HomeSmall Molecule HPLCHPLC Analysis of DMB-Labeled Sialic Acids on Ascentis® Express RP-Amide: Comparison of Biosimilars to Reference Materials
HPLC Analysis of DMB-Labeled Sialic Acids on Ascentis® Express RP-Amide: Comparison of Biosimilars to Reference Materials

Conditions | |
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column | Ascentis® Express RP-Amide, 10 cm x 2.1 mm I,D., 2.7 µm particles (53913-U) |
column temp. | 30 °C |
mobile phase | [A] Water, 0.1% formic acid, [B] acetonitrile, 0.1% formic acid |
gradient | 0-1 min 6% B; 1.01-4 min 20% B; 4.01-12 min 6% B, total run time 15 min |
flow rate | 0.2 mL/min |
pressure | 1300 psi (89.6 bar) |
sample | Mix of DMB-labeled NGNA and NANAsialic acid, 5 mg/mL each |
injection | 0.5 µL |
detector | fluorescence, lambda excitation = 373 nm, lambda emission = 448 nm |
Description | |
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Analysis Note | Sialic acids affect the bioavailability, function, stability, and metabolism of glycoproteins. Two forms of sialic acid are commonly present in therapeutic glycoproteins: N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA). One of the most common quantification methods involves releasing sialic acids from the glycoprotein, derivatizing NANA and NGNA with 1,2-diamino-4, 5-methylenedioxybenzene (DMB), and analyzing by C18-HPLC with fluorescence detection. This procedure is subject to interference from peaks originating from excess reagent and other derivatized impurities, limiting sensitivity and reproducibility. The objectives of this study were to develop a significantly improved HPLC-fluorescence method for DMB-NANA and DMB-NGNA, and to apply this method to compare two candidate biosimilar therapeutic proteins to their respective reference materials. |
Categories | Analytical Chromatography, Glycosides, Proteins |
Featured Industry | Life Science and Biopharma Pharmaceutical (small molecule) |
suitability | application for HPLC |
Materials
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