title: Profiling of erythropoietin products by capillary electrophoresis with native fluorescence detection.
abstract: The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal-to-noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE-Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO-α and EPO-β) based on the recorded glycoform pattern. The developed CE-Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.
authors.affiliation: Biomolecular Analysis, Utrecht University, the Netherlands.
authors.forename: Bregje J
authors.initials: BJ
authors.lastname: de Kort
authors.forename: Gerhardus J
authors.initials: GJ
authors.lastname: de Jong
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin