Protein-Chromatographic Ligand Interaction
For chromatographic separation, ligands are expected to interact with different regions of the molecules based on their surface properties and inherit affinity for a given region. To answer the question of preferred protein-ligand interactions that lead to separation, different docking studies were performed. Multiple single ligand docking assessments were performed to the entire antibody surface, the Fab region, the CDR region, and the Fc region. The contact sites for protein/ligand and associated affinities were ranked based on interaction energy. Figure 1A shows that the region with the highest (more negative ΔG) protein-ligand interaction energies for the top three binding sites differed for the three antibodies. There is a correlation between in silicobinding affinities (ΔG) and experimentally determined protein retention. Previous studies have demonstrated that a single post translational modifications (PTM) in the CDR region not only affect antibody potency but can have significant effect on chromatographic separation (Kern, Mende, Denefeld, Sackewitz, & Chelius, 2014). Thus, chromatographic ligand binding to CDR region can elucidate potential chromatographic separation (Figure 1B).