Skip to Content
MilliporeSigma

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 application for HPLC

Materials

analytical column

Product No.
Description
Pricing

Ascentis® Express RP-Amide, 2.7 μm HPLC Column

2.7 μm particle size, L × I.D. 10 cm × 2.1 mm

CONDITIONS

column

Ascentis® Express RP-Amide, 10 cm x 2.1 mm I,D., 2.7 μm particles (53913-U)

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)

column temp.

30 °C

detector

fluorescence, λ excitation = 373 nm, λ emission = 448 nm

injection

0.5 μL

sample

Mix of DMB-labeled NGNA and NANAsialic acid, 5 mg/ml each

Description

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.

Legal Information

Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany