Method of obtaining fibrinogen concentrate

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology of obtaining haemostatic medications. Claimed is method of separating purified fibrinogen concentrate, free of viruses and ballast proteins. Solubilisation of cryoprecipitate of fresh frozen human plasma is realised. Fibrinogen is precipitated with 20-30% PEG solution. Separated sediment is dissolved in buffer with sodium citrate and sodium chloride. Virus inactivation of solution by solvent-detergent method is carried out in presence of 1-3% Tween-80 and 0.1-1.5% of tri-n-butylphoshate. Obtained concentrate is purified from products of virus inactivation and solvent-detergents by triple extraction with liquid paraffin. After that, obtained fibrinogen concentrate is re-precipitated with 1.0-2.5 M glycine solution. Sterile filtration and lyophilic drying with further corking of lyophilisate under vacuum and thermal inactivation are performed.

EFFECT: invention makes it possible to obtained lyophilised form of fibrinogen concentrate with approximately 55% output.

 

The invention relates to pharmaceutical industry, namely to the biotechnology production of hemostatic drugs. Fibrinogen is synthesized in the liver and secreted in plasma in concentrations sufficient for coagulation. Fibrinogen is a soluble glycoprotein that is converted under the action of thrombin in the fibrin network of fibres which is the basis of the bunch, providing hemostasis. Besides the hemostatic function of fibrinogen is involved in wound healing, tumor development and metastasis (Handbook for medical laboratory technicians in research methods of plasma hemostasis. The blood coagulation factors. 2008, Moscow, p. 7).

Congenital deficiency of fibrinogen (hypofibrinogenemia) can disrupt homeostasis and lead to bleeding. More common is the acquired deficiency of fibrinogen, which may be a consequence of hemodilution, blood loss, disseminated intravascular coagulation (DIC) and sepsis (Bratchikov A. M. Clinical problems of fibrinolysis. 1993, Kiev, pp. 151-172).

Hereditary or acquired fibrinogen deficiency causes the development of hypofibrinogenaemia, the main method of treatment and prevention which is replacement therapy with preparations of fibrinogen" (Zubairov D. M. Fibrinogen and fibrin. In: Molek�regular basis of blood coagulation and clot formation. 2000, Kazan, pp. 1-32).

Substitution therapy in patients with hypofibrinogenemia and its prevention using preparations containing fibrinogen.

In addition to replacement therapy drugs based on the received fibrinogen concentrate can be used in surgical practice as a component of fibrin glue. The composition of this glue allows you to perform operations on large wound surfaces, whether the operation in areas with a high degree of vascularity, or processing of large surfaces burns that accelerates the regeneration of epithelial tissues (Zilbert A. P. Blood loss and blood transfusion. Principles and methods of bloodless surgery. 1999, Petrozavodsk, pp. 55-62).

To the problems of production of drugs on the basis of fibrinogen from human blood plasma include:

- the possibility of infecting the recipient with hepatitis viruses (A, B, C), human immunodeficiency, parvovirus, syphilis and other pathogens,

- the possibility of transfer to the recipient with a certain group of blood group present in the preparations (concentrates average purity) of antibodies to antigens of red blood cells from other blood groups,

- contamination of drugs on the basis of fibrinogen ballast proteins,

To solve these problems develop appropriate technological approaches. Thus, the preparation of the pool of plasma is carried out OTB�R carefully screened healthy donors, which determine possible infection with hepatitis viruses, human immunodeficiency, etc. However, to fully guarantee the absence of viral infection in the process of obtaining concentrates of fibrinogen carry out viral inactivation.

A method of producing a concentrate of fibrinogen by salting out using alcohol-salt cold deposition" (blood products. Instructional and methodological materials for the control and production. - M., 1976, pp. 145-202). This method requires a large time and energy costs, preparation of complex buffer salt solutions and the need for their subsequent laundering, making the process cumbersome, and the duration of the procedure leads to a significant loss of product during receipt. Method can not be recommended for the production of a preparation from small amounts of blood.

Deposition of fibrinogen ammonium sulphate also requires subsequent purification of the product from salt solutions, which leads to additional technological difficulties" (Patent RU №2062103). Concentrates of fibrinogen obtained by cryoprecipitate, successfully applied in foreign surgical practice" (Patent RU №2062103), however, achieved the concentration of fibrinogen remains relatively low. The closest to the claimed method is a method of producing drug concentrations�ATA fibrinogen, presented in the Patent US 4960757.

The method consists in obtaining a concentrate of fibrinogen precipitate containing fibrinogen, obtained in the process for preparation of factor VIII is carried out according to the patent German Patent No. 2916711. The feedstock is dissolved in a buffer containing NaCl, by heating to 37°C and stirring to maintain pH=7,5 2N NaOH. Further to the fibrinogen concentrate was added a solution of CaCl2.2H2O and sucrose to a concentration of 60% in the final solution. Then, the solution of fibrinogen with stabilizers subjected to thermal pasteurization in a water bath at 60°C, to inactivate viruses and other pathogens. After pasteurization, the fibrinogen solution is diluted with buffer containing sodium chloride and sodium citrate and dwukrotnie periostat glycine. The disadvantage of this method is to reduce the concentration of fibrinogen in the final product of the partial polymerization at the stage of thermal inactivation. Formed at this stage, the fibrin is separated double-glycine precipitation. Thus the yield of fibrinogen in the final stage becomes smaller.

