Efficient recovery of recombinant human erythropoietin from milk of transgenic pigs by two-step pretreatment

The selective removal of impurity proteins and colloidal particles from milk prior to chromatographic purification processes presents a crucial issue in the production of therapeutic proteins from transgenic animals with high recovery yield and purity. We have developed an efficient two-step precipitation method for the recovery of the recombinant human erythropoietin (rhEPO) of interest from transgenic sow milk. Here, rhEPO was partially purified from transgenic sow milk via a two-step precipitation method consisting of ammonium sulfate and divalent metal precipitations, with a yield of approximately 82.1% and a purification fold of 10.4 at a copper concentration of 30 mM. Copper proved to be the strongest flocculating agent among the divalent ions tested for the aggregation of milk proteins under 35%, with ammonium sulfate, zinc, nickel, and calcium demonstrating increasing flocculating capability in the given order. Copper and zinc proved to be appropriate divalent metals for the recovery of rhEPO at high yield and purity, and the optimal concentration ranges of copper and zinc were 20~40 and 40~80 mM, respectively.