Conclusions
In this paper, a combination of NMR and MD simulations was employed to
develop a fundamental understanding of how single mode CEX and MM CEX
chromatographic ligands interact with the FC domain of
an IgG1 mAb. The NMR results provided insights into the interaction
sites of these ligands on the FC. Preliminary data
analysis of the NMR results based on CSPs revealed that, while the
single mode ligand interacted across the entire FCsurface, the MM ligands appeared to interact with specific clusters of
residues forming a concentrated binding region. In order to obtain more
quantitative information, the CSP data at different ligand
concentrations for each FC residue were fit to the
Langmuir equation to determine the residue specific binding dissociation
constants (KD). While the single mode CEX ligand showed
weak interactions over the FC surface with high
KD values (> 150 mM), the Nuvia cPrime MM
ligand exhibited up to two orders of magnitude smaller
KD values indicating significantly stronger binding to
the FC. The MM ligand binding sites on the
FC were concentrated in the hinge region and near the
interface of the CH2 and CH3 domains
which are relatively flexible regions of the FC. These
sites were also observed to consist of primarily positively charged,
polar and aliphatic residues on the FC surface.
Interestingly, histidine residues in these regions exhibited some of the
strongest binding to the MM ligand. Comparisons of the NMR results with
protein surface properties suggested that the MM ligand binding regions
were associated with overlapping regions of positive charge and
hydrophobicity on the FC surface. Finally, we presented
MD simulations to provide further insights into the binding mechanisms
these interactions. As was determined from the NMR analysis, the
simulations also indicated that the histidine residues in the interface
of the CH2 and CH3 domains were “high
affinity” binders. The simulations also indicated that these histidines
interacted with the MM ligands via electrostatic and pi-pi interactions.
This combined biophysical and simulations approach elucidates the MM
ligand interaction sites on the FC and sets the stage
for future analyses of even more complex biotherapeutics such as Fabs
and mAbs. While the current work was focused on only two MM ligands,
this method can readily be extended to other MM ligands that have been
recently shown to exhibit unique chromatographic selectivities for the
separation of complex multi-domain proteins (Robinson, Roush, 2018). The
molecular insights gained from this work can also potentially lead to
new protein molecular descriptors that may be useful for predicting the
multimodal chromatographic behavior of FC containing
molecules such as mAbs, bispecific Abs and fusion proteins. Future work
will also examine the interactions of the Fc with MM ligand-coated
nanoparticles in order to more fully account for co-operativity and
avidity effects in these systems.