After the last blog post on improving monoclonal antibody analysis using a newly released column and buffer solution (link to post), I am pleased to add two recently released application notes on the HILIC HPLC analysis of human and bovine fetuin glycans that do not have ionizable groups to the growing library of biopharma posts on this blog!
As has been mentioned before on this blog, glycans are key biomarkers for disease states, such as cancer, and may also have the potential to reveal predisposition to other diseases. As minor changes in glycan structure can result in dramatic differences in cell function, it is vital to be able to efficiently separate all isomeric and branching variants present in a glycan sample to achieve maximum structural elucidation. This structural elucidation of glycans is required for the development of biopharmaceutical drugs using monoclonal antibodies.
Both application notes below were developed by chemists from our Runcorn, UK, Applications Lab in collaboration with one of our customers: Ludger Ltd, Oxford, UK.
HILIC HPLC Separation of Human IgG Glycans
In Application Note 20703, Analysis of Human IgG Glycans on a Solid Core Amide HILIC Stationary Phase, (downloadable PDF) the chemists describe a method that is simple, robust, and highly efficient using our rapid-separation HPLC system (Thermo Scientific Dionex UltiMate 3000 RSLC HPLC system) equipped with our fluorescence detector (Thermo Scientific Dionex FLD Fluorescence Detector) with one of our solid-core HPLC columns (Thermo Scientific Accucore 150-Amide-HILIC HPLC column).
This particular column is designed for the separation of hydrophilic biomolecules in HILIC mode and provides fast and highly efficient separations as a result of the column particles having a solid core and a porous outer layer. Another advantage of these columns is that they can be used with a system backpressure that is compatible with conventional HPLC systems.
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The chromatograph shows excellent separation of the 7 glycans in the sample at under 20 mins.
HILIC HPLC Analysis of Bovine Fetuin Glycans
In Application Note 20702, A HILIC Method for the Analysis of Bovine Fetuin Glycans, (downloadable PDF) the chemists use the same system setup as in the previous application note above to separate 8 bovine fetuin glycans in less than 25 mins. Again, the method is simple, robust, and highly efficient.
NOTE: As mentioned in both the application notes, for glycans with ionizable groups, you can use one of our mixed mode HILIC HPLC glycan columns (Thermo Scientific GlycanPac AXH-1 column) which provides the simultaneous separation of glycans by charge, size, and polarity. Here’s a poster note that you can download showing the use of the column in a method for the analysis of charged glycans: Novel Glycan Column Technology for the LC-MS Analysis of Labeled and Native N-Glycans Released from Proteins and Antibodies. (Downloadable PDF).
If you have any questions on this column or type of analysis, do enter your questions in the Comments box below; our experts look forward to hearing from you.