N-glycans profiling is essential for the production of biopharmaceuticals. Yaohai Bio-Pharma provides N-glycan profiling service using Liquid Chromatography-Mass Spectrometry (LC-MS) of released and labeled glycans. The general steps for preparing N-glycans sample include digestion of the glycoprotein (GP), de-glycosylation, purification, labeling/derivatization, and Solid Phase Extraction (SPE).
We have been involved in the Protein Structural Characterization of various large molecules, including Recombinant Subunit Vaccines, Nanobodies/VHHs/ Single-domain Antibodies (sdAbs), Antibody Fragments, Hormones/Peptides, Cytokines, Growth Factors (GF), Enzymes, Collagens, etc.
Regulatory Requirements for N-Glycan Profiling
According to ICH Q6B guideline, “for glycoproteins, the carbohydrate content (neutral sugars, amino sugars, and sialic acids) is determined. Additionally, the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile), and the glycosylation site(s) of the polypeptide chain are analyzed to the extent possible.
Analytical Method
| Analysis |
Methods |
| N-glycan profiling |
Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of released and labeled glycans |
Procedures
1. Enzymatic glycan release with Peptide N-Glycosidase F (PNGase F)
2. Derivatization of released glycans by 2-aminobenzamide (2-AB)
3. Purification of the harvested N-glycans on a TSK Amide - 80 column if large quantities of detergents, nonvolatile salts, or materials with free amino groups are present
4. Labelling by fluorescent 2-aminobenzamide (2-AB)
5. LC-MS analysis of labeled N-glycans
6. Data analysis
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