Evidence for reliable and consistent glycosylation of glycoprotein therapeutics is typically obtained through LC-based analysis of N-linked glycans. On occasion, regulatory agencies request information of monosaccharide content as an orthogonal technique for confirming the glycan composition. Beyond profiling changes in total glycan composition, monosaccharide analysis can also be used as an exploratory technique for identifying various monosaccharide modifications, including phosphorylation and sulfation, which can be particularly challenging to discern when analyzing at the released glycan level in the absence of mass spectrometry.
To date, HPLC has been sufficient and reliable for performing monosaccharide analyses, using 2-aminobenzoic acid (2AA)-derivatized glycan hydrolysates where each of the individual monosaccharides were successfully separated using reversed-phase chromatography. More recently, the analytical demands placed on development and QC labs that are tasked with characterizing and monitoring glycoprotein therapies have increased dramatically. Assays used to monitor changes in glycosylation therefore need to be updated in order to maximize productivity through improved analytical efficiency. UPLC offers distinct advantages for monosaccharide analysis in terms of improving analytical quality and reducing assay time. The ACQUITY UPLC H-Class Bio System can be used to perform both HPLC and UPLC methods.