Separation-based glycoprofiling approaches using fluorescent labels.
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.
Domann
Paula Jane
PJ
LGC Ltd, Teddington, Middlesex, UK. paula.domann@lgc.co.uk
Pardos-Pardos
Ana Carmen
AC
Fernandes
Daryl Ludger
DL
Spencer
Daniel Ian Richard
DI
Radcliffe
Catherine Mavis
CM
Royle
Louise
L
Dwek
Raymond Allen
RA
Rudd
Pauline Mary
PM
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019075:Electrophoresis, Capillary / Q000379:methods; D005453:Fluorescence; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007834:Lasers; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results; D000509:alpha-Fetoproteins / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000737:chemistry
D005456:Fluorescent Dyes; D006023:Glycoproteins; D011134:Polysaccharides; D000509:alpha-Fetoproteins; D062367:ortho-Aminobenzoates; D006026:Glycoside Hydrolases; C000219:anthranilamide