Calorimetry for studying the adsorption of proteins in hydrophobic interaction chromatography.

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.

Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry.

Heat of adsorption is an excellent measure for adsorption strength and, therefore, very useful to study the influence of salt and temperature in hydrophobic interaction chromatography. The adsorption of bovine serum albumin and ß-lactoglobulin to Toyopearl Butyl-650 M was studied with isothermal titration calorimetry to follow the unfolding of proteins on hydrophobic surfaces. Isothermal titration calorimetry is established as an experimental method to track conformational changes of proteins on stationary phases. Experiments were carried out at two different salt concentrations and five different temperatures. Protein unfolding, as indicated by large changes of molar enthalpy of adsorption Δhads , was observed to be dependent on temperature and salt concentration. Δhads were significantly higher for bovine serum albumin and ranged from 578 (288 K) to 811 (308 K) kJ/mol for 1.2 mol/kg ammonium sulfate. Δhads for ß-lactoglobulin ranged from 129 kJ/mol (288 K) to 186 kJ/mol (308 K). For both proteins, Δhads increased with increasing temperature. The influence of salt concentration on Δhads was also more pronounced for bovine serum albumin than for ß-lactoglobulin. The comparison of retention analysis evaluated by the van't Hoff algorithm shows that beyond adsorption other processes occur simultaneously. Further interpretation such as unfolding upon adsorption needs other in situ techniques.

Hydrophobic interaction chromatography of proteins: thermodynamic analysis of conformational changes.

For BSA and beta-lactoglobulin adsorption to hydrophobic interaction chromatography (HIC) stationary phases leads to conformational changes. In order to study the enthalpy (DeltaH(ads)), entropy (DeltaS(ads)), free energy (DeltaG(ads)) and heat capacity (Deltac(p,ads)) changes associated with adsorption we evaluated chromatographic data by the non-linear van't Hoff model. Additionally, we performed isothermal titration calorimetry (ITC) experiments. van't Hoff analysis revealed that a temperature raise from 278 to 308K increasingly favoured adsorption seen by a decrease of DeltaG(ads) from -12.9 to -20.5kJ/mol for BSA and from -6.6 to -13.2kJ/mol for beta-lactoglobulin. Deltac(p,ads) values were positive at 1.2m (NH(4))(2)SO(4) and negative at 0.7m (NH(4))(2)SO(4). Positive Deltac(p,ads) values imply hydration of apolar groups and protein unfolding. These results further corroborate conformational changes upon adsorption and their dependence on mobile phase (NH(4))(2)SO(4) concentration. ITC measurements showed that DeltaH(ads) is dependent on surface coverage already at very low loadings. Discrepancies between DeltaH(ads) determined by van't Hoff analysis and ITC were observed. We explain this with protein conformational changes upon adsorption which are not accounted for by van't Hoff analysis.

Hydrophobic interaction chromatography of proteins V. Quantitative assessment of conformational changes.

Protein adsorption during hydrophobic interaction chromatography (HIC) may induce conformational changes. We analyzed conformational changes in three model proteins, bovine serum albumin (BSA), beta-lactoglobulin, and lysozyme by attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and pulse response experiments. Conformational changes occurred in the secondary structure of BSA, the tertiary structure of beta-lactoglobulin, and no changes occurred in lysozyme under the adsorption conditions investigated. Protein unfolding varied substantially among proteins, caused incomplete isocratic elution in HIC, and was confirmed by in situ assessments. Lower temperatures and binding capacities significantly reduced protein unfolding; the activation energy for unfolding ranged from 47 to 125 kJ/mol.