Establishment of a method for mapping of N-linked oligosaccharides and its use to analyze industrially produced recombinant erythropoietin.
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.
D000818:Animals; D016466:CHO Cells; D002848:Chromatography, DEAE-Cellulose; D006224:Cricetinae; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000737:chemistry*; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization