Evaluation of preferred binding regions on ubiquitin and IgG1 FC for interacting with multimodal cation exchange resins using DEPC labeling/mass spectrometry.

There is significant interest in identifying the preferred binding domains of biological products to various chromatographic materials. In this work, we develop a biophysical technique that uses diethyl pyrocarbonate (DEPC) based covalent labeling in concert with enzymatic digestion and mass spectrometry to identify the binding patches for proteins bound to commercially available multimodal (MM) cation exchange chromatography resins. The technique compares the changes in covalent labeling of the protein in solution and in the bound state and uses the differences in this labeling to identify residues that are sterically shielded upon resin binding and, therefore, potentially involved in the resin binding process. Importantly, this approach enables the labeling of many amino acids and can be carried out over a pH range of 5.5-7.5, thus enabling the protein surface mapping at conditions of interest in MM cation exchange systems. The protocol is first developed using the model protein ubiquitin and the results indicate that lysine residues located on the front face of the protein show dramatic changes in DEPC labeling while residues present on other regions have minimal or no reductions. This indicates that the front face of ubiquitin is likely involved in resin binding. In addition, surface property maps indicate that the hypothesized front face binding region consists of overlapping positively charged and hydrophobic patches. The technique is then employed with an IgG1 FC and the results indicate that residues on the CH 2-CH 3 interface and the hinge are significantly sterically shielded upon binding to the resin. Further, these regions are again associated with significant overlap of positively charged and hydrophobic patches. On the other hand, while, residues on the CH 2 and the front face of the IgG1 FC also exhibited some changes in DEPC labeling upon binding, these regions have less distinct charged and hydrophobic patches. Importantly, the hypothesized binding patches identified for both ubiquitin and FC using this approach are shown to be consistent with previously reported NMR studies. In contrast to NMR, this new approach enables the identification of preferred binding regions without the need for isotopically labeled proteins or chemical shift assignments. The technique developed in this work sets the stage for the evaluation of the binding domains of a wide range of biological products to chromatographic surfaces, with important implications for designing biomolecules with improved biomanufacturability properties.

Probing IgG1 FC-Multimodal Nanoparticle Interactions: A Combined Nuclear Magnetic Resonance and Molecular Dynamics Simulations Approach.

In this study, NMR and molecular dynamics simulations were employed to study IgG1 FC binding to multimodal surfaces. Gold nanoparticles functionalized with two multimodal cation-exchange ligands (Capto and Nuvia) were synthesized and employed to carry out solution-phase NMR experiments with the FC. Experiments with perdeuterated 15N-labeled FC and the multimodal surfaces revealed micromolar residue-level binding affinities as compared to millimolar binding affinities with these ligands in free solution, likely due to cooperativity and avidity effects. The binding of FC with the Capto ligand nanoparticles was concentrated near an aliphatic cluster in the CH2/CH3 interface, which corresponded to a focused hydrophobic region. In contrast, binding with the Nuvia ligand nanoparticles was more diffuse and corresponded to a large contiguous positive electrostatic potential region on the side face of the FC. Results with lower-ligand-density nanoparticles indicated a decrease in binding affinity for both systems. For the Capto ligand system, several aliphatic residues on the FC that were important for binding to the higher-density surface did not interact with the lower-density nanoparticles. In contrast, no significant difference was observed in the interacting residues on the FC to the high- and low-ligand density Nuvia surfaces. The binding affinities of FC to both multimodal-functionalized nanoparticles decreased in the presence of salt due to the screening of multiple weak interactions of polar and positively charged residues. For the Capto ligand nanoparticle system, this resulted in an even more focused hydrophobic binding region in the interface of the CH2 and CH3 domains. Interestingly, for the Nuvia ligand nanoparticles, the presence of salt resulted in a large transition from a diffuse binding region to the same focused binding region determined for Capto nanoparticles at 150 mM salt. Molecular dynamics simulations corroborated the NMR results and provided important insights into the molecular basis of FC binding to these different multimodal systems containing clustered (observed at high-ligand densities) and nonclustered ligand surfaces. This combined biophysical and simulation approach provided significant insights into the interactions of FC with multimodal surfaces and sets the stage for future analyses with even more complex biotherapeutics.

Identification of preferred multimodal ligand-binding regions on IgG1 FC using nuclear magnetic resonance and molecular dynamics simulations.

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 15 N-labeled FC domain indicated that while single-mode ion exchange ligands interacted very weakly throughout the FC surface, multimodal ligands containing negatively charged and aromatic moieties 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 CH 2 and CH 3 domains. Furthermore, 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 π-π 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.

A thermodynamic evaluation of antibody-surface interactions in multimodal cation exchange chromatography.

In this study, the thermodynamics of binding of two industrial mAbs to multimodal cation exchange systems was investigated over a range of buffer and salt conditions via a van't Hoff analysis of retention data. Isocratic chromatography was first employed over a range of temperature and salt conditions on three multimodal resins and the retention data were analyzed in both the low and high salt regimes. While mAb retention decreased with salt for all resins at low salts, retention increased at high salts for two of the resins, suggesting a shift from electrostatic to more hydrophobic driven interactions. The retention data at various temperatures were then employed to generate non-linear van't Hoff plots which were fit to the quadratic form of the van't Hoff equation. At low salts, retention of both mAbs decreased with increasing temperature and the van't Hoff plots were concave downward on Capto MMC and Nuvia cPrime, while being concave upward on Capto MMC ImpRes. Different trends were observed on some of the resins with respect to both the concavity of the van't Hoff plots as well as the impact of temperature on the favorable enthalpies in the low salt regime. Interestingly, while increasingly favorable enthalpy with temperature was observed with Capto MMC and Nuvia cPrime at low salt, favorable enthalpy decreased with temperature for Capto MMC ImpRes. At high salts, binding of both mAbs on the two Capto resins were consistently entropically driven, consistent with desolvation. While the negative heat capacity data at low salts indicated that desolvation of polar/charged groups were important in Capto MMC and Nuvia cPrime, the positive data suggested that desolvation of non-polar groups were more important with Capto MMC ImpRes. Finally, the data at high salts indicated that desolvation of non-polar groups was the major driver for binding of both mAbs to the Capto resins under these conditions.

Network topology of LMWG cross-linked xyloglucan hydrogels for embedding hydrophobic nanodroplets: mechanistic insight and molecular dynamics.

Indigenous polymers have functional implications in biomedicine due to the presence of an inherent favorable structural architecture that supports hydrogel formation. In this study, we present the molecular level characterization of xyloglucan hydrogels using experimental and molecular simulation methods. We studied supramolecular self-assembly of tamarind seed-derived xyloglucan induced by low molecular weight gelators to form dense networks and rationalized its capabilities as a multifunctional and multiresponsive matrix for holding hydrophobic nanometric oleic acid globules intact for extended periods, preventing coalescence triggered instability using computational methods and imaging. Computational studies using molecular dynamics simulations provided mechanistic evidences of molecular associations supported by strong hydrogen bonding interactions responsible for fundamental gel network formation. Real-time imaging studies revealed that the gel matrix immobilizes intact oleic acid globules within its framework preventing coalescence. Molecular dynamics studies further showed key interactions of xyloglucan with the surfactant polysorbate 60 involved in sustaining oleic acid globules within the gel matrix, which corroborate with FE-SEM results. This study is an interesting approach to understand how molecular level associations in xyloglucan-based hydrogels can control and preserve nanosized hydrophobic oils imparting tenability and long-term droplet stability. The study also brings new insights into the conformational flexibility of xyloglucan around molecules with favorable and unfavorable interactive chemistries. Graphical abstract .

In silico modeling of functionalized poly(methylvinyl ether/maleic acid) for controlled drug release in the ocular milieu.

Controlling structurally defined properties of drug-bound macromolecules such as surface adhesion and interaction with endogenous proteins in the surrounding environment using prior data from computer-assisted simulation can be of great use in designing controlled release macromolecular therapeutic systems. In this paper, we describe experimental correlation of real-time properties of a polymer with pendant drug molecules, with predicted values obtained from studying in silico molecular interactions of this polymer with ocular surface proteins (mucin) for formulating an ophthalmic in situ gel. Mucoretention of the drug (norfloxacin) within the eye sac is closely associated with binding interactions occurring on the ocular surface, and covalent association of the drug with the mucoadhesive polymer, poly(methylvinyl ether/maleic acid), can largely reduce dosing frequency eliciting prolonged antibacterial action much required in treating conjunctival infections. The physicochemical properties and 3D conformation of the drug-polymer conjugate were predicted by computational studies. Molecular docking of the drug-polymer conjugate with ocular surface mucin (MUC-1) suggested that amino acid residues Arg1095, Asn1091, and Gln1070 of mucin are involved in hydrogen bonding with carboxyl residues in the polymer structure. The orientation of the drug-polymer conjugate in solution profoundly depends on the properties of the drug. The studies further reveal that molecular interactions of MUC-1 with the drug in the drug-polymer conjugate influence the binding orientation of the drug-polymer to mucin. Computationally predicted solvation energies revealed a significant difference in energy values between drug molecule alone (- 113.04 kcal/mol) and the drug-polymer (- 492.44 kcal/mol) suggesting higher aqueous solvation of the drug-polymer conjugate compared with less-soluble drug, and that interactions between polymer chains and ocular aqueous environment dictate the drug-polymer conjugate's free energy. Our results demonstrate the fabrication of a macromolecular therapeutic gel using drug-polymer with controlled release properties and mucoadhesion guided by information predicted from computational software. Notably, in silico studies reveal that even small variations in molecular composition, in this case, an antibacterial drug that contributes less than half of the entire molecular weight can considerably change the drug's presentation to the ocular environment. Graphical abstract Table of contents graphic.