Sialic Acid and Monosaccharide Analyses

Sialic Acid Analyses

A diverse range of sialic acids are found in nature, but the two major sialic acid species found on N- and O-linked glycans contained in biopharmacuticals are N-acetyl-neuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc). Since sialylation can enhance serum half-life, as well as affect biological activity, it is important to accurately monitor both the quantitative levels and types of sialic acids during all stages of the product life cycle.

Many LC-based methods begin with the release of the targeted sialic acids under milder acid hydrolysis conditions (e.g., 2 M acetic acid for 2 hours at 80 °C). The released sialic acids then can be derivitized with 1,2-diamino-4,5-methylenedioxybenzene-2HCl (DMB) dye.  

Monosaccharide Analyses

The primary monosaccharides that are found in N- and O-linked glycans are the neutral monosaccharides N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), galactose (Gal), glucose (Glc), mannose (Man), and fucose (Fuc). The analysis of non-charged monosaccharides frequently begins by acid hydrolysis of the glycan by incubation with trifluoroacetic acid (TFA) or hydrochloric acid (HCl). Usually, a three-hour incubation at 100 °C with 2 M TFA will release all of the monosaccharides. However, it should be noted that during hydrolysis, the N-acetyl groups on GlcNAc and GalNAc are hydrolysed to glucosamine (GlcN) and galactosamine (GalN). Following hydrolysis, the released monosaccharides are derivatized using 2-aminobenzoic acid (2-AA).