Development and verification of an N-glycans identification method for therapeutic monoclonal antibody SIBP-A
10.13200/j.cnki.cjb.004396
- VernacularTitle:治疗性单克隆抗体SIBP-A N-糖谱鉴定方法的建立及验证
- Author:
ZHANG Lin
- Publication Type:Journal Article
- Keywords:
Therapeutic monoclonal antibody;
Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-Tof-MS);
Ultra-high performance liquid chromatography-fluorescence
- From:
Chinese Journal of Biologicals
2025;38(1):27-33
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and verify an ultra-high performance liquid chromatography-fluorescence detection(UPLCFLD) system for the measurement of N-glycans of therapeutic monoclonal antibody SIBP-A, in order to validate the method for the release and stability detection of the monoclonal antibodies.Methods The therapeutic monoclonal antibody SIBP-A was selected as the research subject, the N-glycans of which were identified by using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-Tof-MS), and then were quantitatively analyzed by UPLCFLD. The specificity, repeatability, intermediate precision, linearity and range, as well as robustness of the developed UPLCFLD method were verified. The stability tests of samples at -30 and 10 ℃ were performed, and the detection results were compared with those of samples treated at room temperature.Results Ten main types of glycan molecules were detected by UPLC-FLD, which were identified as M3B, F(6)A1, A2, F(6)A2, Man5, F(6)A2[6]G(4)1, F(6)A2[3]G(4)1, A2[6]G(4)1, A2[6]G(4)1, A2[3]G(4)1 and F(6)A2G(4)2 by mass spectrometry. The peak of SIBP-A was normal, and 80% acetonitrile solution registered no significant interference peak at the sample peak. The relative standard deviation(RSD) of the sum of galactosefree oligosaccharides' peak area percentage was 0. 8% among six parallel samples. In the three tests by two operators, the RSD of the sum of galactose-free oligosaccharides' peak area percentage was 1. 4%. There existed a linear relationship between the protein content within a sample range of 320 to 480 μg and the sum of peak areas for galactose-free oligosaccharides with the R~2of 0. 97. The RSDs of the sum of galactose-free oligosaccharide peak area percentage were determined to be0. 2%, 0. 1%, and 0. 1% at pH 4. 4, 4. 5, and 4. 6 mobile phase A, respectively, and the RSD of the percentage of overall galactose-free oligosaccharide peak area across different pH mobile phase A was found to be 0. 8%. Additionally, the RSDs of the sum of galactose-free oligosaccharide peak area percentage were 0. 5% and 0. 6% respectively after the treated samples were subjected to -30 or 10 ℃ for 24 hours in darkness.Conclusion Based on the UPLC-Q-Tof-MS technique that effectively enables the identification of N-glycans of the therapeutic monoclonal antibody SIBP-A, the developed UPLC-FLD detection method enables accurate quantitative analysis of N-glycans of SIBP-A, exhibiting excellent specificity, repeatability, intermediate precision, linearity and range, robustness, as well as stability for the detection of samples stored at-30and 10 ℃, which can be utilized for the release and stability testing of this antibody.
