Method of making preparation of human blood coagulaton factor viii

FIELD: technological processes.

SUBSTANCE: preparation of human blood coagulation factor VIII is made by producing cryoprecipitate out of blood plasma, virus inactivating treatment, purification of cryoprecipitate solution, concentration, sterilizing filtration, bottling and drying. At the same time cryoprecipitate is produced with the help of running water with the temperature of (4±2)°C during unfreezing of fresh frozen plasma, virus inactivating treatment is carried out by means of solvent-detergent method, chromatographic purification of virus inactivated cryoprecipitate solution is carried out with application of sorbent with fractionation interval up to 20,000 kilodaltons, at the flow rate of (20±15) cm/hr, with further concentration by means of ultrafiltration on hollow fibers. Stabilizers are added, such as albumin in final concentration of (5±3) g/l, sugars and amino acids up to final concentration of (10±3) g/l. During drying initial preparation temperature is (25±15)°C below zero, final temperature is (25±8)°C, total duration of preparation drying process is (45±7) hours.

EFFECT: coagulation factor yield stability is increased and technological production design is simplified.

2 ex

 

The invention relates to biotechnology and relates to methods for producing preparations of factor VIII clotting of human blood.

This method allows to obtain the drug, which is a factor VIII clotting person, selected by the method of cryofracture followed by chromatographic purification of plasma for fractionation (FS 42-0091-02) and used as a medicine.

A method of obtaining purified preparations of factor VIII blood clotting system by obtaining cryoprecipitate from blood plasma, virusinaktivierung processing solution cryoprecipitate, chromatographic purification solution cryoprecipitate by the method of ion exchange chromatography, sterile filtration, filling, drying by sublimation (Epifanov, Wongpanich, Avellino, Iaiasa; SRW first quarter 1996 - VI sq. 1997; New in Transfusiology, issue 26, Wolfberry TL, Fetisov L.V., Ajigasawa M.A.).

However, this method has the following disadvantages.

1. Unstable output of factor VIII in relation to the content of factor VIII in the original cryoprecipitate (65-100%).

2. When the chromatographic purification is applied expensive sorbent.

The task of the invention is to increase the stability of the output of factor VIII, the simplification of the technological scheme of production is VA, the use of cheaper chromatographic sorbent, the increasing profitability of the drug.

The problem is solved by developing a new method that includes receiving cryoprecipitate from blood plasma, the cleaning solution cryoprecipitate, concentrating, sterilizing filtration, bottling and drying.

New in the proposed method is that cryoprecipitate is obtained using running water when defrosting frozen plasma cleaning is performed by gel-chromatography, followed by concentration by ultrafiltration in hollow fibers, to add stabilizers, which are used albumin, sugar and amino acids, drying sterile concentrate the eluate containing factor VIII is carried out by freezing the product to a temperature of minus 25±15)°and subsequent increase in temperature (5±2)°With every hour to a temperature of (25±8)°C.

The method consists in the following.

Fresh frozen plasma is thawed in running water with a temperature of (4±2)°C. After complete disappearance of the ice and sediment in the containers with the plasma containers immediately reveal and plasma with sludge pumped into the reactor. The mixture from the reactor under pressure of sterile compressed air (0,022±0,002) MPa ove cnie centrifuges. After centrifugation the precipitate is extracted from the rotors supercentrifuge and immediately frozen at a temperature of minus 70±10)°C. the precipitate, hereinafter referred to as cryoprecipitate consists of blood plasma proteins, insoluble at a temperature plasma (4±2)°C. one liter of plasma get from 9 to 11 g cryoprecipitate.

