| Monoclonal antibodies separation and purification is the key issues of the monoclonal antibody production process, this paper describes the use of surface plasmon resonance (SPR) spectroscopy to study antibody ligand interactions for hydrophobic charge-induction chromatography (HCIC), and its versatility in investigating the surface and solution factors affecting the interactions.Two density model surfaces presenting HCIC ligand (mercapto-ethyl-pyridine, MEP) were prepared on Au using self assembly technique. The surface chemistry and structure, ionization and protein binding of such model surfaces were characterized by X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS), contact-angle titration and SPR, respectively. The influences of the surface and solution factors, e.g., ligand density, salt concentration and solution pH, on protein adsorption were determined by SPR. Our results showed that ligand density affects both equilibrium and dynamic aspects of the interactions. Specifically, dense ligand leads to increase in binding strength, rapid adsorption, slow desorption and low specificity. In addition, both hydrophobic interaction and hydrogen bonding contribute significantly to the protein adsorption at neutral pH, while the electrostatic repulsion is overwhelmed at acidic condition. The hydrophobic interaction in high concentration of lyotropic salt would cause the drastic conformational changes of the adsorbed protein.In addition, a novel antibody separation system was built using magnetic nanoparticles modified with HCIC ligand. Our results suggested that, this system is suitable for rapid separation of antibody with good selectivity. This antibody-ligand interaction study would strongly support and promote the production process optimization of monoclonal antibodies separation and purification. |
