Adsorption Properties And Mechanism Of Hydrophobic Charge-Induction Resins

As the important therapeutic and diagnostic drug,antibody has been widely used with a broad market demand.The antibody industry calls for cheaper and efficient chromatographic resins for the purification of monoclonal antibodies(mAbs),as Protein A-based affinity chromatography has some drawbacks,such as high cost and ligand leakage.Hydrophobic charge-induction chromatography(HCIC)is one of the promising techniques for antibody purification,which has the advantages of large adsorption capacity,high selectivity,mild operation condition and lower cost.The understanding of interaction mechanism of HCIC will help improving antibody adsorption performance of HCIC.In-this work,the adsoption properties and ligand-potein interaction mechanism of one commercial HCIC resin(MEP HyperCel)and three new home-made HCIC resins(MMI,ABI and W-ABI)were studied through static adsorption experiment and isothermal titration calorimetry(ITC).Firstly,the adsorption peoperties of MEP HyperCel resin were studied.The static adsorption experiments showed that the adsorption capacities of IgGs(hIgG,bIgG,mAb)binding to MEP HyperCel resin were much higher than that of albumins(HSA and BSA).It results indicated that MEP-based resin had high adsorption selectivity to IgG,which would be beneficial in antibody purification from complex feedstock.In addition,MEP HyperCel showed pH-dependent adsorption behaviors and salt-tolerant property during the binding of IgG.The addition of sodium caprylate could obviously reduce the adsorption of BSA on MEP HyperCel,and thus improced the binding selectivity to IgG.The influence of sodium caprylate became more evident when NaCl was added or the temperature increased.The interactions between sodium caprylate,MEP ligand or MEP resin and protein were studied by ITC.It was found that the sodium caprylate-BSA interaction was domained by hydrophobic force and stronger than the sodium caprylate-IgG interaction.The hydrophobic interactions between sodium caprylate and BSA became more powerful when NaCl was added or the temperature increased.ITC analysis demonstrated that the interaction between MEP ligand and IgG was mixed mode,including hydrophobic interaction,electrostatic interaction,hydrogen bond,Van der Waals' force and so on,which enhanced the affinity of MEP-IgG.The solution pH had an obvious influence on the electrostatic interaction and hydrophobic force between MEP ligand andIgG,leading to the fact that the binding capacity varied significantly with the change of pH value.The enthalpy-entropy compensation(EEC)phenomenon was found during MEP-IgG binding at various salt concentrations,which revealed the thermodynamic mechanism of salt-tolerant property of MEP-based resin.The adsorption characteristics of three new HCIC resins(MMI,ABI and W-ABI)were investigated and the results showed that all the three resins had high binding capacity of IgG.The binding capacity of W-ABI resin was higher than other two resins and MMI resin showed the lowest capacity.MMI,ABI and W-ABI resin had pH-dependent and salt-tolerant adsorption property as MEP HyperCel resin,while W-ABI resin was less sensitive to pH value than MMI and ABI resins.The adsorption capacities of MMI and ABI resins were significantly decreased in the buffer with 50%ethylene glycol(v/v),while the influence of ethylene glycol on W-ABI resin was slight.The binding behaviors of IgG with MEP,MMI,ABI and W-ABI resins changed little when arginine concentration varied from 0.25 M to 1.0 M.ITC studies of the interaction between three new HCIC resins and IgG was carried at different pH values and various concentrations of NaCl,ethylene glycol or arginine.It showed that pH value had significant influence on HCIC resin-IgG interaction and the influences of NaCl on HCIC resins differed from one another.Ethylene glycol affected the hydrophobic force between HCIC resin and IgG,while arginine influenced the electrostatic interaction between HCIC resin and IgG.At neutral pH condition,IgG bound to MMI,ABI or W-ABI resin through mixed mode interactions.The ITC results indicated that the thiophilic interaction and hydrophobic force of MMI resin-IgG were stronger than electrostatic interaction.The interaction mechanism of ABI rein was similar to MEP HyperCel resin,while the contribution of hydrophobic force during IgG binding to ABI resin was higher than that of IgG binding to MEP HyperCel.The interaction between W-ABI resin and IgG was so strong that IgG adsorption on W-ABI resin was less pH-dependent than that on MEP,MMI and W-ABI resins.In order to better understand the adsorption property and interaction mechanism of HCIC resin,ITC analysis was carried at different pH,salt and additive conditions.The results revealed the molecular interactions between HCIC resins and IgG and the binding mechanism of HCIC resin,which would be helpful in the process optimization and design of new HCIC ligands.