RU2253475C1 - Method of preparing factor viii preparation - Google Patents

Abstract

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

Description

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

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

Hemophilia A or classical hemophilia is a genetic disorder associated with the sex of the transmitter and causing a lack of coagulation factor VIII activity. Distinguish between severe, moderate and mild forms of hemophilia in accordance with the level of factor VIII. Severe patients have about or less than 1% (one unit of factor VIII per deciliter of blood) of factor activity. They are prone to frequent hemorrhages with minor or imperceptible injuries, especially to joints and muscles. In patients with moderate hemophilia, the level of factor VIII is from 2 to 4 units / dl, and bleeding occurs with moderate injuries. In people with mild hemophilia, from 5 to 30 units / dl of factor VIII and bleeding occur with severe injuries or surgery. The average normal level of factor VIII is 100 units / dl. The norm ranges from 50 to 180 units / dl.

Factor VIII is involved in the activation of factor X in a sequence that leads to the formation of a fibrin clot. Its function is measured by the activity of factor VIII, the result of which is the formation of fibrin thickening sites. Factor VIII circulates in conjunction with von Willebrand factor (fV factor), which stabilizes it and is necessary for the normal adhesion of platelets to damaged vessel walls and for connecting platelets to each other. Factor VIII is isolated and used both separately and as a complex with von Willebrand factor.

Factor VIII concentrates are made from blood plasma obtained in a commercial process from donors. The term “specific activity” is used to characterize the concentrates, which is expressed in units per mg of protein. As a rule, albumin is added to stabilize, the final indicator of specific activity is low and therefore does not give a correct idea of the effectiveness of the drug. "Specific activity excluding albumin" is one of the last indicators of the level of purification, however, with the development of concentrates of highly purified factor VIII, in which Willebrand factor remains, they speak of "specific activity excluding albumin and vW".

Plasma for receiving drugs is used from donors of different blood groups. In “medium purity” concentrates with a specific activity of up to 10, isoagglutinins to the red cells of antigen A and B are present. T-1/2 of these antibodies is long, therefore they accumulate during long courses of intensive care (for example, after surgery) and can cause hemolysis in recipients whose blood group is A, B or AB. Some recipients are much more sensitive to hemolysis than others. In highly purified concentrates, the level of isoagglutinin is very low.

Concentrates of factors are the main way to treat severe hemophilia A. Allergic reactions are rare, and the material can easily be used in the home administration program. Therefore, researchers are constantly interested in this group of drugs.

Factor VIII is a protein, therefore, like other proteins used for medicinal purposes, it can be unstable during storage. Stability is one of the problems that is solved when developing new drugs. According to existing requirements, the reconstituted concentrate must be stable for at least 12 hours.

Another problem associated with the use of a blood donor group is the need to protect recipients from infection, which is contained in the plasma of donors. In some countries, coagulation factor concentrates are made from plasma obtained from a small, carefully selected group of donors.

Different countries use different plasma and whole blood tests. Blood plasma is tested for hepatitis B virus, for HIV, for transaminases, for antibodies to hepatitis C, whole blood tests for antibodies to hepatitis C. However, if the tests show a negative result, this does not guarantee the absence of infections, as donors may it’s just a low level of antibodies, or the exacerbation of the infection last occurred without antibodies (“infection window”). Some factor concentrate manufacturing companies began to check the concentrates for viruses as an intermediate cleaning step.

To date, methods for removing dangerous viruses from concentrates have been greatly improved. HIV is removed by heat. Hepatitis viruses are more resistant to heat than HIV. The concentrates are heated after lyophilization and packaged in vials with a “dry” heat of 60-100 ° C, or steamed at a temperature of 60-80 ° C after lyophilization, but before filling into bottles, or when the concentrate itself is liquid - at a temperature of 60 ° C to lyophilization ("pasteurized"). The duration of heating depends on the method chosen. Plasma can be processed by the solvent-detergent method (DM), which is very effective against viruses with a lipid membrane, including HIV, and hepatitis B and C. Viruses without a membrane like hepatitis A and B-19 parvovirus are not destroyed by this method. The diabetes method is more common because it produces a good effect and has the least negative effect on coagulation factors. Since cases of transmission of hepatitis A virus through concentrates treated with DM technology were noted, a double inactivation technique was introduced that combines heating with diabetes. Double inactivation concentrates (DM and pasteurization) began to provoke inhibitors in those patients who were previously often treated with blood products, but belonged to a low risk group of inhibitors. Currently, for the concentrates of factor VIII, the SD method and dry heat are mainly used.

The virus content in factor concentrates can also be reduced by more intensive cleaning or separation of other plasma components from coagulation factors. For this purpose, monoclonal antibodies can be used. These concentrates are also processed by high temperature and DM method.