Centrifugal representing griebenow the blood plasma is collected in a container and further used for the manufacture of other blood products. The output from this stage is 38-43% of the content of factor VIII in the original plasma. Then make a cleaning solution cryoprecipitate as follows: in a container with frozen cryoprecipitate add (40±20) mm citrate buffer, pH 6.8 and 7.5. Then cryoprecipitate with citrate buffer was thawed in a water bath at a temperature of (25±1)°C. the Thawed solution cryoprecipitate centrifuged, separating the supernatant solution cryoprecipitate and transmit it to clarifying filtration. Brightening filtering is performed on the filter, filled membranes with a pore diameter of from 3.0 to 10 μm. From the tank supernatant solution cryoprecipitate filtered under pressure (by 0.055±0,005) MPa. In capacity with the clarified supernatant solution cryoprecipitate add a solution of heparin in the ratio of 0.3 kg cryoprecipitate / 7000-10000 IU of heparin and PE is amerivault. Then spend virusinaktivierung processing using solvent-detergent method. This supernatant solution cryoprecipitate treated with tri-n-butylphosphate and Tween 80 in the final concentrations of 0.3% and 1%, respectively, for (6±2) hours at a temperature of (30±2)°and With constant stirring. Also virusinaktivierung processing is carried out with the use of copper phenantroline buffer. This supernatant solution cryoprecipitate is treated with a solution of copper phenantroline buffer in the ratio of 1 part of copper phenantroline buffer/40 parts of the supernatant solution cryoprecipitate. Copper phenantroline buffer (CuPH) is prepared by mixing 10 ml of 0.1 M histidine, 8 ml of 0.01 M copper sulfate 5-water and 8 ml of 0.5 M 1,10-phenanthroline. The volume was adjusted to 200 ml with water for injection. Then carry out the chromatographic purification virusinaktivierung solution cryoprecipitate using sorbent with an interval fractionation up to 20,000 kDa at a flow rate of 20±15 cm/h

After chromatographic purification, the eluate containing factor VIII, collected in sterile vials and add albumin to a final concentration (5±3) g/l, alanine or arginine, or glycine or histidine to the final concentration (10±3) g/l, maltose, or glucose, or sorbitol or sucrose to ConECs the th concentration (10± 3) g/l and frozen at a temperature of minus 60±30)°C.

Next, the eluate containing factor VIII is thawed at a temperature of (20±1)°and poured it into the tank. Then spend brightening filtration through membranes with pore size of 3.0 to 10.0 μm under pressure (by 0.055±0,005) MPa. The output from this stage is 80-85% of the content of factor VIII in the original cryoprecipitate. This was followed by concentration.

Concentration carried out by the method of ultrafiltration plant for membrane filtration using membrane where the cutoff is not more than 200 kDa. For this ultrafiltration installation fill the eluate containing factor VIII, and begin the process of concentration. The eluate containing factor VIII, when installation of the tank, circulates in a closed path, creating a tangential flow along the fibers on the inner part thereof. Part of the eluate containing factor VIII, leaves without a filter and returned to the bottle. This part of the solution is called concentrate and contains all the material that stays in accordance with the size of the pores. In each cycle of transmission of the eluate containing factor VIII, part passes through the membrane and is sent in a separate container. This part is called the permeate, which is a waste product and contains water with dissolved salts. Perme the t periodically (not less than 15 min) tested for protein content. To do this, mix in the tube is equal to the volume of permeate and 20% solution of trichloroacetic acid. Precipitate formation in vitro indicates a violation of the integrity of membranes. The eluate containing factor VIII concentrate to values from 10 to 15 IU of factor VIII in 1 ml of the eluate, or from more than 20 IU of factor VIII in 1 ml of the eluate. The output from this stage is 95-99% of the content of factor VIII in the original eluate containing factor VIII. Then spend a sterilizing filter simultaneously with the filling process of the drug vials. The filtration system consists of a tank, filter, and capsules. Installation of the system is performed in compliance with the rules of asepsis. Then concentrate the eluate containing factor VIII from the tank under pressure (by 0.055±0,005) MPa filtered through a filter filled with membranes with a pore size of 3.0 to 10.0 μm, and then through the filter, filled membranes with pore size of 0.45 μm and 0.22 μm. Sterile dilution of the eluate is poured into bottles.

The output from this stage is 83-85% of the content of factor VIII in the original concentrate the eluate containing factor VIII.

