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

doi:10.3724/SP.J.1123.2019.03019

Chinese Journal of Chromatography ›› 2019, Vol. 37 ›› Issue (10): 1090-1097.DOI: 10.3724/SP.J.1123.2019.03019

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

LIU Rangdong, XU Xinyao, WANG Weiwei, WANG Yan, YAN Chao

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2019-03-16 Online:2019-10-08 Published:2020-12-11
Supported by:
National Natural Science Foundation of China (Nos. 21874088, 81874307), Shanghai Science and Technology Commission Scientific Research Project (Nos. 16142200300, 17142201000, 18142200700); Shanghai Standardization Promotion Project (No. 17DZ2201500).

Abstract

Abstract: 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 (pI) of trastuzumab and etanercept. In parallel, using the newly built capillary isoelectric focusing platform, the pI 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 pI values of monoclonal antibodies and fusion proteins in protein drugs. Moreover, the measured pI values were consistent with those estimated using the HPC capillary.

Key words: capillary isoelectric focusing (cIEF), immobilized pH gradient (IPG), isoelectric point (pI), online platform, protein separation

CLC Number:

O658

LIU Rangdong, XU Xinyao, WANG Weiwei, WANG Yan, YAN Chao. Preparation of capillary isoelectric focusing with monolithic immobilized pH gradient and its application in determination of isoelectric point of proteins[J]. Chinese Journal of Chromatography, 2019, 37(10): 1090-1097.

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

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