FIGURE LEGENDS:
FIGURE 1 Surface representation of antibodies indicating different binding sites for chromatographic ligands. (A) Shows the highest affinity sites for Capto MMC to three different antibodies. The typical interaction energy values for the top three binding sites range from -5 to -7 kcal/mol. The region with the highest interaction energy differs as a function of molecule. (B) The CDR region showed the highest interaction for Capto MMC ligand outside of the top three binding sites for each molecule. The interaction energies ranged from -4 to -5 kcal/mol. In addition, the interaction site, interacting residues, and surface area explored are diverse as should be expected for a hypervariable region.
FIGURE 2 Structure of agarose base matrix and attached ligands.(A) A surface representation of the agarose base matrix and the attached ligands. The ligands are spaced evenly around the resin from a single ligand attachment to six ligands per resin. (B) A cartoon representation of the attachment site of a chromatographic ligand to resin. The head group of the ligand is separated from the base by a linker (backbone). (C) Structure of the ligands used in this study. CaptoTM adhere and CaptoTM MMC are produced by GE Healthcare, USA and CEX (HyperDTM F) is produced by PALL Life Science, USA.
FIGURE 3 Comparison of the interaction energy between different agarose-ligand densities against mAb-1 compared to un-functionalized agarose. The mAb-ligand docking was performed at pH 5.5 and compared between modalities. All agarose-ligand complex had an improved interaction energy over un-functionalized agarose (base matrix). The general trend is that, increasing the number of ligands increase the interaction energy.
FIGURE 4 The interaction site of functionalized agarose (agarose-ligand complex) bound to mAb-1. (A) A close-up view of the interaction site between agarose-ligand complex bound to antibody (mAb-1). There is a range of interaction energies that is attributed to the head group of each attached ligand making interactions with specific sites instead of the entire ligand (head group and backbone) (B) Highest affinity interaction site of agarose-ligand complex in the context of the entire antibody. A solvation droplet was designed to cover the area in blue to simulate any changes in interaction energy as a function of molecular dynamics.
FIGURE 5 Comparison of experimental binding affinities with in silico interaction energies for mAb-1. (A) Interaction energy of Free Ligand (MMC) (Capto MMC ligand un-attached to a base matrix), agarose, and agarose with Capto MMC attached (Agarose MMC also called agarose-ligand complex). As the number of Capto MMC ligands attached to the agarose increase, the overall interaction energy of the functionalized agarose increases. (B) Chromatographic retention (k’ ) determined from retention mapping assessment with increasing ligand density shows a similar trend as docking interaction energies.
FIGURE 6 Molecular dynamics simulation of mAb-1 ligand complex compared to docking score. The interaction energies from the molecular simulation was slightly lower than the docking score but remained constant for the entire length of the simulation.