Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials

A range of studies were carried out to investigate the underlying reason for differences in dynamic binding capacities observed with various antibodies and Fc-fusion proteins during Protein A chromatography. Dynamic binding capacities were determined for these biomolecules using different protein A stationary phase materials. SEC was carried out to determine the relative sizes of the antibodies and fusion proteins. Pore diffusivities and static binding capacities were also determined on these Protein A resin materials. Trends in the dynamic binding capacities for these molecules did not correlate with differences in pore diffusion coefficients as might be expected for a mass transfer limited system. Instead, dynamic binding capacities were seen to follow the same trends as the static binding capacities and the apparent size of the molecules. Differences in static binding capacities were attributed to be due to differences in steric factor between the molecules. Solution binding stoichiometry studies were employed to estimate intra-Protein A steric effects while binding to the various domains within a Protein A ligand. In addition, steric hindrance was also found to exist between adjacent immobilized Protein A ligands on the chromatographic surface. The combination of intra and inter Protein A steric hindrances can explain differences in binding capacities observed between various antibody and Fc fusion proteins. The effect of Protein A ligand density on these supports was also examined and the results indicate that increasing Protein A ligand density leads to a situation of diminishing returns for binding capacity due to increased steric hindrance on the resin surface. The results presented in this paper show that steric hindrances can dominate over mass transfer effects in causing capacity variation between different molecules on the same stationary phase. This can lead to the development of more cost-efficient chromatographic stationary phases as well as provide information during the selection of Protein A media for preparative purification of monoclonal antibodies and Fc fusion proteins.