Global characterization of modifications to the charge isomers of IgG antibody
- Author:
Xinling CUI
1
;
Wei MI
;
Zhishang HU
;
Xiaoyu LI
;
Bo MENG
;
Xinyuan ZHAO
;
Xiaohong QIAN
;
Tao ZHU
;
Wantao YING
Author Information
1. State Key Laboratory of Proteomics
- Keywords:
Antibody;
Charge isomers;
Mass spectrometry;
Posttranslational modification;
Glycopeptide
- From:
Journal of Pharmaceutical Analysis
2022;12(1):156-163
- CountryChina
- Language:Chinese
-
Abstract:
Posttranslational modifications of antibody products affect their stability,charge distribution,and drug activity and are thus a critical quality attribute.The comprehensive mapping of antibody modifications and different charge isomers(CIs)is of utmost importance,but is challenging.We intended to quanti-tatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity.The CIs of antibodies were fraction-ated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection,followed by stepwise structural characterization at three levels.First,the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach;this showed differences in glycoforms and deamidation status.Second,at the peptide level,common modifications of oxidation,deamidation,and glycosylation were identified.Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs.In total,10 N-glycoforms were detected by peptide mapping.Finally,an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides.Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms.The results revealed that sialic acid modification is a critical factor ac-counting for charge heterogeneity,which is otherwise missed in peptide mapping and intact molecular weight analyses.This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.
