Complete and Rapid Desialylation of Glycoproteins using Immobilized SialEXO®

Sialic acids are negatively charged sugar residues present on both N- and O-linked glycans. The inherent charge of sialic acids might complicate analytical workflows, masking other important modifications. The removal of sialic acids therefore facilitates the study of underlying variants in the protein. Here, we present a robust and general workflow for desialylation of glycoproteins using the new Immobilized SialEXO column and demonstrate the enzymatic activity on several therapeutic proteins using orthogonal analytical methods. Immobilized SialEXO is a microspin column for complete removal of sialic acids on 0.5 mg native glycoprotein after a 30 min incubation at room temperature. The activity of Immobilized SialEXO was studied using released glycan analysis, antibody middle-up LC-MS and capillary electrophoresis.

Figure 1. Immobilized SialEXO desialylates 0.5 mg glycoprotein in 30 min at room temperature.

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Removal of Sialic Acids from Native Glycoproteins

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

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Immobilized SialEXO Shows Efficient Desialylation of a Complex Glycoprotein

When setting up reaction conditions there is a trade-off between required reaction time and the amount of enzyme needed to complete the reaction. Higher enzyme amounts guarantee a complete turnover within a shorter time but can complicate down-stream analysis. To avoid such problems, we have immobilized active SialEXO in a spin column format. This allows for the incubation of a glycoprotein substrate with significantly higher amounts of sialidase with shorter incubation times.

We tested the performance of SialEXO and Immobilized SialEXO on the human C1 inhibitor. This glycoprotein is a challenging substrate with 6 N- and up to 26 O-glycans modified with both α2,3 and α2,6-linked sialic acid. Analysis of released N-glycans demonstrated complete desialylation by SialEXO in solution and Immobilized SialEXO (Fig. 2)

Figure 2. C1 inhibitor was either treated with SialEXO in solution for 2 hours at 37°C or with Immobilized SialEXO for 30 min at room temperature. N-glycans were released from the resulting desialylated protein using PNGase F and the resulting free glycans could be labeled with 2-AB and analyzed by HILIC-FLD HPLC (Thermo Scientific™ Vanquish™ Duo UHPLC system with an Accucore™ 150 Amide HILIC column, 2.1 x 150 mm).

Complete Desialylation of a Monoclonal Antibody

Antibodies with additional glycans in their Fab regions are often sialylated to a significant extent. Cetuximab carries a Fab glycan which is partially modified with N-Glycolylneuraminic acid (Neu5Gc). These sialic acids can be removed rapidly using immobilized SialEXO (Fig. 3). This demonstrates the utility of such an enzyme for analysis of this important class of biologics and its activity towards non-human sialic acids.

Figure 3. Top: For a fast and simple analysis of cetuximab glycosylation, the mAb was digested below the hinge using FabRICATOR protease and reduced to yield Fc/2, LC and Fd’ fragments. These fragments were separated by reverse phase HPLC (Waters BioResolve RP column, 2.1 x 100mm) and analyzed by ESI-Q-TOF mass spectrometry (Bruker Impact II).
Bottom: Deconvoluted mass spectra of the Fd’ fragment showing the Fab glycosylation profile. Cetuximab (top, black) and cetuximab treated with Immobilized SialEXO (bottom, teal) are compared. Sialylated structures (Neu5Gc, light blue diamond) are highlighted in purple and are absent in the lower spectrum. The asterisk marks peaks originating through neutral loss during ionization rather than remaining sialylated Fd’ fragments.

Simplified Charge Variant Analysis of Biologics

Capillary electrophoresis is commonly used to determine charge variants during characterization and quality control of biologics. The inherent charge of sialic acids might complicate charge variant profiles, masking other important modifications. The removal of sialic acids therefore facilitates the study of underlying charge variants in the protein. Cetuximab (a), tPA (b) and etanercept (c) were desialylated using Immobilized SialEXO and improved the separation in imaged isoelectric focusing (icIEF; Fig. 4), using Maurice from ProteinSimple.


Figure 4.
Instrumentation: ProteinSimple Maurice CE instrument and icIEF cartridge. The samples were analyzed with Compass for iCE software. All samples were diluted to 0.8 mg/mL. Focusing conditions: 1 minute at 1500 V, 8 minutes at 3000 V. a) (Maurice icIEF Biosimilar Platform Method, https://www.proteinsimple.com/technical_library.html) Ampholyte solution: 4% Pharmalytes (3% 8–10.5, 1% 5-8) containing 3.2 M urea, 5 mM iminodiacetic acid (IDA) and 10 mM Arginine. pI markers: 5.85 and 10.17. b) Ampholyte solution: 4% Pharmalytes (3% 8–10.5, 1% 5-8) containing 3.2 M urea, 5 mM IDA, 10 mM Arginine and 5% SimpleSol Protein Solubilizer. pI markers: 5.85 and 10.17. Samples desalted using a 10kDa MW centrifugal filter and 4M urea. c) Ampholyte solution: 1% Servalyts (0.5% 2-9, 0.5% 4-7) containing 6.4 M urea, 0.1% poloxamer 188, 10 mM IDA and 10 mM Arginine. pI markers: 4.05 and 8.4

 

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Immobilized SialEXO Brochure

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