Drying sterile concentrate the eluate containing factor VIII, starting with the freezing of the product at a temperature of minus 25±5)°C. Then increase the temperature to (5±2)°With every hour to a temperature of (25±8)#x000B0; C. the Final temperature of the product should be (25±8)°C. the Total duration of the drying process of the preparation 45±7 p.m residual moisture of the product should be 2,2±0,2%.

The output from this stage is 80-90% of the content of factor VIII in sterile concentrate the eluate containing factor VIII.

Example 1.

Fresh frozen plasma was thawed in running water with a temperature of 2°C. After complete disappearance of the ice and sediment in the containers with the plasma containers immediately dissected and plasma with sediment was pumped into the reactor. The mixture from the reactor under pressure of sterile compressed air at 0.020 MPa were received on flow-through centrifuge. After centrifugation the precipitate was removed from the rotors supercentrifuge and immediately frozen at minus 60°C.

Centrifugal collected in a container and further used for the manufacture of preparations of immunoglobulin and albumin.

The output from this stage was 38% of the content of factor VIII in the original plasma. Then made cleaning solution cryoprecipitate as follows: in a container with frozen cryoprecipitate was added 20 mm citrate buffer, pH 6.8. Then cryoprecipitate with citrate buffer was thawed in a water bath at a temperature of 24°C. the Thawed solution cryoprecipitate centrifuged, separated supernatant solution is cryoprecipitate and handed it to clarifying filtration. Brightening filtering was performed on the filter, filled membranes with a pore diameter of 3.0 mm. From the tank supernatant solution cryoprecipitate was filtered under a pressure of 0.05 MPa in a sterile container. In capacity with the clarified supernatant solution cryoprecipitate was added to the heparin solution in the ratio of 0.3 kg cryoprecipitate/7000 IU of heparin and stirred. Then there was virusinaktivierung processing using solvent-detergent method. This supernatant solution cryoprecipitate was treated with tri-n-butylphosphate and Tween 80 in the final concentrations of 0.3% and 1%, respectively, for (6±2) hours at the temperature of (3±2)°and With a constant weak mixing. Then there was the chromatographic purification virusinaktivierung solution cryoprecipitate using sorbent with an interval fractionation up to 20,000 kDa at a flow rate of 5 cm/h

After chromatographic purification, the eluate containing factor VIII, were collected in sterile vials and added albumin to a final concentration of 2 g/l, glycine to a final concentration of 7 g/l sucrose to a final concentration of 7 g/l and were frozen at minus 40°C. Next, the eluate containing factor VIII, was thawed at a temperature of 19°and poured it into the tank. Then there was brightening filtration through membranes with dia is slow then 3.0 mm under a pressure of 0.05 MPa in a sterile container. The output from this stage was 80% of the content of factor VIII in the original cryoprecipitate. Then there was some concentration.

Concentration was performed by the method of ultrafiltration plant for membrane filtration using membrane where the cutoff is not more than 200 kDa. For this ultrafiltration installation filled the eluate containing factor VIII, and began the process of concentration. The eluate containing factor VIII, when installation of the tank, circulated through a closed path, creating a tangential flow along the fibers on the inner part thereof. Permeate every 15 min was tested for protein content. This was mixed in a test tube and an equal volume of permeate and 20% solution of trichloroacetic acid. The eluate containing factor VIII, was concentrated to 13 IU of factor VIII in 1 ml. Output from this stage was 99% of the content of factor VIII in the original eluate containing factor VIII. Then spent sterilizing filter simultaneously with the filling process of the drug vials. The filtration system consisted of a tank, filter, and capsules. Installation of the system was performed in compliance with the rules of asepsis. Then concentrate the eluate containing factor VIII from the tank under pressure 0.05 MPa was filtered through a filter filled with membranes with a pore size of 3.0 μm, and then through the filter, napravle the hydrated membranes with pore size of 0.45 μm, 0.22 μm. Sterile dilution of the eluate was poured into vials.

The output from this stage was 83% of the content of factor VIII in the original concentrate the eluate containing factor VIII.

