Method for producing concentrated factor viii of human blood plasma

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and describes a method for recovery of factor VIII from human blood plasma not identified by related analysis of hepatitis and HIV1/2 viruses consisting in sequential cryoprecipitation, dissolution in an aqueous solution of heparin and solubilisation of a cryoprecipitate, sorption of a prothrombin-converting complex factor by aluminium hydrate, removal of fibrinogen, fibronectin and associated protein by polyethylene glycol-4000, viral inactivation with solvent detergents and preliminary filtration, anion-exchange chromatography, preferentially with EDM-TMAE Fractogel, with elution by a sodium chloride buffer, stabilisation by albumine solution, sterile filtration in membrane filters of pore diameter 0.22 mcm, bottling (200-300 IU/bottle), lyophilisation and second thermal viral inactivation with purification using the aqueous solution of unfractionated heparin of the concentrations equal to 5-100 international units (IU)/ml, preferentially 10-25 IU/ml, polyethylene glycol-4000 in the final concentration 3.5% and acidification of the medium, preferentially to pH 6.6, strong TMAE anion exchangers.

EFFECT: method substantially provides higher effectiveness of purification and specific activity of factor VIII.

1 tbl, 4 dwg, 2 ex

 

The invention relates to the field of pharmaceutical industry, namely biotechnology obtain hemostatic drugs. Quantitative or functional deficiency of factor (f) VIII clotting causes the development of hereditary haemorrhagic disease of hemophilia a, the primary method of treatment and prophylaxis which is substitution therapy drugs F. VIII.

F VIII is a glycoprotein plasma ecovalence associated with von Willebrand factor, which significantly improves the stability of f VIII. Gene F. VIII localized on the X chromosome, and its mutations lead to the development of hemophilia And inherited in a recessive trait. Activated f VIII (f VIIIa) is labile, protein without enzymatic co-factor f IXa. F VIII is a necessary component of the internal path of blood clotting (Zubairov D.M. Factor VIII. In: Molecular basis of blood coagulation and clot formation. 2000, Kazan, p.101-110).

Substitution therapy in patients with hemophilia a and for its prevention, use preparations containing f VIII. Currently the need of health of the Russian Federation in the preparations f VIII is about 300 million International units (ME), but large-scale domestic industrial production in the country is missing (AMCEN AV and other Method of preparation and the properties of the drug factor VIII light is tawania of human blood plasma // Siberian honey. journal, 2009, No. 2, p.17-20). Getting f VIII is carried out or the fractionation of human blood plasma or animals, or recombinant methods of genetic engineering. Obtained from blood plasma preparations may contain, in addition to f VIII, von Willebrand factor, which suggests their use in von Willebrand disease. To the problems of production of drugs F. VIII from human blood plasma are as follows:

- the possibility of infecting the recipient with hepatitis viruses (a, b, C), HIV, parvovirus, syphilis and other pathogens

- possibility of transfer to the recipient with certain groups present in the products (concentrates average purity) of antibodies to erythrocyte antigens of other blood groups,

- contamination of drugs F. VIII ballast proteins,

- stability of the protein f VIII in its concentrates (based on existing requirements F. VIII should be stable for at least 12 hours after reconstitution of the lyophilisate) (Burnouf T. Plasma fractionation in the world: current status // Transfus. Clin. Biol., 2007, v.14 (1), p.41-50).

To solve these problems, develop appropriate technological approaches. So, when preparing a pool of blood plasma filter carefully screened healthy donors, which determine possible infection with hepatitis viruses, human immunodeficiency etc. However, for complete assurance of the absence of viral infecti is in the process of obtaining concentrates of antihemophilic factor carry out viral inactivation. Due to the different nature of viruses (with or without lipid membranes) inactivation is most often carried out comprehensively, namely solvent-detergent and appropriate heating (dual inactivation) (Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products: Annex 4. - WHO Technical Report, Series No. 24, 2004, p.177).

To increase the purity of the obtained concentrates F. VIII consistently use a set of procedures fractionation (precipitation, various types and combinations of high-performance chromatographic methods). The increased purity of the product ensures the increase of its specific specific activity (koagulologicheskaja activity f VIII/mg protein) and reduces the impurity content of undesirable components.

