Establishment of a Method for Mapping of N-Linked Oligosaccharides and Its Use to Analyze Industrially Produced Recombinant Erythropoietin

• Published in: Biological & pharmaceutical bulletin Vol. 22; no. 4; pp. 339 - 346
• Main Authors: KANAZAWA, Koki, ASHIDA, Kyoko, ITOH, Masao, NAGAI, Hiroshi, SASAKI, Hiroshi, FUKUDA, Minoru
• Format: Journal Article
• Language: English
• Published: Tokyo The Pharmaceutical Society of Japan 01.04.1999; Maruzen; Japan Science and Technology Agency
• Subjects: 2-aminobenzamide; Analysis; Animals; Biological and medical sciences; CHO Cells; Chromatography, DEAE-Cellulose; Cricetinae; Culture Media; Electrophoresis, Polyacrylamide Gel; Erythropoietin - chemistry; General pharmacology; Humans; MALDI-TOF-MS; Medical sciences; N-Acetylneuraminic Acid - chemistry; N-linked oligosaccharide analysis; Oligosaccharides - chemistry; Oligosaccharides - isolation & purification; Pharmacology. Drug treatments; recombinant human erythropoietin; Recombinant Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; tetrasialylated oligosaccharide; Human; Separation method; Fluorescence detector; Erythropoietin; Photoionization; Oligosaccharide; HPLC chromatography; Matrix effect; Recombinant protein; Mass spectrometry; Laser desorption; Quantitative analysis
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.22.339

A rapid and convenient method for N-linked oligosaccharide structure analysis was developed and applied to the quality examination of commercially manufactured recombinant human erythropoietin (rEPO). Oligosaccharides released from rEPO expressed in Chinese hamster ovary cells were labeled with 2-aminobenzamide, and analyzed by a combination of diethylaminoethyl (DEAE) column HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This newly developed method allowed us to analyze sialylated oligosaccharides directly. The results showed that these oligosaccharides are composed of neutral-mono-di-tri-tetrasialyl oligosaccharides in a ratio of<1 : 2 : 13 : 34 : 51%, and that the core structure was a mixture of complex type bi-, tri- and tetra-antennary forms with one to three polylactosaminyl chains. Further analysis showed clearly that the N-linked oligosaccharide structure of rEPO has not changed throughout the past ten years of manufacturing by our company. The analysis was carried out using a small quantity (1 nmol) of rEPO, demonstrating the efficacy of the newly developed method. Further, the N-linked oligosaccharides of rEPO manufactured by our company almost coincided with those of the erythropoietin European Pharmacopoeia Biological Reference Preparation (Eur.Ph.BRP), a standard product.