Purification of Monoclonal Antibodies by Ion-Exchange Displacement Chromatography

本课题旨在研究单克隆抗体（MAbs）的纯化。这项工作将集中在小鼠IgG1和IgM的纯化上，并将小鼠IgG分离成IgG1、IgG2a、IgG2b和IgG3。单克隆抗体是通过收集腹腔内注入杂交瘤细胞的小鼠的腹水或收集体外培养杂交瘤细胞的营养培养基产生的。无论采用何种方法，抗体都必须从复杂的蛋白质混合物中分离出来。建议采用离子交换置换色谱法进行纯化和分馏。在这种方法中，进料首先通过一个柱，抗体吸附到柱上。实验将在以弱阴离子交换剂PAE-1000为吸附剂的制备型高效液相色谱系统上进行。下一步是置换被吸附的分子。PI建议使用葡聚糖硫酸盐作为替代物。PI将研究调整葡聚糖硫酸盐的分子量和电荷的潜力，以改变硫酸盐相对于抗体对吸附剂的亲和力。由于商业上只有两种分子量的硫酸盐，而且每种硫酸盐含量和电荷密度都只有一种，因此这种裁剪将涉及合成。将对单克隆抗体，硫酸盐和两种代表性的污染蛋白，转铁蛋白和白蛋白进行纯成分等温线测量。这些等温线测量将用于确定合适的置换剂，然后确定置换剂、单克隆抗体、转铁蛋白和白蛋白的多组分等温线。最后进行柱内位移试验。

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.