Quantitative Sialic Acid Analysis

Sialic acids are terminal, negatively charged monosaccharides present on many N- and O-glycans. Biopharmaceuticals often contain two main types of sialic acid; N-acetyl-neuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc). Neu5Ac is found in both human and non-human cells, whereas Neu5Gc is not present on human glycoproteins. The biopharmaceuticals efficacy, serum half-life and immunogenicity are impacted by both the abundance and the type of sialylation (including O-acetylation). Consequently, sialylation is a glycosylation critical quality attribute (GCQA).

Sialic acid analysis is a regulatory requirement (ICHQ6B) and should be performed throughout the product lifecycle to specify the following:

• Overall degree of drug sialylation – absolute quantitation of sialic acid residues per molecule
• Relative quantities of Neu5Ac:Neu5Gc
• Relative percentage of O-acetylated sialic acids

This module provides absolute amounts of Neu5Ac and Neu5Gc and relative amounts of Neu5,9Ac₂. The analysis will be performed on single or triplicate releases of samples (typically 50-200 µg for each release), an equivalent amount (by volume) of sample buffer negative control, and alongside Ludger's system suitability standards, positive (including Ludger's quantitative sialylated glycopeptide standard BQ-GPEP-A2G2S2-10U) and negative controls.

This module is suitable for:

• sialylated samples
• quality control
• monitoring batch to batch consistency
• comparability studies
• research

In order to gain more information on relative proportion of the mono-, di-, tri- and tetra-sialylated glycan structures present in the samples WAX-HPLC module is required.

Sample types:

• Biopharmaceuticals: glycoprotein hormones (e.g. follicle stimulating hormone (FSH) and erythropoietin (EPO), Fc fusion proteins, monoclonal antibodies (mAbs), vaccines).
• Cells: mammalian cell lines, bacterial cell components
• Biological fluids, tissues and others
• COVID-19 patient samples (e.g. plasma, tissues)
• SARS-CoV-2 infected cell lines

Quantitative Sialic Acid Release and DMB Labelling

Sialic acids are released from glycoproteins by mild acid hydrolysis, labelled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) and analysed by LudgerSep R1 HPLC with fluorescence detection.

The absolute amounts of Neu5Ac & Neu5Gc are calculated by reference to standard curves from Ludger's quantitative Neu5Ac & Neu5Gc standards. The presence of O-acetylated sialic acids is determined by comparison to a Neu5,9Ac₂ standard and to a reference panel containing Neu5Ac, Neu5Gc, Neu5,7Ac₂, Neu5,Gc9Ac, Neu5,9Ac₂ and Neu5,7,(8),9Ac₃Gc. The relative proportions of these different sialic acids are determined from the peak areas.

Report

Final report contains:

• HPLC profiles for system suitability standards and Ludger positive controls
• HPLC profiles for client samples and buffer negative controls
• Quantitation of Neu5Ac & Neu5Gc as nmoles/mg protein
• Relative quantitation of the O-acetylated sialic acids (e.g. Neu5,9Ac₂)