Sialylation levels influence oligosaccharide quantitation: analyzing response variability using high-pH anion-exchange chromatography and pulsed amperometric detection

A protein's carbohydrate side chains affect its solubility, how it folds, its in vivo biological functions, and its pharmacokinetic profiles. Because of the complex nature of glycoproteins and the various recombinant DNA technology processes used to produce them, it remains a challenge to asses...

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Bibliographic Details
Published inBiopharm International Vol. 16; no. 6; p. 44
Main Authors Ganesa, Chandrashekar, Granda, Brian W, Mattaliano, Robert J
Format Magazine Article Trade Publication Article
LanguageEnglish
Published Monmouth Junction Intellisphere, LLC 01.06.2003
MultiMedia Healthcare Inc
Subjects
Acids
Analysis
Caustic soda
Cells
Cellulose
Chromatography
Composition
Electrodes
Ions
Molecular weight
Numerical analysis
Oligosaccharides
Proteins
Sensors
Sodium
United States
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Summary:A protein's carbohydrate side chains affect its solubility, how it folds, its in vivo biological functions, and its pharmacokinetic profiles. Because of the complex nature of glycoproteins and the various recombinant DNA technology processes used to produce them, it remains a challenge to assess batch-to-batch consistency by evaluating glycosylation patterns. Glycans can be characterized by releasing the oligosaccharides from the protein using either chemicals or enzymes. The released oligosaccharides are then characterized using HPLC, fluorophore-assisted carbohydrate electrophoresis, capillary electrophoresis, mass spectrometry, or high-pH anion-exchange chromatography. High-pH anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) resolves neutral and charged oligosaccharide species that differ in branch, linkage, and positional isomerism. One advantage of this method is that the oligosaccharide structures can be detected without prior derivatization. PAD cells, however, have finite lifetimes because the working electrode is subject to surface recession and fouling. That means the electrodes must be polished or replaced when baseline noises increase or when oligosaccharide peak responses begin to change. Our investigation indicates that the variability between detector cells may limit the usefulness of HPAEC-PAD as a rugged method for quantifying glycosylation patterns.
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ISSN:1542-166X
1939-1862