title: A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals.
abstract: The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Harazono
authors.forename: Tetsu
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Satsuki
authors.initials: S
authors.lastname: Itoh
authors.forename: Minoru
authors.initials: M
authors.lastname: Tada
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hashii
authors.forename: Akiko
authors.initials: A
authors.lastname: Ishii
authors.forename: Teruyo
authors.initials: T
authors.lastname: Arato
authors.forename: Shigehiro
authors.initials: S
authors.lastname: Yanagihara
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yagi
authors.forename: Akiko
authors.initials: A
authors.lastname: Koga
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Tsuda
authors.forename: Mikiko
authors.initials: M
authors.lastname: Kimura
authors.forename: Masashi
authors.initials: M
authors.lastname: Sakita
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kitamura
authors.forename: Hideto
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Hisashi
authors.initials: H
authors.lastname: Mimura
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Murata
authors.forename: Yasuki
authors.initials: Y
authors.lastname: Hamazume
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sato
authors.forename: Shunji
authors.initials: S
authors.lastname: Natsuka
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Kakehi
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Kinoshita
authors.forename: Sakie
authors.initials: S
authors.lastname: Watanabe
authors.forename: Teruhide
authors.initials: T
authors.lastname: Yamaguchi
mesh_terms: D000606:Amino Sugars / Q000032:analysis / Q000592:standards; D001688:Biological Products / Q000737:chemistry* / Q000592:standards; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D002852:Chromatography, Ion Exchange / Q000379:methods / Q000592:standards; D004921:Erythropoietin / Q000737:chemistry; D005079:Excipients; D006031:Glycosylation; D009005:Monosaccharides / Q000032:analysis* / Q000592:standards; D011994:Recombinant Proteins; D012015:Reference Standards; D015203:Reproducibility of Results; D012794:Sialic Acids / Q000032:analysis / Q000592:standards; D010959:Tissue Plasminogen Activator / Q000737:chemistry
chemical_list: D000606:Amino Sugars; D001688:Biological Products; D005079:Excipients; D009005:Monosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D004921:Erythropoietin; D010959:Tissue Plasminogen Activator