Drying sterile concentrate the eluate containing factor VIII started with freezing the product at a temperature of minus 10°C. Then raised the temperature on 3°With every hour to a temperature of 17°C. the Final temperature of the product was 17°C. the Total duration of the drying process of the drug was 38 PM residual moisture of the product amounted to 2.4%.

The output from this stage was 80% of the content of factor VIII in sterile concentrate the eluate containing factor VIII.

Example 2.

Fresh frozen plasma was thawed in running water with a temperature of 6°C. After complete disappearance of the ice and sediment in the containers with the plasma containers immediately dissected and plasma with sediment was pumped into the reactor. The mixture from the reactor under pressure of sterile compressed air 0,024 MPa were received on flow-through centrifuge. After centrifugation the precipitate was removed from the rotors supercentrifuge and immediately frozen at minus 80°C.

Centrifugal collected in a container and further used for the manufacture of preparations of immunoglobulin and albumin.

The output of this stage is raised 43% of the content of factor VIII in the original plasma. Then made cleaning solution cryoprecipitate as follows: in a container with frozen cryoprecipitate was added 60 mm citrate buffer, pH 7.5. Then cryoprecipitate with citrate buffer was thawed in a water bath at a temperature of 26°C. the Thawed solution cryoprecipitate centrifuged, separated supernatant solution cryoprecipitate and handed it to clarifying filtration. Brightening filtering was performed on the filter, filled membranes with a pore size of 10.0 μm. From the tank supernatant solution cryoprecipitate was filtered under a pressure of 0.06 MPa in a sterile container. In capacity with the clarified supernatant solution cryoprecipitate was added to the heparin solution in the ratio of 0.3 kg cryoprecipitate/10000 IU of heparin and stirred. Then there was virusinaktivierung processing using copper phenantroline buffer. This supernatant solution cryoprecipitate was treated with a solution of copper phenantroline buffer in the ratio of 1 part of copper phenantroline buffer/40 parts of the supernatant solution cryoprecipitate. Copper phenantroline buffer (CuPH) was prepared by mixing 10 ml of 0.1 M histidine, 8 ml of 0.01 M copper sulfate 5-water and 8 ml of 0.5 M 1,10-phenanthroline. The volume was brought to 200 ml with water for injection. Then there was the chromatographic purification virusinfection what about the solution cryoprecipitate using sorbent with an interval fractionation up to 20,000 kDa at a flow rate of 35 cm/h

After chromatographic purification, the eluate containing factor VIII, were collected in sterile vials and added albumin to a final concentration of 8 g/l, glycine to a final concentration of 13 g/l, sucrose to a final concentration of 13 g/l and frozen at minus 80°C. Next, the eluate containing factor VIII, was thawed at a temperature of 21°and poured it into the tank. Then there was brightening filtration through membranes with pore size of 10.0 μm under a pressure of 0.06 MPa in a sterile container. The output from this stage was 85% of the content of factor VIII in the original cryoprecipitate. Then there was some concentration.

Concentration was performed by the method of ultrafiltration plant for membrane filtration using membrane where the cutoff is not more than 200 kDa. For this ultrafiltration installation filled the eluate containing factor VIII, and began the process of concentration. The eluate containing factor VIII, when installation of the tank, circulated through a closed path, creating a tangential flow along the fibers on the inner part thereof. Permeate every 15 min was tested for protein content. This was mixed in a test tube and an equal volume of permeate and 20% solution of trichloroacetic acid. The eluate containing factor VIII, was concentrated to 22 ME in 1 ml of the eluate. Output Yes the Noi stage was 95% of the content of factor VIII in the original eluate, containing factor VIII. Then spent sterilizing filter simultaneously with the filling process of the drug vials. The filtration system consisted of a tank, filter, and capsules. Installation of the system was performed in compliance with the rules of asepsis. Then concentrate the eluate containing factor VIII from the tank under pressure of 0.06 MPa was filtered through a filter filled with membranes with a pore size of 10.0 μm, and then through the filter, filled membranes with pore size of 0.45 μm and 0.22 μm. Sterile dilution of the eluate was poured into vials.

