Hydrophobic Charge Induction Chromatography: Ligand Screening, Coupling And Its 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 and thereby decreases the cost of purication significantly. The current work focused on the design, screening and coupling of new ligand for HCIC, the application of SH-Sepharose on the adsorption of model protein, thermodynamic analysis of interaction between model protein and free or immobilized ligand.Firstly, a novel HCIC ligand was screened in the commercial MDL database by using heterocyclic nitric compound, imidazole, as leading compound. According to the structure similarity theorem, a sub-database has been obtained and then a novel HCIC SLC-HA was screened from this sub-database based on the disassociate constant of compound calculated by the Marvinsketch software.Secondly, Sepharose CL-6B was used as a medium to coupling SLC-HA obtained from above virtual screening process and the optimal coupling condition was achieved which the density of epoxy groups reached 110μmol/mL. The temperature for SLC-HA coupling is 60°C. After reaction at 170 rpm for 10 h, the SLC-HA density can achieve 90-95μmol/mL.Thirdly, the adsorption of Lysozyme on adsorbent was performed at different ionic strengths and pHs. The results exhibited that the static adsorption capacity of HCIC matrices did not vary greatly in ionic strength from 0.0 to 1.5 mol/L whilst the dynamic adsorption capacity decreased significantly with a decreasing pH from 7.0 to 4.5, which possessed excellent HCIC property.Finally, the highly accurate apparatus isothermal titration calorimetry (ITC) is used to investigate the interaction between SLC-HA and protein, protein and resin. We can build a new road by comparing the thermodynamic constant obtained from these two kinds of experiments.