This module is an extension of the Level 1 and Level 2 N-glycan characterization work to gain more detailed information on the individual N-glycan sites. This can be achieved by site occupancy analysis and detailed site-specific glycosylation analysis.
Level 3 N-glycan Site-Specific Glycosylation Analysis
The number of potential N-glycosylation sites is determined by the amino acid sequence of the glycoprotein. This module provides HILIC-UPLC-FLR profiles for the N-glycans at each site occupied by N-glycans. The glycoprotein is digested into peptides and glycopeptides using proteases. These are then separated by C18 column chromatography and fractions collected. Each fraction is treated with PNGase F to release the N-glycans that are then fluorescently labelled and analysed by HILIC-UPLC-FLR to obtain an individual site glycosylation profile.
Determination of site glycosylation is a non-standard glycoprofiling module because the exact conditions required will differ for each glycoprotein analysed.
The amino acid sequence and the formulation buffer can both have an effect on the exact protocol followed as well as on generated data.
The analysis will be performed on a single glycoprotein sample, alongside Ludger positive and negative controls, and system suitability standards.
Glycan profiles for each individual site are identified by comparison of the data obtained from Level 1 and Level 2 analyses.
This module is suitable for:
- quality control – profile comparisons to monitor known structures
- monitoring batch to batch consistency
- comparability studies
In order to gain more detailed information on N-glycan % occupancy at each site, site occupancy analysis is recommended.