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Separation‐based Glycoprofiling Approaches using Fluorescent Labels
Author(s) -
Domann Paula Jane,
PardosPardos Ana Carmen,
Fernandes Daryl Ludger,
Spencer Daniel Ian Richard,
Radcliffe Catherine Mavis,
Royle Louise,
Dwek Raymond Allen,
Rudd Pauline Mary
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700640
Subject(s) - capillary electrophoresis , biopharmaceutical , glycan , chemistry , chromatography , glycoprotein , mass spectrometry , fluorescence , gel electrophoresis , biochemistry , biology , microbiology and biotechnology , physics , quantum mechanics
Glycoprotein analysis is essential within the biopharmaceutical industry, as the structure of the different glycans present can affect the safety and efficacy of products. However analysis of cleaved glycans presents a major analytical challenge, due to their inherent complexity, lack of chromophore and the existence of various isoforms (both position and linkage). In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants, so the released glycan pool contains a range of structures. Both normal phase chromatography and capillary gel electrophoresis offer excellent selectivity for the analysis of fluorescently labelled glycans. The normal phase (NP) chromatographic approach is sensitive, reliable and well established, with databases available for searching structures assigned relative to retention times. Capillary gel electrophoresis with laser induced fluorescence (CGE‐LIF) offers faster analysis times, though currently no databases are available to search mobilities against structures, therefore data has to be cross‐correlated with either normal phase chromatography or mass spectrometry approaches when developing and validating methods. The principles of both methods are described and a review is presented that includes evaluation against a set of criteria established through consultation with the biopharmaceutical industry.