Stabilisation of liquid solutions of recombinant protein for storing in frozen state

FIELD: chemistry.

SUBSTANCE: invention represents method of stabilisation of liquid solution of coagulation and/or clotting factors for storing in frozen state, which contains: providing liquid solution of coagulation and/or clotting factor, where said solution has concentration NaCl and/or KCl, at least, 100 mM; addition of carbohydrate to said solution until said solution reaches in freezing temperature of vitrifying -56°C or higher; and freezing of said solution for storage.

EFFECT: invention ensures stabilisation of liquid solution of coagulation or clotting factors for storing in frozen state.

20 cl, 1 tbl, 2 dwg

 

This application claims the benefit of provisional patent application 60/926,698, filed on April 26, 2007, the disclosure of which is incorporated by reference.

The scope of the invention

The invention relates to the freezing and storage of liquid solution of recombinant protein, preferably bulk solutions.

The level of technology

The production of recombinant proteins in cell culture usually includes a number of stages of purification, by which the desired protein product isolated from the recombinant host cells and/or associated cultural environment. Many important recombinant proteins get in a large industrial scale. In the case of pharmaceutical proteins, for example, often need more than one stage of purification to achieve the desired level of purity of the product.

It may be necessary storage of bulk solution of recombinant protein, which was initially cleared, but not completely, before final purification for the preparation of a pharmaceutical product. For example, the protein product of the reaction of recombinant fermentation can be purified by affinity or ion-exchange column. After the initial passage through a column of protein product is only partially purified, and the solution still contains components, such as the remains of the cell is culture and other proteins. Before final conversion in pharmaceutical product bulk solution must be further processed to obtain protein a satisfactory degree of purity.

Typically, the solution for example, lucindy buffer, which is used for separation of protein after the first stage of cleaning is a highly concentrated salt solution. In the case of elution of column with high salt concentration needed to isolate the protein from the column. Accordingly the bulk solution obtained after the first stage of cleaning may contain a solution having a high concentration of monovalent salts, usually sodium chloride, but it is possible and potassium chloride and other salts.

Storage of bulk solution of recombinant protein is a complex task due to the high salt concentration and very low concentrations of protein solution. Ideally, proteins are stored at temperatures below the glass transition temperature to ensure stability, as in the glassy state inactivation and protein denaturation are extremely slow in pharmaceutical temporal scale. On the other hand, the presence of a high concentration of salt in the solution leads to lower its glass transition temperature, and in solutions with high salt concentration and low concentration of protein very low temperature the measures necessary to achieve such status.

Storing in a frozen state at a higher temperature is desirable for bulk solutions in large bulk quantities for reasons of cost and efficiency, but at the same time maintain the stability and activity of the protein.

Brief description of the invention

The invention is a method of stabilizing a liquid solution of recombinant protein for storage in a frozen state, which includes: providing a solution of recombinant protein, where this solution has a concentration of monovalent salt, such as NaCl and/or KCl at least 100 mm; adding carbohydrate to the specified solution in a quantity sufficient to give the solution at the freezing temperature of vitrification -56°C or above; and freezing the specified solution for storage.

The invention also provides a liquid solution of recombinant protein, which is stabilized for storage in a frozen state, which contains a carbohydrate in an amount sufficient to give the solution at the freezing temperature of vitrification -56°C or higher.

Description of the drawings

Figure 1 shows the effect on the saving activity of the recombinant protein from adding carbohydrate, with other excipients or without other excipients, as described in the text, to the bulk solution of recombinant the actor VIII. Four different compositions designated as F1, F2, F3 and F4. The activity of Factor VIII was evaluated after freezing and storage at -30°C at different points in time, as shown, for up to 24 weeks. To add carbohydrate bulk solution contained about 600 mm NaCl, 10 mm CaCl2, 20 mm imidazole and 0.1% Triton X-100, except F3, which was diluted as described here. The graph depicts the dependence of the efficiency of coagulation time (IU/ml).

