Evidence for reliable and consistent glycosylation of glycoprotein therapeutics is typically obtained through LC or LC-MS analysis of released N-linked glycans. However, drug discovery and development organizations, as well as regulatory agencies, frequently find value using orthogonal techniques to determine and/or confirm quantitative levels of sialic acids and monosaccharides contained in a therapeutic protein due to potential effect on biological activity and stability of the drug.
Waters offers UPLC, UHPLC, and HPLC-based C18 columns and defined application conditions that can assist scientists with the analysis and quantitation of these important glycan derived components.
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.
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).