Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry |
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Authors: | Chen Xiaoyu Flynn Gregory C |
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Institution: | Process and Analytical Sciences, Amgen, Inc., Thousand Oaks, CA 91320, USA. |
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Abstract: | An on-line reversed-phase (RP) high-performance liquid chromatography/mass spectrometry (MS) method has been developed for profiling and characterizing N-glycans from recombinant immunoglobulin G antibodies. In this method, released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2AB) and separated on a RP column with on-line fluorescence and MS detection. The method achieves good resolution of all major glycans and segregates glycan types (high-mannose, hybrid, and complex) to different regions of the chromatogram, thus allowing accurate quantification of N-glycans from the fluorescent signal alone. Moreover, the mobile phase used allows high quality on-line MS detection. The 2AB-labeled N-glycans demonstrate good ionization efficiency in electrospray and generate primarily doubly charged M+2H](2+) ions. The mass and structural information can be readily obtained from the on-line MS and tandem MS data. As little as 70 fmol glycan species can be detected and identified. |
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Keywords: | N-linked glycans Oligosaccharides Antibody LC/MS Tandem MS |
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