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.