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Cross Validation of Liquid Chromatography-Mass Spectrometry and Lectin Array for Monitoring Glycosylation in Fed-Batch Glycoprotein Production

Journal article
Authors Catherine A Hayes
R. Doohan
D. Kirkley
K. Leister
B. Harhen
A. V. Savage
Niclas G. Karlsson
Published in Molecular Biotechnology
Volume 51
Issue 3
Pages 272-282
ISSN 1073-6085
Publication year 2012
Published at Institute of Biomedicine
Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 272-282
Language en
Links dx.doi.org/10.1007/s12033-011-9465-...
Keywords Glycosylation, High mannose, Acetylation, N-linked oligosaccharides, Lectin array, Mass spectrometry, hamster ovary cells, protein, microarray, glycomics, strategy, glycans, oligosaccharides, expression, antibodies, ammonia
Subject categories Medical Biotechnology

Abstract

Glycosylation analysis of recombinant glycoproteins is of importance for the biopharmaceutical industry and the production of glycoprotein pharmaceuticals. A commercially available lectin array technology was evaluated for its ability to present a reproducible fingerprint of a recombinant CTLY4-IgG fusion glycoprotein expressed in large scale CHO-cell fermentation. The glycosylation prediction from the array was compared to traditional negative mode capillary LC-MS of released oligosaccharides. It was shown that both methods provide data that allow samples to be distinguished by their glycosylation pattern. This included information about sialylation, the presence of reducing terminal galactose beta 1-, terminal N-acetylglucosamine beta 1-, and antennary distribution. With both methods it was found that a general trend of increased sialylation was associated with an increase of the antenna and reduced amount of terminal galactose beta 1-, while N-acetylglucosamine beta 1- was less affected. LC-MS, but not the lectin array, provided valuable information about the sialic acid isoforms present, including N-acetylneuraminic acid, N-glycolylneuraminic acid and their O-acetylated versions. Detected small amounts of high-mannose structures by LC-MS correlated with the detection of the same epitope by the lectin array.

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