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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

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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 \r\nPharmaceutical (small molecule)
suitability	application for HPLC

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