Calorimetrical Measurement Of Protein Adsorption On Hydrophobic Interaction Adsorbent

Hydrophobic interaction chromatography (HIC), as one of important techniques in downstream bioprocessing, has been extensively applied in the purification of biological macromolecules and nanoparticles, such as proteins. The experimental evidences indicated that protein adsorption on HIC adsorbent may give rise to a loss of protein activity and conformational changes. In the current research, the conformational change of BSA on HIC adsorbents, Phenyl Sepharose 6 Fast Flow series, was investigated by calorimetrical analysis on a basis of isothermal titration calorimeter and van't Hoff method.Firstly, the static adsorption of BSA on Phenyl Sepharose 6 FF was investigated at different conditions to assess the effect of salt types and concentration,ligand density, temperature on the adsorption equilibria. The results showed that a stronger adsorption of BAS on HIC adsorbents could occur in presence of sodium sulfate compared with that in ammonium sulfate. The protein adsorption was more promoted by increasing salt concentration and ligand density. At a temperature range of 18oC to 25oC, the adsorption of BSA on Phenyl Sepharose 6 FF was an adsorption-dominated process while it transformed to a partition-dominated process at higher temperatures over 25oC.Secondly, calorimetry of BSA adsorption on the HIC adsorbent was analyzed by ITC, and then the Gibbs free energy changeΔG, enthalpyΔH, entropy changeΔS and heat capacity changeΔC_p could be calculated from thermodynamic data collected in ITC experiments. The result showed that adsorption of BSA on HIC adsorbent was typical of enthalpy-entropy compensation (EEC). During protein adsorption, the unfavorable entropy change was compensated by a part of favorable enthalpy change and thus adsorption was driven by partial enthalpy change. In a stronger adsorption of protein on HIC adsorbent, the EEC became to weaken. During the adsorption process, moreover, adsorbed BSA on HIC adsorbent experienced a great conformational change while the release of water on the surfaces of both adsorbent and protein played an important role in the adsorption.Finally, the heat capacity change of BSA during protein adsorption was calculated from static adsorption data using van't Hoff analysis and ITC experiments, respectively. By comparing the result of heat capacity change obtained from van't Hoff analysis and ITC data, it could be concluded that heat capacity change obtained from ITC experiments had a better correlation with conformational change affected by temperatures. Furthermore, salt concentration had the most significant influence on conformational change and the release of waters, thereby promoting the adsorption of more BSA on HIC adsorbent. The similar tendency could be achieved at higher temperatures. The higher salt concentration was, the less enhancement of temperature was.