RU2556804C2 - Method of obtaining fibrinogen concentrate - Google Patents

Abstract

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.

Description

The invention relates to the field of pharmaceutical industry, namely to biotechnology for producing hemostatic drugs. Fibrinogen is synthesized in the liver and secreted into plasma in concentrations sufficient for blood coagulation. Fibrinogen is a soluble glycoprotein that is converted by thrombin to fibrin, the fiber network of which is the basis of a clot that provides hemostasis. In addition to hemostatic function, fibrinogen is involved in wound healing, tumor development, and metastasis (Manual for laboratory assistants using methods for studying plasma hemostasis. Blood coagulation factors. 2008, Moscow, p.7).

Congenital fibrinogen deficiency (hypofibrinogenemia) can disrupt hemostasis and lead to bleeding. More common is acquired fibrinogen deficiency, which may be due to hemodilution, blood loss, disseminated intravascular coagulation (DIC), as well as sepsis (A.M. Brother. Clinical problems of fibrinolysis. 1993, Kiev, p. 151-172).

Hereditary or acquired fibrinogen deficiency causes the development of hypofibrinogenemia, the main method of treatment and prevention of which is replacement therapy with fibrinogen preparations ”(Zubairov DM Fibrinogen and fibrin. B: Molecular basis of blood coagulation and thrombosis. 2000, Kazan, pp. 1-32) .

In replacement therapy for patients with hypofibrinogenemia and for its prevention, drugs containing fibrinogen are used.

In addition to substitution therapy, preparations based on the obtained fibrinogen concentrate can be used in surgical practice as a component of fibrin glue. The composition of this glue allows operations on large wound surfaces, whether operations in places with a high degree of vascularization, or treatment of large surfaces of burns, which accelerates the process of regeneration of integumentary tissues (Zilbert A.P. Blood loss and blood transfusion. Principles and methods of bloodless surgery. 1999 , Petrozavodsk, p. 55-62).

The problems of production of drugs based on fibrinogen from human blood plasma include:

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

- the possibility of transferring to a recipient with a certain blood group the groups present in the preparations (medium-purity concentrates) of antibodies to red blood cell antigens of other blood groups,

- contamination of drugs based on fibrinogen with ballast proteins,

To solve these problems, appropriate technological approaches have been developed. So, when preparing a pool of blood plasma, carefully selected healthy donors are selected for possible infection with hepatitis viruses, human immunodeficiency viruses, etc. However, to completely guarantee the absence of a viral infection, virus inactivation is carried out in the process of obtaining fibrinogen concentrates.

A known method of producing a fibrinogen concentrate by salting out using alcohol-salt cold deposition "(Blood products. Guidance materials for control and production. - M., 1976, pp. 145-202). This method requires a lot of time and energy, preparation of complex buffered saline solutions and the need for their subsequent washing, which makes the process cumbersome, and the duration of the procedure leads to significant product losses during production. The method cannot be recommended for the production of the drug from small amounts of blood.

The precipitation of fibrinogen with ammonium sulfate also requires subsequent purification of the product from saline solutions, which leads to additional technological difficulties ”(RU Patent No. 2062103). Fibrinogen concentrates obtained by cryoprecipitation are successfully used in foreign surgical practice ”(Patent RU No. 2062103), however, the achieved fibrinogen concentration remains relatively low. Closest to the claimed method is a method of obtaining a preparation of fibrinogen concentrate, presented in Patent US 4960757.

The method consists in obtaining a fibrinogen concentrate from a precipitate containing fibrinogen obtained in the process of isolation of a factor VIII preparation carried out according to German Patent No. 2916711. The feed was dissolved in a buffer containing NaCl, heated to 37 ° C and stirred while maintaining pH = 7.5 with 2N NaOH. Then, a CaCl ₂ .2H ₂ O solution and sucrose are added to the fibrinogen concentrate to a concentration of 60% in the final solution. Next, the fibrinogen solution with stabilizers is subjected to thermal pasteurization in a water bath at a temperature of 60 ° C in order to inactivate viruses and other pathogens. After pasteurization, the fibrinogen solution is diluted with a buffer containing sodium chloride and sodium citrate and reprecipitated with glycine twice. The disadvantage of this method is the decrease in the concentration of fibrinogen in the final product due to partial polymerization at the stage of thermal inactivation. The fibrin formed at this stage is separated by double reprecipitation of glycine. Thus, the yield of fibrinogen at the final stage becomes less.

