The Ligand Screening And Fabrication Of Adsorbent For Hydrophobic Charge Induction Chromatography

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 ligands 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. Thereby, the cost of purication significantly decreased. The cureent work focused on the design, screening and coupling of new HCIC ligand and its application in the purification of model protein.Firstly, a novel HCIC ligand was screened in the commercial MDL database by using indole as leading compound. According to the structure similarity theorem, a sub-database has been obtained from MDL database using indole as the leading compound and then a novel HCIC ligand was screened from this sub-database based on the disassociate constant of compound calculated by the Marvinsketch software. Finally, a potential HCIC ligand, 5-amino indole, was obtained in our works.Secondly, Sepharose CL-6B was used as a medium to coupling new HCIC ligand obtained from above virtual screening process and the optimal coupling condition was achieved which the density of epoxy groups reached 100μmol/mL. The results showed that the optimal epoxy density could be achieved at the activation temperature of 40 ?C for 2.5 h in 10 % (v/v) epichlorohydrin and 0.8 mol/L of NaOH. The ligand coupling temperature is 49°C, after reacted at 170rpm for 20 h, the ligand density can achieve 85μmol/mL.Thirdly, the adsorption of Lysozyme on adsorbent was performed at different ionic strengths and buffer pHs. The results exhibited that the static adsorption capacity of HCIC matrices with a ligand of 5-amino ranged from 40mg/g wet resin to 51 mg/g wet resin in ionic strength from 0.0 to 3.0 mol/L. While the dynamic adsorption capacity increased with an elevated buffer pH from 3 to 9, ranged from 2.43 mg/mL to 9.05 mg/mL.Finally, the recovery of Lysozyme samples with different sodium chloride concentrations ranged from 50 mmol/L to 1000 mmol/L kept at approximately 90% during the chromatography procedure. As demonstrated, it is indicated that the 5-AI-Sepharose CL-6B chromatographic matrice possessed excellent HCIC property.