Structural characterization of recombinant human erythropoietins by fluorophore-assisted carbohydrate electrophoresis.
Routine analysis of the carbohydrate moieties of a glycoprotein is critical for ensuring the consistent quality of biopharmaceutical products. Fluorophore-assisted carbohydrate electrophoresis (FACE) is a recently introduced method for the separation, by polyacrylamide gel electrophoresis, of oligosaccharides labeled with 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS). In this study, we have evaluated the applicability of the FACE method to analysis of the carbohydrate moieties of three different recombinant human erythropoietins (rHuEPOs), two Chinese hamster ovary (CHO) cell-derived rHuEPOs, and one baby hamster kidney (BHK) cell-derived rHuEPO. The N-linked oligosaccharides released from the rHuEPOs were labeled with ANTS. Enzymic sequence analysis of the N-linked oligosaccharides was performed by the FACE method. The results showed that the FACE method was useful for the analysis of sialo-, asialo-, and agalacto-oligosaccharides in the same gel. Its usefulness for rapidly and reliably revealing the oligosaccharide profiles of given glycoproteins, and its reproducibility were also confirmed in this study. In conclusion, the method can be used for evaluating the quality consistency of recombinant glycoprotein products in terms of the carbohydrate moiety.
Morimoto
K
K
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan.
Maeda
N
N
Abdel-Alim
A A
AA
Toyoshima
S
S
Hayakawa
T
T
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000737:chemistry*; D005456:Fluorescent Dyes; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology; D008969:Molecular Sequence Data; D009281:Naphthalenes; D009844:Oligosaccharides / Q000737:chemistry; D011786:Quality Control; D011994:Recombinant Proteins