Characterization of recombinant human granulocyte colony stimulating factor (rHuG-CSF) by capillary zone electrophoresis, capillary isoelectric focusing electrophoresis and electrospray ionization mass spectrometry

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) is a hematopietic cytokine that stimulates and regulates the proliferation and differentiation of neutrophils. Glycosylated and non-glycosylated forms of rHuG-CSF cannot be distinguished by traditional biological assays. In addition, it is very difficult to characterize impurities of the same molecular weight in biologicals. In this study, non-glycosylated rHuG-CSF, two glycosylated rHuG-CSF isoforms and their commercial dosages were successfully separated by capillary zone electrophoresis (CZE) using 50 mM Tricine containing 20 mM NaCl and 2.5 mM 1,4-diaminobutane (DAB) at pH 8.0, which could be employed for the qualitative discrimination assay of rHuG-CSF related products. CZE, capillary isoelectric focusing electrophoresis (CIEF), and mass spectrometry (MS) were used to effectively characterize non-glycosylated rHuG-CSF. It was found that proteins in the samples with different pIs in the CIEF profile could not be detected by CZE, while no difference was observed between these proteins and rHuG-CSF. Further analysis by electrospray ionization mass spectrometry with the resolution of 2000 showed that the components with different pIs in the non-glycosylated rHuG-CSF bulk sample are nearly equal in molecular weight. Therefore, it is necessary to combine several modem analytical techniques for quality control to get well-characterized biologicals. (C) 2004 Elsevier B.V. All rights reserved.