Purification and characterization of recombinant human erythropoietin from milk of transgenic pigs

Abstract

BACKGROUND: Human erythropoietin (hEPO), a hydrophobic acidic glycoprotein responsible for the regulation of red blood cell production in mammals, is used for the treatment of anemia. In general, the purification of transgenic animal-derived therapeutic proteins is not easy due to their low titer concentrations and abundant contaminant proteins. For the first time, here the purification and characterization of rhEPO from themilk of transgenic pigs are described. RESULTS: The rhEPO was purified by heparin chromatography, reverse-phase chromatography, and gel filtration chromatography, resulting in a 16.5% yield and >98% purity. The rhEPO purified from the milk of transgenic pigs contained less acidic isoforms andwas underglycosylated in contrast to CHO-derived rhEPO. Cell proliferation of the F-36/EPO-dependent cell line was proportional to the dose of transgenic pig-derived rhEPO. CONCLUSION:Transgenicpig-derivedrhEPOwithhighpuritywas achieved after three-step chromatographyfollowing two-step precipitation. The transgenic pig-derived rhEPO was demonstrated to have comparable potency with CHO-derived rhEPO. Transgenic pig-derived rhEPO may not be therapeutically feasible because of different glycosylation, and thus further studies are required to elucidate the effect of this aberrant glycosylation on the biological activity and stability in vivo.