Ligand Density and Resin Surface Design
Previous studies demonstrated a strong correlation between chromatographic ligand docking binding affinities (surrogate for ΔG) and chromatographic separation (k’) (Insaidoo et al., 2015). While these results may hold true for a smaller biological molecule, the complexity of possible ligand interaction sites on an antibody makes such an approach challenging (Hanke & Ottens, 2014). To address the complexity in chromatographic separation, a molecular model of agarose ligand complex was formulated to capture a broader range of ligand densities and interaction sites. 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 resin area. From our internal calculation, these agarose models span the range of low to medium and high-density resin that is commercially available (Figure 2A).