Sialic Acid Analysis

Sialic Acids (neuraminic acids) are usually found as terminal structures of both N-linked and O-linked glycans.

Sialyated N-linked and O-linked glycans image

Being located at the terminal position of glycans, sialic acids are a likely point of contact for many glycoprotein interactions. They are also important for the stability and 3D conformation of glycoproteins and are involved in many biological interactions:

  • Sialyation of IgG reduces ADCC and increases anti-inflammatory activity
  • Neuraminic acids increase the serum half-life of glycoproteins by preventing uptake by the liver’s asialoglycoprotein receptor
  • NGNA(Neu5Gc), a glycan not found in humans, can illicit an immune response and lead to increased neutralization of biopharmaceuticals.
  • Cell line choice can greatly influence the type of neuraminic acids present on a biopharmaceutical, for example a large portion of the sialic acids on mouse IgG are often NGNA.

Biopharmaceutical Sialylation

Biopharmaceutical sialylation refers to the type and distribution of neuraminic acids in the glycans of therapeutic glycoproteins. It is imperative for drug safety and efficacy to monitor both the level and types of sialic acids during all stages of the product life cycle.

The sialylation pattern can be a very useful measure of product consistency during manufacturing. The glycan charge profile shows distribution of neutral, mono-, di-, tri-, and tetra-charged glycans.

Weak anion exchange (WAX)-HPLC Charge separation of 2AB labeled Glycans on a LudgerSep-C3 column.
Top trace (red): fetuin N-glycan. Bottom trace(blue): EPO N-glycans

Oligosaccharide Profiling

In addition to the charge profile, a detailed oligosaccharide profile can be obtained by normal phase analysis of fluorescently labeled glycans.

IgG oligosaccharide profile image
HILIC-UPLC profiles of procainamide labelled N-glycans released from IgG-1 mAb.
Structures have been assigned by exoglycosidase sequencing and mass spectrometry

Sialic Acid Characterization

A diverse range of sialic acids are found in nature, but the two main types of neuraminic acid residues found in biopharmaceuticals produced in mammalian expression systems are N-acetylneuraminic acid (Neu5Ac or NANA) and N-glycolylneuraminic acid (Neu5Gc or NGNA), having a glycolyl group in place of the acetyl.

Neu5,9Ac2, having an additional acetyl group, is found on EPO and other human glycoproteins. O-acetylated sialic acids can change ligand interactions and affect degradation of therapeutic glycoproteins.

Sialic Acids image

The LudgerTag DMB Labeling Kit contains all the reagents necessary to release, label, identify and quantitate sialic acids.

Sialic Acid Analysis of EPO
Identification and quantitation of DMB labeled sialic acids in EPO. Analysis on LudgerSep-R1 HPLC column

Standards included in the kit:
Sialic Acid Reference Panel
Neu5Ac -quantitative
Neu5Gc -quantitative
  (Neu5,9Ac2 available separately)

Release of Sialic Acids The release of sialic acids from the glycoprotein occurs by a mild acid hydrolysis. Samples and controls are incubated with 2M acetic acid at 80°C for 2 hours.

Sialic Acid Labeling Reaction The released sialic acids are fluorescently labelled with 1,2-diamino-4,5-methylenedioxybenzene-2HCl (DMB). The acid hydrolysed samples and process controls plus Ludger sialic acid standards: Neu5Ac, Neu5Gc, sialic acid reference panel, and Neu5,9Ac2 if using, are incubated with DMB at 50°C for 3 hours.

Analysis via HPLC or UHPLC A standard curve is calculated using the Neu5Ac and Neu5Gc standards. The sialic acid reference panel and Neu5,9Ac2 are used for sialic acid identification. Analysis can be performed using the LudgerSep R1x150mm column for HPLC or via UHPLC using a LudgerSep uR2x100mm column.

NANA vs NGNA - Quantification is Key

Controlling the ratio of Neu5Ac(NANA) to Neu5Gc(NGNA) is critical for biomanufacturers: humans cannot synthesize 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 neutralize any Neu5Gc containing biopharmaceuticals, thereby lowering the drugs efficacy.

The LudgerTag DMB Labeling Kit allows quantitation of Neu5Ac and Neu5Gc. The two monosaccharides are NIST-F and USP traceable quantitative standards. 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

NANA and NGNA image
Identification and quantitation of DMB labeled Neu5Ac and Nue5Gc in mouse and human IgG. Analysis on LudgerSep-R1 HPLC column.