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Identification of Preferred Multimodal Ligand Binding Regions on IgG1 FC using Nuclear Magnetic Resonance and Molecular Dynamics Simulations
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  • Ronak Gudhka,
  • Camille Bilodeau,
  • Scott McCallum,
  • Mark McCoy,
  • David Roush,
  • Mark Snyder,
  • Steven Cramer
Ronak Gudhka
Rensselaer Polytechnic Institute
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Camille Bilodeau
Rensselaer Polytechnic Institute
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Scott McCallum
Rensselaer Polytechnic Institute
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Mark McCoy
Merck and Co., Inc.
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David Roush
Merck and Co., Inc.
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Mark Snyder
Bio-Rad Laboratories
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Steven Cramer
Rensselaer Polytechnic Institute
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Peer review status:UNDER REVIEW

30 Jul 2020Submitted to Biotechnology and Bioengineering
30 Jul 2020Assigned to Editor
30 Jul 2020Submission Checks Completed
04 Aug 2020Reviewer(s) Assigned

Abstract

In this study, the binding of multimodal chromatographic ligands to the IgG1 FC domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated 15N-labeled FC domain indicated that while single mode ion exchange ligands interacted very weakly throughout the FC surface, multimodal ligands interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the Fc. The multimodal ligand binding sites on the FC were concentrated in the hinge region and near the interface of the CH2 and CH3 domains. Further, the multimodal binding sites were primarily composed of positively charged, polar and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on FC corroborated molecular level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand-FC binding in these preferred regions was shown to be electrostatic interactions and pi-pi stacking of surface exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular level understanding of multimodal ligand-FC interactions and sets the stage for future analyses of even more complex biotherapeutics.