Purification of Monoclonal Antibodies by Ion-Exchange Displacement Chromatography

本研究对单克隆抗体的纯化进行了研究， 抗体（MAb）。这项工作将集中在净化 小鼠igg 1 和IgM，以及小鼠IgG的亚分级 IgG 1、IgG 2a、IgG 2b和IgG 3。 单克隆抗体由以下任一种方法生产： 从小鼠收集腹水， 在其腹膜腔中注射杂交瘤细胞，或 通过收集其中杂交瘤 在体外培养细胞。 不管是什么方法， 抗体必须从复杂的蛋白质混合物中分离出来。 该提案建议使用离子交换分散 通过色谱法进行纯化， 亚分馏 在此方法中，首先传递提要 抗体吸附在柱子上。 实验将在制备型HPLC系统上进行 以弱阴离子交换剂PAE-1000为吸附剂。 的 下一步是置换吸附的分子。的PI 建议使用葡聚糖硫酸盐作为置换剂。的PI 将研究定制分子的潜力， 葡聚糖硫酸盐的重量和电荷，以改变 硫酸盐对吸附剂的亲和力相对于 抗体的其中一些裁剪将涉及综合 由于商业上只有两种分子量的硫酸盐 可用，并且其中每一种仅具有一种硫酸盐含量， 因此是电荷密度。纯组分等温线测量 将承担的单克隆抗体，硫酸盐和两个 代表性的污染蛋白、转铁蛋白和 白蛋白。这些等温线测量将用于确定 合适的置换剂，然后是多组分等温线， 将测定置换剂、MAb、转铁蛋白和白蛋白。 最后，将在柱中进行位移实验。 进行。

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.