| Human fibrinogen (Fg) is the most abundant coagulation factor in plasma, and the key protein in the coagulation system. The final stage of coagulation is the convert of Fg into fibrin with the action of thrombin, where fibrin and other blood cell components aggregate into insoluble substance groups and stop the bleeding. In addition to the direct participation in the clotting process, Fg also mediates platelet aggregation, affects blood viscosity, and it is an important risk factor of the cardiovascular and cerebrovascular diseases. Fg deficiency-related diseases include congenital fibrinogen decrease or deficiency, acquired fibrinogen deficiency due to liver severe damage, cirrhosis, disseminated intravascular coagulation, trauma, postpartum hemorrhage, major surgery, and internal bleeding. For the congenital deficiency reduction, Fg supplement is the only reliable method of treatment, and for the reduction and deficiency caused by different acquired factors, different doses of Fg infusion preparations are also needed. What is more, a certain reserve of Fg must be maintained each year, because Fg is a hemostatic drug used in military strategic reserves. However, so far, only a few of blood products companies have Fg production capacity in our country, and their total production were very limited, that is less than100,000bottles between2008and2010year, which causes Fg preparations are always in short supply in the clinical applications. Therefore, this project intends to develop the production process of Fg preparations using the company’s existing plasma, prepare products that meet the requirements of the Chinese Pharmacopoeia, and relieve clinical urgent situation. The research contents and the results obtained are as follows.1Determined the raw material according to each component of the existing process of blood productsWe carried out a detailed study on the source plasma, cryoprecipitate, acid precipitation, and Cohn’s fraction Ⅰ+Ⅲ, analyzing the protein content, Fg purity, and Fg coagulation activity. Meanwhile, we assessed the influence of selection of raw materials on the company’s existing product line, and ultimately, we selected acid precipitation as the raw materials. Acid precipitation is the Fg-rich acidic precipitation enriched from the production of human coagulation factor VIII (human coagulation factor Ⅷ, F Ⅷ) cryoprecipitation, which is discarded in current production systems. The route choosing acid precipitation as the raw materials can further improve the comprehensive utilization of human plasma, to generate greater economic and social benefits.2Determined the process route and obtained a high-purity liquid of FgWe obtained a mature production process for Fg:first, healthy fresh frozen plasma was certrifugated to prepare cryoprecipitate, and then the cryoprecipitate was dissolved, and centrifuged to obtain Fg-rich acid precipitation after adjusting pH. Second, redissolved after acid precipitation, the sample was put through the DEAE-650M ion-exchange column chromatography to remove other clotting factors and fibronectin, and then was inactivated of lipid-enveloped viruses by S/D method. Third, after two glycine precipitation to remove S/D, and ultrafiltration, we obtained the Fg liquid. After verification, Fg liquid purity is greater than90%, and the solidification time is less than30s. The process does not select low-temperature ethanol method as commonly used, avoiding the influence of ethanol on protein denaturation, while reducing the temperature of the process itself accuracy requirements.3Selected suitable viral inactivation methods of Fg production process, and the results were validatedFor security, after the promulgation of the2005edition of <Chinese Pharmacopoeia>, the new standards require blood products especially clotting factor category products to go through effective virus inactivation step against lipid-enveloped viruses and non-lipid-enveloped viruses. Compared with different purification processes, we made a detailed and reliable test on the viral inactivation process, and determined the use of organic solvent/detergent method and the dry heat method to inactivate lipid-enveloped viruses non-lipid-enveloped viruses, respectively. We have entrusted Chinese Military Academy of Medical Sciences to verify the virus inactivation effect of our three batches of human Fg, batches were:200908S01,200909S02,200909S03. Validation results show that the three batches of human Fg intermediate solution prepared through the S/D inactivation method, can not be detected of HIV, PRV and Sindbis, and the titers of HIV, PRV and Sindbis are decreased4log or more. Meanwhile, the effect of EMCV inactivated by dry heat has also been validated, which is in line with national requirements.4Prepared samples through pilot scale, carried out stability studies, and obtained clinical trial approvalsWe researched the25℃accelerated test and2～8℃long-term stability about the Fg preparations with specification of0.5g/bottle, batches were:201001S02,201002S03,201003S04. Compared with the listed products, we studied the product appearance, vacuum degree, reconstitution time, visible foreign matter, pH and stability (no clot or fibrin deposition when incubated at37℃for60min after reconstitution), Fg content, clotting activity. The results showed:the indicators of our three batches of products are all qualified; comparative study with the reference standard indicated that both our products and listed products are within the scope of qualified, and some indicators of the test products also better than those of the reference.We have gotten clinical trial approvals from State Food and Drug Administration on March,2012, and the clinical trials are underway. |
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