Method of human blood coagulation viii factor concentrate production and related product

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

 

The invention relates to the field of preparative biochemistry, pharmacy and medicine and relates to a method of obtaining a concentrate of factor VIII clotting of blood and the product it contains.

Factor VIII plays an important role in the blood clotting system and is a major component of pharmaceuticals for the treatment of hemophilia. In plasma its content is small at ten-thousandths of a percent relative to the total protein. This determines the main problem in creating preparations containing this factor - the need for high efficiency of technological processes of their production, taking into account the main component of cost is the cost of human blood plasma. All preparations containing this factor, divided by the way they are received on genetic engineering and the drugs derived from human blood plasma. The last group represents the concentrates of different purity. Maximum clearance is achieved using monoclonal antibodies attached to a particular carrier, and the minimum is two or three non-chromatographic purification stages of cryoprecipitate. Drugs intermediate degree of purification obtained using a combination of those or other chromatographic methods. Such a variety of preparations containing factor VIII, due to the lack of the receiving consensus about the necessary and sufficient treatment from other components of blood plasma.

The fundamental difference technology is based on a different approach to the spectrum of proteins that are in the final dosage form of factor VIII. First of all it concerns the question of the necessity of cleaning from von Willebrand factor. In fact, on the one hand, von Willebrand factor, while in complex with factor VIII, stabilizes the last, with another subunit of von Willebrand factor form a huge complexes among themselves. Therefore, even if be cleansed from all other proteins in blood plasma, the specific activity of factor VIII in this drug will be far from optimal (when von Willebrand factor in the product is not at all or molar ratio of factor VIII: von Willebrand factor 1:1) and is determined by the ratio of factors. It is known that such an attitude in vitro can reach 1:100. And, considering the high molecular weight von Willebrand factor in relation to factor VIII is a clear need for the destruction of such a supramolecular complex with von Willebrand factor. In this case, regardless of the use of other techniques you can expect a higher yield and high specific activity of factor VIII in the final preparation both high and low purity.

The question of how and at what stage of the determination of factor VIII needed to destroy the Assembly of von Willebrand factor, closely svazas problem of maintaining the stability of factor VIII in the process of isolation and purification. After removal of the factors that lower the stability of factor VIII, such as prothrombin and thrombin, the most inactivating effect on it may have procedures for viral inactivation. The fact that it's pretty hard procedures and their implementation in one way or another requires stabilizing factors. Partly the complex of factor VIII and von Willebrand factor performs the same function. Therefore, the destruction of supramolecular aggregates of von Willebrand factor should be carried out either after the procedure, viral inactivation, or to artificially introduce additional stabilizers.

Known methods of obtaining a concentrate of factor VIII, in particular described in patent RU 2025129, EN 2148411, EN 2055593.

The closest is the method described in patent RU 2253475.

The essence of the present invention is that a method was developed to obtain preparations containing factor VIII, wherein using thiol-disulfide exchange was destroyed supramolecular complexes of von Willebrand factor, the activity of factor VIII were not addressed. The proposed method allowed us to develop a method to obtain preparations containing factor VIII, which are more efficient and specific content in comparison with similar methods, but does not include this stage.

Another aspect of the claimed invention I have is the product for use in medicine, veterinary medicine and/or research purposes, containing a concentrate of active factor VIII clotting person with a specific activity of not less than 300 IU/mg and 0.1% albumin, and purity more than 98%, obtained as described in the description and claims.

The product may be a solution or a lyophilized powder (for which the above concentrate is transferred into vials and dried by freezing under vacuum). If necessary, it may further contain conventional pharmaceutical excipients, solvents and/or excipients that are acceptable for parenteral (e.g. intravenous bolus or infusion, intramuscular, intranasal) administration. Listed excipients are mixed with a dry concentrate or added to the solution according to conventional methods. Fillers are, for example, sugars (glucose, lactose, sucrose, maltose, or mixtures thereof), sugar recovery (aritra, mannitol and other) in a physiologically acceptable concentrations, if necessary, antioxidants, traditional injectable pharmaceutical compositions. Optionally, the product may contain inorganic and/or organic salts and their combinations to maintain an acceptable pH values.

The solvent for the lyophilized product can service shall be distilled pyrogen-free water, saline, buffered saline solution.

You can also liofilizirovanny the finished product containing a concentrate of factor VIII and any of the following, auxiliary substances, in particular albumin, or a combination thereof.

The product can have an appropriate package and, optionally, instructions for use.

The implementation of the method is illustrated by the following examples.

Example 1.

1 kg of cryoprecipitate blood plasma was dissolved for 1 hour in water containing heparin (3 units/ml) at a temperature of 25°C. was Added aluminum hydroxide, brought the pH to 7.0 and after 15 minutes of incubation was centrifuged. To the obtained supernatant was added heparin to a final concentration of 3 units/ml, was then added 2-mercaptoethanol to a final concentration of 2 mm and incubated for one hour, then add the polyethylene glycol - 4000 to a final concentration of 2%. After adjusting the pH to 6.6 incubated for 15 minutes and centrifuged. The precipitate was discarded and the supernatant were dialyzed against water containing heparin (3 units/ml)was added glycine to a final concentration of 13%, and incubated for one hour. Then centrifuged, the precipitate was discarded. To the supernatant was added tributyl phosphate to a final concentration of 0.3% tween 80 to a final concentration of 1%, brought the pH to 7.2, and incubated at on the th temperature for 6 hours. After this was added sodium chloride to a final concentration of 14%, incubated for one hour and centrifuged. The precipitate was washed with sodium chloride solution and were dialyzed against a solution containing 200 mm sodium chloride, 2.5 mm calcium chloride, 20 mm sodium citrate, 10 mm lysine and 100 mm glycine. The specific activity was about 60 IU/mg of total protein. Added albumin to a concentration of 0.1%, sterile filtered and subjected to freeze-drying in combination with a subsequent heat treatment. The output process of the activity of factor VIII was about 60%.

