Hydrophobic Charge Induction Chromatography: Adsorption And Thermodynamic Analysis

In hydrophobic charge induction chromatography (HCIC), the adsorption of protein to a moderately hydrophobic surface could be achieved in a wide range of ionic strengths and then elution was performed at an acidic pH, at which the ligand on the hydrophobic surface is positive charged. Thus, purification of target protein could be easily obtained without the complicated pre-treatment, such as the adjustement of ionic strength or/and pH in feedstock. Therefore, the cost of purication is lower. This work focused on the coupling of new HCIC ligand and its application in the purification of model protein.Firstly, Sepharose CL-6B was used as the medium to coupling new HCIC ligand obtained from above virtual screening process and the optimal coupling condition was achieved. The results showed that the preferred Allyl density could be obtained at an allyl bromide concentration of 30% (v/v) and 4 mol/L of NaOH after reaction was performed in an incubator with a speed of 170 rpm at 25°C for 24 h.The results showed that the best reaction condition: the concentration of Allyl Bromide is 30%(v/v); the concentration of NaOH is 4 mol/L; the reaction time is 24 h; the temperature is 25℃; the rotate speed is 170 rpm.Secondly, SLC-AN was choosed to be coupled to Seppharose CL-6B and the optimal coupling condition was achieved. The temperature for SLC-AN coupling is 50 . After reaction at 170 rpm for 36 h, the SLC-AN can be coupled to the medium.And adjusting the concentration of SLC-AN, the density can be changed. In this experiment, the density can achieve 94, 71, 39μmol/ml.Thirdly, the adsorption ofγglobulin, bovine serum albumin on adsorbent was performed in different conditions. It was found thatγglobulin is salt-independent adsorption, but BSA is sensitive to ionic strength. The results indicated the hydrophobic interaction may play more important role in the adsorption betweenγglobulin and the ligand whereas the electrostatic interaction is dominant in the adsorption of BSA onto adsorbent.Finally, the highly accurate apparatus isothermal titration calorimetry (ITC) is used to investigate the interaction between the protein and resin. We can build a new road by analyzing the thermodynamic constant from this kind of experiment.