Structural Characterisation of Monoclonal Antibody Based on LC-MS: A General Method
Huairu Tian, Chen Zhang, Qian Li, Siyuan Wang, Junjie Shen, Shuqiang Zhao, Le Guo, Guolin ZhangIntroduction:
To establish a universal method based on LC-MS for the structural characterization of mAbs. This method includes determining the intact molecular weight, deglycosylated intact molecular weight, reduced molecular weight, and deglycosylated-reduced molecular weight of mAbs, along with peptide mapping (amino acid sequence coverage) and glycan profiling.
Methods:
LC-MS analysis was performed using mAb injections as test samples. A MAbPac™ RP column was coupled to intact mass spectrometry for molecular weight measurement. For peptide mapping, trypsin-digested samples were analyzed using an ACQUITY UPLC Peptide CSH C18 column with tandem mass spectrometry (MS/MS) to assess amino acid sequence coverage. Glycan profiling was performed using an ACQUITY UPLC Glycan BEH Amide column, with 2-ABlabeled glycans analyzed via secondary mass spectrometry.
Results:
For bevacizumab (three manufacturers), sintilimab, and camrelizumab, the intact molecular weight, deglycosylated molecular weight, reduced molecular weight, and deglycosylatedreduced molecular weight of mAbs showed errors of less than 10 ppm when compared with theoretical sequences, confirming consistency with theoretical values. Peptide mapping demonstrated that the amino acid sequence coverage of bevacizumab and sintilimab matched the theoretical sequences. Glycan profiling identified distinct glycan profiles for the tested mAbs.
Discussion:
A universal LC-MS-based method was developed for comprehensive structural characterization of monoclonal antibodies, including molecular weight, peptide mapping (sequence coverage), and glycan profiling. The method is efficient, straightforward, and validated across three mAb therapeutics, providing reliable support for the development and analysis of mAbbased therapeutics.
Conclusion:
This method allows companies to directly apply it to quality control. It has been validated with several target biologics, demonstrating its versatility and general applicability.