Stable, free from albumin liofilizovannye composition of recombinant factor viii


(57) Abstract:

The invention relates to medicine and sustainable, not containing albumin preparation of recombinant Factor VIII (rFVIII) in dried form, which has both crystalline and amorphous components, which after dilution water contains from about 65 to 400 mm glycine, 50 mm histidine, from 15 to 60 mm sucrose, 1 to 50 mm sodium chloride, 5 mm calcium chloride and from 50 to 1500 IU/ml of rFVIII. The most preferred formulation contains after dilution with water to about 290 mm glycine, 20 mm histidine, 30 mm sucrose, 30 mm sodium chloride, 2.5 mm calcium chloride and from 50 to 1500 IU/ml of rFVIII. The residual water content in the lyophilized drug is 1 to 3 wt.%, preferably about 1 wt.%. The invention improves the shelf life. 3 C.p. f-crystals, 1 Il., table 1.

The invention relates to pharmaceutical preparations in General and, in particular, to the obtained freeze-dried stabilized recombinant Factor VIII (rF VIII) that does not contain albumin.

Factor VIII is a well-known plasma protein that plays an essential role in the blood clotting process. Although Factor VIII can come combinatii DNA (rF VIII) to avoid potential contamination, related substances in the blood. In addition, the sources of recombinant Factor VIII provide an almost unlimited number of coagulation factor that avoids the limitations in supply associated with the use of plasma donor blood as its source.

As one of the benefits derived recombinant Factor VIII (rF VIII) is that it is not from human blood plasma and, therefore, free from potential impurities from it, to produce rF VIII was to obtain a stable composition rF VIII, which can be described as absolutely free from any contamination by the products of human activity. However, unfortunately, the Factor rF VIII is a protein and, like many other proteins used in therapy, may become unstable during storage. To overcome this instability, the usual practice in the product as a stabilizer add serum albumin human. It was found that albumin is a good stabilizer rF VIII, and it is used in many commercially available industrial products. However, the use of human albumin as a stabilizer rF VIII lost pervonachalnika substances).

Recently described several preparations of Factor VIII in environments with low and medium ionic strength, with the use of sodium chloride, calcium chloride and histidine as ion buffer. In addition, applied basic amino acids such as lysine, and sugars such as mannitol, sucrose or maltose. In order to improve the stability does not contain albumin preparation of Factor VIII, while maintaining the required isotonicity of the solution required for therapeutic purposes, not containing albumin preparations used about 10% sugar (see, for example, U.S. patent 4877608 (low ionic strength). European patent 0314095 describes the other not containing albumin composition with high ionic strength and histidine as BufferedReader agent.

The above U.S. patent 4877608 considers a liquid solution, and not liofilizirovanny product, which must withstand lyophilization cycles required to obtain the product. Used first liquid composition with a relatively high content of sodium chloride is described in European patent 314095.

Other patents relating to preparations of Factor VIII, include U.S. patent 5399670, which describes the use of arginine in Sarat, does not contain sucrose or glycine, and the U.S. patents 4440679 and 4623717, which include the application of at least 30 wt.% sugars in combination with amino acids for stabilization of F VIII in the liquid state under conditions of pasteurization (60oWith at least 10 hours).

In addition to the above preparations of Factor VIII there are several other patents relating to treatment and/or stabilization of Factor VIII. These include U.S. patent 5288853, which describes a multistage production process, consisting in the use column to bind heparin followed by the addition of glycine to form as a product of purified Factor VIII.

U.S. patent 5399670 describes the process of obtaining a lyophilized Factor VIII with increased stability, which before lyophilization requires the addition of arginine to the solution of Factor VIII.

U.S. patent 5259951 describes a multi-stage method of purification of Factor VIII from plasma using columns for ion exchange chromatography.

U.S. patent 4758657 describes a multistage process for separating Factor VIII: C from the plasma, wherein at least one of the steps necessary adsorption of Factor VIII:C in hydrophobic media.

