Differences between the N-glycans of human serum erythropoietin and recombinant human erythropoietin.

The differences between the N-glycans of human erythropoietin (hEPO), extracted from serum, and recombinant human erythropoietin (rhEPO) reported by Skibeli et al1 have received attention in the editorials of 2 other journals. One suggested that these differences might be involved in the etiology of the autoimmune pure red cell aplasia found in some patients treated with rhEPO.2 The other suggested that these differences might form the basis for a test to detect the doping of athletes with rhEPO.3 A review of the data of Skibeli et al, however, indicates that the differences they have reported between the N-glycans of hEPO and rhEPO are not consistent with the differences between the physicochemical properties of these EPOs found in other laboratories. This discrepancy may be a consequence of the effects on the N-glycans of hEPO of the conditions used by Skibeli et al to extract hEPO from serum in order to compare it with unextracted rhEPO. Skibeli et al reported that hEPO lacked the tetra-acidic (tetrasialylated) N-glycans found in the rhEPOs, and was also lower in its content of tri-acidic N-glycans.1 These findings are summarized in their Table1,1 which also permits comparison of the N-glycan charges of different EPOs, as calculated, for example, by the method of Hermentin et al.4 This suggests that the total negative charge of the N-glycans of hEPO is 70% that of the rhEPOs, and implies that hEPO is less acidic than the rhEPOs. Thus the sialic acid residues of the N-glycans of EPO represent up to 12 of a possible total of 14 sialic acid residues in EPO, and make a major contribution to the net negative charge of EPO, as indicated by the fact that the pI of intact EPO is in the range of about 2.5 to 4.0,5 and the pI of desialylated EPO is about 8.5.6 However, other studies have consistently shown hEPO to be more acidic than rhEPO,7-9 and, indeed, this is the basis for an established test for doping.9 The discrepancy between the findings of Skibeli et al and those of others is probably due to the conditions used to extract the hEPO from serum to compare it with unextracted rhEPOs. Although the data in Figure 8 of Skibeli et al is said to demonstrate “the similarity of sugar profiles from rhEPO with or without a beadextraction step,” comparisons of the areas under the 2 elution profiles indicates that the extraction procedure has reduced the recovery of all oligosaccharides with elution times of 60 minutes or more, representing triand tetrasialylated N-glycans, and has increased the relative recovery of most of the neutral, monoand di-sialylated N-glycans.1 This effect of the extraction procedure probably represents some desialylation of the N-glycans of EPO, since the 20min-treatment with 10mmol/l HCl, used to dissociate the antibody-bound EPO, is also commonly used, albeit at 80°C rather than ambient temperature, for the quantitative desialylation of N-glycans.10