Separation of monoclonal antibody charge state variants by open tubular capillary electrochromatography with immobilised protein as stationary phase.
Monoclonal antibodies (mAbs) are highly heterogeneous and complex glycoproteins requiring powerful analytical tools for characterization and quality control. In this work, we utilize adsorbed bovine serum albumin (BSA) as a stationary phase in open tubular (OT) capillary electrochromatography for separation of charge state variants of mAbs. Poly(diallydimethylammonium chloride) (PDDA) was used to assist fabrication of BSA coated OT column by electrostatic self-assembly. Scanning electron microscopy and electroosmotic flow measurement were carried out to characterize the as-prepared BSA coated PDDA OT columns. The electrochromatographic performance of the OT columns was evaluated by separation of basic proteins and different charge state variants of mAbs. The effects of background solution pH and concentration on separation were investigated. A rapid separation of charge state variants of mAbs was successfully achieved in the BSA coated PDDA OT column. Separation of seven variants of the mAb cetuximab was achieved using the prepared column. Two basic variants and one acidic variant of rituximab, and two basic variants and four acidic variants of trastuximab were successfully distinguished from the main forms. In addition, the columns demonstrated good repeatability and stability with the run-to-run, day-to-day and batch-to-batch relative standard deviations of migration times less than 3.7%.
Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
Yamin
Y
Zhang
Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
Wentao
W
Wang
Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
Xue
X
Xiao
Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China. Electronic address: jiali@scnu.edu.cn.
Li
L
Jia
D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D053801:Capillary Electrochromatography; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D053841:Electroosmosis; D055767:Immobilized Proteins / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry; D055672:Static Electricity
D000911:Antibodies, Monoclonal; D055767:Immobilized Proteins; D012710:Serum Albumin, Bovine
Charge heterogeneity; Monoclonal antibody; Open tubular capillary electrochromatography; Separation