Purification of Monoclonal Antibodies by Ion-Exchange Displacement Chromatography

点击翻译按钮获取中文摘要

This proposal studies the purification of monoclonal antibodies (MAbs). The effort will focus on the purification of mouse IgG1 and IgM, and a subfractionation of mouse IgG into IgG1, IgG2a, IgG2b and IgG3. MAbs are produced by either the collection of ascites fluid from mice which have been injected with hybridoma cells in their peritoneal cavity, or by the collection of the nutrient medium in which hybridoma cells are cultured in vitro. Whatever the method, the antibody must be separated from a complex mixture of proteins. The proposal suggests using ion exchange dispacement chromatography to effect the purification and subfractionation. In this method, the feed is first passed through a column, and the antibodies adsorb onto the column. Experiments will be conducted on a preparative HPLC system using the weak anion exchanger PAE-1000 as the adsorbent. The next step is to displace the adsorbed molecules. The PI suggests using dextran sulphates as the displacers. The PI will investigate the potential of tailoring the molecular weight and charge of dextran sulphates in order to vary the affinity of the sulphates to the adsorbent relative to the antibodies. Some of this tailoring will involve synthesis since only two molecular weight sulphates are commercially available, and each of these at only one sulphate content and therefore charge density. Pure component isotherm measurements will be undertaken of the MAbs, sulphates and two representative contaminating proteins, transferrin and albumin. These isotherm measurements will be used to determine suitable displacers, and then multicomponent isotherms of the displacers, MAbs, transferrin and albumin will be determined. Lastly, displacement experiments in the column will be undertaken.