Comparison of different capillary electrophoresis methods for analysis of recombinant erythropoietin glycoforms

Human erythropoietin (hEPO), an endogenously produced glycoprotein, plays a fundamental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant erythropoietin (rEPO) by molecular biology techniques has made possible its therapeutic use together with its abuse in competitive sports. Since the glycosylation pattern of rEPO depends, among other things, of the cell line used for production, it presents a glycoform scheme different from manufacturer to manufacturer and different also from endogenous hEPO. The separation of these different glycoforms is interesting since it could be useful: (i) for determining the glycoform distribution in recombinant products from different batches and/or manufacturers, (ii) for studying the link between glycosylated forms and their activity, and (iii) for helping to discriminate between endogenous and recombinant EPO. This paper desribes the development of three different Capillary Electrophoresis methods that use UV detection to separate rEPO glycoforms. The special features of the three methods are discussed in terms of resolution of bands of rEPO glycoforms, method reproducibility, and compatibility with other more sensitive detection systems such as laser induced fluorescence using on-column derivatization protocols or more selective ones such as mass-spectrometry.