Pilot production of recombinant human clotting factor IX from transgenic sow milk

Valuable pharmaceutical proteins produced from the mammary glands of transgenic livestock have potential use in the biomedical industry. In this study, recombinant human clotting factor IX (rhFIX) produced from transgenic sow milk for preclinical animal studies have been established. The transgenic sow milk was skimmed and treated with sodium phosphate buffer to remove abundant casein protein. Then, the γ-carboxylated rhFIX fraction was segregated through the Q Sepharose chromatography from uncarboxylated one. For safety issue, the process included virus inactivation by solvent/detergent (S/D) treatment. Subsequently, the S/D treated sample was loaded into the Heparin Sepharose column to recover the rhFIX fraction, which was then reapplied to the Heparin Sepharose column to enhance rhFIX purity and lower the ratio of activated form rhFIX (rhFIXa) easily. This was possible due to the higher affinity of the Heparin affinity sorbent for rhFIXa than for the rhFIX zymogen. Furthermore, an IgA removal column was used to eliminate porcine IgA in purified rhFIX. Finally, nanofiltration was performed for viral clearance. Consequently, a high-quality rhFIX product was produced (approximately 700 mg per batch). Other values for final rhFIX preparation were as follows: purity, >99%; average specific activity, 415.6±57.7 IU/mL and total milk impurity, <0.5 ng/mg. This is the first report that described the whole process and stable production of bioactive rhFIX from transgenic sow milk. The overall manufacturing process presented here has the potential for industrial production of rhFIX for treatment of hemophilia B patients.