Figure 2 shows the loss of activity of recombinant FVIII after freezing and storage at -70°C and -30°C bulk solution of Factor VIII used in the experiments is illustrated in figure 1, but without the stabilizing of the excipients. The designation "LN2 to -70°C" indicates that the samples were frozen in liquid nitrogen before storage at -70°C, and LN2 to -30°C" indicates that the samples were frozen in liquid nitrogen before storage at -30°C. the EVA to -70°C" indicates that the samples were frozen in plastic bags for storage and stored at -70°C.

Description of the preferred embodiment of the invention,

A liquid solution of recombinant protein can contain any solution of recombinant protein obtained from cultures of recombinant cells using affinity chromatography, ion exchange chromatography or the like. In predpochtitelnye the execution of the invention the solution is a bulk solution, which contains the solution, which was originally cleared. All versions of the invention, a liquid solution is a solution with a high salt concentration, preferably an aqueous solution.

Recombinant proteins include, for example and without limitation to this, coagulation factors, viral antigens, bacterial antigens, fungal antigens, protocollie antigens, peptide hormones, chemokines, cytokines, growth factors, enzymes, blood proteins such as hemoglobin, α-1-antitripsin, fibrinogen, serum albumin human prothrombin/thrombin antibodies, coagulation factors and/or blood clotting and their biologically active fragments, such as Factor V, Factor VI, Factor VII, Factor VIII and their derivatives, such as FVIII with a remote In-domain, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, Fletcher Factor, Fitzgerald Factor, and the Factor a background of Villebranda; milk proteins such as casein, lactoferrin, secrete lysozyme, α-1 antitripsin, protein factors, immune proteins and their biologically active fragments; and antibodies, including monoclonal antibodies, single-chain antibodies, antibody fragments, chimeric antibodies, humanized antibodies and other molecules of antibodies that can be produced in a culture of recombinant cells.

Preferred in the present recombinant baie is OK is a recombinant Factor VIII. Factor VIII as used here includes engineered variants of Factor VIII, such as variants of Factor VIII with a remote In-domain.

Bulk solution in the sense in which it is mentioned in the present invention, contains partially, but not completely purified liquid solution of recombinant protein that contains at least 100 mm monovalent salt. Monovalent salt preferably represents NaCl, which is usually used for elution of recombinant proteins, purification columns. However, NaCl may be replaced, wholly or partially, on KCl. Bulk solution may also contain various amounts of other salts, such as ferrous salts, including calcium chloride.

Using the term "partially, but not completely purified" refers to a liquid solution, subjected to at least one stage of purification, but this liquid solution still contains sufficient residual impurities, so that at least one stage of purification is required before preparation of the final product. For example, a bulk solution of recombinant Factor VIII must be further purified before final preparation of the product, which in the case of Factor VIII and other proteins may include lyophilization.

The liquid solution contains at least 100 mm monovalent salt, predpochtitelno 100 mm NaCl, more preferably, at least 300 mm NaCl, more preferably at least 500 mm NaCl, more preferably at least 560 mm NaCl, and even more preferably at least 600 mm NaCl. Often bulk solutions of recombinant protein have such a high concentration of monovalent salt after the initial stage of purification.

The following versions of the invention a liquid solution contains 100-200 mm NaCl, 100-300 mm NaCl, 200-300 mm NaCl, 100-400 mm NaCl, 100-500 mm NaCl, 100-600 mm NaCl, 100-800 mm NaCl, 300-500 mm NaCl, 300-600 mm NaCl, 300-800 mm NaCl, 400-600 mm NaCl, 400-800 mm NaCl, 500-600 mm NaCl, 560-700 mm NaCl and 500-800 mm NaCl.

Bulk solutions according to the invention, moreover, has a very low concentration of protein in them. In versions of the invention, the concentration of recombinant protein in bulk solution can be as low as 0.0001 mmol/l, 0.001 µmol/l or 0.01 µmol/l In versions of the invention, the concentration of recombinant protein in bulk solution can be as high as 10 µmol/l, 1 μmol/l or 0.1 μmol/L. Any protein concentration lying within any combination of upper and lower limits, is an embodiment of a bulk solution value according to the invention.

