(9e) Modeling the Synergies of Saline and Organic Modifiers On Rpc Separation

During the last decades, great advances have been made regarding both modeling and understanding of preparative ion exchange chromatography (IEC). The Langmuir mobile phase modifier (MPM) [1] and the steric mass action (SMA) [2] models each give an accurate description of the effect of the salt concentration and recent developments in high throughput screening [3] as well as molecular dynamics simulations [4] have greatly increased the knowledge of the underlying mechanisms. In comparison, the chromatographic modes based on hydrophobic interactions, i.e. hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC), have been left in the shade. The complexity of the interaction between fatty ligands and hydrophobic patches on proteins has made it elusive. Although some serious attempts have been made, e.g. using a thermodynamic approach [5], many aspects of these hydrophobic phenomena are still not well understood.

As a step towards a better understanding of RPC and a model that applies for both HIC and RPC, an RPC separation of three insulins has been studied. This is a continuation of a project which was initiated with a study of the corresponding HIC separation. In both these cases, the effects of salt as well as organic modifiers have been investigated. This study included two different resins with C4 and C18 ligands, respectively, both on silica backbone.

The linear range of the isotherm was mapped by isocratic pulse experiments, performed at various concentrations of salt and organic modifier. The concentration of organic modifier was limited by nature; either by the lack of retention or by the salt solubility at the higher end, and by low UV signal-to-noise ratio, due to very high retention, at the lower end. For either resin, a series of pulse experiments at varying load within the non-linear range was performed to investigate the capacity properties.

Based on the experimental data, a mechanistic model of the system is developed. Since the resin particles are rather small and since the adsorption kinetics are generally rate-controlling for RPC, the column is described by a reaction dispersive model. The adsorption is described by a combination of thermodynamics [5] and the Langmuir MPM model, with adjustments which account for the observed effects of the modifier concentrations. The model parameters related to these effects as well as Henry’s constant were calibrated against the retention data from the linear range experiments. The model parameters concerning capacity and kinetics were tuned by hand to make the peak shape fit the overloaded and linear range experiments, respectively.

The final model can be used as a tool to design new separation processes, as well as to optimize already existing ones. It is also useful in robustness studies and for filing purposes. Apart from the obvious practical benefits, it provides a new step of insight into the underlying phenomena and gives a deeper understanding of the process.

References

1.         Melander, W.R., S. El Rassi, and C. Horváth, Interplay of hydrophobic and electrostatic interactions in biopolymer chromatography: Effects of salts on the retention of proteins. J. Chromatogr., 1989. 469: p. 3-27.

2.         Brooks, C.A. and S.M. Cramer, Steric Mass-Action Ion Exchange: Displacement Profiles and Induced Salt Gradients. AIChE J., 1992. 38(12): p. 1969-1978.

3.         Nfor, B.K., et al., Rational and systematic protein purification process development: the next generation. Trends Biotechnol., 2009. 27(12): p. 673-679.

4.         Holstein, M.A., et al., Effects of Urea on Selectivity and Protein−Ligand Interactions in Multimodal Cation Exchange Chromatography. Langmuir, 2012. 29(1): p. 158-167.

5.         Mollerup, J.M., A Review of the Thermodynamics of Protein Association to Ligands, Protein Adsorption, and Adsorption Isotherms. Chem. Eng. Technol., 2008. 31(6): p. 864-874.