A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals |
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| Authors: | Akira Harazono Tetsu Kobayashi Nana Kawasaki Satsuki Itoh Minoru Tada Noritaka Hashii Akiko Ishii Teruyo Arato Shigehiro Yanagihara Yuki Yagi Akiko Koga Yuriko Tsuda Mikiko Kimura Masashi Sakita Satoshi Kitamura Hideto Yamaguchi Hisashi Mimura Yoshimi Murata Yasuki Hamazume Takayuki Sato Teruhide Yamaguchi |
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| Affiliation: | 1. Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan;2. Pharmaceuticals and Medical Device Agency, Shin-kasumigaseki Building 3-3-2, Kasumigaseki, Chiyoda-ku, Tokyo 100-0013, Japan;3. Production Division, Kyowa Hakko Kirin Co., Ltd. 100-1, Hagiwara-machi, Takasaki City, Gumma 370-0013, Japan;4. Analytical Technology Research Department, Chugai Pharmaceutical Co., Ltd. 5-5-1, Ukima, Kita-ku, Tokyo 115-8543, Japan;5. Pharmaceutical Analysis, Pharmaceutical Research & Technology Laboratories, Astellas Pharma Inc. 180, Ozumi, Yaizu City, Shizuoka 425-0072, Japan;6. Technology Research and Development, Dainippon Sumitomo Pharma Co., Ltd. 1-3-45, Kurakakiuchi, Ibaraki City, Osaka 567-0878, Japan;7. College of Science, Niigata University, 8050, Ikarashi 2-no-machi, Nishi-ku, Niigata City, Niigata 950-2181, Japan;8. School of Pharmacy, Kinki University, 3-4-1, Kowakae, Higashi-Osaka City, Osaka 577-8502, Japan |
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| 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 4–7 N 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. |
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