Carbohydrate is added in a liquid solution before freezing in a quantity sufficient to impart races the thief, when frozen, the glass transition temperature -56°C or higher, or more preferably, at least -34°C, or any temperature, as expressed by an integer or fractional number lying between these limits. Normal glass transition temperature of bulk solution with a high salt concentration and low concentration protein essentially amounts to less than -56°C, for example from -60 to -70°C. the Amount of the added carbohydrate required to raise the temperature of vitrification to -56°C, should be considered as one of the factors the concentration of protein. Higher concentrations of protein in themselves contribute to the increase of glass transition temperature of bulk solution. As other factors, the number of carbohydrate should not unduly increase the viscosity of the solution, and preferably the viscosity should be maintained lower than about 9.0 SP. The conductivity of the solution may vary, adding carbohydrate and preferably should be maintained below about 39 MSM/see

Freezing of the solution in the context of the present invention means the freezing of bulk liquid solution, and must be distinguished from lyophilization, which includes various technical considerations.

The carbohydrate may be a type of carbohydrate commonly used in the pharmaceutical compositions, the key sugar and di-, oligo - and polysaccharides. Examples include dextrans, cyclodextrine, chitosans, starch, hyaluronic acid, cellulose, raffinose, maltose, lactose, stachyose and combinations thereof. Preferred examples are carbohydrates, which are approved for injection, which include sucrose, trehalose, gidroxiatilkrahmal, dextran, or combinations thereof. Carbohydrates, pharmaceutical grade available commercially from many suppliers.

The exact number of carbohydrate necessary to protect the solution during freezing, can easily be determined, for example, by differential scanning calorimetry and depends on the specific protein and specific carbohydrate. Preferred at the present time the number of carbohydrate are 8-25 wt.% from the mass of liquid solution.

In specific embodiments, the execution of the invention the number of carbohydrate be about 8-15 wt.%, 12-20 wt.%, 16-20 wt.%, 15-25 wt.% and 20-25 wt.% from the mass of liquid solution.

Other components due to the initial purification (e.g., elution), may be present in bulk solution including a surfactant (such as Tween 80 or Triton-X), calcium chloride or imidazole. Other excipients may be added in liquid solutions. As shown in the compositions of no is e, additional surfactant may be added as excipient. As the next excipient can be added amino acid (e.g. glycine).

Examples

The invention is illustrated with the use of recombinant Factor VIII as an example, the recombinant protein. Recombinant Factor VIII receive, using methods known in the art for example as described in U.S. patent No. 5576194; 5804420 and 5733873. In preferred versions of the invention the recombinant Factor VIII produced in mammalian cells in large-scale fermentation reactors, in an environment which can be free from serum and/or protein. Preferably the recombinant Factor VIII is secreted into the environment by recombinant cells.

Recombinant factor VIII (full length) was isolated from the host cells and purified from the clarified tissue culture fluid by chromatography carried out using a membrane adsorber. The use of membrane adsorber allows you to isolate and concentrate the recombinant Factor VIII of the tissue culture fluid by binding and elution (as described in Suck et al., J. Biotechnology, 121: 361-367, 2006.) The eluate was divided into four portions and each portion was transferred into a sterile flask (400 ml in each flask). In addition to recombi is based Factor VIII and residual impurities, left, the eluate (bulk solution) contained about 600 mm NaCl, 10 mm CaCl2, 20 mm imidazole and 0.1% Triton X-100. The concentration of recombinant Factor VIII in the eluate was approximately 0.067 mmol/L.

Carbohydrate, or a combination of carbohydrates along with other excipients, as described, is then added to each flask at room temperature in amounts shown as Composition 1, 2, 3 and 4 in Table 1 below.

