Method for producing concentrated von willebrand factor or factor viii/von willebrand factor complex and applying thereof

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

 

The technical field to which the invention relates.

The present invention relates to therapeutic factor concentrate of von Willebrand's disease or a complex of factor VIII/factor a Background of Villebranda and the method of obtaining medical compounds intended for the treatment of disease Von Willebrand's disease (Von Willebrand''s Disease, VWD) and hemophilia a, in which the concentrate is subjected to nanofiltration through pores smaller than 35 nm, which effectively delays the viruses with shell or without shell, such as, for example, hepatitis a virus or erythrovirus B19.

Prior art

The factor a Background of Villebranda (VWF) is a protein of blood plasma, having a multimeric (oligomeric structure, with molecular weight different isoforms varies from approximately 230000 Da (daltons) for each Monomeric subunit to more than 20 million Yes to multimeric forms with high molecular weights, forming, thus, the largest known soluble protein. Its concentration in plasma is about 5-10 µg/ml [Siedlecki et al., Blood, vol 88, No. 8, 1996: 2939-2950] and is present in plasma form small size corresponds to a dimer with an approximate size of 500000 Yes.

VWF plays an important role in primary hemostasis, responsible for the adhesion of platelets to the damaged surface of blood vessels and, thus, for the Fort is the key platelets from platelet which begins the process of forming fibrin coagulate. It is assumed that a multimeric forms with high molecular weights provide a process for the adhesion of platelets in subendothelial with greater efficiency, and that the clinical efficacy of VWF concentrates associated with the concentration of multimers with high molecular weight [Metzner et al., Haemophilia (1998) 4, 25-32].

In addition, VWF in plasma is involved in the transport and stabilization of factor VIII (FVIII), as discovered that the FVIII molecule in the native state is associated with a multimeric forms of VWF. The complex of factor VIII/factor Von Willebrand's disease (FVIII/VWF) reaches a length of up to 1150 nm [K. Furuya et al., Vox Sanguinis (2006) 91, 119-125]. In addition, VWF in the form of its small molecular forms will have a size of approximately 149×77×3,8 nm and can change its structure depending on the flow velocity (angular power shift), turning into an elongated or linear form [Siedlecki et al., Blood (1996) 88, 2939-2950]. The concentration of FVIII in plasma is approximately 0.05 to 0.1 µg/ml (i.e. about 50-100 times less than the concentration of VWF).

Quantitative or qualitative defects of VWF leads to disorders in primary hemostasis, known as the disease Von Willebrand's disease, which manifests itself in the form of bleeding.

Concentrates purified VWF and FVIII concentrates with a high content of functional VWF found therapeutic application in the treatment of disease Von Willebrand's disease.

Another aspect that must be taken into account is the fact that because VWF is a natural stabilizer for FVIII, FVIII concentrates with a high content of VWF can have many advantages when used for the treatment of hemophilia A, which was noted by many authors, for example: longer life time in vivo for input FVIII, protective effect against inhibitory antibodies to FVIII [Gensana M. et al., Haemophilia, (2001) v.7, 369-374] [Bjorkman S. et al, Clin Pharmacokinet, (2001) v.40, 815-832] [Behrmann K. et al., Thromb Haemost, (2002) v.88, 221-229] and possibly a lower rate of antibody formation, inhibiting the activity of FVIII [Goudemand J. et al., Blood (2006) 107: 46-51].

Were developed analytical methods for the characterization of both content and activity of VWF in its concentrates. Determination of VWF activity as a cofactor of Ristocetin (VWF:RCo) is a widely used method for determining the activity of VWF [Heath et al., Thromb Haemost 1992; 68:155-159]. Measurement of VWF as antigen (VWF:Ag) [Cejka J. Clin Chem. 1982; 28:1356-1358] shows us the number of both active and inactive VWF in the sample.

One of the important parameters to determine the functional quality of VWF concentrates is the ratio between the activity of VWF:RCo and VWF antigen:Ag.

