Measuring the Glycan Occupancy of Intact mAbs using HILIC and Detection by Intrinsic Fluorescence

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Matthew A. Lauber and Stephan M. Koza
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In addition to profiling the heterogeneity of glycosylation, it is also critical to assay glycan occupancy. Here, we demonstrate that a Glycoprotein BEH Amide column, purposefully designed for large molecule HILIC separations, can be used to directly quantify incomplete N-glycan occupancy in intact mAb samples. Unlike a conventional CE-SDS separation of reduced mAbs, this technique provides a non-inferred assessment on the nature (i.e. 1 N-glycan versus 0 N-glycans) of glycan occupancy for the intact mAb. The proposed HILIC methodology is also MS-compatible, making it possible to readily confirm the assignments of observed peaks.

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