RU2445974C2 - Method for producing concentrated factor viii of human blood plasma - Google Patents

Abstract

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.

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Description

The invention relates to the field of pharmaceutical industry, namely to biotechnology for producing hemostatic drugs. A quantitative or functional deficiency of coagulation factor (F) VIII causes the development of a hereditary hemorrhagic hemophilia A disease, the main method of treatment and prevention of which is replacement therapy with F VIII drugs.

Ф VIII is a blood plasma glycoprotein non-covalently associated with von Willebrand factor, which significantly increases the stability of Ф VIII. Gene F VIII is located on the X chromosome, and its mutations determine the development of hemophilia A, inherited by a recessive trait. Activated F VIII (F VIIIa) is a labile, non-enzymatic protein co-factor F IXa. Ф VIII is a necessary component of the internal pathway of blood coagulation (Zubairov D.M. Factor VIII. B: Molecular basis of blood coagulation and thrombosis. 2000, Kazan, pp. 101-110).

In the replacement therapy of patients with hemophilia A and for its prevention, drugs containing F VIII are used. At present, the need for healthcare in the Russian Federation in drugs F VIII is about 300 million international units (ME), but there is no large-scale domestic industrial production in the country (Yamkin A.V. et al. Method for the preparation and properties of the drug VIII coagulation factor human blood plasma // Siberian Medical Journal, 2009, No. 2, pp. 17-20). Obtaining F VIII is carried out either by fractionation of the blood plasma of humans or animals, or by recombinant methods of genetic engineering. Preparations obtained from blood plasma may contain, in addition to F VIII, von Willebrand factor, which suggests their use in von Willebrand disease. The problems of the production of drugs F VIII from human blood plasma include:

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

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

- contamination of drugs F VIII with ballast proteins,

- stability of the protein F VIII in its concentrates (according to existing requirements, F VIII must be stable for 12 hours after dissolution 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, appropriate technological approaches have been developed. So, when preparing a pool of blood plasma, carefully selected healthy donors are selected for possible infection with hepatitis viruses, human immunodeficiency viruses, etc. However, to completely guarantee the absence of viral infection, virus inactivation is carried out in the process of obtaining antihemophilic factor concentrates. Due to the different nature of viruses (with or without lipid membrane), inactivation is most often carried out in a complex, namely solvent detergents and appropriate heating (double 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 N24, 2004, p. 177).

To increase the degree of purification of the obtained concentrates, F VIII, a series of fractionation procedures (precipitation, various types and combinations of highly efficient chromatographic methods) are sequentially used. The increase in the purity of the drug provides an increase in its specific, specific activity (coagulological activity f VIII / mg protein) and reduces the content of impurity undesirable components.

In order to increase the stability of Ф VIII concentrates, in the process of its preparation, the prothrombin complex proteins are removed, albumin, heparin, lysine and other substances are added.

Currently, the following drugs F VIII from plasma are widely used:

Table 1 Characterization of antihemophilic drugs A drug Production method The specific activity of f VIII, IU / mg protein Manufacturer Note Koate-dvi Cohn-Orly Fractionation, Precipitation, Chromatography fifty Barer, USA + PV, s / d and heating Immunate Ooh 70 Baxter, USA + PV, s / d and heating Octanate Ooh one hundred Octapharma, Austria + PV, s / d and heating Factor 8Y Precipitation heparin / glycine 2.5-4 Bio Pro Lab., England + PV, dry heat, Emoclot Ooh 80 Kedrion, Italy + PV, s / d and dry heat Note: s / d - solvent / detergent, + PV - the presence of von Willebrand factor, IOX - ion-exchange chromatography.

The following methods of isolation from plasma f VIII are known.

By the method presented in RF Patent No. 232,495, F VIII was obtained by cryofractionation of blood plasma, dissolution of cryoprecipitate, viral inactivation of cryoprecipitate by the solvent-detergent method using tri-n-butylphosphate and Triton X-100, chromatography on Sepharose 4FF, ultrafiltration and lyophilization.

US patent 5252709 A1 describes a method for producing concentrate Ф VIII, which consists in suspending plasma cryoprecipitate in a solution of sodium heparin, precipitating ballast proteins with aluminum hydroxide, sterilizing the filtration of the obtained supernatant, viral inactivation by solvent detergents, adsorption of the target product on a chromatographic column with Fractogel followed by Fractogel buffer followed by a solution containing sodium chloride, and lyophilization.

