Capillary electrophoretic separation of human recombinant erythropoietin (r-HuEPO) glycoforms.
Free zone capillary electrophoresis was investigated for the separation of glycoforms in recombinant erythropoietin (r-HuEPO). Reduction of electroendoosmotic flow through the addition of 1,4-diaminobutane and in the presence of urea resulted in optimum separation of all of the major glycoforms. Incubation of r-HuEPO with neuraminidase caused the slowest migrating species to diminish first in response, indicating that these contained the highest numbers of sialic acids. Additional evidence was obtained from spiking r-HuEPO with individual glycoforms isolated by preparative isoelectric focusing. These results indicate that the separation occurs in a predictable manner in order of increasing number of sialic acids contained in the glycoforms. As a consequence, the electropherograms provide the same information as that obtained from conventional gel isoelectric focusing, although separating by a different mechanism.
Watson
E
E
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Yao
F
F
D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004586:Electrophoresis / Q000379:methods; D004921:Erythropoietin / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D009439:Neuraminidase / Q000494:pharmacology; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012794:Sialic Acids / Q000737:chemistry / Q000302:isolation & purification; D014508:Urea / Q000494:pharmacology
D009844:Oligosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D004921:Erythropoietin; D014508:Urea; D009439:Neuraminidase