NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarsegrained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems.ligand binding site | mixed mode chromatography | protein-ligand interactions | binding site mapping | pseudoaffinity T he development of efficient bioseparation processes for the production of high-purity biopharmaceuticals is one of the most pressing challenges facing the pharmaceutical and biotechnology industries today. In addition, high-resolution separations for complex bioanalytical applications are becoming increasingly important. Although it is generally accepted that nonspecific interactions can often complicate single mode chromatographic separations (e.g., ion-exchange, reversed-phase), these additional interactions can also result in unexpected selectivities (1, 2).Recent advances in the design of multimodal (MM) chromatographic systems have produced previously undescribed classes of chromatographic materials that can provide alternative and improved selectivities as compared to traditional single mode chromatographic materials (3-9). Johansson et al. have developed a library of MM ligands that can be employed for the capture of charged proteins under high salt conditions (4, 5). Liu et al. have developed a silica-based MM resin capable of weak anion-exchange and reversed-phase interactions for the simultaneous separation of acidic, basic, and neutral pharmaceutical compounds (9). Small ligand pseudoaffinity chromatographic materials such as those used for hydrophobic charge induction chromatography have resulted in previously undescribed classes of MM ligands that offer unique selectivities due more to multiple low affinity MM interactions than to specific binding to certain classes of proteins (10). In addition, several libraries of MM ligands have been recently developed and employed on chro...

Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. MD simulations also identify additional binding sites that were not observed in the NMR experiments. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.

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A library of cold shock protein B (CspB) mutant variants was employed to study protein binding affinity and preferred orientations in cation exchange chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography. While the expected trends were observed with increasing or decreasing positive charge on the protein surface, the degree of change was a strong function of the location and microenvironment of the mutated amino acid. Quantitative structure-property relationship (QSPR) models were generated using a support vector regression technique that was able to give good predictions of the retention times of the various mutants. Molecular descriptors selected during model generation were used to elucidate the factors affecting protein retention. Electrostatic potential maps were also employed to provide insight into the effects of protein surface topography, charge density and charge distribution on protein binding affinity and possible preferred binding orientations. The use of this protein mutant library in concert with the qualitative and quantitative analyses presented in the article provides an improved understanding of protein behavior in ion exchange systems.

Hydrophobic interaction chromatography (HIC) is known to be potentially denaturing to proteins, but the effects of mobile phase conditions on chromatographic behavior are not well understood. In this study, we apply a model describing the effects of secondary protein unfolding equilibrium on chromatographic behavior, including the effects of salt concentration on both stability and adsorption. We use alpha-lactalbumin as a model protein that in the presence and absence of calcium, allows evaluation of adsorption parameters for folded and unfolded species independently. The HIC adsorption equilibrium under linear binding conditions and solution phase protein stability have been obtained from a combination of literature and new experiments. The effect of salt concentration on protein stability and the rate constant for unfolding on the chromatographic surface have been determined by fitting the model to isocratic chromatography data under marginally stable conditions. The model successfully describes the effects of added calcium and ammonium sulfate. The results demonstrate the importance of considering the effects on stability of mobile phase modifiers when applying HIC to marginally stable

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A lysozyme library was employed to study the effects of protein surface modification on protein retention and to elucidate preferred protein binding orientations for cation exchange chromatography. Acetic anhydride was used as an acetylating agent to modify protein surface lysine residues. Partial acetylation of lysozyme resulted in the formation of a homologous set of modified proteins with varying charge densities and distribution. The resulting protein charge ladder was separated on a cation exchange column, and eluent fractions were subsequently analyzed using capillary zone electrophoresis and direct infusion electrospray ionization mass spectrometry. The ion exchange separation showed a significant degree of variation in the retention time of the different variants. Several fractions contained coelution of variants, some with differing net charge. In addition, several cases were observed where variants with more positive surface charge eluted from the column prior to variants with less positive charge. Enzymatic digest followed by mass spectrometry was performed to determine the sites of acetylation on the surface of the variants eluting in various fractions. Electrostatic potential maps of these variants were then generated to provide further insight into the elution order of the variants.

In this study, predictive quantitative structure-property relationship (QSPR) models that employed a support vector machine regression algorithm and a set of novel pH-dependent descriptors were employed for the prediction of protein chromatographic behavior at any pH. The calculated pH-dependent descriptors were based on protein crystal structures and sequence information and represent charge and electrostatic potential properties on the protein surfaces. With this set of pH-dependent descriptors, proteins at different pH were treated as distinct molecules, thus enabling the generation of integrated QSPR models, which allow the prediction of chromatographic behavior of test set proteins across a wide range of mobile-phase pH conditions. The predictions from these integrated QSPR models in general showed good agreement with the experimental results. For proof of concept, the steric mass action adsorption isotherm parameters of a binary test set of proteins (lysozyme and aprotinin) at a pH not employed in the training set were predicted from the integrated QSPR models. Further, the predicted parameters were used in a macroscopic transport model to simulate the chromatographic separation of this binary protein mixture at this new pH. The simulated column behavior of these proteins showed good agreement with experimental results. The use of pH-dependent descriptors in this multiscale modeling approach now enables the prediction of various modes of protein chromatography at any mobile-phase pH, which may have significant implications for downstream bioprocessing.