Quantitative Sialic Acid Analysis
Importance of sialic acid analysis
Sialic acids are negatively charged monosaccharides found on the non-reducing termini of glycans.They are important for the stability and 3D conformation of glycoproteins and are involved in many biological interactions. Sialic acids often have a pivotal functional impact: for example sialylation of the N-glycans on IgG increases anti-inflammatory activity; O-acetylated sialic acids can change ligand interactions and affect degradation (O-acetylated sialic acids including Neu5,9Ac2 are found on EPO); and the presence of sialic acids also increases the serum half-life of glycoproteins by preventing uptake by the asialoglycoprotein-receptor located on liver cells.
A diverse range of sialic acids are found in nature, but the two major sialic acids found on N-glycans and O-glycans in biopharmaceuticals are N-acetyl-neuraminic acid (Neu5Ac, or NANA) and N-glycolyl-neuraminic acid (Neu5Gc, or NAGA). Humans cannot synthesise Neu5Gc and its presence on a drug can lead to immune reactions such as chronic inflammation. Anti-Neu5Gc antibodies have been detected in normal human sera, and can neutralise any Neu5Gc containing biopharmaceutical, thus lowering the drug’s efficacy. It is important to be aware that the choice of cell line can greatly influence the type of sialic acids present on a biopharmaceutical, for instance a large proportion of the sialic acids on mouse IgG are often Neu5Gc.
It is therefore imperative, for drug safety and efficacy, to monitor both the level and types of sialic acids during all stages of the product life cycle as well as QC batch to batch consistency. Hence, sialic acid analysis is a regulatory requirement laid out in the ICH Q6B guidelines for characterisation of biopharmaceuticals.
Information that makes kits and standards available
Quantitation of sialic acids (Neu5Ac & Neu5Gc) as nmol/mg protein
Relative quantitation of the O-acetylated Neu5,9AC2
The following kits and standards are used for sialic acid quantitative analysis
Release of Sialic Acids
Sialic acids are released by mild acid hydrolysis from the glycoprotein samples; typically from triplicate 50 µg amounts, although larger amounts may be required for samples with very low levels of sialylation e.g. MAbs. We recommend a number of process controls are also taken through the process: sample buffer or water as negative controls; fetuin glycoprotein and/or a quantitative glycopeptide as positive controls. Samples and process controls are incubated with 2M acetic acid at 80°C for 2 hours.
DMB Labelling
To enable sensitive detection the released sialic acids are then fluorescently labelled with 1,2-diamino-4,5-methylenedioxybenzene.2HCl (DMB) – see figure 1. When the sialic acid ring opens it exposes a ketone group next to the carboxylic acid, the DMB binds across these two adjacent keto groups producing a fluorophore (other monosaccharides do not have these adjacent keto groups, so they are not fluorescently labelled). The acid hydrolysed samples and process controls plus Ludger sialic acid standards: Neu5Ac (B491176),Neu5Gc (B491217), sialic acid reference panel (S491526) and Neu5,9Ac2 (N491192) if using, are incubated with DMB at 50°C for 3 hours.
LC Analysis
The Neu5Ac and Neu5Gc standards are diluted to produce a standard curve for quantitation of these two sialic acids. The sialic acid reference panel (S491526) and Neu5,9Ac2 are used for sialic acid identification.
LC Conditions
Solvent A = acetonitrile:methanol:water 9:7:84
Solvent B = acetonitrile
Fluorescence: Excitation ƛ: 373 nm; Emission ƛ: 448 nm
Column temp = 30°C; Sample temp = 10°C
Results
The peak areas of Neu5Ac and Neu5Gc in samples are compared to standard curves to provide quantitative data on the amounts of the sialic acids in your samples and in the process control. Figure 2 shows data for sialic acids released from mouse and human IgGs. The mouse IgG has high levels of the non-human sialic acid Neu5Gc, and in this case the human IgG has 4 times more sialic acids than the mouse IgG. Figure 3 shows data for the sialic acids in EPO. The absolute amounts of the Neu5Ac and Neu5Gc can be determined by comparison to the standard curve. However, EPO also contains the O-acetylated sialic acid Neu5,9Ac2. Under aqueous conditions the acetate on the 9 position can migrate to the 8 position, so it is difficult to have a fully quantitative standard for Neu5,9Ac2 . The sialic acids are therefore identified by comparison to the retention times of the standards, and the relative proportions of the Neu5Ac, Neu5Gc and Neu5,9Ac2 are determined from their peak areas.
Figure 2: Identification and quantitation of Neu5Ac and Nue5Gc in mouse and human IgG.
Figure 3: Identification and quantitation of sialic acids in EPO.