Method of preparing factor viii preparation

FIELD: pharmaceutical industry and biotechnology.

SUBSTANCE: method consists in the following: kryoprecipitate of donor blood plasma is suspended in heparin solution, resulting suspension is consecutively treated with aluminum hydroxide and polyethylene glycol-400 to remove ballast proteins until content of aluminum hydroxide in solution achieves 0.3% and that of polyethylene glycol-400 2%. Thus treated material is subjected to viral inactivation by solvent-detergent technique in presence of Tween and microfiltration followed by chromatographic fractionation on DEAE-containing carrier using buffer solutions, pH 6.75-6.85, with different ionic forces. Resulting concentrate of factor VIII is stabilized with albumin solution to provide final albumin concentration in the preparation 0.1%, sterilized by microfiltration, and transferred into lyophilized form, in which viruses are additionally inactivated via heat treatment.

EFFECT: preserved specific activity of factor VIII with high degree of purification and increased storage stability.

5 cl, 1 tbl

 

The invention relates to the field of pharmaceutical industry and biotechnology and relates to methods of obtaining lyophilized purified preparation of factor VIII from blood plasma.

Factor VIII (antihemophilic factor) is a well-known plasma protein that plays an essential role in the blood clotting process. Its main purpose is the prevention and treatment of hemophilia A.

Hemophilia a or classic hemophilia is a genetic disorder that is associated with the floor of the transmitting and causing the lack of activity of factor VIII clotting. Distinguish between severe, moderate and mild forms of hemophilia in accordance with the level of factor VIII. Patients with severe are about or less than 1% (one unit of factor VIII per deciliter of blood) activity factor. They tend to frequent hemorrhages with little or no visible injuries, especially in the joints and muscles. In patients with moderate hemophilia a level of factor VIII from 2 to 4 units/DL, they have bleeding occurs when the injuries of moderate severity. People with mild hemophilia from 5 to 30 units/DL factor VIII and bleeding occur in severe injury or surgical intervention. The average normal level of factor VIII is 100 units/DL. Rainfall varies from 50 to 180 units/DL.

Factor VIII participates in the activation of factor X in a is sledovatelnot, which leads to the formation of a fibrin clot. Its function is measured by the activity of factor VIII, which results in the formation of the places fibrin thickening blood. Factor VIII circulates in conjunction with the von Willebrand factor (factor PV), which it stabilizes and is necessary for normal adhesion of platelets to the damaged vessel wall and to connect the platelets together. Factor VIII is isolated and used both separately and in the form of a complex with von Willebrand factor.

Concentrates of factor VIII manufactured from plasma obtained in the commercial process from donors. To characterize concentrates use the term "specific activity", which is expressed in units of one mg of protein. Typically, albumin is added to stabilize, the ultimate measure of specific activity is low and therefore does not give a faithful representation about the efficacy of the drug. "Specific activity without regard to albumin is one of the last indicators of the level of treatment, however, with the development concentrates of factor VIII high purification, which remains the von Willebrand factor, say "specific activity without regard to albumin and PV".

Plasma to obtain drugs used from donors with different blood groups. In concentrates "average purity", which have a specific activity of up to 0, there are isoagglutinin to a red cell antigen a and B. T-1/2 these antibodies are long, so they accumulate during long courses of intensive therapy (e.g., after surgery) and can cause hemolysis in recipients whose blood group a, b or AB. Some recipients are much more sensitive to hemolysis than others. In concentrates high cleaning level isoagglutinin very low.

Concentrates of factors - the main method of treatment of severe hemophilia A. Allergic reactions when this occur rarely, the material can easily be used in the program of introduction of preparations at home. Therefore, this group of drugs, the researchers are of permanent interest.

Factor VIII is a protein, therefore, like other proteins used for therapeutic purposes, it may be unstable during storage. Stability is one of the problems that can be solved with the development of new drugs. According to the existing requirements of the recovered concentrate should be stable, at least within 12 hours.

Another problem associated with the use of donor blood group is the need to protect recipients against infection, which is found in the plasma of donors. In some countries, the concentrates of clotting factors in the blood are produced from plasma collected from a small, carefully selected g is uppy donors.

