DMB Sialic Acid Labeling Kit
Sialic acid analysis is a regulatory requirement laid out in the ICH Q6B guidelines for characterisation of biopharmaceuticals. In particular, determination of relative levels of human and non-human type sialic acids (NeuAc/NANA and NeuGc/NGNA respectively) is important since some patients have high levels of anti- NeuGc antibodies which could lead to neutralization and rapid clearing of NeuGc-containing biopharmaceuticals (Nguyen et al, 2005). The significance of NeuAc and NeuGc when analysing glycoproteins means that it is often considered a critical quality attribute (CQA) for biotherapeutics.
A widely used method for determining the ratio of NeuAc to NeuGc is as follows:
The LudgerTag DMB kit (Cat No. LT-KDMB-A1) provides all that is required to release sialic acids from glycoproteins and label them with DMB. The kit contains reagents and materials for up to 22 glycoprotein samples (50-100 Î_g of glycoprotein per sample). Each labeling should work with approximately 10 pmol up to 2.5 nmol sialic acids per sample.
- Release of sialic acid residues from the glycoprotein by mild acid hydrolysis.
- Fluorescent labeling of released sialic acids with1,2-diamino-4,5-methylenoxybenzene (DMB).
- Relative quantitative analysis of DMB-labelled sialic acids by HPLC or UHPLC.
Included in the kit are a sialic acid reference panel (containing Neu5Ac, Neu5Gc, Neu5,7Ac2, Neu5,Gc9Ac, Neu5, 9Ac2 and Neu 5,7, (8), 9Ac3Gc) and both NeuAc and NeuGc quantitative standards. These three standards can be labelled with DMB alongside the released sialic acid samples. Preparation of serial dilutions of the NeuAc and NeuGc quantitative standards enables quantitative analysis. Information on how to do this is explained in the LudgerTag DMB kit guide. The NeuAc and NeuGc quantitative standards are also available to purchase separately if required.
For a positive control, we recommend using fetuin glycoprotein which contains both NeuAc and NeuGc sialic acids. Water can be used as a negative control.
Nguyen DH, Tangvoranuntakul P, and Varki A. (2005) Effects of Natural Human Antibodies against a Nonhuman Sialic Acid That Metabolically Incorporates into Activated and Malignant Immune Cells. J Immunol 175:228-236.