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).