Genovis / SmartEnzymes / Applications / Sialic Acid Linkage Analysis

SmartEnzymes™

 

Complete Desialylation of Biopharmaceuticals

Efficient Removal of Sialic Acids

Exoglycosidases are enzymes that hydrolyze glycosidic bonds of terminal monosaccharide residues. Genovis provides exoglycosidases for removal of specific monosaccharides for analysis of glycoproteins (Figure 1). The enzymes vary in their substrate specificity and can be used to specifically remove different types of monosaccharides such as sialic acids, galactose and more. The enzymes may also differ in what glycosidic linkage they hydrolyze, for example SialEXO 2-3 specifically hydrolyzes sialic acids linked with an alpha-2,3 bond and no other type of linkages. The exoglycosidases are available in immobilized format to increase performance and provide preparation without enzyme in the sample. Exoglycosidases are important tools for analytical workflows to study both the present glycoforms and the underlying glycoprotein.

Sialic Acid Linkage Analysis

Sialic Acid Linkage - SialEXO

Figure 1. HILIC analysis of released glycans with α2-3 and α2-6 linked sialic acids released by SialEXO 2-3 and SialEXO Lyophilized respectively. Defined N-glycan libraries consisting of biantennary glycans modified with either α2,3 (left) or α2,6-linked sialic acid (right) were treated with SialEXO for 2 h at 37°C and analyzed by HILIC-FLD HPLC (ThermoScientific™ Accucore™ 150 Amide HILIC column, 2.1 x 150 mm).

The enzymatic specificities of exoglycosidases can be used to determine the glycosidic linkages of the glycoforms on biopharmaceuticals. Sialic acid is primarily present on biopharmaceuticals linked as alpha2,3 or alpha2,6. SialEXO is a broad acting sialidase mix that will hydrolyze all sialic acid linkages including alpha2,3, alpha2,6, and alpha2,8. The enzymatic activity will result in a complete desialylated sample that can be further studied using analytical technologies. SialEXO 2-3 is an enzyme that display high specificity for alpha2,3 linked sialic acids, as demonstrated on the released glycans in Figure 1.

 

Hydrophilic interaction liquid chromatography (HILIC) was used to analyse two glycan libraries. One library contained released glycans with α2-3 linked sialic acids and the other had α2-6 linked sialic acids. Treatment with SialEXO Lyophilized or SialEXO 2-3 Lyophilized was compared (Fig. 2). The shift in peaks clearly shows the release of α2-3 and α2-6 linked sialic acids from the glycans by SialEXO 2-3 Lyophilized and SialEXO Lyophilized respectively.

 

The specificities of SialEXO Lyophilized and SialEXO 2-3 Lyophilized were further studied using the synthetic substrate sialyllactose and fluorescence intensity as readout. Sialyllactose was analysed with an enzyme coupled reaction where the oxidation of free sialic acid creates an intermediate. The intermediate reacts stoichiometrically with a probe, generating a product that can be detected. Figure 2 shows that both SialEXO 2-3 Lyophilized and SialEXO Lyophilized perform equally for removing α2-3 linked sialic acids from α2-3 sialyllactose. However, for α2-6 sialyllactose which has α2-6 linked sialic acids, then only SialEXO Lyophilized shows activity whereas SialEXO 2-3 Lyophilized does not. This demonstrates SialEXO 2-3 specificity for α2-3 linked sialic acids.

SialEXO 2-3 - Sialic Acid Linkage

 
Figure 2. SialEXO 2-3 specificity demonstrated by its' activity on synthetic substrates. Measurement was done using a a microplate reader with fluorometric detection of released sialic acids.

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