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at 10,000Da. The remaining glycans were then converted to free reducing end glycans by dilution of the sample with 1% FA to obtain an antibody concentration of 0.1mg/ml; incubation for 2h at 37ºC.
Conclusion: This article provides a technical description of the mAb-Glyco Chip Kit that was designed for fast and automated characterisation of N- glycans from monoclonal antibodies (mAb). It was demonstrated that the complete workflow including on-chip deglycosylation of the mAb as well as chromatographic separation, and Q-TOF detection of the
cleaved glycans, and data processing can be completed within a 20 min time period.
Data have verified chip stability, reproducibility and lifetime as well as excellent comparability of results obtained from chip analysis to those obtained from a typical standard workflow (that in this case took 5-6 hours to complete). It can be concluded that the mAb-Glyco Chip Kit provides a robust workflow solution that helps to remove a major bottleneck during the development phase of mAb-based biological drugs allowing the analyst to quickly provide answers.
References [1] R. Jefferis, Nature Reviews Drug Discovery 8 (2009) 226.
[2] C. Huhn, M. Selman, L. Ruhaak, A. Deelder, Wuhrer, Proteomics, 9 (2009) 882.
[3] mAb-Glyco Chip User’s Guide; 2010; G4240-90020 (or 2011; G4240-90022).
[4] Technical Note, (2005) Publication Number 5989-3627EN.
[5] H. Yin, K. Killeen, R. Brennen, D. Sobek, M. Werlich, T. van de Goor, Anal. Chem., 77 (2005) 527.
[6] H. Yin, K. Killeen, J. Sep. Sci., 30 (2007) 1427. [7] M. Bynum, H. Yin, K. Felts, Y. Lee, C. Monell, K. Killeen, Anal. Chem., 81 (2009) 8818.
[8] J.R.Rasmussen, J. Davis, J. Risley, R. Van Etten, J. Am. Chem. Soc., 114 (1992) 1124.
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