Production method of fibrinogen

Abstract

The invention relates to a production method of fibrinogen. The production method comprises the following steps of collecting plasma and quickly freezing plasma; melting plasma and removing cryoprecipitate; preparing a component I precipitate; dissolving the component I precipitate; inactivating virus by adopting an S/D virus inactivation method; performing ethanol precipitation for the first time, and performing centrifuging and dissolving; performing ethanol precipitation for the second time, performing centrifuging and separating a precipitate; preparing a semi-finished product; performingsterilizing and sub-packaging; performing freeze drying; and carrying out dry heat inactivation of viruses. The method has the advantages that the purity of the obtained fibrinogen is greatly improvedand can reach 92% or above; the solidification activity is high and is 18-22 seconds; the re-melting time is short and is 5-10 minutes; after freeze-drying, the appearance is more uniform and stable,the yield is more than 2000 bottles per ton of plasma, and the yield is obviously improved; the long-term stability of the product is good, and the good inherent quality can still be guaranteed after36-month long-term stability test; and due to double virus inactivation, the clinical medication is safer, and no adverse reaction is caused.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a method for producing fibrinogen. Background technique [0002] Human fibrinogen (fibrinogen, Fg) is coagulation factor I, which is the coagulation factor with the highest content in plasma, and is also a "central" protein in the coagulation system. The final stage of coagulation is the formation of thrombin, which converts fibrinogen to fibrin. In addition to directly participating in the coagulation process, fibrinogen can also mediate platelet aggregation and affect blood viscosity. It is an important risk factor for cardiovascular and cerebrovascular diseases. It also plays an important role in pathological processes such as the formation of atherosclerosis and tumor metastasis. Fg gene defects can lead to congenital hypofibrinogenemia (or no) and dysfibrinogenemia. [0003] Fibrinogen directly participates in the coagulation process mainly as coagulation factor I. In t...

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Problems solved by technology

[0004] The purity of Fg is an important indicator affecting its internal quality. Low purity will affect the coagulation activity of the product, and its coagulation activity will be significantly reduced. At the same time, the reconstitution time will also be affected. It has been reported that the purity of Fg is lower than 80%. The dissolution time will exceed 20 minutes, and protein precipitation and protein particle suspension will easily occur at the same time, which brings inconvenience to clinical use

At the same time, if the purity is too low, it will affect the stability of Fg and cause problems for its preservation.

[0005] At present, there are 11 blood product units in my country with the production capacity of Fg. Most blood product companies use low-temperature ethanol to extract Fg from component I. The disadvantage is that the purity of the product is low, generally not more than 80%, and the appearance is poor. , poor resolubility (20-30 minutes), low yield (800-1000 bottles / ton of plasma)

Cryoprecipitate is also used as a raw material to extract Fg from it, but the Fg content in the cryoprecipitate is low, and at the same time, it will cause the final product to contain human coagulation factor VIII impurities, which in turn will cause Fg to be activated and denatured, and the output of human coagulation factor VIII will also be accordingly affected

There is also the co-precipitation of cryoprecipitation and component I, that is, adding a certain concentration of ethanol to the melted plasma and cooling down to make the cold gel and component I precipitate together. The precipitation contains 8% ethanol, which will cause blood coagulation in the precipitate. Denaturing inactivation of factor VIII

[0006] In recent years, it has also been reported that Fg is prepared by chromatography, such as Q Sepharose Fast Flow anion exchange chromatography in CN105504046A "A Preparation Method of Human Fibrinogen"; CN107827974A "A Preparation Method of Human Fibrinogen" What adopts is lysine affinity chromatography and cation exchange chromatography; CN107540743A "A kind of method for preparing human fibrinogen by double-layer chromatography" adopts Q Sepharose Fast Flow anion exchange chromatography and heparin affinity chromatography Chromatographic methods can improve the purity of Fg to a certain extent, but the problem is that the chromatographic packing has a service life. With the increase of the number of uses, the purification effect will be weakened, and whether the long-term stability of the product will be affected is still unknown. It needs to be studied. At the same time, the chromatography filler is expensive, which increases the production cost, will cause the price of medicines to be expensive, and increase the treatment pressure of patients.

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