LC-MS/MS BIOPHARMACEUTICAL GLYCOANALYSIS
John E. Schiel, Jennifer Au, Hua-Jun He, Karen W. Phinney
Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications. The quantity, location, and size of glycans attached can vary, potentially resulting in glycoforms with different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry-based techniques, each of which provides various aspects of characterization. The current report describes the development and comparison of two LC-MS/MS protocols for biopharmaceutical glycoanalysis.
The first technique utilized pronase, a mixture of proteases capable of cleaving any peptide bond, to digest the glycoprotein into amino acids and short glycopeptides. The resultant glycopeptides were analyzed using a combination of MALDI-TOF MS and graphitized carbon LC-MS/MS.
In the second method, glycans were enzymatically released from the protein, labeled with 2-aminobenzamide (2-AB), and analyzed using LC-Fluorescence-MS/MS. Interpretation of the combined methods allowed for identification of glycan identity, glycosylation site, and relative quantification. The relative advantages, disadvantages, and similarities between each method will be discussed. These methods are widely applicable and show promise for the rapid analysis of biopharmaceutical glycosylation.