Glycosylation can have significant effects on the clinical safety and efficacy of biopharmaceuticals [1,2]. As a result, both innovator drugs’ and biosimilars’ glycan profiles need to be fully characterized during the product lifecycle, from development to commercialization including batch-to-batch consistency and release to the market [2]. Additionally, a vast amount of research has been performed on the identification of disease biomarkers based on the glycosylation of biological samples [3,4,5]. Due to the high demand and institutional pressure for biopharmaceutical characterization [6] and the huge number of samples that a clinical setting is exposed to [7], there is a need for the development of a rapid, sensitive and selective method for glycan and glycopeptide analysis. Glycosylation analysis both at the N-glycan and glycopeptide level provide complementary information. N-glycan analysis gives specific information about the glycan structure and linkages by exoglycosidases analysis while glycopeptide analysis is a more favourable approach for the characterization of site-specific glycan compositions.
A new label for both N-glycan and glycopeptide analysis has been investigated. This label has an imidazolium group with a permanent positive charge that has been used as MS probe showing greater spectral peak intensities and lower limits of detection [8]. This new label synthesis is achieved in two steps. Glycoanalysis with this tag is quick and easy: it just requires 4 steps and no dry down steps are involved in the process, allowing it to be completed in as little as 2 hours. Final sample separation and identification is performed by tandem HILIC-UHPLC and ESI-MS.
This poster will focus on the details of this new chemical derivatisation label. We will show that reliable data for both N-glycan and glycopeptide identification is generated using this rapid procedure with a small amount of glycoprotein (less than 10 μg for proteins with only one glycosylation site). The results obtained as a proof-of-concept for the analysis of antibodies and fetuin will be presented. As further optimization is required, the plans for future work will also be discussed.
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- Kailemia, M. J.; Park, D.; Lebrilla, C. B. Glycans and Glycoproteins as Specific Biomarkers for Cancer. Analytical and Bioanalytical Chemistry. 2017, pp 395–410. https://doi.org/10.1007/s00216-016-9880-6.
- Costa, J.; Streich, L.; Pinto, S.; Pronto-Laborinho, A.; Nimtz, M.; Conradt, H. S.; de Carvalho, M. Exploring Cerebrospinal Fluid IgG N-Glycosylation as Potential Biomarker for Amyotrophic Lateral Sclerosis. Mol. Neurobiol. 2019. https://doi.org/10.1007/s12035-019-1482-9.
- Pavić, T.; Dilber, D.; Kifer, D.; Selak, N.; Keser, T.; Ljubičić, Đ.; Dugac, A. V.; Lauc, G.; Rumora, L.; Gornik, O. N-Glycosylation Patterns of Plasma Proteins and Immunoglobulin G in Chronic Obstructive Pulmonary Disease. J Transl Med 2018, 16 (323). https://doi.org/10.1186/s12967-018-1695-0.
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- Walsh, G. Biopharmaceutical Benchmarks 2018; 2018; Vol. 36. https://doi.org/10.1038/nbt.4305.
- Galan, M. C.; Tran, A. T.; Bernard, C. Ionic-Liquid-Based Catch and Release Mass Spectroscopy Tags for Enzyme Monitoring. Chem. Commun. 2010, 46 (47), 8968–8970. https://doi.org/10.1039/c0cc04224b.
