Preparation of human erythropoietin for tissue culture

A simple two-step procedure has been developed for the production of human urinary erythropoietin suitable for tissue culture use. Lyophilized human urinary protein was fractionated by phenyl-Sepharose chromatography, the erythropoietin binding in 2 M lithium chloride and being eluted by an ascending linear gradient to 6 M guanidine hydrochloride. The erythropoietin was further purified by ethanol (75% vol/vol) precipitation. This two-step procedure resulted in a 25-fold purification of erythropoietin with a 50% recovery of the starting activity. The erythropoietin preparation was suitable for use in murine or human cultures, being free of protease activity, inhibitory factors, colony-stimulating factors, and erythroid-enhancing activities.