title: Cross validation of liquid chromatography-mass spectrometry and lectin array for monitoring glycosylation in fed-batch glycoprotein production.
abstract: Glycosylation analysis of recombinant glycoproteins is of importance for the biopharmaceutical industry and the production of glycoprotein pharmaceuticals. A commercially available lectin array technology was evaluated for its ability to present a reproducible fingerprint of a recombinant CTLY4-IgG fusion glycoprotein expressed in large scale CHO-cell fermentation. The glycosylation prediction from the array was compared to traditional negative mode capillary LC-MS of released oligosaccharides. It was shown that both methods provide data that allow samples to be distinguished by their glycosylation pattern. This included information about sialylation, the presence of reducing terminal galactose β1-, terminal N-acetylglucosamine β1-, and antennary distribution. With both methods it was found that a general trend of increased sialylation was associated with an increase of the antenna and reduced amount of terminal galactose β1-, while N-acetylglucosamine β1- was less affected. LC-MS, but not the lectin array, provided valuable information about the sialic acid isoforms present, including N-acetylneuraminic acid, N-glycolylneuraminic acid and their O-acetylated versions. Detected small amounts of high-mannose structures by LC-MS correlated with the detection of the same epitope by the lectin array.
authors: School of Chemistry, National University Ireland, Galway, Ireland. catherine.hayes@medkem.gu.se
authors: Catherine A
authors: CA
authors: Hayes
authors: Roisin
authors: R
authors: Doohan
authors: David
authors: D
authors: Kirkley
authors: Kirk
authors: K
authors: Leister
authors: Brendan
authors: B
authors: Harhen
authors: Angela V
authors: AV
authors: Savage
authors: Niclas G
authors: NG
authors: Karlsson
mesh_terms: D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005690:Galactose / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D008358:Mannose / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D012044:Regression Analysis; D015203:Reproducibility of Results
chemical_list: D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; D019158:N-Acetylneuraminic Acid; D008358:Mannose; D005690:Galactose