Mixed Mode Adsorption Chromatography And Its Application For Immunoglobulin Yolk Separation

Mixed mode adsorption chromatography is a novel bioseparation technique, which combines several types of ligands, such as hydrophobic liangd and ion exchange ligand. The electrostatic attractive interactions could enhance the adsorption and the electrostatic repulsive interactions which would be useful for desorption procedure. With high ligand density, mixed mode adsorption shows a salt-independent adsorption property. The sample dilution or other additives could be canceled, which certainly improves the separation efficiency and reduces the separation cost. In this thesis two kinds of typical mixed mode adsorbents, Streamline Direct HST and MEP HyperCel, were investigated with some model proteins. In addition, the adsorbents were used to isolate immunoglobulin from diluted egg yolk.The thesis is divided mainly into four sections.Section â…  is on the isotherm adsorption behavior of Streamline Direct HST. With bovine serum albumin (BSA) and hemoglobin as the model proteins, the influences of pH and salt on the adsorption were investigated. The results indicated that the protein adsorption changed with pH. The optimal pH could be found for highest adsorption capacity. The electrostatic interactions might be the most important contributor to the protein adsorption when the electrostatic attractive interactions exist between protein and adsorbent, and the isotherm adsorption curves could be fitted with Langmuir equation. When there are some amounts of electrostatic repulsion protein-adsorbent interactions, the adsorption process is patch controlled due to the uneven distribution of charge on the surface of protein molecule.Section â…¡ is on the separation of immunoglobulin yolk (IgY) with Streamline Direct HST. The results indicated that Streamline Direct HST could adsorb almost all proteins in the diluted egg yolk. The adsorption capacity was high and the influence of salt could be negelected. The change of pH could achieve desorption of proteins, however the purification factor with Streamline Direct HST was low.Section â…¢ is on the isotherm adsorption behavior of hydrophobic charge inducedchromatography (HCIC) adsorbent, MEP HyperCel. With BSA and lysozyme as the model proteins, the adsorption behavior of HCIC adsorbent MEP HyperCel was complicated. At the pH of 6-12, MEP HyperCel could always adsorb lysozyme, and the adsorption capacity increased with the increase of pH. It was found that the pH is the important factor to affect the adsorption with MEP HyperCel. In addition, sometimes the adsorption ability is so strong that the elution is difficult.Section IV is on the separation of immunoglobulin yolk with MEP HyperCel. The results indicated that the separation selectivity of IgY with MEP HyperCel was perfect. Under the optimized operation conditions, the purity of IgY after one-step MEP HyperCel chromatography could reach more than 95%. In addition, MEP HyperCel showed the salt-independent adorption, so MEP HyperCel HCIC could be used after salt precipitation. The purity could reach 99.8% after salt precipitation and HCIC.Some adsorption behaviors of mixed mode adsorption were obtained in this thesis, which would improve the design of new mixed mode adsorbents and process optimization. With HCIC adsorbent of MEP HyperCel, IgY could be purified with only one chromatographic step, which would be useful for further studies with mixed mode adsorption for antibody purification.