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.