In the journal Pharmaceutical Research, volume 12, 6 p. 31-837, 1995 describes a drug antagonist rezeptfre on past and present efforts to obtain sustained drug rF VIII, which could be successfully liofilizirovanny, and later again quickly be diluted with water, has until now been difficult to obtain the drug, which not only contains products of human activity, such as albumin, but also meets the correct lyophilization and rapid dilution, and isotonicity, and at the same time provides the Factor rF VIII, stable over a long period of time with a pharmaceutically acceptable shelf life.

It has been unexpectedly discovered that such a drug may. In the process of finding this drug have found that the histidine, which, as reported, was used in the previous technique as BufferedReader agent actually had a destabilizing effect on lyophilized, containing no albumin preparations. It was found, however, that the destabilizing effect of histidine can be successfully overcome by using the new formulation, containing salts, glycine and sucrose, a combination which was found to have a beneficial effect on the stabilization Factor rF VIII. This mixture protects Factor rF VIII during repeated cycles of freezing-thawing in the process of freeze-drying and provides quick the coefficients, in contrast to most known in the art formulations which are substantially amorphous. The preparation according to this invention remains stable in the liquid state, at least within 24 hours at room temperature. In detail, the preparation according to the invention and its application are described below.

The drug Factor rFVIII is a pharmaceutically acceptable, not containing albumin lyophilized product, which can be easily (within 30 seconds) diluted with water and is applicable for the treatment of hemophilia. Dried product is a new mixture of salts, amino acids and sugar. It is stable at room temperature and in contrast to drugs at a known level (drugs used in the technique) has a relatively low sugar content.

The product after dilution water contains the following ingredients:

glycine is from about 65 to 400 mm, preferably 290 mm;

histidine is up to about 50 mm, preferably from 1 to 50 mm, most preferably 20 mm;

sucrose is from about 15 to 60 mm, preferably 30 mm;

sodium chloride is approximately 50 mm, preferably from 1 to 50 mm, most preferably 30 mm;

calcium chloride is the use of the LASS="ptx2">

In the preferred lyophilized drug, the amount of residual water must be from 1 to 3 wt.%, preferably about 1 wt.%.

In the drawing in the form of a chart presents a comparison of the activity in time when 40oWith the preparation of this application with a relatively low sugar content (upper curve) with the drug used in the technique, with a relatively high sugar content (lower curve).

The purpose of this invention was to develop not containing albumin preparation, which ensures the stability of recombinant Factor rF VIII (minimum or less than about 20% loss of activity) during different stages of the process such as ultrafiltration/diafiltration, storage of the frozen mass, the processes of freezing and thawing and lyophilization. In addition, it was desirable to quickly dissolving the product was stable in dilute liquid state. Finally, it is also desirable to have acceptable lyophilized product with a suitable shelf life, which could be liofilizirovanny within a short cycle lyophilization (freeze-drying).

Proteins do not crystallize during lyophilization. The purpose of the drying process was to translate raceworthy, caused by crystalline state (or complete loss of water). So, usually do significant amounts of albumin (1%) to create an amorphous phase (component) to stabilize proteins.

On the basis of General considerations was obtained composition as the crystalline component to ensure rapid lyophilization, and an amorphous component for stabilization of recombinant Factor rF VIII. As used below, the terms "crystalline to amorphous component" means that the formulation contains two or more different phases, and at least one of them is crystalline and one amorphous. Solids can exist in crystalline or amorphous state. Crystalline materials are characterized by the fact that they have a definite structure, stoichiometric composition and melting temperature. On the contrary, amorphous substances do not have a pronounced molecular structure and can be characterized as the supercooled liquid of extremely high viscosity, such as viscoelastic "rubber" or more solid brittle glass. I think that other sugars, such as maltose, trehalose and maltotriose can also be included in the amorphous components is jaś for the development of a pharmaceutically acceptable, not containing albumin preparation of recombinant Factor rF VIII, consisted of the following:

a) the Source material was a thoroughly cleaned (high-purity) rF VIII, which is cleaned using processes orthogonal chromatography. They can be defined as chromatographic processes that are carried out in certain ways, in accordance with certain rules and are typically used consistently. In the protein can be quickly cleaned by applying different (Nai)more effective methods of cleaning. Finally, Factor VIII, at least 90% purity (by electrophoresis in gel) with specific activity of more than 2000 IU/mg protein. Theoretical purity rF VIII is a controversial issue, but I suppose that it is equal to approximately 3500-5000 IU/mg protein.