To improve the stability of the concentrates of f VIII in the process of getting used remove proteins prothrombin complex, the addition of albumin, heparin, lysine and other substances.

Currently widely used these drugs F. VIII from blood plasma:

Table 1
Description antihemophilic medications
MedicationMethod getThe specific activity of F. VIII,IU/mg protein ManufacturerNote
Koate-DVIFractionation according to the Cohn-Orly, precipitation, chromatography50Barer, USA+PV, C./D. and heating
ImmunateZIOC70Baxter, USA+PV, C./D. and heating
OctanateZIOC100Octapharma, Austria+PV, C./D. and heating
Factor 8YThe precipitation of heparin/glycine2,5-4Bio Pro. Lab., England+PV, dry heat,
EmoclotZIOC80Manufactured respectively by kedrion, Italy+PV, C./D. and dry heat
Note: s/A. - solvent/detergent, +PV - presence of von Willebrand factor, ioch - ion-exchange chromatography.

Known methods of allocation of plasma f VIII.

The method presented in the Patent of the Russian Federation No. 2324495, the VIII was obtained by cryofracture blood plasma, dissolving cryoprecipitate, viral inactivation of cryoprecipitate solvent-detergent method using tri-n-butylphosphate and Triton X-100 chromatography on Sepharose 4FF, ultrafiltration and lyophilization.

U.S. patent 5252709 A1 describes a method of obtaining a concentrate of F. VIII, consisting in suspendirovanie cryoprecipitate plasma in the solution of heparinate sodium, deposition of ballast proteins aluminum hydroxide, sterilizing filtration of the obtained supernatant, viral inactivation solvent-detergent, adsorption of the target product on the chromatographic column with Fractogel with subsequent elution buffer solution containing sodium chloride, and lyophilization.

In U.S. Patent 5259951 A1 preparation f VIII were obtained from blood plasma by cryosection and dissolving cryoprecipitate in the solution containing sodium chloride, heparin and glycine, treatment with aluminum hydroxide, heating the intermediate product in the presence of the stabilizer, ion exchange chromatography, dialysis of the precipitate, heating and filtration sterilization, followed by lyophilization.

In U.S. Patent 5259951 A1 highly purified f VIII in blood plasma was obtained by consecutive application of cryoprecipitate, dissolution of cryoprecipitate in an aqueous solution containing heparin, treatment with aluminum hydroxide and polyethylene glycol (PEG)-4000, chrome is adopted on the ion exchanger, stabilization of albumin, heparin, PEG-4000, lysine and histidine concentration, diafiltration, heat viral inactivation and lyophilization.

Closest to the claimed method (prototype) is a method of obtaining f VIII, presented in the Patent of the Russian Federation 2253475 C1. The method consists in obtaining from human blood plasma of cryoprecipitate, which are suspended in an aqueous solution of heparin (1-3 IU/ml), in the cleaning of ballast proteins by aluminum hydroxide and PEG-4000 (final concentration of 0.3% and 2%, respectively), in viral inactivation solvent-detergent method in the presence of Twin, microfilaria, chromatographic fractionation on DEAE-containing media with application buffer (pH of 6.75-6,85) solutions of different ionic strength, stabilization of the obtained solution f VIII albumin (final concentration of 0.1%), sterilization by microfiltration, freeze-drying and subsequent heat treatment for the purpose of additional viral inactivation. The disadvantages of this method include a significant loss of target substances by chromatographic fractionation and the insufficient degree of purification allocated F. VIII. On stage chromatographic purification output f VIII was 40-45%, the content and specific activity of factor VIII were equal to 15-20 IU/ml and 80-100 IU/mg protein, respectively.

The purpose of this is part II of the invention is to develop a method for industrial production of stable high-purity F. VIII. This goal is achieved by developing technologies that provide a lyophilized form of concentrate f VIII with high specific specific activity by increasing the degree of purification.

The development of technologies for extraction of f VIII for receiving antihemophilic factor first conducted in laboratory conditions, and then were scaled in terms of pilot production. Production of the drug f VIII comply with the rules of Good Manufacturing Practice (GMP), good rules of industrial activities, standard operating procedures (SOPS) and the necessary conditions of sterility.