Considering the possible values of the multimeric structure of VWF and multimers with high molecular weight for their clinical activity and performance and, characterization of multimeric structure is essential to determine the suitability of concentrates VWF and FVIII concentrates with a high content of VWF. Such multimeric structure is determined with the aid of gelelectrophoresis [Ruggeri et al., Blood 1981; 57: 1140-1143].

Were described different methods of purification of VWF or a complex of FVIII/VWF in which VWF is functional and is in sufficient concentration for use as a therapeutic product for the treatment of VWD, as shown in patents EP 0411810 and EP 0639203 and publishing Ristol P. et al., Sangre (1996) 41:125-130.

Other methods of purification of FVIII give the final product, not containing VWF or containing only trace amounts. These concentrates are not suitable for the treatment of VWD. Although in some cases the residual VWF is present in such FVIII concentrates, it is non-functional, because it loses some of multimers that form, and especially multimers with high molecular weight. These concentrates will not possess the advantages of FVIII concentrates that are rich in VWF in the application for the treatment of hemophilia A.

These concentrates are described (tables 2 and 3) in the List concentrates of clotting factors, created in 1997 and updated in 2006, the world Federation of hemophilia (World Haemophilia Federation, WHF) (Kasper, C.K.; Brooker, M. Registry of clotting factor concentrates, January 2006), where among other things listed met the water fractionation and inactivation of viruses, the concentration of VWF and their functional efficiency.

Among FVIII concentrates, which was subjected to nanofiltration, we must distinguish between those who have been through nanofiltration pore size of 35 nm (or larger), i.e. those for which the nanofiltration not effective against viruses that do not have a shell, such as hepatitis a virus (approximately 24 nm) or virus B19 (between 18 and 24 nm). On the other hand, concentrates of FVIII, which were nanofiltration through pores smaller than 35 nm, do not contain VWF, and if they contain, are deprived of multimers with high molecular weight, with the result that they are not effective in the treatment of VWD and not have the benefits of FVIII concentrates rich in VWF, when their use for the treatment of hemophilia A.

The possibility of removal of pathogenic agents is extremely important to ensure the safety of biological products and, consequently, in the production process this purpose include various methods. Among them, it should be noted chemical inactivation, based on the action of organic solvents in combination with a detergent, which has been shown to be highly effective against viruses with a lipid envelope, although they are not effective against viruses, deprived of lipid membranes. Other methods of physical treatment such as heat treatment, are effective is passed, regardless of whether the virus lipid membrane, but their effectiveness depends on the rigidity of processing, which, in turn, depends on the stability of the protein, which undergoes this process of inactivation. Other ways that help to reduce viral contamination include separation using precipitation or chromatography.

One of the methods which have been proven highly effective for removing viruses, regardless of the presence or absence of lipid membranes is filtration through filters with pore size, which provide a delay of viral particles (nanofiltration). It was also shown that this method is effective for removal and other infectious particles such as prions. However, the effectiveness of this method is determined by the pore size, which, in turn, depends on the size of the protein, which should be subjected to nanofiltration.

There are nanofilter with different pore size, typically from 15 to 75 nanometers (nm) and, as a rule, the smaller the pore size, the higher the efficiency of the filters in the retention of pathogens: nanofilter with pore size less than 35 nm, preferably from 15 to 20 nm, are filters that trap the smallest viruses ranging in size from 18 to 23 nm, such as erythrovirus B19 and hepatitis a virus (a size of about 24 nm). Thanks to its characteristics these nanofilter are the Xia physically fit only for proteins smaller which, thus, can be filtered to yield acceptable for industrial production (usually the protein yield after nanofiltration should be 60% or more).

Probably because the molecular structure of VWF or a complex of FVIII/VWF in principle not able to pass through the pores of nanofilters with a diameter of less than 35 nm, especially multimeric forms of VWF with high molecular weight. To date, the nanofiltration VWF or a complex of FVIII/VWF, comprising a multimeric form high molecular weight, through nanofilter with a pore diameter of less than 35 nm was impossible.

