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

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.