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SmartEnzymes™

 

Unmasking Charge Variants

Remove Sialic Acids to Unmask Charge Variants

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. This application shows a fast and easy way of sample preparation to remove all sialic acids prior to charge variant analysis.

 

Improved Separation in Charge Variant Analysis

SialEXO Immobilized is a microspin column for complete removal of sialic acids on 0.5 mg native glycoprotein after a 30 min incubation at room temperature. To study the underlying charge variants on etanercept, a general workflow for desialylation of glycoproteins using SialEXO Immobilized column was developed. Capillary electrophoresis is commonly used to determine charge variants during characterization and quality control of biologics. The glycoproteins were desialylated using SialEXO Immobilized and analyzed in imaged isoelectric focusing using Maurice from ProteinSimple (Fig. 1). Combined, this analytical workflow improved the separation and enabled charge variant analysis of glycoproteins. Etanercept, would be very hard to analyze without first removing sialic acids, but using SialEXO Immobilized separated peaks could be obtained.

Desialylation of Biopharmaceuticals

 

 

Figure 1. Instrumentation: ProteinSimple Maurice CE instrument and icIEF cartridge. The samples were analyzed with Compass for iCE software. 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: 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

SialEXO Immobilized for Desialylation of Complex Glycoproteins

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.

 

Fig 2. We tested the performance of SialEXO Lyophilized and SialEXO Immobilized 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 SialEXO Immobilized (Fig. 2)

 

Charge Variant with SialEXO

 

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