TABLE 1
Composition 1Composition 2Composition 3Composition 4
8% Sucrose15% Sucrose10% Hydroxy-15% Dextran
3% GlycineEmilceramica8% Trehalose
8% Trehalose
80 ppm Tween
80 ppm ween

All of the components in Table 1 are shown in mass% of the total mass of the solution. Carbohydrates and other excipients were obtained commercially. From freshly prepared each portion of sample was collected. Samples of Compositions(1), (2), (3) and (4) were evaluated on the activity of Factor VIII using standard coagulation analysis.

Composition 3 was prepared from the same eluate, but was diluted with buffer containing 20 mm imidazole and 10 mm CaCl2to reduce the NaCl concentration to half. The dilution was carried out to investigate the suitability of the method according to the invention for a solution with a lower, but still relatively high concentration of monovalent salt.

The glass transition temperature of each sample was determined using differential scanning calorimetry (DuPont Modulated DSC). Compositions 1, 2, 3 and 4 showed the glass transition temperature -56°C, -34.9°C and 35.5°C, respectively. In each case, the glass transition temperature was considerably higher than the glass transition temperature observed in the absence of added carbohydrate (which for the same bulk solution without carbohydrate, as defined, was in the range of from -60 to -70°C). The composition had the following viscosity: Composition 1: 1.8428 SP; Song 2: 3.1089 SP; Song 3: 6.8076 JV and the Composition is 4: 7.2123 SP. The composition had the following conductivity: Composition 1: 27.8 MS/cm; Song 2: 25.57 MS/cm; Song 3: 21.05 MS/cm and Composition 4: 32.1 MSM/see

As it was discovered, all the compositions were stable after storage in a frozen state at temperatures of -80, -30, -18 and -14°C for up to 24 weeks, as determined using coagulation analysis for the activity of Factor VIII in different moments of time, without significant loss of activity.

As shown in figure 1, all four compositions according to the invention supported coagulation activity of Factor VIII is essentially the initial level after storage at -30°C for up to 24 weeks.

As shown in figure 2, in the absence of additional excipients solution loses essentially all of coagulation activity of Factor VIII after a day of storage at -30°C.

1. A method of stabilizing a liquid solution of clotting factors and/or collapse for storage in a frozen state, which contains:
a. providing a liquid solution of clotting factors and/or coagulation, where this solution has a concentration of NaCl and/or KCl at least 100 mm;
b. add carbohydrate to the specified solution to achieve the specified solution at the freezing temperature of vitrification -56°C or higher and stameriena specified solution for storage.

2. The method according to claim 1, in which the Jew is the second solution of the factors of coagulation and/or clotting represents the bulk solution.

3. The method according to claim 1, wherein the solution has a concentration of NaCl of at least 100 mm.

4. The method according to claim 1, wherein the carbohydrate is added in the amount of 8-25 wt.% by weight of solution.

5. The method according to claim 1, wherein the carbohydrate is selected from the group consisting of sucrose, trehalose, hydroxyethylamine, dextran, and combinations thereof.

6. The method according to claim 1, in which the bulk solution has a concentration of NaCl of at least 300 mm.

7. The method according to claim 1, in which the bulk solution has a concentration of NaCl of at least 600 mm.

8. The method according to claim 1, in which the factor of coagulation and/or clotting is a Factor VIII.

9. The method according to claim 1, wherein the glass transition temperature is between -56°C and
-35°C.

10. The method according to claim 1, additionally containing an addition of amino acids and/or surfactants.

11. A liquid solution of clotting factors and/or coagulation, which is stable for storage in a frozen state, and which has a concentration of NaCl and/or KCl at least 100 mm, and which contains carbohydrate in such quantity that the solution reaches the freezing temperature of vitrification -56°C or higher.

12. A liquid solution of the factors of coagulation and/or clotting in claim 11, which contains the bulk solution.

13. A liquid solution of the factors of coagulation and/or clotting in claim 11, and Audi concentration of NaCl, at least 100 mm.

14. A liquid solution of the factors of coagulation and/or clotting in claim 11, containing a carbohydrate in an amount of from 8 to 25 wt.% by weight of solution.

