Cross Validation of Liquid Chromatography–Mass Spectrometry and Lectin Array for Monitoring Glycosylation in Fed-Batch Glycoprotein Production
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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.
KeywordsGlycosylation High mannose Acetylation N-linked oligosaccharides Lectin array Mass spectrometry
Chinese hamster ovary
N-Acetylneuraminic acid or sialic acid
This research was supported by the Industrial Development Authority, Ireland.
- 3.Karlsson, N. G., Wilson, N. L., Wirth, H. J., Dawes, P., Joshi, H., & Packer, N. H. (2004). Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis. Rapid Communications in Mass Spectrometry, 18, 2282–2292.CrossRefGoogle Scholar
- 18.Rosenfeld, R., Rosenberg, R., Olender, R., Plaschkes, I., Dabush, D., Himmelfarb, C., et al. (2007). High-throughput glycoanalysis for use in biopharmaceuticals development and manufacturing in bioseparation and bioprocessing, vol 2. In G. Subramanian (Ed.), Bioseparation and bioprocessing (pp. 663–673). Germany: Wiley-VCH Verlag GmbH & Co.Google Scholar
- 19.Sutcliffe, A., & Smith, V. (2006). Glycosylation patterns during fermentation. Genetic Engineer & Biotechnology News, 26(8), 45–47.Google Scholar
- 23.Maya, R. B., Amor, Y., Rosenberg, R., Byk-Tennenbaum, T., Samokovlisky, A., Olender, R., et al. (2007). Glycoanalysis on a lectin array: applications to the development of biopharmaceuticals and life science research in glycobiology. In C. Sansom & T. Markman (Eds.), Glycobiology (pp. 340–351). UK: Scion.Google Scholar