Enzymatic tools for sialic acid composition analysis

The sialylation of glycans found on biopharmaceuticals is a growing area of interest. Sialidase enzymes with strict specificity help reveal details regarding sialic acid composition. SialEXO is a sialidase mix for complete removal of α2-3, α2-6 & α2-8 linked sialic acids whereas SialEXO 23 is specific for α2-3 linkages. By combining the results obtained with SialEXO and SialEXO 23, an overview of the sialylation state can be obtained for biopharmaceuticals, facilitating the monitoring of critical quality attributes. This page demonstrates how the activity and specificity of SialEXO and SialEXO 23 can be applied in different applications. SialEXO 23, SialEXO and other commercially available sialidase products are compared.



SialEXO is available for ordering. Buy online or visit 'How to order'. 

Sialidase specificity for synthetic substrates

Fluorescence detection on a microplate reader was used to probe the specificity of SialEXO and SialEXO 23. The synthetic substrate 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 1 shows that both SialEXO 23 and SialEXO 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 shows activity whereas SialEXO 23 does not. This demonstrates SialEXO 23 specificity for α2-3 linked sialic acids.

S23 Apps Fig 1 190527

Figure 1. SialEXO 23 specificity demonstrated by its' activity on synthetic substrates. Measurement was done using a a microplate reader with fluorometric detection of released sialic acids.

HILIC analysis of released glycans

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 or SialEXO 23 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 23 and SialEXO respectively.


SialEXO 23 sialic acid

Figure 2. HILIC analysis of released glycans with α2-3 and α2-6 linked sialic acids released by SialEXO 23 and SialEXO respectively.

Sialic acid analysis of intact glycoproteins 

The activity of SialEXO 23 was compared to SialEXO using SDS-PAGE and lectin blotting, Fig. 3. The substrate was an intact native glycoprotein, Bovine Fetuin. Coomassie staining (A), shows the substrate before (control) and after incubation with SialEXO 23 or SialEXO. The SNA blot (B) reveals that treatment with SialEXO 23 does not release α2-6 linked sialic acids since a band is present. SialEXO is active on α2-6 linked sialic acids, confirmed by the absence of a band. Furthermore, the MALII blot (C) which is highly specific for α2-3 linked sialic acids shows no bands after treatment with SialEXO or SialEXO 23. Both enzymes release α2-3 linked sialic acids from intact native glycoprotein, as indicated by the loss of signal. This data highlights the high specificity of SialEXO 23 for α2-3 linked sialic acids and SialEXO's capacity for complete sialic acid removal from the intact glycoprotein substrate Bovine Fetuin.


S23 Apps Fig 3 Lecting gels 190527 sialic acid

Figure 3. SialEXO 23 specificity for intact glycoproteins.

Broad pH range, fast reaction time and compact size

In addition to high specificity, SialEXO and SialEXO 23 have broad working pH ranges and a high enzyme activity. This means the sialic acid release reaction can be run concurrently in the same tube as another reaction, for example with the glycan release. This saves time and resources while avoiding additional unnecessary handling and buffer exchange steps.

The accessibility of the glycans to the sialidase can be impacted by steric hinderances since some glycans may be buried. A smaller enzyme (ie. lower Mw) is favourable to have a better chance of accessing hard to reach glycans.

Table 4 & 5 summarises these attributes for commercially available sialidases. Firstly, those specific for α2-3 linked sialic acids like SialEXO 23 (Table 4) and secondly general sialidases like SialEXO (Table 5).

Table 4. Summary of key attributes for specific α2-3 sialidases like SialEXO 23.

SialEXO® 23 Other Sialidase 1 Other Sialidase 2
Source organism Akkermansia muciniphila Streptococcus pneumoniae Streptococcus pneumoniae
Reaction time 1 hr 1 hr 1 hr
pH range (optimal) 7.0 to 9.0 (7.5) (5.5) (6.0)
Molecular weight (kDa) 66 74 75

Table 5. Summary of key attributes for general α2-3, 6, 8 sialidases like SialEXO.

SialEXO® Other Sialidase 3 Other Sialidase 4
Source organism Akkermansia muciniphila Arthrobacter ureafaciens Arthrobacter ureafaciens
Reaction time 2 hrs 1h or O/N 1 hr or more if branched
pH range (optimal) 6.5 to 9 (6.8) (6.0) 4.5 to 8 (6.0)
Molecular weight (kDa) 43 + 66 100 51-88

Product SialEXO remove sialic acid


Removal of Sialic Acids from Native Glycoproteins

  • Hydrolyzes sialic acids on N- and O-linked glycans
  • 2 h reaction
  • Requires no co-factors
  • α2-3, α2-6 and α2-8-linked sialic acids

Product SialEXO 25 remove sialic acid

S23 Gubbebattle-card-cleavage-sialexo-1024

Removal of α2-3 Sialic Acids from Native Glycoproteins

  • Hydrolyzes sialic acid on N- and O-linked glycans
  • 1 hr reaction
  • Requires no co-factors
  • α2-3-linked sialic acids only

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