Glycosylation Site Analysis
Glycosylation site analysis is used to determine the population profile of the N-glycans or O-glycans present at each glycosylation site in a glycoprotein. This information can be used in all stages of drug development in conjunction with linkage analysis to provide a detailed picture of the glycans present. It can also be used to demonstrate consistency between batches for release during the manufacturing process.
Glycosylation site analysis is a powerful technique in glycoprotein analysis. It can be used for:
Screening of glycoproteins manufactured using different cell lines
Identification of unusual glycosylation profiles
Determination of occupancy of glycosylation sites
Glycosylation sites are initially identified as part of a peptide mapping study using LC-MS. Glycosylation at each individual site is then characterised following release of the glycans from each purified glycopeptide using:
Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI-MS) to give molecular ion data
Electrospray Mass Spectrometry (ES-MS) to give fragment ion information (which is useful for more detailed structural interpretation)
High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) to give a chromatographic profile
Glycosylation site analysis is a key part of the requirements laid out in ICH Q6B guidelines for characterisation and confirmation of biopharmaceuticals in support of new marketing applications.