Sugar Chain Structure, Saccharide Composition

Monosaccharide Composition Analysis

■ Monosaccharide Composition Analysis of Mouse Monoclonal Antibodies

As the sugar chains in glycoproteins such as antibody drugs are changed by the actions of multiple enzymes after protein translation, the diversity and nonuniformity of the sugar chain structure is an unavoidable problem. Nevertheless, the guidelines require determination of the sugar content (neutral sugars, amino sugars, sialic acid).
A refractive index detector is generally used for saccharide composition analysis. However, fluorescence detection is used for saccharide composition analysis of antibody drugs as it offers higher sensitivity. Since sugars are not fluorescent, they undergo derivatization by an appropriate method to make them fluorescent before analysis by a high-performance liquid chromatograph with a fluorescence detector.

This is an example of analysis of mouse monoclonal antibodies.
Test and research flow
1. Conduct enzyme digestion (neuraminidase, etc.), hydrolysis, and N-acetylation on the mouse monoclonal antibodies.
2. Apply fluorescent markers to the reducing terminus of the free monosaccharides using 4-aminobenzoic acid ethyl ester (ABEE).
3. Perform LC analysis of the fluorescent-marked monosaccharides using the Prominence UFLC system.
* Use the Plus S ABEE Saccharide Composition Analysis Kit (manufactured by J-OIL MILLS, Inc., Japan) for pretreatment of the mouse monoclonal antibodies.

A standard sample comprising a mixture of six monosaccharides with fluorescent markers was analyzed using the Prominence UFLC system, and the calibration curve for each monosaccharide was determined from the area values (Fig. 1). Next, one-tenth of the monosaccharide mixture obtained by treating 60 µg (400 pmol) mouse monoclonal antibody was analyzed using the Prominence UFLC system, and the area of each monosaccharide peak was calculated.

Fig. 1 LC Chromatogram of Standard Sample (Gal/Man/Sia/GlcNAc/Fuc/GalNAc Mixture)

The amount of each monosaccharide contained in 1 mol monoclonal antibody was then determined from the calculated monosaccharide peak areas, using the calibration curve obtained from the standard sample.
Table 1 lists the amount of each monosaccharide contained per 1 mol of the commercial mouse monoclonal antibody used for this test.

Table 1 Monosaccharide Content per 1 Mol Antibody