Protein Interactions in Hydrophobic Charge Induction Chromatography (HCIC)

Abstract

A quantitative understanding of how proteins interact with hydrophobic charge induction chromatographic resins is provided. Selectivity on this mode of chromatography for monoclonal antibodies as compared to other model proteins is probed by means of a linear retention vs pH plot. The pH-dependent adsorption behavior on this mode of chromatography for a hydrophobic, charged solute is described by taking into account the equilibrium between a hydrophobic, charged solute and an ionizable, heterocyclic ligand. By analogy, an equation that is seen to adequately describe macromolecular retention under linear conditions over a range of pH is developed. A preparative, nonlinear isotherm that can capture both pH and salt concentration dependency for proteins is proposed by using an exponentially modified Langmuir isotherm model. This model is seen to successfully simulate adsorption isotherms for a variety of proteins over a range of pHs and mobile phase salt concentrations. Finally, the widely differing retention characteristics of two monoclonal antibodies are used to derive two different strategies for improving separations on this mode of chromatography. A better understanding of protein binding to this class of resins is seen as an important step to future exploitation of this mode of chromatography for industrial scale purification of proteins.

A Review of the Thermodynamics of Protein Association to Ligands, Protein Adsorption, and Adsorption Isotherms

“…Eq. (18) shows that the driver of the retention in hydrophobic chromatography is the magnitude of the mobile phase activity coefficient. The activity coefficients of proteins in salt solutions parallel the Hofmeister series [6,7].…”

“…We can therefore conclude that DG at absorption equilibrium must be zero. From experimental measurements of retention data we can determine the thermodynamic equilibrium constant K i from either (18) or (21) and thus estimate the standard Gibbs energy change of adsorption by using (16). However, there are numerous papers where the authors claim to be able to calculate the Gibbs energy change of adsorption from the distribution coefficient [21][22][23][24].…”

“…If we model the adsorption as an association, the ion-exchange model is given in (21), and the hydrophobic interaction can be modelled according to (18). Due to the salts usually applied in ion-exchange chromatography, like for instance NaCl, the activity coefficients play a minor role in agreement with the Hofmeister series.…”

“…Ghose et al [18] applied (47) to model protein interactions in hydrophobic charge induced chromatography. The models, (20) and (49), show that the retention in hydrophobic chromatography can be controlled by altering the magnitude of the activity coefficient of the solutes.…”

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

“…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 chromatographic resins for screening with biological mixtures.…”