N-Glycan Profiling Case Study

Title: Identification of low abundant isomeric N-Glycan structures in biological therapeutics by LC/MS

Journal: Analytical Chemistry

Published: 2016

Background

N-linked glycosylation is one of the most common post-translational modifications (PTMs) in therapeutic proteins such as monoclonal antibodies (mAbs). Methods applied for glycan characterization in general require the release of glycans from proteins and fluorescence labeling of the released glycans. The separation methods play critical role in the quantitation of released glycans. High-performance anion-exchange chromatography (HPAEC), reversed-phase liquid chromatography, normal phase (NP)/hydrophilic interaction chromatography (HILIC), and capillary electrophoresis (CE) are widely used in glycan quantitation. In this paper, we applied a method of online LC-MS characterization of 2-AB-labeled glycans in negative mode.

Materials & Methods

N-Glycan Profiling Service

1. Release of Glycans by PNGaseF

2. Glycan Label with 2-AB and Separation of 2-AB-Labeled Glycans

3. Online LC-MS of HILIC-Separated, 2-AB-Labeled Glycans

4. Endomannosidase Treatment

Results

Figure 1 shows the zoomed fluorescence chromatogram of HILIC UPLC-MS analysis of 2-AB-labeled, N-linked glycans released from a mAb. Structural isomers, such as G1F and G1F′, can be separated by HILIC UPLC and identified by MS. For species coeluted in HILIC UPLC, they can be easily identified and differentiated by their unique masses using the online MS detection. In this paper, we applied this approach on characterization of three low abundant isomeric structures (A1G1F, G1F+GlcNAc, Man8) from a human therapeutic mAb. These three glycans are highlighted in red in Figure 1. Their abundances are all less than 1% of the total glycans, as determined by fluorescence signal. The structures of these low abundant species are shown in Figure 1.

The design of UPLC-HILIC with fluorescence and online mass spectrometry detection enabled the analysis to provide orthogonal resolutions for structural isomers of glycans. The added MS detection further expanded the method capability to resolve and identify unknown glycan species.

N-Glycan Profiling Case Study Figure 1. Zoomed fluorescence chromatogram of HILIC UPLC-MS analysis of 2-AB-labeled, N-linked glycans released from a mAb ^[1]

Reference

Zhao J.; et al. Identification of low abundant isomeric N-glycan structures in biological therapeutics by LC/MS. Analytical Chemistry. 2016, 88(14):7049-59. https://doi.org/10.1021/acs.analchem.6b00636

* For Research Use Only. Not for use in diagnostic procedures.

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