The output from this stage was 85% of the content of factor VIII in the original concentrate the eluate containing factor VIII.

Drying sterile concentrate the eluate containing factor VIII started with freezing the product at a temperature of minus 40°C. Then raised the temperature on 7°With every hour to a temperature of 33°C. the Final temperature of the product was 33°C. the Total duration of the drying process of the drug accounted for 52 hours the residual moisture of the product was 2%.

The output from this stage was 90% of the content of factor VIII in sterile concentrate the eluate containing factor VIII.

Justification of the execution modes of the proposed method

Defrost frozen plasma in the flowing water with temperature (4±2)°provides access 3843% of the content of factor VIII in the original plasma. The use of running water with a temperature below 2°when defrosting frozen plasma leads to a prolongation of the process of defrosting and, as consequence, to decrease in output less than 38% of the content of factor VIII in the original plasma. This causes a reduction in the profitability of production decreases as output factor VIII with a liter of fresh frozen plasma, as well as the complexity of the technological scheme, as it requires additional stress concentration of the eluate containing factor VIII.

The use of running water with a temperature above 6°when defrosting frozen plasma leads to disruption of the process of precipitation factor VIII clotting, resulting in its output cryoprecipitate less than 38% of the content of factor VIII in the original plasma. This leads to lower profitability.

When cleaning cryoprecipitate in capacity with the clarified supernatant solution cryoprecipitate add a solution of heparin in the ratio of 0.3 kg cryoprecipitate/7000-10000 IU of heparin and mix. When adding a solution of heparin to a final concentration of less than 7000 Units 0.3 kg cryoprecipitate the output from this stage is less than 80% of the content of factor VIII in the original cryoprecipitate, which reduces the profitability of the drug. Adding heparin solution until the final concentration of the more than 10,000 IU 0.3 kg cryoprecipitate reduced activity of factor VIII in the eluate, contains factor VIII, resulting in complication of the technological scheme, as it requires additional stress concentration of the eluate containing factor VIII.

When cleaning virusinaktivierung solution cryoprecipitate by gel-chromatography using a sorbent with an interval fractionation up to 20,000 kDa at a flow rate of 20±5 cm/h provides access 80-85% of the content of factor VIII in the original cryoprecipitate. When using sorbent with higher interval fractionation increases the interaction of factor VIII molecules with the sorbent. As a result, the output factor VIII is reduced and is less than 80% of its content in the source cryoprecipitate. At a flow rate of less than 5 cm/h increases the time chromatographic purification that leads to the complication of the process flow, and reduces the profitability of the drug. At a flow rate of 35 cm/h decreases the quality of the chromatographic purification of the eluate containing factor VIII, resulting in complication of the technological scheme due to the need for additional stages of refinement.

When the concentration of the eluate containing factor VIII by ultrafiltration on an installation for membrane filtration using membrane where the cutoff is not more than 200 kDa, output is 95-99% of the content of factor VII in the original eluate, containing factor VIII. When kontsentrirovanii of the eluate containing factor VIII using membranes, in which the cutoff is more than 200 kDa, part of the factor VIII molecules pass through the membrane and into the permeate, which is a waste product. This causes a reduction of the yield of factor VIII less than 95% of the content in the original eluate containing factor VIII.

Adding albumin to a final concentration (5±3) g/l in the eluate containing factor VIII, stabilizes factor VIII molecule, increasing yield stability of factor VIII after concentrating, sterilizing filtration and drying. Adding albumin to a final concentration of less than 2 g/l resulted in reduced stability of the molecules of factor VIII, resulting in a reduced yield of factor VIII after concentrating, sterilizing filtration and drying. Thus, reduced yield stability factor VIII clotting in the manufacturing process of the drug. Adding albumin to a final concentration of greater than 8 g/l does not increase the stability of the factor VIII molecules, but increases the cost of the drug, therefore, reduces the profitability of the drug.