At all stages of production raw materials, intermediate products and targeted drug testing for activity f VIII single-stage coagulopathies method and protein content by the method of Bradford. At various stages of production determine coagulopathies activity of von Willebrand factor and prothrombin, the content of fibronectin was measured by enzyme-linked immunosorbent method. On the level of industrial production is strictly controlled, the degree of microbial infection.

Raw material for production of f VIII is fresh frozen plasma blood activity f VIII is not less than 0.7 IU/ml Necessary condition of plasma is proven in the absence of virus infection and hepatitis b and C and human immunodeficiency virus HIV 1/HIV 2. In the cleaning process f VIII at the stage of deposition used a combination of polyethylene glycol (PEG)-4000 (Merck, Germany) and aluminum hydroxide (Biosector, Denmark). To conduct viral inactivation using 1% solution of tween 80 and 0.03% tri-n-butylphosphate (Merck, Germany). Anion exchange chromatography is carried out on high-exchangers of the group), not containing DEAE, preferably EMD-TMAE Fractogel (Merck, Germany), using chromatographic columns Millipore Vantage S2 and chromatograph BioProcess (GE, USA). The concentration of sodium ions were determined by ion analyzer (CIBA-CORNING, USA). Sterile filtration of the preparation is carried out using 0.22 μm filters (Millipore, USA), and lyophilization - apparatus SERAIL (France), specially developed mode.

The inventive method of obtaining f VIII consists of the following stages:

1. From fresh frozen human blood plasma by the method of controlled thawing and centrifugation produce cryoprecipitate (CP) (detachable cryosupernatant used as raw material for production of f IX, albumin and immunoglobulins).

2. The obtained KP solubilizer with subsequent adjustment of pH. To increase the purity of the target f VIII using an aqueous solution containing 5-100 international units (ME) nefrackzionirovannam heparin in 1 ml, preferably 10-25 IU/ml

3. The solubilized KP treated with hydro gel is sid aluminum, intense absorption at neutral pH factors of the prothrombin complex. A solution of KP without factors of the prothrombin complex was purified from fibrinogen, fibronectin and other proteins by precipitation with PEG-4000 and centrifugation.

Combined application used final concentration of heparin (5-100 IU/ml), aluminum hydroxide (0.3%) and PEG-4000 (3,5%) provides the intermediate product is purified solution of KP - with increased stability and specific activity of f VIII due to more efficient removal of ballast proteins and proteases. The chosen concentrations of heparin (preferably 10-25 IU/ml), the values of pH, preferably pH=6,6) and the final concentration of PEG-4000 (3,5%) provides a significant reduction stage before chromatographic purification of the content of fibrillar protein fibronectin from 2-4 g/l up to 20-30 mg/l (prototype - 200-300 mg/l).

4. The purified solution of the KP is subjected to viral inactivation using solvent-detergent (tween-80) and tri-n-butylphosphate) and pre-filtering with the optimal conditions for virus inactivation: neutral pH values (6,9-7,1), temperature 25°C. and a processing time of 6 hours.

5. A further selection (clean) f VIII of the solution virus-inactivated KP carried out by anion exchange chromatography (AOH) using strong anyoneon nikov, preferably ionoobmennik EMD-TMAE Fractogel. This type of sorbents in combination with the use of optimum ionic strength fractionation reduces the degree of adhesion of von Willebrand factor and to increase the stability of the target drug, F. VIII, containing the von Willebrand factor. The chromatographic purification is carried out at the size of the column is 10×15 cm, the amount of applied sample - 120-200 thousand ME, the starting buffer - Tris-citrate salt with a neutral pH, elution - step NaCl gradient and rate of application and elution - 80-100 cm/hour.

6. The target fraction f VIII obtained in step AOH has a specific activity of at least 140 (150-200) IU/mg protein, which is significantly (1.5-2 times) higher than the specific activity of f VIII known drugs from plasma. This fraction is diluted Tris-citrate buffer with neutral pH to the desired activity F. VIII, stabilize albumin to a final concentration of albumin in the product 0.1% spend sterile filtration and filling solution of f VIII in bottles.

