Liquid chromatographic profiling of monosaccharide concentrations in complex cell-culture media and fermentation broths

Abstract

A solid phase extraction, liquid chromatography and fluorescence (SPE-RPLC-FL) based protocol for the determination of free monosaccharides in highly complex raw material powders and formulated fermentation feedstocks and broths has been developed. Monosaccharides within sample extracts were derivatised pre-column with anthranilic acid and the derivatives separated using reversed-phase LC with fluorescence detection. Using a 2.1 mm × 50 mm 1.8 µm Zorbax Eclipse XDB-C₁₈ column, a flow rate of 0.4 mL min⁻¹ and an acetonitrile gradient in a sodium acetate buffer (pH 4.3; 50 mmol L⁻¹) the baseline resolution of glucosamine, mannosamine, galactosamine, galactose, mannose, glucose, ribose, xylose, fucose and sialic acid within 20 minutes was achieved. Pre-column derivatisation involved combining a 30 mg mL⁻¹ solution of anthranilic acid in a 1 : 1 ratio with an aqueous standard prior to injection. Standard analytical performance criteria were used for evaluation purposes, with the method found to exhibit LOD's as low as 10 fmol, and be linear and precise (%RSD < 2.2% (n = 7). The method was applied to the analysis of a range of highly complex biopharmaceutical production samples, including yeastolate powders, chemically defined media and in-process fermentation broth samples. Sample preparation involved passing an aqueous sample through a C₁₈ solid phase extraction cartridge to trap hydrophobic peptides and vitamins, with recovery of all test sugars exceeding 90%. Finally, standard statistical analysis was performed on samples taken from different lots in order to estimate lot-to-lot variability.