Profiling Released High Mannose and Complex N-Glycan Structures from Monoclonal Antibodies Using RapiFluor-MS Labeling and Optimized Hydrophilic Interaction Chromatography

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Scott A. McCall, Matthew A. Lauber, Stephan M. Koza, Erin E. Chambers
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This application highlights the development of an LC method that optimizes the chromatographic resolution for the released N-glycans that are commonly found on mAbs.

The N-linked glycosylation of mAbs can impact their circulation half-life and efficacy. Therefore, it is particularly important for the N-glycans of a mAb to be well characterized and routinely monitored.

By labeling mAb N-glycans with RapiFluor-MS, high sensitivity detection by both fluorescence and MS is made possible. The sample loading condition, gradient steepness, flow rate, and separation temperature of the universal N-glycan profiling method were adjusted to create a mAb N-glycan profiling method that was able to better resolve the Man5/A2G1+FA1G1 and FA2G2Sg1/FA2G2Ga2 critical pairs. The mAb N-glycan profiling method yielded a half-height resolution of 1.61 for M5/A2G1+ N-glycans and FA2G2Sg1/FA2G2Ga2 of 1.13.

By improving the resolution of these critical pairs of N-glycans, we have provided additional separation space for monitoring high mannose structures. To this end, the RapiFluor-MS High Mannose Standard was used in a series of spiking experiments to demonstrate the quantitative performance of this new gradient for analyzing high mannose N-glycan structures.

Using the mAb N-glycan profiling method in conjunction with the new RapiFluor-MS High Mannose Standard and the RapiFluor-MS Dextran Ladder allows for the easier adoption of this system solution.

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