Different countries use different tests of plasma and whole blood. Analysis of blood plasma for hepatitis b virus, HIV, transaminases, antibody to hepatitis C, the analysis of whole blood for antibodies to hepatitis C. However, if the test shows a negative result, it does not guarantee the absence of infection, as donors may simply be a low level of antibodies, or exacerbation of infection for the last time was spent without antibodies ("window of infection"). Some manufacturers of factor concentrates have to check concentrates on viruses as an intermediate purification step.

To date ways to remove harmful viruses from concentrates significantly improved. HIV is removed using heat. The hepatitis viruses are more resistant to heat than HIV. Concentrates are heated after lyophilization and Packed into vials during the "dry" heat 60-100°With, or steamed at a temperature of 60-80°after drying, but before packing in bottles, or when the concentrate itself is liquid at a temperature of 60°before lyophilization ("pasteurized"). The duration of heating depends on the chosen method. The plasma can be treated solvent-detergent method (DM), which is very effective against viruses with a lipid envelope, including HIV and the hepatitis viruses is in b and C. Viruses without shell, as hepatitis a and b-19 parvovirus, this method is not destroyed. The SD method is more common because it produces a good effect, and has the least negative effect on the clotting factors of the blood. As there were cases of transmission through concentrates, processed DM technology, hepatitis a virus, that was the method of double inactivation of combining heat with diabetes. Concentrates with double inactivation (DM and pasteurization) started to provoke inhibitors in those patients who previously were often treated with blood products, but belonged to the group of low-risk education inhibitors. Currently concentrates of factor VIII mainly used the SD method and the dry heat.

Viruses in factor concentrates can be reduced also more intense purification or separation of other components of the plasma from clotting factors. This purpose can be used monoclonal antibodies. These concentrates are also processed by high temperature and the SD method.

Currently available in various preparations of factor VIII concentrate:

- recombinant - for example, Immunet, firm Baxter,

from human plasma including Hemofil M in the form of powder for preparation of injection solution ME 250, ME 500 and 1000 IU patients in the firm who axter; Coat-CP company Bayer; Octave in the form of powder for preparation of injection solution ME 250, ME 500, 1000 IU in bottles companies Octapharma; Amelot DI in the form of powder for preparation of injection solution 100 ME 250 ME in vials of 5 ml, ME 500 and 1000 IU in 10 ml vials company Italco;

from plasma of animals - factor VIII:C, porcine.

Known methods of production based on the methods of precipitation, gel chromatography, ion exchange chromatography and their various combinations.

For example, a known method of obtaining a preparation of factor VIII, free from isoagglutinin, including the dissolution of cryoprecipitate in the solution containing sodium chloride, glycine and heparin, adding a suspension of aluminum hydroxide, pasteurization of a solution containing factor VIII in the presence of the stabilizer, processing of ion-exchange method using buffer solutions, dialysis of the precipitate, heating and filtration sterilization and subsequent freeze-drying (U.S. patent 5043428 A).

Also known is a method of obtaining high-purity factor VIII from blood plasma by obtaining cryoprecipitate, it dissolved in heparinized water, by treatment with a suspension of aluminum hydroxide and then PEG-4000 at pH 6.4 and 6.6, processing supernatant by ion-exchange resin using a buffer, stabilize the selected factor albumin, heparin is given, PEG-4000 and, optimally, lysine or histidine, concentration, diafiltration, pasteurization and freeze-drying (U.S. patent 5259951 A).

As the closest analogue (prototype) can be mentioned a method of obtaining a concentrate of factor VIII, including the suspension of cryoprecipitate human plasma in aqueous solution of heparinate sodium, when the content of factor VIII specific activity between the 0.6 and 1.1 IU/mg, pH 7-7 .1, cleaning gel, aluminum hydroxide, sterilizing filtration of the supernatant, inactivating viruses processing solvent-detergent method in the presence of tween-TNBP, the adsorption of further purified by solution in the chromatographic column with fractogel, selection using buffer solution, optionally containing sodium chloride, lyophilization (U.S. patent 5252709 A).

The present invention was to optimize the treatment process of the drug from ballast substances and viral inactivation with the aim of preserving the high specific activity of the factor VIII target product and stability during storage.

This task is solved by a new process for the preparation of lyophilized purified preparation of factor VIII from blood plasma.

