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

Abstract

Human erythropoietin (hEPO), an endogenously produced glycoprotein, plays a fun- damental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant erythropoietin (rEPO) by molecular biology techniques has made pos- sible its therapeutic use together with its abuse in competitive sports. Since the glyco- sylation pattern of rEPO depends, among other things, of the cell line used for pro- duction, it presents a glycoform scheme different from manufacturer to manufacturer and different also from endogenous hEPO. The separation of these different glyco- forms is interesting since it could be useful: i) for determining the glycoform distribu- tion 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 detec- tion to separate rEPO glycoforms. The special features of the three methods are dis- cussed 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.