Design of a three-step chromatographic process of recombinant human erythropoietin purification

•RhEPO produced by HEK 293 cells was purified from untreated cultivation broth.•The purification procedure design was based on batch and column experiments.•Multimodal chromatography was an effective capture step.•Hydrophobic interaction chromatography was an effective purification step.•Cation-exchange chromatography was the final purification step. This work deals with the purification of an essential therapeutic protein, recombinant human erythropoietin (rhEPO). The source of rhEPO was the post-harvest medium from the cultivation of recombinant human embryonic kidney cell 293 line (HEK 293) with the protein concentration in the medium of 1.9 mg/mL and the rhEPO fraction of only 2.5%. The medium was directly used for chromatographic separation without any previous pre-treatment. A three-step chromatographic procedure was designed based on the results of both column and batch adsorption/desorption experiments. In the first chromatographic separation step, a multimodal adsorbent with cation exchange group, Capto MMC, was used. Many impurity proteins were removed in the breakthrough during the column loading. Effective desorption of rhEPO from this matrix was achieved using 1 M arginine. RhEPO purity of the intermediate product reached 27%. A hydrophobic interaction chromatography adsorbent, Capto Phenyl, was used in the second purification step. Elution was carried out efficiently, using 30% isopropanol and recovering 95% of rhEPO, which was further purified by cation-exchange chromatography using Fractogel EMD SE HiCap. The designed purification process eliminated over 99% of impurity proteins contained in the HEK post-harvest cultivation medium, resulting in an overall purification factor of about 40.