Determination of the monosaccharide composition of a glycoprotein pharmaceutical is a key quality control assay for glycoprotein-based therapeutics. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is a well-established method for glycoprotein carbohydrate analysis. This work demonstrates an improved HPAE-PAD method using the new Thermo Scientific™ Dionex™ CarboPac™ PA20 column with 4 µm particle size to simultaneously quantify six major monosaccharides present in glycoprotein acid digests. The total run time is 20 minutes as compared to a 32-minute method with a 6.5 µm particle size column. The column’s smaller particle size offers higher peak efficiencies leading to high resolution separations in significantly shorter run times.<br/>Here, two different commercially available proteins, bovine fetuin and alpha1 acid glycoprotein were individually subjected to two different hydrolysis conditions using 1) HCl, which favors release of amino sugars like galactosamine and glucosamine and 2) TFA, which favors release of neutral sugars like mannose, glucose, and galactose. Figure 1 shows a representative chromatogram of a standard mix containing the six common glycoprotein carbohydrates. All peaks are well resolved and the separation is completed within 8 minutes. The method is linear at monosaccharide concentrations between 1.56 to 300 uM (Table 1). The monosaccharide retention time and peak area RSDs are less than 2.66%, indicating excellent method precision. The more efficient peaks also allow the injection of less sample for routine monosaccharide quantification. The method described here requires 0.5 µg injected protein compared to 2 µg for the method previously described. This assay was validated according to the analytical performance characteristics outlined in USP General Chapter &lt;1225&gt;, Validation of Compendial Procedures. The method was shown to measure accurately the monosaccharide concentration in complex matrices like acid-hydrolyzed proteins. Moreover, the method is robust to experimental condition variations that occur during routine use.

Improved HPAE-PAD method for glycoprotein monosaccharide determination

255th National Meeting (2018)

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