Adding alanine or arginine, or glycine or histidine to the final concentration (10±3) g/l in the eluate containing factor VIII, stabilizes factor VIII molecule,increasing yield stability of factor VIII after concentration, sterilizing filtration and drying. Adding alanine or arginine, or glycine or histidine to a final concentration of less than 7 g/l resulted in reduced stability of the molecules of factor VIII, resulting in a reduced yield of factor VIII after concentrating, sterilizing filtration and drying. This reduces the yield stability factor VIII clotting in the manufacturing process of the drug. Adding alanine or arginine, or glycine or histidine to a final concentration of greater than 13 g/l does not increase the stability of the factor VIII molecules, but increases the cost of the drug, therefore, reduces the profitability of the drug.

Adding maltose, or glucose, or sorbitol, or sucrose to a final concentration (10±3) g/l in the eluate containing factor VIII stabilizes factor VIII molecule, increasing yield stability of factor VIII after concentrating, sterilizing filtration and drying. Adding maltose, or glucose, or sorbitol, or sucrose to a final concentration of less than 7 g/l resulted in reduced stability of the molecules of factor VIII, resulting in a reduced yield of factor VIII after concentrating, sterilizing filtration and drying. This reduces the yield stability factor VIII clotting in the manufacturing process of the drug. Added the maltose, or glucose, or sorbitol, or sucrose to a final concentration of more than 13 g/l does not increase the stability of the factor VIII molecules, but increases the cost of the drug, therefore, reduces the profitability of the drug.

When conducting drying, including freezing, at a temperature of minus 25±15)°With the increase of temperature (5±2)°per hour to a final temperature (25±8)°and the total duration of the drying process of the preparation 45±7 h, the output is 80-90% of the content of factor VIII in sterile concentrate the eluate containing factor VIII. Freezing at a temperature of less than 10°leads to the reduction of output less than 80% of the content of factor VIII in sterile concentrate the eluate containing factor VIII. Freezing at a temperature of more than 40°does not increase the yield of factor VIII, but increases the cost of the drug, therefore, reduces the profitability of the drug. When a temperature rise of less than 3°per hour or more than 7°per hour output is reduced and is less than 80% of the content of factor VIII in sterile concentrate the eluate containing factor VIII. The duration of the drying process less than 38 hours the product has a residual moisture content of 2.4% and does not meet the requirements of the Fund applicable to this drug. The duration of the process is and drying over 52 hours of the release of factor VIII clotting is reduced and is less than 80% of the content of factor VIII in sterile concentrate the eluate, containing factor VIII.

Obtained by the claimed method, the preparation of coagulation factor VIII human blood provides increased levels of factor VIII in plasma and temporarily eliminates the defect of coagulation in patients with hemophilia And inherited bleeding disorders characterized by insufficient activity of specific plasma protein, factor VIII clotting) and is intended for the treatment and prevention of bleeding in patients with hemophilia A, including in an emergency or a planned surgical intervention.

A method of obtaining a preparation of factor VIII clotting of human blood, which consists in obtaining cryoprecipitate from blood plasma, virusinaktivierung handling, cleaning solution cryoprecipitate, concentrating, sterilizing filtration, bottling and drying, characterized in that the cryoprecipitate obtained using running water with a temperature of 4±2°when defrosting frozen plasma, virusinaktivierung treatment is carried out using the solvent-detergent method before cleaning solution cryoprecipitate, the chromatographic purification virusinaktivierung solution cryoprecipitate performed using sorbent with an interval fractionation up to 20,000 kDa, at a flow rate of 20±15 cm/h, with further concentrated the eat ultrafiltration in hollow fibers, the addition of stabilizers, which are used albumin at a final concentration of 5±3 g/l of sugar and amino acids to a final concentration of 10±3 g/l, when the initial drying temperature of the drug is minus 25±15°C, the final temperature is 25±8°S, the total duration of the drying process of the drug is 45±7 PM



 

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SUBSTANCE: invention relates to development of novel fields in investigation of blood clot fibrin. Method involves dissolving fibrin isolated from human or animals blood clot in 18-22.5% sodium chloride solution and incubation at temperature 37-38°C for 18-24 h. Prepared fractions are separated in dividing funnel and fibrin physiological solution is prepared by addition of the corresponding amount of distilled water. Invention provides preparing fibrin fraction without using sodium hydroxide but by using neutral salt followed by addition of distilled water up to preparing fibrin fractions in sodium chloride physiological solution.