In US Pat. No. 5,259,951 A1, preparation F VIII was obtained from blood plasma by cryoprecipitation and dissolution of cryoprecipitate in a solution containing sodium chloride, heparin and glycine, purification with aluminum hydroxide, heating of the intermediate in the presence of a stabilizer, ion exchange chromatography, dialysis of precipitate, heating and filtration sterilization followed by lyophilization .

In US Pat. No. 5,259,951 A1, highly purified plasma plasma F VIII was obtained by sequential application of cryoprecipitation, dissolution of cryoprecipitate in an aqueous solution containing heparin, purification with aluminum hydroxide and polyethylene glycol (PEG) -4000, chromatography on an ion exchanger, stabilization with albumin, heparin, PEG-4000, lysine and histidine, concentration, diafiltration, thermal viral inactivation and lyophilization.

Closest to the claimed method (prototype) is a method of obtaining f VIII, presented in RF Patent 2253475 C1. The method consists in obtaining cryoprecipitate from human blood plasma, which is suspended in an aqueous solution of heparin (1-3 IU / ml), in the cleaning of ballast proteins with aluminum hydroxide and PEG-4000 (final concentrations of 0.3% and 2%, respectively), in viral inactivation by the solvent-detergent method in the presence of Tween, in microfiltration, in chromatographic fractionation on a DEAE-containing carrier using buffer (pH 6.75-6.85) solutions of different ionic strength, in the stabilization of the resulting solution F VIII albumin (final concentration 0 1%), sterilization by microfiltration, lyophilization and subsequent heat treatment for the purpose of additional viral inactivation. The disadvantages of this method include a significant loss of the target substance during chromatographic fractionation and an insufficient degree of purification of the isolated Ф VIII. At the stage of chromatographic purification, the yield of Ф VIII was 40-45%, the content and specific specific activity of factor VIII were 15-20 IU / ml and 80-100 IU / mg of protein, respectively.

The aim of the present invention is to develop a method of industrial production of stable highly purified f VIII. This goal is achieved by the development of technology that provides a lyophilized form of concentrate Ф VIII with high specific specific activity by increasing the degree of purification.

The development of the technology of isolation of F VIII to obtain the antihemophilic factor was first carried out in laboratory conditions, and then scaled under experimental production conditions. The manufacture of F VIII was in accordance with Good Manufacturing Practices (GMP), appropriate industrial practices, standard operating procedures (SOPs), and necessary sterility conditions.

At all stages of production, the feedstock, intermediates and the target drug are tested for activity F VIII by a single-stage coagulological method and protein content by the Bradford method. At various stages of production, the coagulological activity of von Willebrand factor and prothrombin is determined, the content of fibronectin is measured by enzyme-linked immunosorbent assay. At the industrial production level, the degree of microbial infection is strictly controlled.

The raw material for obtaining F VIII is freshly frozen plasma of donated blood with an activity of F VIII of at least 0.7 IU / ml. A necessary condition for the use of plasma is the proven absence of infection with hepatitis B and C viruses and HIV 1 / HIV 2 immunodeficiency. In the process of purification of Ph VIII, a combination of polyethylene glycol (PEG) -4000 (Merck, Germany) and aluminum hydroxide (Biosector, Denmark). For viral inactivation, a 1% solution of Tween 80 and 0.03% tri-n-butyl phosphate (Merck, Germany) are used. Anion exchange chromatography was performed on strong anion exchangers of the TMAE group that did not contain DEAE, preferably EMD-TMAE Fractogel (Merck, Germany), using a Millipore Vantage S2 chromatographic column and a BioProcess chromatograph (GE, USA). The concentration of sodium ions was determined on an ion analyzer (CIBA-CORNING, USA). Sterile filtration of the drug is carried out using 0.22 μm filters (Millipore, USA), and lyophilization on a serail device (France) according to a specially developed regime.

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

1. Cryoprecipitate (KP) is isolated from freshly frozen human blood plasma by controlled thawing and centrifugation (detachable cryosupernatant is used as a raw material for obtaining Ф IX, albumin and immunoglobulins).

2. The resulting KP is solubilized with subsequent pH adjustment. To increase the degree of purification of the target F VIII, an aqueous solution is used containing 5-100 international units (ME) of unfractionated heparin in 1 ml, preferably 10-25 IU / ml.

3. The solubilized KP is treated with an aluminum hydroxide gel that intensively sorb prothrombin complex factors at neutral pH. The KP solution without prothrombin complex factors is purified from fibrinogen, fibronectin and other proteins by PEG-4000 precipitation and centrifugation.

