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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

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

Conditions

columnAscentis® 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
gradient0-1 min 6% B; 1.01-4 min 20% B; 4.01-12 min 6% B, total run time 15 min
flow rate0.2 mL/min
pressure1300 psi (89.6 bar)
sampleMix of DMB-labeled NGNA and NANAsialic acid, 5 mg/mL each
injection0.5 µL
detectorfluorescence, lambda excitation = 373 nm, lambda emission = 448 nm

Description

Analysis NoteSialic 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.
CategoriesAnalytical Chromatography, Glycosides, Proteins
Featured IndustryLife Science and Biopharma
Pharmaceutical (small molecule)
suitabilityapplication for HPLC
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