The aim of the present invention was to develop a method for producing purified fibrinogen. This goal was achieved by the development of technology that provides a lyophilized form of fibrinogen concentrate. An advantage of this isolation method is the use of a buffer solution containing an anticoagulating agent, sodium citrate. One of the advantages is the use of Tween-80 (polyoxyethylene sorbitan monooleate) and TnBP (tri-n-butyl phosphate) as detergents during viral inactivation. This method also carries out a procedure for purification of fibrinogen concentrate from the products of the viral inactivation process.

The technology provides for the dissolution of the feedstock (cryoprecipitate of freshly frozen human plasma) in water in the presence of 1-3 units. heparin, treatment of the cryoprecipitate solution with aluminum hydroxide gel, precipitation of fibrinogen with a 20-30% PEG solution, subsequent dissolution of the selected precipitate in a buffer with sodium citrate and sodium chloride, viral inactivation of the solution of solvent / detergent Tween-80 and TnBP for 6-10 hours, removal inactivation products and inactivating agents by extraction of the solution with paraffin oil, purification from ballast proteins by double reprecipitation of glycine with a concentration of 1-2.5 mol / l, subsequent sterile filtration, bottling and lyophilization.

The development of fibrinogen isolation technology was first carried out under laboratory conditions, followed by optimization of the selected technique, and then scaled up under experimental production conditions. The production of the fibrinogen preparation was carried out in accordance with the rules of Good Manufacturing Practice (GMP), standard operating procedures (SOPs) and the necessary sterility conditions. ”

The raw material for the preparation of fibrinogen concentrate was frozen cryoprecipitate of freshly frozen human plasma.

At all stages of production, the feedstock, intermediates and the target drug were tested for fibrinogen content by the Claus method.

The following is a specific embodiment of the invention.

Example

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

2. Then, a 30% PEG solution is added to the resulting mixture to a concentration of 3%, and the pH of the solution is adjusted to a value close to the isoelectric point of fibrinogen (6.6-6.8) with acetic acid solution. Under the action of PEG, fibrinogen precipitates, which are separated from the supernatant by centrifugation. Next, the supernatant is transferred to the production of purified factor VIII.

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

4. Viral inactivation of fibrinogen is 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. The removal of inactivation products and inactivating agents is carried out by extraction of the fibrinogen solution with paraffin oil. To do this, add vaseline oil in a 2: 1 ratio to the virus-inactivated fibrinogen concentrate and mix vigorously for 20 minutes. The mixture is then 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 ballast proteins is carried out by reprecipitation of the obtained fibrinogen concentrate with glycine, bringing its concentration in solution to 1.5 mol / L. In this case, a precipitate of fibrinogen is formed, and most ballast proteins remain in solution. The fibrinogen posture is separated by centrifugation at 4000 rpm for 10 minutes and dissolved in a solution containing 10 mmol of sodium citrate and 0.15 mol of sodium chloride. The procedure is repeated twice, each time washing off the fibrinogen precipitate with a cold 1.5 M glycine solution.

After reprecipitation, fibrinogen is dissolved in a buffer to a concentration of 25 g / l, sterile filtered, poured into vials and lyophilized. The resulting lyophilisate is sealed under vacuum, the vials are crimped with aluminum caps and subjected to thermal inactivation at 80 ° C for 72 hours.

Fibrinogen yield is about 55%.

Claims (1)

A method for isolating a purified virus-free fibrinogen concentrate, which consists in solubilizing the feedstock represented by cryoprecipitate of freshly frozen human plasma, precipitating fibrinogen with a 20-30% PEG solution, dissolving the selected precipitate in a buffer with sodium citrate and sodium chloride, virus inactivation of the solution by solvent-detergent method in the presence of 1-3% Tween-80 and 0.1-1.5% tri-n-butylphosphate, purification of the resulting concentrate from viral inactivation products and solvent detergents by extraction of vaseline m butter conducted three times, followed by reprecipitation of the obtained fibrinogen concentrate 1.0-2.5 M solution of glycine, sterile filtered and freeze dried, followed by capping the lyophilizate under vacuum and heat inactivation.