PURIFICATION AND BIOLOGICAL-ACTIVITY OF A RECOMBINANT-HUMAN-ERYTHROPOIETIN PRODUCED BY LYMPHOBLASTOID-CELLS

A recombinant human erythropoietin (rH-EPO) was obtained from the cult ure supernatants of human B-lymphoblastoid,cells transfected by the hu man EPO gene. rH-EPO was purified by a two-step method based on immuno affinity and ion exchange chromatography. The first step was achieved by an anti-EPO monoclonal antibody (Mab). This Mab, immobilized on Sep harose 4B, allowed a 410-fold purification of the protein. The second step consisted of ion exchange chromatography on DEAF Sephacel. The co mbination of these two steps results in a highly purified rH-EPO with a global yield of about 50%; the specific activity of the protein was 176,000 IU/A(280). The NMR spectrum was characteristic for a well stru ctured, single-conformation protein. The purified protein was analyzed by SDS-PAGE and isoelectric focusing. The biological activity of puri fied rH-EPO was measured in vivo, by the incorporation of Fe-59 into r ed blood cells (RBC) of polycythemic mice and in vitro by the prolifer ative response of an EPO-dependent cell line. The purified protein exp ressed in lymphoblastoid cells of human origin had the same biological activity as that of urinary EPO and rH-EPO produced in other mammalia n cells.