Figure Legends
Figure 1: The Ligands employed in NMR experiments to represent the
chromatographic resins; (a) SP Sepharose, (b) Nuvia cPrime and (c) Capto
MMC ligands.
Figure 2: Snapshot from MD simulation of FC surrounded
by Capto MMC ligands in free solution. Protein is shown in a surface
representation in grey, water is shown in wireframe and colored based on
atom type, and ligands and ions are shown in a licorice representation
and colored based on atom type. Color scheme: hydrogen, white; oxygen,
red; carbon, cyan; nitrogen, blue; and sulfur, yellow.
Figure 3: Chromatographic Retention of the FC domain on
single mode SP Sepharose and MM CEX systems. 40 CV linear salt gradient
from 0 to 1M NaCl at pH 5.
Figure 4: Residue specific ligand-induced changes in combined chemical
shift for binding of (a) SP Sepharose, (b) Capto MMC and (c) Nuvia
cPrime ligands to the FC domain. Secondary structure of
the FC domain at the bottom of a. Numbered regions 2-14;
25-38; 61-70; 87-98; 119-127; 154-157; 211-216; represent the loops &
α-helices. (Note: different y-axis scale for Nuvia cPrime).
Figure 5: (a) A colored cartoon and surface representation of residue
specific ligand induced changes in combined chemical shift upon binding
of SP Sepharose ligand to the FC domain. (b)
Electrostatic Potential (EP) map at pH 5 and (c) Surface Aggregation
Propensity (SAP) map of the FC domain. (Note: numbered
regions 1-13; 25-38; 87-98; 119-127; 211-216; represent the loops &
α-helices).
Figure 6: A colored cartoon and surface representation of residue
specific ligand induced changes in chemical shift for binding of (a)
Capto MMC and (b) Nuvia cPrime ligands to the FC domain.
(Note: numbered regions 1-13; 25-38; 87-98; 119-127; 211-216; represent
the loops & α-helices).
Figure 7: Representative 15N-TROSY peaks from the
Nuvia cPrime titration experiments for residues (a) Aspartate 182, (b)
Valine 178 and (c) Histidine 66 at 0 (purple), 5 (magenta), 10(cyan), 20
(green), 40 (pink) mM ligand concentrations
Figure 8: Nuvia cPrime binding sites on FC domain as
determined by NMR with color coded dissociation constant
(KD) for non-interacting, grey; and strong, red;
intermediate, salmon; and weak, pink; binding residues. Residues located
in the hinge and CH2-CH3 interface
regions are highlighted in green and blue ellipses, respectively.
Figure 9: Nuvia cPrime binding hotspots on the FC as
determined by MD simulations.
Figure 10: The dual role of protonated histidines in the (a) flexible
hinge region and (b) interface of the CH2 and
CH3 domains.
Figure S1: Comparison of the CSP data between (a) Nuvia cPrime and SP
Sepharose, blue; (b) Capto MMC and SP Sepharose, brown; and (c) Capto
MMC and Nuvia cPrime, purple; ligand systems.
Figure S2: Representative ligand induced changes in chemical shift as a
function of Nuvia cPrime ligand concentration and the resulting fits for
residues (a) Aspartate 182, (b) Valine 178 and (c) Histidine 66.
Table S1: A List of residues on the FC surface
interacting with Nuvia cPrime ligand, associated binding dissociation
constant (KD) with the standard error of fitting and the
coefficient of determination (R2). Residues located in
the hinge and CH2-CH3 interface regions
are indicated by * and #, respectively.