15. A liquid solution of the factors of coagulation and/or clotting in claim 11, where the carbohydrate is selected from the group consisting of sucrose, trehalose, hydroxyethylamine, dextran, and combinations thereof.

16. A liquid solution of the factors of coagulation and/or clotting in claim 11, where the liquid solution is a NaCl concentration of at least 300 mm.

17. A liquid solution of the factors of coagulation and/or clotting in claim 11, where the liquid solution has a concentration of NaCl of at least 600 mm.

18. A liquid solution of the factors of coagulation and/or clotting in claim 11, where the factor of coagulation and/or clotting is a Factor VIII.

19. A liquid solution of the factors of coagulation and/or clotting in claim 11, where the glass transition temperature is between -56°C and -35°C.

20. Liquid bulk solution of recombinant Factor VIII, which contains a NaCl concentration of at least 100 mm, contains recombinant Factor VIII in a concentration of between 0.0001 µmol/ml and 10 µmol/l, and stabilized for storage in the frozen state by adding carbohydrate.



 

Same patents:

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

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns area of molecular biology and biochemistry, and can be used in medicine. There is offered mutein conjugate of the blood coagulation factor VIII (FVIII) wherein a residue not being cysteine in position 41, 129, 377, 388, 468, 491, 556, 1804, 1808, 1810, 1812, 1813, 1815 and/or 2118 is substituted by a cysteine residue with polyethylene glycol (PEG) where a PEG molecule is bound with a polypeptide in a mutant cysteine residue.

EFFECT: improved pharmacokinetic properties of the FVIII as an ingredient of the conjugate under the invention with preserved a procoagulant factor activity allows presenting new FVIII PEG-muteins for producing of a pharmaceutical compositions for treating hemophilia.

12 cl, 38 dwg, 8 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: declared invention refers to chemical-pharmaceutical industry. A method involves the following stages: preparing a solution of von Willebrand factor or von Willebrand factor/factor VIII complex which contains VWF in concentration up to 12 IU VWF:RCoAui and has the von Willebrand factor/factor VIII ratio equal to 0.4 or more; nanofiltering through a filter of pore size less than 35 nanometres at pressure less than or equal to 0.5 bar and in the presence of 0.05 to 0.2 M of calcium ions.

EFFECT: development of the effective method for producing concentrated von Willebrand factor or factor VIII/von Willebrand factor complex to be applied for treating hemophilia And or von Willebrand disease.

11 cl, 8 ex, 6 tbl, 1 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.

EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.

41 cl, 3 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention claims compositions which can include one or several mammary gland tumour proteins, their immunogenic parts or polynucleotides encoding such parts. Alternatively the therapeutic composition can include antigen-presenting cell expressing mammary gland tumour protein, or T-cell specific to cells expressing such protein. These compositions can be applied in prevention and treatment of such diseases as mammary gland cancer. Invention also claims diagnostic methods based on determination of mammary gland tumour protein or mRNA encoding such protein in sample.

EFFECT: use of peptides obtained from protein expressed from mammary gland by tumour in diagnostics and therapy of mammary gland cancer.

37 cl, 6 ex, 1 dwg

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to method of human blood coagulation VIII factor production and related product. Method includes blood serum as cryoprecipitate, heparine added, PEG-4000, centrifugation, supernatant is added with tributyl phosphate and Twin-80, repeated centrifugation, sediment washed with sodium chloride, then it is dissolved in tris-HCl buffer with additives, let through column filled with gel and attached antibodies to Willebrand's factor, factor VIII elution, dialysis. Produced concentrate does not contain Willebrand's factor and has activity not less than 300 ME/mg of protein with purity not less 98% and contains albumin of concentration of 0.1%. Product is lyophilized with further processing.

EFFECT: product does not display any toxicity and cause allergic reaction.