The aim of the present invention to provide a method for producing a purified fibrinogen. This goal was achieved by the development of a technology for providing a lyophilized form of a concentrate FiBr�of novena. The advantage of this method of allocation is the use of a buffer solution containing anticoagulate agent is sodium citrate. Just one advantage is the use of Tween-80 (monooleate polyoxyethylenesorbitan) and TnBP (tri-n-butylphosphate) as detergents when conducting viral inactivation. In this method also be in the process of purification of fibrinogen concentrate from the products of the process of viral inactivation.

The technology provides for the dissolution of the feedstock (cryoprecipitate fresh frozen human plasma) in water in the presence of 1-3 units of heparin, treatment of a solution of cryoprecipitate gel of aluminium hydroxide, the precipitation of fibrinogen 20-30% solution Page, the subsequent dissolution of selected sediment in the buffer with sodium citrate and sodium chloride, viral inactivation solution solvent/detergent Tween 80 and TnBP within 6-10 hours, removal of products of inactivation and inactivating agents extraction solution of liquid paraffin, purified from ballast two proteins by precipitation with glycine concentration of 1-2.5 mol/l, subsequent sterile filtration, filling and lyophilization.

The development of separation technology of fibrinogen first conducted in the laboratory with subsequent optimization of the chosen method, and then scaled textures in the conditions experienced PR�production. The production of the preparation of fibrinogen was carried out in accordance with the rules of good Manufacturing Practice (GMP), standard operating procedures (SOP) and necessary conditions of sterility".

Raw material for production of fibrinogen concentrate was frozen cryoprecipitate fresh frozen human plasma.

At all stages of production raw materials, intermediate products and targeted drug tested on the content of fibrinogen by the method of Claus.

Below is a specific example of the invention.

Example

1. The feedstock (cryoprecipitate fresh frozen plasma) in an amount of 100 g was dissolved in three times the volume of distilled water containing 1-3 units of heparin and add 3% gel Al(Oh)3(ratio: 1 kg suspended cryoprecipitate to 10 g of aluminum hydroxide).

2. Next, to this mixture was added 30% solution of Page, to a concentration of 3%, and the pH of the solution was adjusted to a value close to the isoelectric point of fibrinogen (6,6-6,8) acetic acid. Under the action of Paga fibrinogen precipitate, which was separated from the supernatant by centrifugation. Next, the supernatant is transferred to the production of purified factor VIII.

3. The precipitate containing fibrinogen was dissolved with vigorous stirring in a tenfold volume of buffer containing 10 mmol sodium citrate and 150 mmol of sodium chloride, maintaining the pH at 7.8 to 8.0 with 0.5 M solution of Tris-buffer, and a temperature of 30-35°C. the solution was Then centrifuged for clarification at 4000 rpm for 10 minutes.

4. Viral inactivation of fibrinogen carried out in the presence of 1% Tween-80®and 0.3% TnBP, incubating the resulting solution at room temperature with continuous stirring for 6-10 hours.

5. Removal of products of inactivation and inactivity agents is carried out by extraction of fibrinogen solution paraffin oil. For this to virusinaktivierung fibrinogen concentrate is added mineral oil in the ratio of 2:1 and intensively stirred for 20 min. the mixture was centrifuged at 2000 rpm for 10 minutes and the aqueous layer is separated. This procedure is carried out three times.

6. The separation of fibrinogen from the ballast proteins is carried out by precipitation of the obtained concentrate of fibrinogen glycine, bringing its concentration in the solution to 1.5 mol/L. thus, a precipitate of fibrinogen, and most of the ballast proteins remain in solution. Of postural fibrinogen was separated by centrifugation at 4000 rpm for 10 minutes and dissolved in a solution containing 10 mmol sodium citrate and 0.15 mol of sodium chloride. The procedure was repeated twice, each time washing the precipitate fibrinogen cold 1.5 M solution of glycine.

After the resultant deposition of fibrinogen dissolved in the buffer to the observed concentration�radio 25 g/l, sterile-filtered, poured into bottles and lyophilizers. The obtained lyophilisate sealed under vacuum, vials crimped aluminum caps and subjected to thermal inactivation at 80°C for 72 hours.

The yield of fibrinogen is about 55%.

A method of separating a purified concentrate of fibrinogen, free from viruses, namely, to solubilize the starting material, represented by cryoprecipitate fresh frozen human plasma, deposition of fibrinogen 20-30% solution of PEG, selected by dissolving the precipitate in buffer with sodium citrate and sodium chloride, virus inactivation of a solution solvent-detergent method in the presence of 1-3% Tween-80 and 0.1 to 1.5% tri-n-butylphosphate, purifying the resulting concentrate products from viral inactivation and solvent-detergent extraction liquid paraffin conducted three times, subsequent precipitation of the obtained concentrate of fibrinogen 1,0-2,5 M solution of glycine, sterile filtration and freeze-drying with subsequent capping of the extract under vacuum and terminatively.



 

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