Effect of Ligand Density on Chromatographic Retention
Despite the correlation between single chromatographic ligand binding affinities and experimental data, chromatographic separation is much more complex than a single ligand-protein interaction. A single ligand binding utilizes both the head group and ligand backbone that attaches to the resin (Figure 2 B and C). Yet, on a chromatographic scale, ligand density, separation distance from resin to head group, multiple interaction effect (potential avidity and binding optimization), and compensatory protein surface properties could all be critical contributions to protein retention and separation (Hanke & Ottens, 2014). The flexibility of the spacer arm could also contribute to interaction of the ligand and the protein. To understand this mechanism and demonstrate the effect of the aforementioned factors on protein binding, we utilized an agarose surface model which was functionalized with different chromatographic ligands. This mimic a patch of chromatographic resin and how it interacts with proteins. In addition, the ligands were attached to the resin surface with a separation distance of 10-14 Å, ranging from one attached ligand to six ligands per section of resin (supplementary figure 1). These agarose models span the range of low to medium and high-density resins that are commercially available (Figure 2A). The entire agarose-ligand complex could scan the surface of the antibody using the sub-region classification outlined above to identify potential binding sites and cumulative binding affinities – effectively a combination of avidity and binding/interaction energies. A control binding study was done using only the agarose resin to investigate the contribution of the agarose to the calculated protein-ligand binding affinities as agarose has been reported to support protein purification previously in the literature (Crone, 1974).
Figure 3 shows a plot of the change in binding affinity over the un-functionalized agarose. For each chromatographic modality, thein silico binding affinities (ΔG) to the antibody-ligand complex increased as the number of attached ligands per agarose increased. Further, the different chromatographic ligands had different binding affinities with Capto MMC showing the strongest interactions (Figure 3).