6 cl, 2 ex

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

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a novel stable ready formulation of pharmaceutical composition containing VIII factor and can be used in treatment of hemophilia. Invention relates to a solid pharmaceutical composition prepared by lyophilization of an amino acid-free solution and comprising the following components: (a) VIII factor in the concentration 50-10000 IU/ml for human VIII factor or human recombinant VIII factor, or 50-10000 U/ml for swine VIII factor or swine recombinant VIII factor; (b) surfactant in the concentration above critical micellar concentration up to 1% (vol./vol.); (c) calcium chloride; (d) sucrose; (e) sodium chloride; (f) trisodium citrate, and (g) amino acids-free buffer in the concentration 1-50 mM with pH 6-8 before lyophilization and after dissolving in water for injection. Also, invention relates to a liquid pharmaceutical composition prepared after dissolving indicated stable solid pharmaceutical composition in sterile water optionally containing sodium chloride. Invention provides preparing a stable ready formulation of pharmaceutical composition of VIII factor wherein albumin is replaced with other stabilizing agents.

EFFECT: improved and valuable properties of pharmaceutical composition.

25 cl, 3 tbl, 3 ex

FIELD: medicine, pharmaceuticals.

SUBSTANCE: invention relates to pharmaceutical agent containing factor VII or factor VII-related polypeptide and factor VIII or factor VIII-related polypeptide. Disclosed are application of factor VII or factor VII-related polypeptide and factor VIII or factor VIII-related polypeptide in production of drug, kit for episodic bleeding treatment, as well as methods for prophylaxis and treatment of episodic bleeding in subject.

EFFECT: accelerated coagulation of FVIII-deficit plasma.

43 cl, 1 tbl, 2 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to methods for preparing biologically active substances from donor blood. Method for enriching donor blood plasma cryoprecipitate with factor VIII involves defrosting freshly frozen donor blood plasma at +1-4°C to obtain cryofractions from plasma cryosupernatant and cryoprecipitate. After formation of the donor blood freshly frozen plasma dry calcium chloride is added in the amount from 0.001 to 0.004 mole/l followed by addition of sodium citrate in the amount from 0.05 to 0.10 mole/l and stirring at +1-4°C for 10-15 min. Then prepared cryoprecipitate deposit is separated by centrifugation. For preparing cryoprecipitate freshly frozen plasma is used prepared by usual method with using conventional preserving agents. Using the method provides significant increasing the content of factor VIII in cryoprecipitate being without appreciable co-precipitation of inert proteins.

EFFECT: improved enriching method.

2 tbl, 3 ex

FIELD: medicine, hematology, pharmacy.

SUBSTANCE: invention relates to the composition of factor VIII composed without addition of albumin and comprising the following excipients of composition in addition to factor VIII: from 4% to 10% of filling agent taken among group consisting of mannitol, glycine and alanine; from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose, arginine; from 1 mM to 5 mM of calcium salt, from 100 mM to 300 mM of NaCl, and buffer agent for pH value maintenance about between 6 and 8. Alternatively, the composition can comprise from 2% to 6% of hydroxyethylstarch; from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose, arginine; from 1 mM to 5 mM of calcium salt, from 100 mM to 300 mM of NaCl, and buffer agent for pH value maintenance between 6 and 8. In additional variant of realization of invention the composition can comprise: from 300 mM to 500 mM of NaCl, from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose and arginine; from 1 mM to 5 mM of calcium salt, and buffer agent. The composition provides stability in the absence of albumin or other proteins.

EFFECT: valuable properties of compositions.

35 cl, 11 tbl, 7 ex

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

FIELD: medicine.

SUBSTANCE: invention relates to methods for preparing biologically active substances from donor blood. Method for enriching donor blood plasma cryoprecipitate with factor VIII involves defrosting freshly frozen donor blood plasma at +1-4°C to obtain cryofractions from plasma cryosupernatant and cryoprecipitate. After formation of the donor blood freshly frozen plasma dry calcium chloride is added in the amount from 0.001 to 0.004 mole/l followed by addition of sodium citrate in the amount from 0.05 to 0.10 mole/l and stirring at +1-4°C for 10-15 min. Then prepared cryoprecipitate deposit is separated by centrifugation. For preparing cryoprecipitate freshly frozen plasma is used prepared by usual method with using conventional preserving agents. Using the method provides significant increasing the content of factor VIII in cryoprecipitate being without appreciable co-precipitation of inert proteins.

