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

Citation

Alwael, H and Connolly, D and Paull, B, Liquid chromatographic profiling of monosaccharide concentrations in complex cell-culture media and fermentation broths, Analytical Methods: advancing methods and applications , 3, (1) pp. 62-69. ISSN 1759-9660 (2010) [Refereed Article]


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Copyright Statement

Copyright 2011 Royal Society of Chemistry

DOI: doi:10.1039/c0ay00516a

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 m 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. © 2011 The Royal Society of Chemistry.

Item Details

Item Type:Refereed Article
Research Division:Chemical Sciences
Research Group:Analytical Chemistry
Research Field:Separation Science
Objective Division:Expanding Knowledge
Objective Group:Expanding Knowledge
Objective Field:Expanding Knowledge in the Chemical Sciences
Author:Paull, B (Professor Brett Paull)
ID Code:72041
Year Published:2010
Web of Science® Times Cited:6
Deposited By:Research Division
Deposited On:2011-08-18
Last Modified:2015-01-20
Downloads:0

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