EFFECT: improved separation method of fibrin.

1 dwg

FIELD: medicine, hematology, polypeptides, pharmacy.

SUBSTANCE: invention relates to composition containing factor VII or factor VII-related polypeptide and factor XI or factor XI-related-polypeptide, sets containing indicated compounds and to using composition used in treatment of bleeding. Pharmaceutical composition contains the effective dose of factor VII or factor VII-related polypeptide, and factor XI or factor XI-related polypeptide. Invention provides the following effects: (1) design of composition that can be used effectively in treatment or prophylaxis of bleeding and blood coagulation disorders being oral composition preferably; (2) design of compositions as the single dosed formulation that can be used effectively in treatment or prophylaxis of bleeding or as a procoagulant agent; (3) design of compositions possessing the synergetic effect, design of treatment methods or sets that promote to the development of synergetic effect; (4) design of compositions, methods for treatment and sets without adverse effects, such as the high level of systemic activation of the blood coagulation system.

EFFECT: valuable medicinal properties of pharmaceutical composition.

43 cl, 1 tbl, 4 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: the present innovation deals with methods and compositions for targeted supply of circulation -overlapping solid-phase thrombocytes-binding preparations for treating vascularized tumor or hyperplastic tissue. The solid-phase preparation covered except polystyrene with thrombocytes-binding preparation, such as Willebrand's factor, should be purposefully directed towards vascular net-target in vivo, where it binds and activates thrombocytes which, in their turn, bind and activate other thrombocytes. Variant of composition for inducing thrombogenesis in vivo and containing a solid-phase thrombocytes-binding preparation, except polystyrene, could be consisted of the first binding component and the second binding component, where the first binding component has got binding area for binding a solid-phase preparation with a ligand-receptor complex, and not with a ligand or receptor separately, as for the second binding component it has got binding area for thrombocytes. The innovation provides rapid formation of a dense thrombus that leads to overlapping the circulation in the desired area due to thrombocytic fixation towards the surface of a solid-phase preparation along with increasing its sizes and developing a multi-layer structure of activated thrombocytes as a dense matrix.

EFFECT: higher efficiency.

29 cl, 12 ex, 2 tbl

FIELD: biochemistry.

SUBSTANCE: invention relates to pharmaceutical agents contain factor VII polypeptides and factor IX polypeptides or related polypeptides. Moreover polypeptides for drug production are characterized and methods for prophylaxis and treatment of episodic hemorrhage in subject.

EFFECT: more effective method for treatment of episodic hemorrhage in subject.

43 cl, 1 tbl, 5 ex, 1 dwg

FIELD: veterinary science.

SUBSTANCE: on proving the diagnosis one should introduce immune serum of animals-donors for sick calves till clinical recovery at the titers of hemagglutinins to IRT virus being 1:256, to PG-3 virus being 1:1280 and to VD-BC virus being 1:1024, and, additionally, it is necessary to apply a probiotic preparation lactobifadol at 32x108 microbial cells bifido- and 4x107 lactobacteria. The innovation increases the quantity of recovered animals, shortens the terms of therapy and the number of relapses.

EFFECT: higher efficiency of therapy.

2 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: separation of the mononuclear leukocyte cell suspension from patient's blood is performed by density fractionation. The cell suspension is used immediately after centrifugation of 4 mL blood. The suspension is washed out and diluted by 3.0 mL isotonic sodium chloride. The suspension is instilled 6-8 times daily with 3-4 days break, the course consists of 2-3 procedures.

EFFECT: method increases efficiency of treatment by reduction of treatment time.

1 tbl, 1 ex

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