title: A microcalorimetric study of molecular interactions between immunoglobulin G and hydrophobic charge-induction ligand.
abstract: Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification. In this study, isothermal titration calorimetry (ITC) was used to evaluate the molecular interactions between MEP ligand and immunoglobulin G (IgG). Three types of IgG molecules including human IgG (hIgG), bovine IgG (bIgG) and a monoclonal antibody (mAb) were investigated with human serum albumins (HSA) and bovine serum albumin (BSA) as the comparison. The thermodynamic parameters obtained from ITC were compared with the adsorption data. The results indicated that MEP binding to protein at neutral pH was entropy driven and induced by multimodal molecular interactions that was dominated by hydrophobic forces. The interactions between MEP and IgGs were stronger than that of albumins, which resulted in high binding affinity of IgGs. Moreover, the effects of pH and salt addition on MEP-hIgG binding were studied. The change of enthalpy increased obviously with the decrease of pH, which revealed that the electrostatic forces dominated the MEP-hIgG interactions at acidic condition and caused typical charge-induced elution of HCIC. Salt addition influenced both hydrophobic and electrostatic interactions. With the increase of salt concentration, the hydrophobic interactions decreased first and then increased, while the electrostatic interactions showed the opposite trend. This resulted in trade-off between the multimodal interactions, which caused the salt-tolerant property of MEP resin. In general, ITC studies revealed the molecular mechanism of three critical characteristics of HCIC, multimodal interactions, pH-dependent and salt-tolerant properties.
authors.affiliation: State Key Laboratory of Chemical Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Chemical Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: lindq@zju.edu.cn.
authors.forename: Dong-Qiang
authors.initials: DQ
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Chemical Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Qi-Lei
authors.initials: QL
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Synthetic and Natural Functional Molecular Chemistry of Ministry of Education, Institute of Modern Separation Science, Northwest University, Shaanxi Key Laboratory of Modern Separation Science, Xi'an 710068, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Chemical Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Shan-Jing
authors.initials: SJ
authors.lastname: Yao
mesh_terms: D000327:Adsorption; D000906:Antibodies / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002151:Calorimetry; D002845:Chromatography; D019277:Entropy; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008024:Ligands; D011725:Pyridines; D012710:Serum Albumin, Bovine; D012965:Sodium Chloride; C023666:pyridine
keywords: Adsorption; Hydrophobic charge-induction chromatography; Immunoglobulin G; Isothermal titration calorimetry; Molecular interactions