| Human coagulation factor Ⅷ(FⅧ) is a very important human plasma clotting factor whose absence or deficiency can cause varying degrees of hemophilia A, an X-linked recessive bleeding disorder, even endangering the lives of patients. Hemophilia A is a lifelong disease with no cure method up to now, and the only way is replacement therapy by infusing of FⅧ products. As long as a timely infusion of F Ⅷ products, patients with hemophilia A can have a normal life even in intense sport. However, the serious shortage of Ⅷ products on current domestic market can not meet the needs of patients with hemophilia A. Some patients suffering from hemophilia can not get FⅧ products from the market.FⅧ is a national standard drug included in the Chinese Pharmacopoeia. With the great growth in the domestic economy and the rapid development of protein separation technology, a few domestic manufacturers began producing FⅧ products. Taibang company began developing the separation and purification of FⅧ, with a view to supply more security and effective drugs to hemophilia patients.FⅧ is a large plasma protein with a molecular weight of330kDa and complex structure. Separation and purification of FⅧ is very difficult since the low content of about0.1-0.2μg/ml plasma and a half-life of only12hours. Here we show a scaled-up production process through a lot of experimental data. Cryoprecipitate centrifuged from human fresh frozen plasma was used as the initial material. It was dissolved with appropriate proportion of heparin sodium solution, and most of fibrinogen and fibronectin were precipitated by lowering the dissolved solution pH at appropriate temperature conditions. The supernatant was treated with sovent/detergent (S/D) virus inactivation method for6hours. Then the supernatant with S/D was subjected to flow through Toyopearl DEAE650M weak anion-exchange gel, firstly eluting impurity proteins with washing buffer, and then eluting FⅧ with elution buffer. The eluate was treated with ultrafiltration, then formulated, freeze-dried, dry-heated at100°C for30min, and the final FⅧ product was got.Our FⅧ products have been completed clinical trials with good results. The FⅧ activity levels were significantly increased to a high efficiency level (10minutes after administration reached161.89%,1hour after treatment to112.98%), and after treatment, clinical symptoms have been greatly improved with a improvement rate of93%(of which31.6%excellent,61.4%improvement).According to the test results of National Institutes for Food and Drug Control, a number of quality criteria of Our FⅧ products are better than the minimum standard requirements of "Chinese Pharmacopoeia". Some of the tested items were higher than that in "European Pharmacopoeia" standards. The redissolving time is less than two minutes, only1/15of "Chinese Pharmacopoeia" maximum standard. The specific activity is as high as41.7IU/mg protein,41.7-fold higher than the "Chinese Pharmacopoeia" minimum standard. The anti-A&anti-B hemagglutinins is less than1:4, only1/16of "Chinese Pharmacopoeia" maximum standard. Tween-80residues do not exceed6μg/ml, within1/16of "Chinese Pharmacopoeia" minimum standard. TNBP residues do not exceed3μg/ml, within1/3of "Chinese Pharmacopoeia" minimum standard. In addition, the quality criteria of our FⅧ products are not lower than the levels of commercially available products.Calculated according to the amount of300tons plasma per year,150,000bottles of FⅧ product containing200IU/bottle can be produced, about50million yuan value of output for the company can be got, and about30million IU of FⅧ activity for the patients with hemophilia can be used.This will greatly ease the domestic tension of F Ⅷ products in medication. |
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