Labeling conditions using a 2-aminobenzamide reagent for quantitative analysis of sialo-oligosaccharides.
During 2-aminobenzamide (2-AB)-labeling of sialo-oligosaccharides the authors encountered a significant loss of sialic acids under the reported conditions, at 65 degrees C for 2 h. By examination of the relationship between labeling temperature and recovery of tetra-sialylated oligosaccharides, 2-AB-labeling at 37 degrees C for 16 h resulted in a maximum yield with practically no loss of sialic acid. Under the newly optimized conditions, the reproducibility of 2-AB-labeling was examined in triplicate with three lots of recombinant human erythropoietin (r-HuEPO). The present conditions gave analytical data comparable to those obtained by the established NaB[3H]4 labeling procedure. The modified method has been applied to oligosaccharide structural analysis of sialoglycoprotein therapeutics.
Watanabe
T
T
Chemistry and Quality Control Group, Pharmaceutical Development Laboratory, Kirin Brewery Co., Ltd., Takasaki, Gunma, Japan.
Inoue
N
N
Kutsukake
T
T
Matsuki
S
S
Takeuchi
M
M
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004921:Erythropoietin / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D011786:Quality Control; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D007202:Indicators and Reagents; D009844:Oligosaccharides; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; C016735:sialooligosaccharides; D004921:Erythropoietin; C000219:anthranilamide