Example 2. A preferred variant of the method.

Precipitate factor VIII concentrate prepared as in example 1. After washing the precipitate with a solution of sodium chloride was dissolved in 10 mm Tris HCl buffer containing 100 mm sodium chloride, 2.5 mm calcium chloride, 10 mm lysine and 5 units/ml heparin. The solution was passed through the affinity column containing 1 l of gel with sewn antibody to von Willebrand factor (anti-vWF gel, Immunotech, France). The elution of factor VIII was performed after washing 8 column volumes of equilibrating buffer. The composition of the eluting buffer consisted of 10 mm Tris HCl, 250 mm sodium chloride, 100 mm glycine, 2.5 mm calcium chloride, 250 mm ammonium sulfate, 2,55% glycerol. The resulting solution were dialyzed against a solution containing 200 mm sodium chloride, 2.5 mm calcium chloride, 20 mm C the waste sodium, 10 mm lysine and 100 mm glycine. The specific activity was about 300 IU/mg) was Added albumin to a concentration of 0.1%, sterile filtered and subjected to freeze-drying in combination with a subsequent heat treatment. The output process of the activity of factor VIII was about 70%.

Product purity not less than 98%.

The resulting product is pyrogen-free, does not show toxicity in experiments on laboratory animals (rats, rabbits), does not cause allergic reactions and can be used in medicine, animal health, research purposes.

1. A method of obtaining a concentrate of factor VIII clotting of the blood, characterized by the fact that cryoprecipitate plasma is dissolved in water containing 3 units/ml heparin at a temperature of 25°add the aluminum hydroxide to bring the pH to 7.0, incubated, centrifuged, the resulting supernatant add heparin, then 2-mercaptoethanol to a final concentration of 3 units/ml and 2 mm, respectively, incubated for one hour, add the polyethylene glycol - 4000 to a final concentration of 2%, adjusted pH to 6.6, incubated for 15 min, centrifuged, supernatant cialiswhat against water containing 3 units heparin/ml, then add glycine to a final concentration of 13%, incubated for one hour, centrifuged, the supernatant add tributyl phosphate to the end the end of the ation 0.3% tween 80 to a final concentration of 1%, bring the pH to 7.2, incubated at room temperature for 6 h, then add sodium chloride to a final concentration of 14%, incubated for one hour, centrifuged, the precipitate washed with a solution of sodium chloride, dissolved in 10 mm Tris HCl buffer containing 100 mm sodium chloride, 2.5 mm calcium chloride, 10 mm lysine and 5 units/ml heparin solution passed through a column Packed with gel with sewn antibody to von Willebrand factor, elute buffer consisting of 10 mm Tris HCl, 250 mm sodium chloride, 100 mm glycine, 2.5 mm calcium chloride, 250 mm ammonium sulfate, 2,55% glycerol, and the resulting solution deleteroute against a solution containing 200 mm sodium chloride, 2.5 mm calcium chloride, 20 mm sodium citrate, 10 mm lysine and 100 mm glycine, to the obtained concentrate, having a specific activity of not less than 300 IU/mg protein, add albumin to a concentration of 0.1%, sterile filtered, lyophilizers and subjected to heat treatment.

2. The product represents a concentrate of factor VIII clotting of the blood, characterized by the fact that obtained by the method according to claim 1, has a specific activity of not less than 300 IU/mg protein with a purity not less than 98% and contains albumin to a concentration of 0.1%.

3. The product according to claim 2, characterized in that it is a solution or lyophilized powder.

4. The product according to claim 2, characterized in that before omnitele contains pharmaceutical excipients, carriers, solvents and/or excipients that are acceptable for parenteral administration.

5. Product according to any one of claim 2 through 4 for use in medicine, veterinary medicine and/or research purposes.

6. The product according to claim 5 for the treatment or prevention of conditions associated with a lack of factor VIII.



 

Same patents:

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: 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, hematology, pharmacy.

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EFFECT: valuable properties of compositions.

35 cl, 11 tbl, 7 ex

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EFFECT: considerable reduction of prospective immunological response of human body for foreign protein.

1 ex, 1 tbl

FIELD: medicine; chemistry-pharmaceutical industry.

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EFFECT: increased output and purity of end product.

2 ex

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EFFECT: higher efficiency of therapy.

1 ex

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EFFECT: improved, enhanced and valuable medicinal properties of pharmaceutical composition.

2 tbl, 5 ex

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3 ex

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2 ex

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5 cl, 9 ex

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EFFECT: higher efficiency of therapy.

1 ex

FIELD: medicine.

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FIELD: medicine, pharmacy.

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EFFECT: improved and valuable medicinal properties of composition.

12 cl, 9 dwg, 15 ex

FIELD: medicine, ophthalmology.

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EFFECT: higher efficiency of therapy.

2 cl, 7 dwg, 2 ex

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