| Human antithrombin â…¢ (AT â…¢) in human blood, is an important factor involved in physiological coagulation and coagulation, as a major inhibitor of blood coagulation system and play its anticoagulant effect accounts for about 80% of the total anticoagulant effect in vivo, is the main anticoagulant components in human body.Lack of AT â…¢ will show a variety of symptoms in clinical, it is always applied in the patients, whose AT III activity in body below the normal level of 70%. The number of people who has a deficiency of AT â…¢ accounts for about 1/2000-1/5000 of the total. AT â…¢ is mainly used in hereditary antithrombin deficiency and acquired antithrombin deficiency. At present, the European Pharmacopoeia, United States Pharmacopoeia and Japan drug agency have all recorded AT â…¢ products. Three quarter of 20 mainly blood products factories are producing AT â…¢ for clinical use now, while, there is no product listed in China. In this study based on the plasma as materials, to determine the production process of AT â…¢, then produce products that meet the needs of the clinical patients, will bring huge social benefits and economic benefits.The main research contents and achievements of this project are briefly introduced as follows:(1) Through analysis of the distribution of AT â…¢ in the existing blood products, the raw materials of AT â…¢ were identified.Firstly, we collected information from the literature, compared with the product process in foreign countries. Then comprehensive considerated the available raw materials, through the analysis of the AT III potency of the removal cryoprecipitate plasma and after DEAE Sephadex-A50 adsorption plasma, at the same time, the selection of raw materials impact to the company’s existing product line is evaluated, finally selected after DEAE Sephadex-A50 adsorption plasma as the raw materials. Affinity chromatography was used to capture the target product, and the high recovery rate of AT III product was obtained, and does not affect the company whole process line of products Fâ…§ and downstream products of human serum albumin and static injection of human immunoglobulin and other production. This can make full use of the existing resources, to further improve the comprehensive utilization of plasma, on the one hand, it will bring economic benefits to the enterprise, and it will also produce a certain social benefits.(2) After study on separation and purification and the formulation of the prescription, determine the production process route of AT III, obtained high purity AT III product.The study on the preparation and purification of AT III from the raw materials to prepare liquid, then the process of preparation, using different experimental design, determine the parameter range and prescription process steps in the production of liquid, ultimately determine the production process:plasmaâ†'centrifugal removing of cryoprecipitate â†' DEAE Sephadex-A50 adsorption â†' filtrationâ†' capto Heparin affinity chromatographyâ†'ultrafiltrationâ†'organic solvent / detergent (S/D) viral inactivation â†' capto Q ion exchange chromatography â†' ultrafiltration â†' nano membrane filtration â†' liquid â†' preparation â†' aseptic filtration, subpackage â†' freeze-driedâ†'come out, cappingâ†'dry heat inactivated virusâ†'visual inspectionâ†' storage. The finished product AT III potency ratio was higher than that of 6 IU/mg protein.(3) Determine the best virus inactivation method for AT III production process, and the advanced nano membrane filtration technology was used to remove the virus, verify the virus inactivation effect.In order to ensure the safety of blood products, a variety of different mechanisms are commonly used in the production process. This research ultimately selected for S/D treatment method and heat dry (lyophilized product), respectively, for lipid enveloped and non lipid enveloped viruses. In addition, the advanced technology of nano filtration membrane was used to remove the virus, and the parvovirus was effectively removed, to further ensure the safety of products. Virus inactivation/ removal effect was commissioned by the Chinese Academy of Medical Sciences, Chengdu blood transfusion Institute for verification, the effect can reached the national requirements.(4) Through the pilot, samples were prepared and study on quality and stability.The feasibility of the whole production process was verified by the experiment of three batches of pilot scale. Carry out a comprehensive quality test about the three batches of products and comparative study on the quality of listed products in foreign countries. We studied on the 25 ℃ accelerated test and 2-8 ℃ long-term stability about the three batchs of AT III products. The results show that the production process established in this study is feasible and suitable for scale-up production. All the quality indicators meet the requirements of the quality standard and the results of the key quality indicators are better than those of listed products in foreign countries. Products stability is good in the process of acceleration and long-term stability. |
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