A microcalorimetric study of molecular interactions between immunoglobulin G and hydrophobic charge-induction ligand

• Published in: Journal of Chromatography A Vol. 1443; pp. 145 - 151
• Main Authors: Yuan, Xiao-Ming, Lin, Dong-Qiang, Zhang, Qi-Lei, Gao, Dong, Yao, Shan-Jing
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 22.04.2016
• Subjects: Adsorption; Antibodies - isolation & purification; Antibodies, Monoclonal - metabolism; Binding; Calorimetry; Chromatography; Electrostatics; Entropy; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Hydrophobic charge-induction chromatography; Immunoglobulin G; Immunoglobulin G - metabolism; Immunoglobulins; Isothermal titration calorimetry; Ligands; Molecular interactions; Protein Binding; Pyridines - chemistry; Serum albumin; Serum Albumin, Bovine - chemistry; Serum Albumin, Bovine - metabolism; Sodium Chloride - chemistry; Static Electricity; Thermodynamics; Isothermal titration calorimetry; Adsorption; Hydrophobic charge-induction chromatography; Molecular interactions; Immunoglobulin G
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2016.03.041

•Isothermal titration calorimetry was used to evaluate ligand-protein interactions.•Molecular interactions between HCIC ligand and IgG were studied.•Thermodynamic parameters were analyzed and compared with adsorption data.•Typical molecular mechanism of HCIC was verified. 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.