Molecular Biotechnology

, Volume 51, Issue 3, pp 272–282 | Cite as

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

  • Catherine A. Hayes
  • Roisin Doohan
  • David Kirkley
  • Kirk Leister
  • Brendan Harhen
  • Angela V. Savage
  • Niclas G. Karlsson
Research
First Online:

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 β1-, terminal N-acetylglucosamine β1-, 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 β1-, while N-acetylglucosamine β1- 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.

Keywords

Glycosylation High mannose Acetylation N-linked oligosaccharides Lectin array Mass spectrometry 

Abbreviations

CHO

Chinese hamster ovary

LC

Liquid chromatography

Gal

Galactose

GlcNAc

N-Acetyl glucosamine

HexNAc

N-Acetylhexosamine

Hex

Hexose

NeuAc

N-Acetylneuraminic acid or sialic acid

MS

Mass spectrometry

NeuGc

N-Glycolylneuraminic acid

Man-5

Mannose-5

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Notes

Acknowledgments

This research was supported by the Industrial Development Authority, Ireland.

Supplementary material

12033_2011_9465_MOESM1_ESM.xlsx (53 kb)
Supplementary material 1 (XLSX 52 kb)

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Catherine A. Hayes
    • 1
    • 3
  • Roisin Doohan
    • 1
  • David Kirkley
    • 2
  • Kirk Leister
    • 2
  • Brendan Harhen
    • 1
  • Angela V. Savage
    • 1
  • Niclas G. Karlsson
    • 1
    • 3
  1. 1.School of ChemistryNational University IrelandGalwayIreland
  2. 2.Bristol-Myers SquibbSyracuseUSA
  3. 3.Department of Medical BiochemistryUniversity of GothenburgGothenburgSweden

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