title: Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides.
abstract: Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α1-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.
authors.affiliation: Thermo Fisher Scientific, Lakeside Drive, Sunnyvale, CA 94085, USA.
authors.forename: Udayananth
authors.initials: U
authors.lastname: Aich
authors.affiliation: Thermo Fisher Scientific, Oakmead Parkway, Sunnyvale, CA 94085, USA.
authors.forename: Deanna C
authors.initials: DC
authors.lastname: Hurum
authors.affiliation: Thermo Fisher Scientific, Oakmead Parkway, Sunnyvale, CA 94085, USA.
authors.forename: Lipika
authors.initials: L
authors.lastname: Basumallick
authors.affiliation: Thermo Fisher Scientific, Lakeside Drive, Sunnyvale, CA 94085, USA.
authors.forename: Srinivasa
authors.initials: S
authors.lastname: Rao
authors.affiliation: Thermo Fisher Scientific, Lakeside Drive, Sunnyvale, CA 94085, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Pohl
authors.affiliation: Thermo Fisher Scientific, Oakmead Parkway, Sunnyvale, CA 94085, USA. Electronic address: jeff.rohrer@thermofisher.com.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Rohrer
authors.affiliation: GlycoThera, 30625 Hannover, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kandzia
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D060748:Fetuins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000032:analysis* / Q000592:standards; D009844:Oligosaccharides / Q000032:analysis* / Q000592:standards; D009961:Orosomucoid / Q000737:chemistry / Q000378:metabolism; D012015:Reference Standards; D062367:ortho-Aminobenzoates / Q000737:chemistry*
chemical_list: D060748:Fetuins; D007074:Immunoglobulin G; D009844:Oligosaccharides; D009961:Orosomucoid; D062367:ortho-Aminobenzoates; D001216:Asparagine; D019158:N-Acetylneuraminic Acid; C000219:anthranilamide
keywords: 2-Amino benzamide; Glycoprotein; HPAE–PAD; N-linked glycans; Oligosaccharides; Sialic acids