The combined use of the used final concentrations of heparin (5-100 IU / ml), aluminum hydroxide (0.3%) and PEG-4000 (3.5%) provides an intermediate product - a purified solution of KP - with increased stability and specific specific activity f VIII due to more efficient removal of ballast proteins and proteases. The use of selected concentrations of heparin (preferably 10-25 IU / ml), acidity (preferably pH = 6.6) and a final concentration of PEG-4000 (3.5%) provides a significant reduction before the stage of chromatographic purification of the content of fibrillar fibronectin protein from 2 -4 g / l to 20-30 mg / l (in the prototype - 200-300 mg / l).

4. The purified solution of KP is subjected to viral inactivation using a solvent detergent (Tween-80 and tri-n-butylphosphate) and preliminary filtration with optimal conditions of viral inactivation: neutral pH values (6.9-7.1), temperature 25 ° С and processing time 6 hours.

5. Further isolation (purification) of F VIII from the virus-inactivated KP solution is carried out by anion exchange chromatography (AOX) using strong anion exchangers, preferably EMD-TMAE Fractogel ion exchanger. This type of sorbents in combination with the use of optimal ionic fractionation strength allows to reduce the degree of adhesion of von Willebrand factor and increase the stability of the target drug f VIII containing von Willebrand factor. Chromatographic purification is carried out with a column size of 10 × 15 cm, the amount of applied sample is 120-200 thousand ME, the starting buffer is a tris citrate salt with a neutral pH, the elution is performed using a stepwise NaCl gradient, and the application and elution rates are 80-100 cm / hour .

6. The target fraction F VIII obtained at the AOX stage 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 plasma preparations. This fraction was diluted with a neutral pH tris-citrate buffer to the desired F VIII activity, stabilized with albumin to a final concentration of albumin in the preparation of 0.1% and sterile filtration and filling of the F VIII solution into vials were performed.

7. The vial solution F VIII is lyophilized dried and subjected to additional thermal viral inactivation; loss of activity during thermal inactivation does not exceed 5%.

After lyophilization and thermal viral inactivation under conditions of industrial scaling of the developed technology, 70-130 bottles (20 ml) of lyophilized dried Ф VIII for injection solution with specific activity Ф VIII 200-300 IU / bottle with a yield of specific activity Ф VIII of the whole process are obtained 13-20%.

The following are specific examples of carrying out the invention.

Example 1

1. 180 l of Freshly frozen donor plasma that has been certified for infections with an activity of Ф VIII equal to 0.75 IU / ml (the content of the main product is 135000 IU) is thawed in the reactor at a temperature of -0.5 ° С and cryoprecipitate is released (KP ) using a flow centrifuge at 1800 rpm and 3 ° C. The mass of the KP was 2.0 kg, the content of the target product 80,000 ME (yield on the target product 59%).

2. The resulting KP is dissolved in an aqueous solution of heparin (10 IU / ml of purified water) and solubilized at 25 ° C. The pH of the dissolved and solubilized KP is adjusted to 6.8. The volume of KP is 8.0 L, the activity of the target in the product stage is 72000 ME (yield at the stage of 90%).

3. Sorption of factors of the prothrombin complex is carried out by adding 3% of the aluminum hydroxide gel to the KP in an amount equal to 1/10 of the mass of the KP, with stirring at 25 ° C for 15 minutes. The precipitation of fibrinogen, fibrinectin and ballast proteins is carried out by adding 32% PEG-4000 to a final concentration of 3.5%. The pH was adjusted to 6.6 and the mixture was centrifuged at 4000 rpm at 2-4 ° C. The volume of the target supernatant of 8.0 l, the activity of the target product 70140 ME (yield at the stage of 97%).

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

4. Viral inactivation by the solvent-detergent method is carried out by adding an 11% Tween 80 solution to a final concentration of 1%, followed by slow addition of tri-n-butyl phosphate to a final concentration of 0.3%; The pH was adjusted to 6.8. The virus-inactivated intermediate is filtered through Millipore filters with a pore diameter of 2-8 μm. The volume of the pre-filtered solution is 9.0 L, the content at the stage of the target product is 69300 ME (yield at the stage of 99%).

