固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用

Preparation of capillary isoelectric focusing with monolithic immobilized pH gradient and its application in determination of isoelectric point of proteins

通过聚合物原位聚合反应,制备了部分填充的毛细管整体柱。pH3~10的载体两性电解质被固化在该毛细管整体柱上。在引入八通进样阀、三通阀和四通连接单元的基础上,构建了适用于固化pH梯度毛细管等电聚焦整体柱(M-IPG)的平台。在蛋白质药物测定过程中,用M-IPG柱和羟丙基纤维素(HPC)涂层毛细管柱同时对曲托珠单抗和依那西谱的等电点进行了测定。结果表明,两种等电聚焦柱都能够同时分离混合蛋白质样品并测定蛋白质类药物中单抗和融合蛋白质的等电点(pI),M-IPG柱所测的pI值与HPC涂层毛细管柱测定的结果基本一致,表明了该柱在进一步构建多维分离平台进行蛋白质组学研究方面的潜力。

A partially filled monolith was prepared by in situ polymerization, and then carrier ampholytes (CAs, pH 3-10) were immobilized on its surface.For effective utilization of capillary isoelectric focusing (cIEF) with the monolithic immobilized pH gradient (M-IPG), a new online platform was established by the introducing of an eight-way injection valve, a three-way valve and a cross-shaped unit.Besides, a capillary coated with hydroxypropyl cellulose (HPC capillary) was prepared and used to determine the isoelectric points (p I ) of trastuzumab and etanercept.In parallel, using the newly built capillary isoelectric focusing platform, the p I values of trastuzumab and etanercept were measured with the M-IPG column, and compared with the results obtained using the HPC capillary.It was found that these two cIEF columns can be effectively used to separate proteins and determine the p I values of monoclonal antibodies and fusion proteins in protein drugs.Moreover, the measured p I values were consistent with those estimated using the HPC capillary.