LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics.

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TitleLC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics.
Publication TypeJournal Article
Year of Publication2012
AuthorsSchiel, JE, Au, J, He, H-J, Phinney, KW
JournalAnal Bioanal Chem
Volume403
Issue8
Pagination2279-89
Date Published2012 Jun
ISSN1618-2650
KeywordsAntibodies, Monoclonal, Murine-Derived, Chromatography, Liquid, Fluorescent Dyes, Glycoproteins, Glycosylation, Humans, Polysaccharides, Pronase, Proteolysis, Rituximab, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Streptomyces, Tandem Mass Spectrometry
Abstract

Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.

DOI10.1007/s00216-012-5749-5
Alternate JournalAnal Bioanal Chem
PubMed ID22354569