analysis of Glycans & Glycan IsomersThis follow up blog post features more two application notes and a poster note that use a newly released HPLC/UHPLC column for the fast analysis of challenging glycans & glycan isomers (link to blog post). The previous post featured an application for the separation of charged and neutral glycans present in glycoproteins, glycolipids and glycopolymers.

Separation of Glycans Based on Charge, Isomeric Structure and Size

Application 20910, Separation of 2AA-Labeled N-Linked Glycans from Glycoproteins on a High Resolution Mixed-Mode Column, (downloadable PDF), describes the sensitive separation of 2AA-labeled N-linked glycans released from bovine fetuin using the new HPLC/UHPLC glycan column (Thermo Scientific GlycanPacAXR-1, 1.9 μm, 150 × 2.1 mm) which is a reversed-phase, weak anion-exchange mixed-mode column.

The method exhibits excellent separation of glycans based on charge, isomeric structure and size. The results show that 2AA-labeled neutral glycans eluted between 4 and 12 min, monosialylated glycans eluted between 12 and 27 min, disialylated glycans eluted between 27 and 45 min, trisialylated glycans eluted between 45 and 58 min, and tetrasialylated glycans eluted between 58 and 70 min. More than 70 peaks were identified in less than 70 mins! The column substantially increased the resolution of complex N-linked glycan structures, and helped differentiate isomeric structures not resolved by other approaches.

Our rapid separation HPLC system (Thermo Scientific Dionex UltiMate 3000 RSLC system) equipped with one of ourfluorescence detectors (Thermo Scientific Dionex UltiMate 3000 Rapid Separation Fluorescence Detector FLD-3400) was used in the method development.

Separation of 2AA-Labeled N-linked Glycans from Human Immunoglobulin

Application Note 20909, Separation of 2AA-Labeled N-Linked Glycans from Human IgG on a High Resolution Mixed-Mode Column, (downloadable PDF), also uses the same instrument, detection, and column setup as the application note above for a sensitive method for 2AA-labeled N-linked glycans released from human immunoglobulin G (IgG).

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In the method, 2AA-labeled neutral glycans eluted between 5 and 22 mins, monosialylated glycans eluted between 30 and 45 mins and disialylated glycans elute between 45 and 55 mins. More than 40 peaks were identified from the sample. As in the previous application above, the purified 2AA-labeled N-glycans were directly injected under fully aqueous conditions.

Separation of Fluorescently-Labeled Glycans

Poster Note, An Ultra High Resolution Glycan Column for Isomeric Separation and the Structural Identification of Labeled N-Glycans from Proteins Including Antibodies, (downloadable PDF) describes an LC-MS method in which the fluorescently-labeled glycans from various proteins were separated and analyzed on the new glycan column that was coupled to one of our mass spectrometers intended for biological research (Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometer). Overall, 135 unique glycan structures were identified using a combination of the column and the mass spectrometer.

One of our UHPLC systems (Thermo Scientific Dionex UltiMate 3000 UHPLC) was used in the method development.

Check out our blog for more monoclonal antibodies, proteomics, and biopharma chromatography applications.

If you have any questions on the applications or the column, do enter them in the Comments box below; our experts look forward to hearing from you.