7. Poured the vials of solution f VIII freeze-dried and subjected to additional heat viral inactivation; loss of activity when thermoinactivation not exceed 5%.

After lyophilization and heat viral inactivation in industrial scale developed technologies get 70-130 vials (20 ml) and life is Ino dried f VIII for solution for injection with a specific activity of f VIII 200-300 IU/vial with the output value of the specific activity of f VIII of the whole technological process 13-20%.

Below are specific examples of carrying out the invention.

Example 1.

1. 180 l of fresh Frozen plasma donors, held certification for infections, with the activity of F. VIII, equal to 0.75 IU/ml (containing the main product 135000 IU), thawed in the reactor at a temperature of-0.5°C and produce cryoprecipitate (CP) using a flow-through centrifuge at 1800 rpm and 3°C. the Mass of the CP was 2.0 kg, the content of the target product 80000 ME (the output of the target product 59%).

2. The obtained KP is dissolved in an aqueous solution of heparin (10 IU/ml of purified water) and solubilization at 25°C. pH Dissolved and solubilizing KP adjusted to 6.8. Volume KP 8,0 l, the activity of the target on the stage of the product 72000 ME (the output stage 90%).

3. Sorption factors of the prothrombin complex is realized by adding to the CP 3% gel of aluminum hydroxide in the amount equal to 1/10 of the mass of the CP, with stirring at 25°C for 15 minutes. The deposition of fibrinogen, fibronectin and ballast proteins carried out by adding 32% PEG-4000 to a final concentration of 3.5%. the pH was adjusted to 6.6 and the mixture is centrifuged at 4000 rpm at 2-4°C. the Volume of the target supernatant 8,0 l, the activity of the target product 70140 ME (stage 97%).

The sorption efficiency of the factors of the prothrombin complex and removal of impurity proteins is presented in figure 1 (Sorption factors prothrombin is the first complex by hydoxide aluminum) and figure 2 (Removal of fibrinogen PEG-4000), respectively.

4. Virus inactivation by solvent-detergent method carried out by adding 11% solution of tween 80 to a final concentration of 1% followed by slow addition of tri-n-butylphosphate to a final concentration of 0.3%; pH adjusted to 6.8. Virus-inactivated intermediate filtered through Millipore filters with pore diameter of 2-8 μm. The amount of pre-filtered solution is 9.0 l, the content on the stage of the target product 69300 ME (the output stage 99%).

5. Chromatographic fractionation is carried out using an ion-exchanger EMD-TMAE Fractogel, starting, intermediate, and eluting buffers with concentrations of sodium ions 130, 170 and 450 mmol/l and conductivity 15, 18 and 48 MSM/see the Amount of eluting buffer 3.5 l, the rate of elution of 4.7 l/h. Volume of product 0.4 l, the number 45000 ME. The output stage of the target product is 65%, the activity of f VIII in the target fraction equal to 110 IU/ml and specific activity of f VIII increased from 45 to 150 IU/mg protein. The data presented in figure 3 (Chromatographic purification of f VIII).

6. Sterile filtering the target faction chromatographic purification after dilution to the desired activity and stabilization of albumin (final concentration of 0.1%) is carried out using Millipore filters with a pore diameter of 0.22 μm and a sterile product is poured into vials. At the output stage after testwuide analyses the amount of the target product is 2.2 l, the total activity of F. VIII - 44000 ME (98%).

7. The target product is frozen at -50°C and pressure of 1 bar, for 75 hours and then lyophilizer in conditions -20°C-+30°C at a pressure of 60 mcbar for 40.5 hours. Then conduct heat viral inactivation at 80°C and a pressure of 60 mcbar within 72 hours. The yield of the target drug f VIII equal 40000 ME. The output stage 90%. The output of the process - 30%.

Example 2.

1. 220 l of fresh Frozen plasma donors, held certification for infections, with the activity of F. VIII, 0.9 IU/ml (containing the main product 198000 ME), thawed in the reactor at a temperature of-0.5°C and produce cryoprecipitate (CP) using a flow-through centrifuge at 18000 rpm and 3°C. the Mass of the CP is 2.2 kg, the content of the target product 109470 ME (the output of the target product 55,3%).

