Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor-binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogs were developed to test this hypothesis. Darbepoetin alfa (Aranesp), which was engineered to contain five N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, darbepoetin alfa is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximate threefold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. Darbepoetin alfa is currently being evaluated in human clinical trials for treatment of anemia and reduction in its incidence.