A2P affinity ligand was chosen as a fixed point, while spacer and support were varied. The investigated spacers were 1-2-diaminoethane (2LP), 1,3-propanedithiol (SS3), 3,6-dioxo-1,8-octanedithiol (DES), and a 1,4-substituted [1,2,3]-triazole spacer (TRZ). The support media considered were the agarose (AG) resins, PuraBead, the polyvinylether, Fractoprep, the polymethacrylate, Fractogel, and the porous silica, Fractosil. All materials were tested with pure IgG standard solution, with a mock feed solution as well as real cell culture supernatant. The interaction between IgG and A2P linked through the investigated spacers to AG was studied using molecular dynamics. The effect of a modification of the support chemical structure or of the protein-ligand binding site on the material performances was studied through target oriented simulations. Dynamic binding experiments (DBC) revealed that the performances of materials containing 2LP spacers were significantly decreased in the presence of Pluronic F68. The simulations indicated that this is probably determined by the establishment of intermolecular interactions between the 2LP charged amino group and the ether oxygen of Pluronic F68. The spacer giving the highest IgG dynamic binding capacity when Pluronic F68 was present in the feed was TRZ. The simulations showed that, among the investigated spacers, TRZ is the only one that prevents the adsorption of A2P on the support surface, thus suggesting that the mobility and lack of interaction of the ligand with the support is an important property for an affinity material. Both experiments and calculations agree that the chemistry of the support surface can have a significant impact on IgG binding, either affecting the IgG DBC, as found experimentally for materials having similar ligand densities and spacer arms but different supports, or competing with the affinity ligand when hydrophobic groups are added to the model surface, as was computationally predicted.