2. The obtained KP is dissolved in an aqueous solution of heparin (20 IU/ml, purified water) and solubilizing at 25°C. pH Dissolved and solubilizing KP adjusted to 6.8.

3. Sorption factors of the prothrombin complex is carried out by adding to the KP 3% gel of aluminum hydroxide in the amount equal to 1/10 of the mass of the CP, with stirring at 25°C for 15 minutes. The deposition of fibrinogen, fibronectin and ballast proteins carried out by adding 32% PEG-4000 to a final concentration of 3.5%. the pH was Adjusted to 6.6 and the mixture centrifuged at 400 rpm at 2-4°C. The content of the target product 88450 ME (the output stage 81%).

4. Virus inactivation by solvent-detergent method carried out by adding 11% solution of tween 80 to a final concentration of 1% followed by slow addition of tri-n-butylphosphate to a final concentration of 0.3%; pH adjusted to 6.8. Virus-inactivated intermediate filtered through Millipore filters with pore diameter of 2-8 μm. The content on the stage of the target product 87 565 ME (99%).

5. Chromatographic fractionation is carried out using an ion-exchanger EMD-TMAE Fractogel, starting, intermediate, and eluting buffers with concentrations of sodium ions 130, 170 and 450 mmol/l and conductivity 15, 18 and 48.0 MSM/see the Amount of eluting buffer 3.5 l, the rate of elution of 4.7 l/h. The content of the target product 68 400 ME. The output stage of the target product is 78%, specific activity of f VIII is equal to 165 IU/mg protein.

6. Sterile filtering the target faction chromatographic purification after dilution to the desired activity and stabilization of albumin (final concentration of 0.1%) is carried out using Millipore filters with a pore diameter of 0.22 μm and a sterile product is poured into bottles.

The content of factor VIII - 65830 ME, the yield of the target product at the stage reached 96%.

7. The target product is frozen at -50°C and pressure of 1 bar, for 75 hours and then lyophilizer in conditions -20°C-+30°C the ri pressure 60 mcbar for 40.5 hours. Then conduct heat viral inactivation at 80°C and a pressure of 60 mcbar within 72 hours. The yield of the target drug f VIII equal 59100 ME. The output stage 90%. The output of the process - 30%.

The obtained concentrates of antihemophilic f VIII were sterile, was characterized by total protein content (endogenous protein and exogenous albumin) of 1.5-1.8 mg/ml, the concentration of sodium ions 165-175 mmol/l; target f VIII (after chromatographic purification) possessed high specific specific activity (not less than 140 IU/mg protein). The pharmacokinetics of the drug received f VIII in plasma did not differ from that of antihemophilic drug Immunate (Baxter, USA); data presented in figure 4 (the Pharmacokinetics of the drug received f VIII and drug Immunate).

The resulting preparation F. VIII, according to the permission for medical use, used in the clinic Hematological scientific center of the Russian Academy of medical science for the treatment of 20 patients with hemophilia A. it is Shown high therapeutic efficiency and a good degree of recovery of the drug received f VIII.

The method of separation of factor VIII from blood plasma of a person who is not infected according to the corresponding analysis of hepatitis viruses and HIV 1/2, which consists in sequential cryosurgery, dissolved in an aqueous solution of heparin and solubilization of cryoprecipitate, marblebathroom prothrombin complex aluminum hydroxide, the removal of fibrinogen, fibronectin and impurity proteins by polyethylene glycol-4000, viral inactivation using solvent-detergent and pre-filtration, anion exchange chromatography, preferably using EDM -) Fractogel, with elution of sodium chloride-containing buffer, stabilizing solution of albumin, sterile filtered on membrane filters with a pore diameter of 0.22 μm, filling into vials (200-300 IU/vial, lyophilized and re-heat viral inactivation, characterized in that when cleaning using an aqueous solution nefrackzionirovannam heparin concentrations equal 5-100 International (IU)/ml, preferably 10-25 IU/ml, peg-4000 at a final concentration of 3.5% and the acidification of the environment, preferably until the pH of 6.6, a strong anion-exchangers of the group), that is a new combination of conditions that substantially increase the cleaning efficiency and specific activity of factor VIII.



