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.