(b) protein Recipe was developed using the ultrafiltration/diafiltration (UF/DF), and explored the regeneration during the UF/DF, sensitivity to freeze-thaw and stability in the liquid state at different temperatures temperature control.

c) Potential drugs were additionally characterized by their relationship to heat by using DSC (differential scanning ka is critical melting temperature (Te'). With this information determined the formulation of the drug, which can be quickly liofilizirovanny and which was used in further studies.

d) Drugs with the best recipe liofilizirovanny using rapid cycle lyophilization and analyzed the stability at standard and elevated temperature storage.

e) Sustainable drugs just selected among the samples stored at 40oWith through various time intervals.

When analyzing the results of numerous studies that have led to the formulation of the preparation according to this invention, applied multidimensional strategy (with many variables) experimental design and program to search for a group of ingredients, which was a mixture of amino acids, salts and sugars. The results were analyzed utilizing a sophisticated program to consider all interactions between the ingredients, and perform the analysis of multivariate functions (surfaces) of the response in the data space. It has been unexpectedly found that histidine (usually used in the technique) actually destabilizes the rF drugs VIII. This has led to the need to critically examine the criteria for the various ingredients in n the LASS="ptx2">

Example 1

The influence on the stability of lyophilized recombinant Factor rF VIII studied by titration of varying amounts of histidine in mixtures containing rF VIII, as well as containing 150 mm sodium chloride, 2.5 mm calcium chloride and 165 mm mannitol. The results are presented in the table (see the end of the description).

From the above data shows that the increase in the number of histidine leads to a decrease in activity diluted with water, dried rF VIII depending on the dose. This result confirms that Gastein does not play a role in the stabilization of F VIII in dried condition.

Example 2

Resistance rF VIII in formulations with high and low sugar content.

Were composed of two formulations of recombinant Factor VIII. Instability was studied under conditions of storage at elevated temperature (40oC).

The drug formulation with a high sugar content that is similar to the formulation of the prior art, was an amorphous composition containing, after dilution in water, 50 mm sodium chloride, 2.5 mm calcium chloride, 5 mm histidine and 10 wt.% maltose.

The drug formulation with a low content of sugar in dandouras (30 mm sucrose). This product, after dilution WFI, containing 30 mm sodium chloride, 2.5 mm calcium chloride, 20 mm histidine, 290 mm glycine and about 200 IU/ml rF VIII. Comparing this drug with drug is used in the technique (see drawing), shows that the drug is low in sugar according to this invention is significantly more stable in time than a drug with a high sugar content used in the technique.

It should be borne in mind that various modifications, known to specialists in this field, also belong to this invention. Therefore, the above examples are for illustration only, and the scope of the invention is defined only by the following claims.

1. The composition in the form of a lyophilized product containing recombinant coagulation factor VIII (without albumin), sodium chloride, calcium chloride and sucrose, characterized in that it further comprises glycine, while these components are taken in the following amounts (after dilution with water):

Recombinant coagulation factor VIII - About 50-1500 IU/ml

Glycine - About 65-400 mmol

Histidine - Approximately 50 mmol

Sodium chloride is About 1-50 mmol

Chlorine, however, the residual water content is about 1 to 3 weight. %.

3. The composition according to p. 1, characterized in that the specified components are taken in the following amounts (after dilution with water):

Recombinant coagulation factor VIII - About 50-1500 IU/ml

Glycine - About 290 mmol

Sodium chloride is About 30 mmol

Calcium chloride is About 2.5 mmol

Histidine - About 20 mmol

Sucrose - About 30 mmol

4. The composition according to p. 3, characterized in that the residual water content is about 1 weight. %.


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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

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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.

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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

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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.

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2 ex