The method of producing drug is a clean cryoprecipitate donor blood plasma from ballast protein treatment is th aluminum hydroxide and polyethylene glycol-4000 (PEG-4000), followed by chromatographic fractionation on DEAE-containing media using buffer solutions of different ionic strength to obtain a concentrate of factor VIII, which stabilize, sterilized and transferred to the freeze-dried form. Virus inactivation is performed by the processing solution cryoprecipitate solvent-detergent and heat treatment of lyophilized.

The proposed combination and sequence of the stages of purification, the conditions of processing and fractionation, the proposed method can solve the difficulties associated with carrying out lyophilization, namely the process itself, remained active during drying, stability during further storage, as well as the ability to get a fast solution when diluted freeze-dried in the application.

The method includes the following stages:

- getting cryoprecipitate from blood plasma;

- dissolution of cryoprecipitate in an aqueous solution containing an anticoagulant, such as heparin, pH 6,3-7,3;

The lower threshold of the content of factor VIII in cryoprecipitate 30-35 IU/g

treatment with a solution of aluminium hydroxide;

- centrifugation;

treatment with a solution of PEG-4000 and correction of pH to 6,55-6,65 without further cooling and separation of the supernatant;

virus inactivation by solvent-detergent;

- microfiltration;

- fractionation in a chromatographic column with DEAE-containing media, including loading, elution buffer solution with addition of sodium chloride solution with an initial concentration of sodium ions in the morning the nom solution 106-112 mmol/l, increase it to 132-137 mmol/l final concentration 256-270 mmol/l;

- to stabilize the resulting solution of factor VIII by adding a solution of albumin;

- sterile microfiltration;

- freeze drying and heat treatment of virus inactivation.

To regulate the pH in the method preferably using a 0.5 M solution of acetic acid.

When making cleanup a preferred final concentration in solution of cryoprecipitate of aluminum hydroxide and 0.3%, PEG-4000 - 2%.

As solvent-detergent type, as a rule, tributyl phosphate of sodium and twin.

The microfiltration after viral inactivation carried out optimally in 3 stages through the membrane microfilters with a pore size of 2-8 µm, 1.2 µm and, finally, 0.65 micron.

The fractionation can be used as ion-exchange media of various resins having fixed DEAE groups, such as Fractogel Merck, DEAE-650M company Toson Biosep.

The buffer solution used for fractionation, optimally contains Tris, sodium citrate, calcium chloride, lysine, glycine, sodium chloride, water for injections, pH of 6.75-6,85. It may also contain additional histidine, may be injected glucose. Preferably, the buffer contains 0,014-0,M lysine and 0.11-0,15M glycine.

One of the conditions of fractionation is a change in the concentration of sodium chloride is increased. This technique was used in the known methods. However, the conditions for fractionation according to the proposed method allowed us to optimize the process, to increase output without losing potency.

The following table 1 presents the parameters used buffers in the process of allocation of factor VIII.

Table 1
The bufferpHThe concentration of Na+, mmol/lConductivity, Simens
initial6,75-6,85106-112to 12.5
intermediate6,75-6,85132-13714-15
end6,75-6,85256-27024-26

Heat viral inactivation is carried out after receipt of the lyophilisate, enables to increase the degree of purification of the final product.

Below is a specific example embodiment of the invention.

