Separation Of Immunoglobulin From Porcine Plasma With Mixed-Mode Adsorption Chromatography

The new technology of mixed-mode adsorptive chromatography was applied for the purification of immunoglobulin (Ig) from porcine plasma. Combining the electrostatic and hydrophobic interactions, mixed-mode adsorptive chromatography has a high adsorption capacity and shows the salt-independent property. The target protein can be adsorbed under both high and low salt concentrations. The elution is induced with the electrostatic repulsion between the protein and adsorbents by adjusting the pH value. In China, there is about 1 million tons porcine blood every year, which is mostly abandoned, resulting in the waste of resources and some environmental pollution. Porcine blood is rich in some useful proteins, especially it contains up to 2% of immunoglobulin. Most of immunoglobulin has antibacterial, antiviral and other biological activities. Therefore, developing a suitable process for the economic and efficient separation of porcine blood proteins is necessary.Firstly, several precipitation methods were explored to separate the fibrinogen from porcine plasma, including freezing and thawing, addition of ethanol, PEG and ammonium sulphate. After comparison, the precipitation method with ammonium sulphate was chosen and optimized. The saturation of ammonium sulphate was 20%, and no need to adjust pH of porcine plasma. The supernate was rich in immunoglobulin, which is benefit for the following separation.Secondly, the commercial mixed-mode adsorbent MEP-HyperCel was explored for the separation of porcine immunoglobulin. The adsorption prosperities were investigated, including the adsorption isotherm and kinetics. The influence of pH and salt concentration were studied, and the appropriate conditions for sample loading and elution were chosen. The porcine immunoglobulin was separated from the supernate of ammonium sulphate precipitation with MEP-HyperCel. The purity could reach 77.4%, and the yield was about 11.5mg/ml porcine plasma.Finally, three kinds of home-made thiophilic mixed-mode adsorbents were investigated, including Cell-TuC-DVS-MMI, Cell-TuC-DVS-MEP and Cell-TuC-DVS-MBI. It was found that pH was the key factor to affect adsorption. Cell-TuC-DVS-MMI was best for immunoglobulin purification with high adsorption capacity, good pH sensibility and high selectivity for immunoglobulin. Moreover, the separation conditions were optimized. With the supernate of ammonium sulphate precipitation as sample loading at pH 5, the albumins could be removed with the elution at pH 7, and the porcine immunoglobulin was obtained with the elution at pH3.8. The purity reached 94.3%, and the purification factor was about 5.6.In the present work, the precipitation with ammonium sulphate and mixed-mode chromatography were combined. The fibrinogen was separated with ammonium sulphate precipitation from porcine plasma, the supernate was loaded directly for mixed-mode chromatography and the immunoglobulin with high purity was obtained. This technology provided a new approach for the comprehensive development and utilization of porcine blood resources.