Unusual N-glycosylation of a recombinant human erythropoietin expressed ina human lymphoblastoid cell line does not alter its biological properties

Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycos ylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser -126) and involved in the regulation of the level of red blood cells, Today , only one recombinant human Epo (rHuEpo), produced in CHO cell line, is ex tensively used in therapy to cure severe anemia. The structure of the glyca n chains of this rHuEpo slightly differ of those of the urinary human Epo ( uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a hum an lymphoblastoid cell line, named RPMI 1788. In order to fully characteriz e the Epo-RPMI, structural characterizations of the protein skeleton as wel l as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of so me N-glycan chains, as mainly determined by ESI-MS, revealed some unusual c haracteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a si alyl Le(x) motif, and 13% contain more than three LacNAc repeats (up to fir e per molecule). Despite these unusual: structural characteristics, the dat a concerning the in vitro and in vivo biological activities were not impair ed when compared to Epo-CHO and uHuEpo.