Guide to Sialylation: II Highly Sialylated Glycoproteins

Glycan sialylation is important for the safety and efficacy of a drug

In humans the major sialic acid on glycans is Neu5Ac, however Neu5Gc (which has a glycolyl group instead of an acetyl) can be added to glycoproteins that are expressed in different cell lines. This non-human Neu5Gc may elicit an immunological response which can lead to inflammation and/or neutralisation (and therefore loss of efficacy) of the biopharmaceutical. Sialylation of glycans can also have a major effect on the pharmacokinetic properties of a drug: for example, glycans with terminal galactose (without sialic acids) are removed from circulation by the asialoglycoprotein receptor in the liver. Glycoproteins such as EPO can have sialic acids with extra acetylation (e.g. Neu5,9Ac2) which may affect drug efficacy. Sialylation can also be important for receptor binding and signal transduction; for instance the presence of sialic acid on IgG Fc glycans reduces ADCC (Antibody Dependent Cellular Cytotoxicity) activity and is anti-inflammatory. 

 Identification and quantification of sialic acids
a)DMB Sialic Acid Labelling Kit (Cat# D491322) to release, identify and obtain relative quantitation of Neu5Ac, Neu5Gc and Neu5,9Ac2 by (U)HPLC analysis. We use the fluorescent tag DMB to specifically label sialic acids, then use (U)HPLC with fluorescent detection to detect and quantitate them  (see Figure 1) The use of a fluorescent tag makes the assay more sensitive than PAD. As the DMB tag only becomes fluorescent when labelling the sialic acids (across 2 adjacent keto groups), this specificity also overcomes issues due to other components in the sample buffer (which can be a concern with PAD detection).

b) The absolute amounts of Neu5Ac and Neu5Gc on a glycoprotein can also be determined by comparison to Ludger quantitative standards (Cat # B491176,B491217).

c)Quantitative sialylated glycopeptide standards are recommended to check the efficiency of glycan release, labeling, and recovery.

Figure 1:RP-HPLC Analysis of DMB Fluorescent Labelled Sialic Acids (released by mild acid hydrolysis) from the Sialic acid Reference Panel. This Reference Panel is part of the D491322 kit .

Analysis and characterisation of sialylated glycans

N-glycans are removed from the biopharmaceutical by PNGase F(Cat #P420186).The glycans are fluorescently labelled with 2AA or 2AB using using LudgerTag kits incorporating 2-picoline borane reductant and cleaned up using cartridges. 

a) Analysis by HILIC- (U)HPLC provides GU values which can be matched to database and standard GU values to obtain preliminary structure assignments.

b) Further analysis following exoglycosidase sequencing can be used to characterise the structures present (Figure 2). For example peaks that disappear (or reduce) following digestion with sialidase (Cat# N489858) will have contained sialylated structures.

 

Figure 2: Exoglycosidase sequencing of EPO; 2AB labelled glycans run on a LudgerSep-N2 Column Enzyme specificities: a3Sialic = 1-3 sialic acid; a36Sialic = 1-3 & 6 sialic acids; Fuc = 1-6 fucose; bGal = b1-4 galactose; GlcNAc = b1-2,4,6 N-acetyl-glucosamine.

c) Charge separation of labelled glycans on a  weak anion exchange (WAX) column can provide data on the relative proportions of the mono-, di-, tri- and tetra-sialylated glycans. (Figure 3).c) Charge separation of labelled glycans on a  weak anion exchange (WAX) column can provide data on the relative proportions of the mono-, di-, tri- and tetra-sialylated glycans. (Figure 3).

Figure 3: Weak anion exchange (WAX)-HPLC Charge separation of 2AB labelled Glycans on a column. Top trace (red): fetuin N-glycan. Bottom trace(blue): EPO N-glycans
For complex mixtures, these differently charged fractions can be separated and analysed further by HILIC-(U)HPLC with exoglycosidase sequencing to fully characterise the structures (Figure 4):

Figure 4: Charge fractionation of EPO N-glycans by WAX-HPLC followed by HILIC-UPLC.
d) Mass composition data can be obtained by MALDI analysis following permethylation to stabilise the sialic acids (Figure 5).

Figure 5: MALDI analysis of permethylated EPO N-glycans

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