The Preparation Of Hydrophobic Charge-induction Magnetic Adsorbent And Its Application To Antibody Purification

Hydrophobic charge-induction magnetic adsorbent is a dual-mode adsorbent for protein purification, which combined the magnetism of magnetic nanoparticles and the specificity of hydrophobic charge-induction ligands for the antibodies. Compared with the single model, it has higher separation efficiency, less environmental pollution and low production cost and so on. In recent years, researchers have paid more and more attention for the application of magnetic substrate in protein purification. However, it is particularly important for the screening of ligands to synthesize magnetic adsorbent. So, a series of ligands based on hydrophobic charge-induction properties have been studied and applied in the research of adsorption, retention mechanism and the separation of proteins. Based on the results of electrophoresis, a suitable ligand was selected and the effect of the dual-mode adsorbent on the separation of protein was discussed in detail, and then, with y-immunoglobulin and HSA simulating human serum, we optimized the purification conditions and applied it to the separation and purification of immunoglobulin from actual biological samples. Results show that our preparation of magnetic adsorbent has good specificity, which can complete one step purification of immunoglobulin from actual biological samples and can obtain very good separation effect.The dissertation includes the following four chapters:1. Literature ReviewThis chapter introduced the structure, classification and function of immuneglobulin, especially in human serum and chicken egg yolk. The comments focused on the purification methods of immuneglobulin at present and the application of magnetic adsorbent in the field of biomedical applications and the advantages of the application of HCIC mode in protein purification..2. Ligand Screening and Coupling and its application in antibody purificationA series of hydrophobic charge-induction magnetic adsorbent were prepared using different ligands, such as imidazole,4-mercapto-ethyl pyridine and Amitrole. We tested the morphology, structure, size and bonding situation of microspheres with various test methods, the results showed that the synthesized Fe3O4 nanoparticles retained a smooth spherical morphology and the microspheres of Fe3O4@SiO2 nanoparticles synthesized with TEOS as silicon source was core-shell structure, the saturation magnetization rate reached to 58.1 emu/g. Then, we tested the separation performance to actual biological samples of these magnetic adsorbent in the condition of dual-mode induced hydrophobic adsorbent, respectively. The results indicated that the specific binding ability and the elution ability to protein was diverse. In addition, it is possible that the selectivity of adsorbent was influenced by the hydrophobicity of ligand. Under two modes, the dual-mode magnetic adsorbent, using 4-mercapto-ethyl pyridine (MEP) as ligand, could display the best performance to the target protein purification.3. Adsorption mechanism and adsorption properties of the dual-mode magnetic adsorbentIn this work, ?-immunoglobulin and HSA was used as the template to simulate human serum, And, the magnetic composite microspheres with a typical MEP ligand was studied to optimize purification conditions in order to improve ?-immunoglobulin purity from serum albumin containing feedstock in the future. The essential feature of HCIC is the pH-dependent behavior and salt-independence of dual-mode ligands, which combines hydrophobic and electrostatic interactions together. The influence of NaCl addition and pH change on the adsorption behavior of ?-immunoglobulin and HSA in the adsorbent, as well as the dynamic binding, displacement behaviors and reusability were investigated. the results showed that adding 0.5 mol/L NaCl in the liquid phase could effectively reduce the adsorption of HSA on the MEP adsorbent, but the same treatment had little influence on the adsorption of ?-immunoglobulin. In addition, the saturated adsorption quantity could reach 92.50 ?g/mg finished in near one hour and and the reusability of the MEP adsorbent could reach to over 5 times. The results indicated that magnetic adsorbent with MEP ligand could be a promising resins to improve the separation efficiency of antibodies with the HCIC process.4. The purification of the active proteins from human serum and chicken egg yolk using magnetic adsorbent with MEPThe magnetic adsorbent with 4-mercaptoethyl-pyridine (MEP) as ligand was a promising technology for antibody purification, which had been proved in previous. The adsorbent was selected for separation and purification of IgG and IgY from human serum and chicken egg yolk. It was found that the conditions of pH 6 without NaCl or under pH 6.0 could be used to effectively rich IgG without interference of HSA from the MEP resin. And, IgY could be effectively adsorbed under pH 4.0 with the presence of urea. the mechanism of the selective adsorption between IgG and HSA on the surface of MEP ligands was discussed. We found that isoelectric point played an important role in selective adsorption. And later, Two methods, reversed-phase Chromatography and MODOL-TOF analysis, were used to verify the purity of IgG from HSA containing feedstock, and the purity of IgG was improved to about 98% and IgY could reach to 96%. The results demonstrated that the hydrophobic charge-induction magnetic adsorbent with 4-merca ptoethyl-pyridine (MEP) as ligand was effective for the separation of antibodies from different feedstocks.