5. Chromatographic fractionation is carried out using an EMD-TMAE Fractogel ion exchanger, start, intermediate and elution buffers with sodium ion concentrations of 130, 170 and 450 mmol / L and conductivity of 15, 18 and 48 mS / cm. The volume of the elution buffer is 3.5 l, the elution rate is 4.7 l / h. The volume of the product is 0.4 l, the amount is 45000 ME. The yield at the target product stage is 65%, the activity of Ф VIII in the target fraction is 110 IU / ml, and the specific specific activity of Ф VIII increased from 45 to 150 IU / mg of protein. The data are presented in figure 3 (Chromatographic purification f VIII).

6. Sterile filtration of the target fraction of chromatographic purification after dilution to the desired activity and stabilization with albumin (final concentration 0.1%) is carried out using Millipore filters with a pore diameter of 0.22 μm and the sterile product is poured into vials. At the exit of the stage, after appropriate analyzes, the volume of the target product is 2.2 L, the total activity of F VIII is 44000 ME (98%).

7. The target product is frozen at -50 ° C and a pressure of 1 bar for 75 hours and then lyophilized under conditions of -20 ° C - + 30 ° C at a pressure of 60 μbar for 40.5 hours. Then carry out thermal viral inactivation at 80 ° C and a pressure of 60 μbar for 72 hours. The yield of the target drug F VIII is 40,000 ME. Yield 90%. The output of the entire technological process is 30%.

Example 2

1. 220 L of freshly frozen donor plasma that has been certified for infections with an activity of Ф VIII equal to 0.9 IU / ml (main product content 198000 ME), thawed in the reactor at a temperature of -0.5 ° С and cryoprecipitate is isolated (KP ) using a flow centrifuge at 18,000 rpm and 3 ° C. The mass of the KP is 2.2 kg, the content of the target product is 109470 ME (yield for the target product is 55.3%).

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

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

4. Viral inactivation by the solvent-detergent method is carried out by adding an 11% Tween 80 solution to a final concentration of 1%, followed by slow addition of tri-n-butyl phosphate to a final concentration of 0.3%; The pH was adjusted to 6.8. The virus-inactivated intermediate is filtered through Millipore filters with a pore diameter of 2-8 μm. The content at the stage of the target product 87 565 ME (99%).

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

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

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

7. The target product is frozen at -50 ° C and a pressure of 1 bar for 75 hours and then lyophilized under conditions of -20 ° C - + 30 ° C at a pressure of 60 μbar for 40.5 hours. Then carry out thermal viral inactivation at 80 ° C and a pressure of 60 μbar for 72 hours. The yield of the target drug F VIII is 59100 ME. Yield 90%. The output of the entire technological process is 30%.

The obtained antihemophilic concentrates F VIII were sterile, characterized by a total protein content (endogenous protein and exogenous albumin) of 1.5-1.8 mg / ml, a concentration of sodium ions of 165-175 mmol / l; Target F VIII (after chromatographic purification) had a high specific specific activity (at least 140 IU / mg protein). The pharmacokinetics of the obtained preparation F VIII in the plasma practically did not differ from that of the antihemophilic preparation Immunate (Baxter, USA); the data are presented in Figure 4 (Pharmacokinetics of the resulting preparation F VIII and Immunate).

The obtained preparation F VIII, according to the permission for medical use, was used in the clinic of the Hematological Scientific Center of the Russian Academy of Medical Sciences for the treatment of 20 patients with hemophilia A. High therapeutic efficacy and a good degree of recovery of the obtained preparation F VIII are shown.

Claims (1)

The method of isolating factor VIII from human blood plasma that is not infected according to the corresponding analysis with hepatitis and HIV 1/2 viruses, which consists in successive cryoprecipitation, dissolution of heparin in an aqueous solution and solubilization of cryoprecipitate, sorption of prothrombin complex factors with aluminum hydroxide, removal of fibrinogen, fibronectin and impurity polyethylene glycol-4000 proteins, viral inactivation using solvent detergents and preliminary filtration, anion exchange chromatography, preferably using EDM-TMAE Fractogel, with elution with sodium chloride-containing buffer, stabilization with albumin solution, sterile filtration on membrane filters with a pore diameter of 0.22 μm, filling into bottles (200-300 IU / bottle), lyophilization and repeated thermal viral inactivation, characterized in that during cleaning use an aqueous solution of unfractionated heparin with concentrations equal to 5-100 International (IU) / ml, preferably 10-25 IU / ml, polyethylene glycol-4000 at a final concentration of 3.5% and acidification of the medium, preferably to pH 6, 6, strong ani noobmenniki TMAE groups, i.e. a new combination of conditions, significantly enhancing cleaning efficiency and specific activity of the specific factor VIII.

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