Example 1

Native plasma donor blood, which has a negative reaction for antibodies to HIV, hepatitis C, syphilis and b-antigen, are cryosurgery at a temperature of +3°S, is separated and cooled. Cryoprecipitate allowed to stand for 1 hour at room temperature, crushed and zali is with a heparin solution in water for injection (2 IU/ml). Stirred at 25°15 minutes with a magnetic stirrer. Establish a pH of 6.75-6,85 and thermostatic 45 minutes. Add 3% solution of aluminium hydroxide to a final concentration of 0.3%, followed by stirring at 23-27°15 minutes. The solution is centrifuged in a centrifuge C with a speed of 4000 rpm for 10 minutes at a temperature of 20°C. Enter the PEG solution in 0.01 M sodium citrate to a final concentration of 2%, then the pH is brought to 6,55-6,65 using 0.5 M acetic acid. Cetrifugation. Add a solution of tributyl phosphate of sodium and tween, incubated for 6 hours, add a solution of 2 M sodium chloride to the conductivity 11,6 Simens, set pH to 6.8 with 0.5 M Tris solution. Filtered through a membrane microfilters: 2-8 µm, 1.2 µm 0.65 micron. Transfer the solution to the stage of fractionation. Stage involves loading pump; start washing buffer (0.01 M Tris, 0.02 M sodium citrate, 0,0025 M calcium chloride, 0,016 M lysine, 0.12 M Glycine, 0,063 M sodium chloride, water for injection); washing buffer from the rest of ballast proteins (0.01 M Tris, 0.02 M sodium citrate, 0,0025 M calcium chloride, 0,016 M lysine, 0.12 M Glycine, 0,091 M sodium chloride, water for injection); elution of the target product (0.01 M Tris, 0.02 M sodium citrate, 0,0025 M calcium chloride, 0,016 M lysine. 0.12 M Glycine, 0,220 M sodium chloride, water for injection); washing the column with 2 M solution of sodium chloride. Speed ELS and 3.2 l/h, at a pressure not higher than 1 ATM at room temperature. Add a 10% solution of albumin to a concentration of 0.1%. The output on the base material 26820 ME (40,5%). Sterilize through a sterilizing capsule with a pore diameter of 0.22 μm. Total protein 0.5-1 mg/ml, specific activity 15-20 IU/ml, sodium ions 150-220 mmol/l, pH of 6.75-6,85. The output on the base material 26284 IU (98%). Spend freezing -50°C, pressure 1 bar, 7.5 hours, lyophilization - 20 - +30°at a pressure of 60 mcbar within 40,5 hours, inactivation at 60°and a pressure of 60 mcbar within 72 hours. Exit concentrate on the basic substance 17280 IU (66%).

To determine the specific activity of factor VIII is used one-step method for the determination of factor VIII in plasma using standard factor. To do this, mix equal volumes of a suspension of kaolin in erythropoietine, substrate plasma, standard factor VIII, incubated the mixture at a temperature of 37°and add calcium chloride, determining the time of formation of a dense clot. The calculation of the concentration of factor VIII in % depending on the time of the formation of a clot to produce a calibration curve. It is built on the basis of the data obtained in the determination of clotting time in samples with different content of the standard factor VIII.

The specific activity of the obtained concentrate was not changed after liof the implementation and viral inactivation and amounted to 15-20 IU/ml

The proposed method allows to obtain the drug, its effectiveness is not inferior to the best foreign analogues, and can be recommended for industrial use.

1. A method of obtaining a preparation of factor VIII concentrate, including cryosurgery of human blood plasma, the suspension of the selected cryoprecipitate in an aqueous solution of an anticoagulant, a cleaning treatment with aluminum hydroxide, inactivating viruses processing solvent-detergent method in the presence of the twin, the adsorption of further purified solution by chromatography using ion-exchange resin containing DEAE fixed group, the elution buffer solution containing sodium chloride, lyophilization, characterized in that as an anticoagulant use heparin at pH 6,3-7,3 add the aluminum hydroxide to its concentration in solution cryoprecipitate of 0.3%, followed by additional treatment with a solution of PEG-4000 in his final concentration of 2% with the subsequent establishment of pH 6,55-6,65 after viral inactivation are microfiltration, elute the adsorbed factor VIII buffer solution, pH of 6.75-6,85, with the addition of sodium chloride solution with an initial concentration of sodium ions in the buffer solution 106-112 mmol/l, increase it to 132-137 mmol/l final concentration 256-270 mmol/l, stabilize receive the significant concentrate of factor VIII by adding albumin to its final concentration in the drug of 0.1%, filtered through a sterilizing filter, the obtained freeze-dried concentrate is subjected to additional heat viral inactivation.

2. The method according to claim 1, wherein the microfiltration after viral inactivation carried out optimally in 3 stages through the membrane microfilters with a pore size of 2 to 8 μm, and 1.2 μm and 0.65 μm, respectively.

3. The method according to claim 1 or claim 2, characterized in that the buffer solution used for elution, contains Tris, sodium citrate, calcium chloride, lysine, glycine, sodium chloride, water for injection.

4. The method according to claim 3, wherein the buffer solution contains 0,014-0,027 M lysine and 0.11-0.15 M glycine.

5. The method according to claim 1, characterized in that the content of factor VIII in cryoprecipitate is not less than 30-35 IU/g



 

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