EFFECT: improved enriching method.

2 tbl, 3 ex

FIELD: medicine, pharmaceuticals.

SUBSTANCE: invention relates to pharmaceutical agent containing factor VII or factor VII-related polypeptide and factor VIII or factor VIII-related polypeptide. Disclosed are application of factor VII or factor VII-related polypeptide and factor VIII or factor VIII-related polypeptide in production of drug, kit for episodic bleeding treatment, as well as methods for prophylaxis and treatment of episodic bleeding in subject.

EFFECT: accelerated coagulation of FVIII-deficit plasma.

43 cl, 1 tbl, 2 dwg, 6 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a novel stable ready formulation of pharmaceutical composition containing VIII factor and can be used in treatment of hemophilia. Invention relates to a solid pharmaceutical composition prepared by lyophilization of an amino acid-free solution and comprising the following components: (a) VIII factor in the concentration 50-10000 IU/ml for human VIII factor or human recombinant VIII factor, or 50-10000 U/ml for swine VIII factor or swine recombinant VIII factor; (b) surfactant in the concentration above critical micellar concentration up to 1% (vol./vol.); (c) calcium chloride; (d) sucrose; (e) sodium chloride; (f) trisodium citrate, and (g) amino acids-free buffer in the concentration 1-50 mM with pH 6-8 before lyophilization and after dissolving in water for injection. Also, invention relates to a liquid pharmaceutical composition prepared after dissolving indicated stable solid pharmaceutical composition in sterile water optionally containing sodium chloride. Invention provides preparing a stable ready formulation of pharmaceutical composition of VIII factor wherein albumin is replaced with other stabilizing agents.

EFFECT: improved and valuable properties of pharmaceutical composition.

25 cl, 3 tbl, 3 ex

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

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to method of human blood coagulation VIII factor production and related product. Method includes blood serum as cryoprecipitate, heparine added, PEG-4000, centrifugation, supernatant is added with tributyl phosphate and Twin-80, repeated centrifugation, sediment washed with sodium chloride, then it is dissolved in tris-HCl buffer with additives, let through column filled with gel and attached antibodies to Willebrand's factor, factor VIII elution, dialysis. Produced concentrate does not contain Willebrand's factor and has activity not less than 300 ME/mg of protein with purity not less 98% and contains albumin of concentration of 0.1%. Product is lyophilized with further processing.

EFFECT: product does not display any toxicity and cause allergic reaction.

6 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention claims compositions which can include one or several mammary gland tumour proteins, their immunogenic parts or polynucleotides encoding such parts. Alternatively the therapeutic composition can include antigen-presenting cell expressing mammary gland tumour protein, or T-cell specific to cells expressing such protein. These compositions can be applied in prevention and treatment of such diseases as mammary gland cancer. Invention also claims diagnostic methods based on determination of mammary gland tumour protein or mRNA encoding such protein in sample.

EFFECT: use of peptides obtained from protein expressed from mammary gland by tumour in diagnostics and therapy of mammary gland cancer.

37 cl, 6 ex, 1 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.

EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.

41 cl, 3 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: declared invention refers to chemical-pharmaceutical industry. A method involves the following stages: preparing a solution of von Willebrand factor or von Willebrand factor/factor VIII complex which contains VWF in concentration up to 12 IU VWF:RCoAui and has the von Willebrand factor/factor VIII ratio equal to 0.4 or more; nanofiltering through a filter of pore size less than 35 nanometres at pressure less than or equal to 0.5 bar and in the presence of 0.05 to 0.2 M of calcium ions.

EFFECT: development of the effective method for producing concentrated von Willebrand factor or factor VIII/von Willebrand factor complex to be applied for treating hemophilia And or von Willebrand disease.

11 cl, 8 ex, 6 tbl, 1 dwg

Up!