Various factor VIII concentrate preparations are currently available:

- recombinant - for example, Immunate, Baxter company,

- from human plasma - including Hemofil M in the form of a powder for the preparation of an injection solution of 250 ME, 500 ME and 1000 ME in Baxter bottles; Coate-HP company Bayer; Octavi in powder form for the preparation of an injection solution of 250 ME, 500 ME, 1000 ME in Octapharma bottles; Emoclot DI in the form of a powder for the preparation of an injection solution of 100 ME and 250 ME in 5 ml vials, 500 ME and 1000 ME in 10 ml Italco vials;

- from animal plasma - factor VIII: C, porcine.

Known production methods are based on precipitation methods, gel permeation chromatography, ion exchange chromatography, and all kinds of combinations thereof.

For example, there is a method for producing a preparation of factor VIII, free of isoagglutinin, including dissolving cryoprecipitate in a solution containing sodium chloride, glycine and heparin, adding a suspension of aluminum hydroxide, pasteurizing a solution containing factor VIII in the presence of a stabilizer, processing the ion-exchange method using buffer solutions, dialysis of precipitate, heating and filtration sterilization, followed by freeze-drying (US patent 5043428 A).

There is also known a method for producing highly purified factor VIII from blood plasma by obtaining cryoprecipitate, dissolving it in heparinized water, treating with a suspension of aluminum hydroxide and then PEG-4000 at pH 6.4-6.6, treating the supernatant with an ion-exchange resin using buffer, stabilization the isolated factor by albumin, heparin, PEG-4000 and, optimally, lysine or histidine, concentration, diafiltration, pasteurization and lyophilization (US patent 5259951 A).

As the closest analogue (prototype) can be specified a method of obtaining a concentrate of factor VIII, including the suspension of cryoprecipitate of human plasma in an aqueous solution of sodium heparin, when the content of factor VIII with specific activity between 0.6 and 1.1 IU / mg, pH 7- 7.1, gel purification of aluminum hydroxide, sterilizing filtration of the supernatant, virus-inactivating treatment with solvent-detergent method in the presence of tween TNBP, adsorption of the pre-treated solution on a chromatographic column with fractogel, isolation using a buffer solution additionally containing sodium chloride, lyophilization (US Pat. No. 5,252,709 A).

The objective of the present invention was to optimize the process of purification of the drug from ballast substances and viral inactivation in order to maintain high specific activity of factor VIII of the target product and increase storage stability.

This problem is solved by a new method of obtaining a lyophilized highly purified preparation of factor VIII from blood plasma.

The method for producing the preparation is the purification of cryoprecipitate of donated blood plasma from ballast proteins by treatment with aluminum hydroxide and polyethylene glycol-4000 (PEG-4000), followed by chromatographic fractionation on a DEAE-containing carrier using buffer solutions of different ionic strength to obtain a factor VIII concentrate that is stabilized, sterilized and converted to lyophilized form. Viral inactivation is performed by treating the cryoprecipitate solution with a solvent-detergent and heat treating the lyophilisate.

The proposed combination and sequence of stages of purification, processing and fractionation conditions in the proposed method can solve the difficulties associated with lyophilization, namely the process itself, maintaining drying activity, stability during further storage, as well as the ability to quickly obtain a solution when diluting the lyophilizate during use.

The method includes the following steps:

- obtaining 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 for the content of factor VIII in cryoprecipitate 30-35 IU / g

- treatment with a solution of aluminum hydroxide;

- centrifugation;

- treatment with a solution of PEG-4000 and pH correction to 6.55-6.65, without subsequent cooling, and separation of the supernatant;

- viral inactivation by solvent detergent;

- microfiltration;

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

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

- sterile microfiltration;

- freeze drying and thermal viral inactivation.

To adjust the pH in the method, it is preferable to use a 0.5 M solution of acetic acid.

When carrying out purification, a final concentration of 0.3% aluminum hydroxide cryoprecipitate in solution, PEG-4000 - 2%, is preferred.

As a solvent detergent, sodium tributyl phosphate and tween are typically added.

Microfiltration after viral inactivation is carried out optimally in 3 stages through membrane microfilters with pore sizes of 2-8 μm, 1.2 μm and, finally, 0.65 μm.

In the fractionation, various resins having fixed DEAE groups, for example Fractogel from Merck, DEAE-650M from Toson Biosep, can be used as ion exchange media.

The buffer solution used for fractionation contains optimally Tris, sodium citrate, calcium chloride, lysine, glycine, sodium chloride, water for injection, pH 6.75-6.85. It may also contain additional histidine, glucose may be administered. Preferably, the buffer contains 0.014-0.027 M lysine and 0.11-0.15 M glycine.

One of the conditions for fractionation is a change in the concentration of sodium chloride in the direction of increase. This technique was used in known methods. However, the fractionation conditions according to the proposed method allowed to optimize the process, increase the product yield and not lose specific activity.

Table 1 below shows the parameters of the buffers used in the process of isolating factor VIII.

Table 1 Buffer pH The concentration of Na +, mmol / l Conductivity, mSiemens elementary 6.75-6.85 106-112 up to 12.5 intermediate 6.75-6.85 132-137 14-15 finite 6.75-6.85 256-270 24-26

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

The following is a specific embodiment of the invention.

Example 1

Native donor blood plasma having a negative reaction to antibodies to HIV, hepatitis C virus, the causative agent of syphilis and the Hbc antigen is subjected to cryoprecipitation at a temperature of + 3 ° C, separated and cooled. Cryoprecipitate is incubated for 1 hour at room temperature, ground and poured with a solution of heparin in water for injection (2 IU / ml). Stirred at 25 ° C for 15 minutes with a magnetic stirrer. Set the pH to 6.75-6.85 and thermostat for 45 minutes. A 3% aluminum hydroxide solution is added to a final concentration of 0.3%, then stirring is carried out at 23-27 ° C for 15 minutes. The solution is centrifuged in a Ts14 centrifuge at a speed of 4000 rpm for 10 minutes at a temperature of 20 ° C. A solution of PEG in 0.01 M sodium citrate is introduced to a final concentration of 2%, then the pH is adjusted to 6.55-6.65 with 0.5 M acetic acid. Centrifuged. A solution of sodium tributyl phosphate and tween is added, incubated for 6 hours, a solution of 2 M sodium chloride is added to a conductivity of 11.6 m Siemens, a pH is adjusted to 6.8 with a 0.5 M Tris solution. Filter through membrane microfilters: 2-8 microns, 1.2 microns, 0.65 microns. Transfer the solution to the fractionation step. Stage includes loading pump; washing with a start 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 with buffer B from the balance 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 a 2 M sodium chloride solution. The elution rate of 3.2 l / h, at a pressure of not higher than 1 ATM, at room temperature. Add 10% albumin solution to a concentration of 0.1%. The yield of the basic substance 26820 ME (40.5%). Sterilized 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 6.75-6.85. The output of the main substance 26284 IU (98%). Freeze at -50 ° C, pressure 1 bar, 7.5 hours, lyophilization - 20 - + 30 ° C at a pressure of 60 μbar for 40.5 hours, inactivation at 60 ° C and a pressure of 60 μbar for 72 hours. The yield of concentrate in the main substance 17280 IU (66%).

To determine the specific activity of factor VIII, a one-step method is used to determine the content of factor VIII in plasma using a factor standard. To do this, equal volumes of kaolin suspension in erythrophosphatide, substrate plasma, factor VIII standard are mixed, the mixture is incubated at a temperature of 37 ° C and calcium chloride is added, determining the time of formation of a dense clot. The calculation of the concentration of factor VIII in% depending on the time of clot formation is performed according to the calibration schedule. It is built on the basis of data obtained when determining the clotting time in samples with different contents of the factor VIII standard.

The specific activity of the obtained concentrate did not change after lyophilization and viral inactivation and amounted to 15-20 IU / ml.

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

Claims (5)

1. A method of producing a factor VIII concentrate preparation, including cryoprecipitation of human blood plasma, suspension of the isolated cryoprecipitate in an aqueous anticoagulant solution, purification by treatment with aluminum hydroxide, virus-inactivating treatment by the solvent-detergent method in the presence of tween, adsorption of the purified solution by chromatography using ion-exchange resin containing DEAE fixed groups, elution with a buffer solution containing sodium chloride, lyophilization, characterized in that in As an anticoagulant, heparin is used at a pH of 6.3-7.3, aluminum hydroxide is added to its concentration in a cryoprecipitate solution of 0.3%, then additional purification with a PEG-4000 solution at a final concentration of 2% is carried out, followed by a pH of 6.55- 6.65, after viral inactivation, microfiltration is carried out, the adsorbed factor VIII is eluted with a buffer solution, pH 6.75-6.85, with the addition of sodium chloride with an initial concentration of sodium ions in a buffer solution of 106-112 mmol / l, increasing it to 132- 137 mmol / L and final concentration 256 -270 mmol / l, stabilize the resulting factor VIII concentrate by adding an albumin solution to its final concentration in the preparation of 0.1%, filter through a sterilizing microfilter, and the resulting lyophilized concentrate is subjected to additional thermal viral inactivation. 2. The method according to claim 1, characterized in that microfiltration after viral inactivation is carried out optimally in 3 stages through membrane microfilters with pore sizes of 2-8 μm, 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, characterized in that 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 the cryoprecipitate is not less than 30-35 IU / g