Investigation of protein ligand interactions in liquid chromatographic systems
2017-01-24T00:52:08Z (GMT) by
The increasing importance of biopharmaceuticals has strengthened the interest in the development of specific analytical and separation technologies to resolve complex mixtures of proteins into highly pure end products at an industrial scale. One of the key elements of these purification processes is the preservation of the secondary and tertiary structure of the protein products to maintain their biological efficiency. The knowledge about possible conformational state(s) and the thermodynamic behaviour of important biomolecules such as peptides and proteins when associated with chromatographic ligands commonly used in these purification processes is crucial for effective productivity steering and quality control. Thermodynamic information can direct the design of new chromatographic methods for the isolation and purification of recombinantly produced proteins increasing the selectivity for the desirable product using ideal combinations of chromatographic ligands and experimental conditions. Chromatographic conditions which specifically eliminate conformationally problematic contaminants are still under-developed as the thermodynamic differences between product and by-product retention behaviour have not been established for many biomolecules. In previous work, Melander and Horvath have established that the salt dependence of solubility and retention in hydrophobic interaction chromatography can be described by the two antagonistic effects of salts on electrostatic and hydrophobic interactions. Because of this latter property, the chemical nature of the applied salts has a determining role in hydrophobic interaction chromatography. However, since proteins can undergo structural changes such as unfolding in solution with different salt composition and be subject to structural rearrangements as they interact with chromatographic ligands, the thermodynamic framework of Melander and Horvath requires a re-evaluation and further development to include an interactive term. The changes in thermodynamic parameters can be monitored by reversed-phase liquid chromatography (RPC) or hydrophobic interaction chromatography (HIC) and compared to the results obtained in bulk solution. Differences in thermodynamics may reveal different protein unfolding routes in the two different systems as well as shed light on the validity of the underlying assumptions incorporated into the van`t Hoff equation. In this PhD thesis RPC and HIC were used to assess the thermodynamics of the interaction of hen egg white lysozyme (HEWL) and horse heart myoglobin (HMYO) with n-butyl and n-octadecyl chromatographic ligands commonly used in protein analysis and purification in various organic and aqueous salt solutions and at various temperatures. Thermodynamic parameters derived from their retention times are expected to shed light on the nature of molecular occurrences that lie behind the hydrophobic interaction processes. These parameters such as the Gibbs free energy ∆G_assoc^o, the enthalpy ∆H_assoc^o, the entropy ∆S_assoc^o and the heat capacity ∆C_p^o elucidate how the hydrophobic effect in different solvational environments, such as aqueous-organic or aqueous-salt solutions affects the thermodynamics of protein immobilised-ligand interactions. Solubility of proteins and their retention on hydrophobic surfaces follow similar principles; both are related to the interactions that occur on the hydrophobic-hydrophobic interfaces. However, in a very special case if a protein is close to salting out conditions the hydrophobic interactions are strengthened providing an opportunity for in-depth investigations of the molecular arrangements on a hydrophobic contact area. An elegant tool for such investigations applicable for high-throughput analysis of protein solubility is a fully automated liquid handling robot which also allows obtaining retention data of proteins on HIC ligands in various chromatographic media obtained under identical handling conditions. Correlation of the HIC retention data with the results of the solubility experiments using the corresponding salt solutions within the concentration range of protein elution is expected to deliver information about the unique chromatographic behaviour of proteins from a different aspect as normally used in HIC methods. Solubility data when correlated with the thermodynamic parameters obtained on HIC ligands will shed light on the energetic conditions of the proteins in various solutions which are related to the hydrophobic effect both in solution and on hydrophobic surfaces. Monitoring the expected heat- and salt-induced changes of the shape and structure of HEWL and HMYO with circular dichroism spectroscopy (CDS) is expected to reveal the contribution of the helical, β-sheet, β-turn and unordered structural motifs to the protein ligand interaction under the applied conditions. Furthermore, small angle X-ray scattering (SAXS) experiments can provide valuable information about the outer shape of the proteins in various salt solutions and temperatures. The outcome of the CDS and SAXS experiments will be correlated with the thermodynamic data obtained on butyl and phenyl HIC ligands. This may allow deep insights into the energetics of the globular protein - non-polar surface interactions governed by the physical and chemical properties of the solid phase, eluent and temperature and will reveal variations of the structural elements and the molecular shape of the proteins as a result of the employed experimental conditions. The knowledge of the conformational state(s) that proteins can adopt in association with these chromatographic ligands and the thermodynamic parameters underlying these interactions will allow informed choices on column materials, eluent composition and separation conditions for the preservation of their biologically active secondary and tertiary structures in production and quality control. Once the differences in the interaction thermodynamics between the folded, native protein and its unfolded forms have been determined, improved quality-by-design methods for column-assisted protein refolding methods may be possible. Finally this study is intended to advance knowledge on how to best guide the development of green, tailor-made chromatographic methods for protein analysis and purification in the pharmaceutical industry.