Development Of Chromatography Matrices For Antibody Purification

Chromatographic separation has been widely used in the research of the separation and purification of antibodies. As agarose gel has the advantages of good hydrophilicity, biocompatibility, a suitable construction and easy to modification, it has been widely used in the bioseparation field. However, the high price of the import agarose gel prevent it been applicated in a large scale. Generally, polyethyleneimine precipitation is used to remove the nucleic acids from the supernatant of disrupted cells in the laboratory. However, the higher isoelectric point of basic proteins will combine with the nucleic acids and precipitate by using the polyethyleneimine precipitation, which will cause the protein loss. Based on the previous works, this paper further optimizes the preparation of agarose gel. By using the agarose gel as the matrix, ethanediamine as the link, a strong-base anion exchanger whose functional group is betaine has been successfully synthesized. This paper also optimized the experimental conditions of the remove of nucleic acids. Since different substrate materials have different constructions, functional groups, diameters and anti-pressure levels, each of them has their advantages during the separation and purification. In this paper, MEP have been used as the funcitional group, combining with three substrate materials respectively to synthesize chromatographic packings for the separation and purification of antibodies. The synthesis conditions have been optimized and the separation capability of these packings also have been tested.The main method to prepare agarose gel is reversed suspended agitation. By researching the cross-linking and screening, agarose microspheres which are physicochemical stable and in a certain range of diameters have been gained. It is found that adding water and standing will be better to demulsify. Select200mesh and screening4times can get the ideal range of diameters.The agarose gel is uesd as the substrate material to synthesize the nucleic acid adsorbent. This experiment compared the substrate materials in different densities which are activated respectively by epichlorohydrin and carbonyl diimidazole in different quantities. The ethanediamine linked with betaine by EDC reaction in order to get nucleic acid adsorbent in different densities of aglucons. The experiment tested different conditions to remove the nucleic acid, and when pH8.5, NaCl1.0mol/L, adsorbent0.34g/ml, the effective of the removDifferent kinds of matrixes binding MEP are used to the separation and purification of the antibodies. The performance parameters of the three matrixes have been firstly evaluated. By optimizing the experimental conditions agarose gel and spherical cellulose:activation time8h, NaOH1.0mol/L, activator0.08mL/g gel, N-bromosuccinimide0.08g/mL, bromination reaction time1h, MEP0.06g/g gel, coupling time20h, coupling PH10. Under the above conditions, the more stable synthetic material with a higher density ligand can be gained; Polyvinyl alcohol microspheres:activation time20h, NaOH1.2mol/L, activator0.10mL/g gel, bromination reaction time1h, MEP0.04g/g gel, coupling time20h, coupling PH10. Under the above conditions, the more stable synthetic material with a higher density ligand can be gained. Further researches have been done to evaluate the adsorption selectivity to the antibodies in the serum and the pressure resistance of different synthetic materials. Since the matrix is not able to adsorb antibodies by itself, the selectivity of synthetic materials is similar. But the pressure resistances have a great difference, which shows of nucleic acids is better and less loss of the target proteins.