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Hemophilia B is the congenital bleeding disorder caused by deficiency in functional coagulation factor IX (FIX) and about 28,000 patients worldwide in 2012. And current treatment is restricted to protein-replacement therapy, which required FIX concentrates for patients’ life-time. Approximately 1 billion units FIX were consumed in 2012. However, still about 70-80% patients, mostly in developing countries, received inadequate or no treatment because of the unavailable and/or unaffordable FIX concentrates. Considering safety reasons, e.g. transmission of blood-borne diseases, the recombinant human FIX (rFIX) is recommended other than the plasma-derived FIX. However, only one rFIX is currently available on the market. The complexity of the FIX protein and its post-translational modifications (PTMs) cause the limit quantity and unaffordable high price of the recombinant human FIX. We previously reported successfully expressing recombinant human FIX in the milk by mammary gland of transgenic pig (tg-FIX) and established a lab-scale purification protocol to achieve active tg-FIX. The expression of tg-FIX level was about 2-3 g/L and with 10-20% specific activity. The final purified high acidic tg-FIX had the specific activity closed to the normal human plasma derived FIX. In this study, efforts were aim to further increase the yield of the active tg-FIX from the transgenic pig. First, we investigated degradation/activation of the tg-FIX in the milk by predominant milk-borne protease, plasmin. This provided us the data in decreasing the degradation and activation of final products. Then, to process the excessive non-active pro-peptide attached tg-FIX, we bio-engineered the mammary gland by introducing truncated human furin gene. The co-expressed recombinant furin cleaved pro-peptide of tg-FIX both in mammary epithelium, it also secreted and cleaved the pro-peptide in milk. This bio-engineering has been proved not interfering the over-expression of the tg-FIX and its specific activity. Finally, to mass producing active tg-FIX, we established the large scale purification protocol. The purified active tg-FIX was achieved within four chromatography steps with full specific activity. Not like the previous one, the active tg-FIX was further enriched and the contaminant activated tg-FIX was totally eliminated. This study made progress in producing recombinant human FIX economically.
Advisor: William H. Velander
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