 

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6 cl, 4 dwg

FIELD: medicine.

SUBSTANCE: after operation a section of tumour is taken from patient, placed into diffusion chamber together with solution of chemical preparation, chamber is placed into peritoneal cavity of rat for 1 week. After that animals are removed from experiment, fixation, staining and light microscopy of filter from chamber are performed, sensitivity of tumour to said chemical preparation is determined by presence or absence of tumour cell growth. After that, since the eighth day after operation patient is subjected to intraperitoneal autoplasmachemical therapy for 5 days with the preparation to which tumour is sensitive.

EFFECT: increase of anti-tumour action of intraperitoneally introduced cytostatics, incubated on autoplasma, due to individual selection of chemical preparation.

2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to a set for hypogalactia prevention in women with thyroid diseases. The set for hypogalactia prevention in women with thyroid diseases which includes the preparations: aminophylline; no-spa; a preparation improving blood rheology specified in the group: rheopolyglucinum, fresh frozen plasma, a microdose of aspirin; an energy preparation specified in the group: glucose, actovegin; amino acid specified in the group: methionine, glutamic acid, galascorbinum; antioxidants, a ferriferous preparation specified in the group: Sorbifer Durules, tardiferon; and a vitamin complex containing vitamins A, E, C.

EFFECT: set reduces rate and severity of lactation disorders in women with a thyroid pathology and improves breast milk quality.

2 cl

FIELD: medicine.

SUBSTANCE: invention relates to method of biological material disinfection. Method of removing trialkylphosphate solvent and detergent, which represents polyoxiethylene ether, from mixture of plasma, trialkylphosphate solvent and said detergent, where solvent and detergent were used for virus plasma inactivation, include treatment with synthetic hydrophobic adsorbent from polystyrene and divinylbenzole copolymer in ratio 1 g of adsorbent per 1-8 ml of plasma for 30-60 min at 20-30°C, and collection of treated plasma, where both solvent and detergent are removed by one-stage processing by hydrophobic interaction.

EFFECT: invention ensures safe, efficient and economical method of removing viricidal preparations, that is solvent and detergent from virus-inactivated joined in pool plasma by means of hydrophobic interaction chromatography.

9 cl, 7 tbl, 1 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to oncology, and can be used in treatment of mammary gland cancer (MGC). Method is realised in the following way. After confirmation of malignant character of tumour tissue in patient sampling of 200 ml of blood from peripheral vein into reservoir with hemopreservative is carried out, after that it is centrifuged with separation of plasma, which is combined with cyclophosphane in amount 400 mg/m2, doxorubincin in dose 40 mg/m2 is introduced into erythrocyte mass; both reservoirs are incubated in thermostat for 30 minutes at temperature 37°C. After that, sucking of blood from the place of tumour tissue sampling is carried out with further introduction into it of 200 mg of cyclophosphane diluted in 5 ml of physiological solution. Tissue of mammary gland around tumour is infiltrated with incubated plasma with cyclophosphane, and erythrocyte mass with doxorubicin is introduced intravenously by drop infusion.

EFFECT: application of the invention makes it possible to increase efficiency of MGC treatment due to creation of maximal concentration of chemical preparations in leision focus and prevention of tumour process dissemination.

1 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: one should apply an autohemocomponent preparation being supernatant liquid of patient's autoblood at increased serotonin content obtained due to irreversible thrombocytic aggregation due to the impact of 0.5 mg ATP per 1.0 ml plasma followed by a 30-min-long centrifuging at the rate of 1000, 2000 and 3000 rot./min for 20, 7 and 3 min, correspondingly. In case of no exudative phenomena on patient's eye bottom the obtained preparation should introduced at the quantity of 7-10 ml once in 48 h for 1 mo (totally, 15 injections). In case of exudative-hemorrhagic phenomena it should be introduced parabulbarly at the volume of 0.5 ml and parenterally - 7.0-10.0 ml once in 48 h for 1 mo per 15 injections, correspondingly. The preparation enables to improve visual functions due to decreased tissue hypoxia and normalization of microcirculation in visual analyzer.

EFFECT: higher efficiency of therapy.

2 cl, 7 dwg, 2 ex

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