According to the list concentrates of coagulation factors of the world Federation of hemophilia (WHF), which was mentioned above, there is one concentrate past nanofiltration FVIII (in this case used a pore size of 20 nm), which is a Cross Eight M red cross Japan, which is also described in the publication K.Furaya et al, Vox Sanguinis (2006) 91, 119-125. Despite the fact that the article says that if you filter through the pores of 20 nm in concentrate FVIII VWF is stored and what its multimeric structure is not disturbed, we can see from the list WHF that this concentrate contains a non-functional VWF. Returning to publishing Furuya et al., we see that the content of VWF is at trace level [p.123:... the number of VWF were close to those that Oba is found (0,007-0,015 U VWF/U FVIII:C),...], while this ratio in the plasma is 1 unit activity of VWF for every unit of FVIII activity (ratio 1:1). On the other hand, the characteristic of the multimeric structure of the residual VWF (Figure 4, p.123) demonstrates no more than 10 multimeric bands, whereas it is known that multimer with well-preserved multimeric structure, which have high molecular weight, must contain at least 11 bands (Metzner et al., Haemophilia (1998) 4; page 27, 2nd paragraph). What Furaya reports in its publication, is as follows: FVIII concentrate, purified by affinity chromatography using monoclonal antibodies contains only trace amounts of VWF, which is represented only by multimarine with low molecular weights, and such a composition of VWF is not changed for the worse when nanofiltration through a pore size of 20 nm. Obviously, this concentrate (Cross Eight M) is not suitable for the treatment of VWD and does not have the benefits of the presence of VWF in the proportions (1:1)detected in normal blood plasma of a person.

In the List of concentrates of coagulation factors of the world Federation of hemophilia mentioned above mentioned and other FVIII concentrate, past nanofiltration, which is a LFB's FACTANE. According to the above list, this concentrate was nanofiltration through a pore size of 15 nm and it contains VWF. Dan is the first product corresponds to the product obtained according to patent WO 2005040214, which describes the composition of FVIII held nanofiltration through a filter with a pore size of from 13 to 25 nm, in which the efficiency of trapping the virus combined with the presence of VWF with high molecular weight (more than 10 multimers) in an amount of less than 15%. This again confirms what is evident from the publication Furaya, namely, that multimeric VWF with a low molecular weight can pass through nanofilter with pore size of 20 nm, when they are present in low concentrations compared to FVIII, that is, they ratio of VWF/FVIII far from 1 (0,015 in the case of article Furaya and 0.15 in the case Factane, patent WO 2005040214). Multimer with high molecular weight, on the contrary, delayed on nanofilter. Moreover, in this patent to save FVIII associated with VWF, which also had to be saved, provided the enhanced dissociation of the complex of FVIII/VWF by adding CaCl2at concentrations higher than 0.20 M, which is the minimum concentration at which dissociates the complex of FVIII/VWF. Despite this, in all the examples, in order to ensure dissociation of the complex of FVIII/VWF, and, consequently, to increase the output of FVIII was used, the concentration of CaCl2at least 0,35 M Described in this patent, the composition is intended solely for the treatment of deficiency of FVII, and not for the treatment of VWD. Indeed, this product is not suitable for the treatment of VWD, as specified in the product license issued by the French Agency for the Safety of Health Products” [http://afssaps-prd.afssaps.fr/php/ecodex/frames.php?specid=66716833&typedoc=R&ref=R0093176.htm], section 4.1 Therapeutic indications, where it is clearly indicated that the product does not contain VWF in quantities sufficient for the treatment of VWD. In addition, in dealing with this product publications [Vox Sanguinis (2007) 92, 327-337] the authors confirm that the product (Factane) is not intended for the treatment of VWD (str).

Summarizing all the above, we can conclude that the methods described K.Furuya et al, [Vox Sanguinis (2006) 91, 119-125]patent WO 2005040214 and publishing Vox Sanguinis (2007) 92, 327-337, designed for FVIII concentrate, which has a low content of VWF (<15%, or the ratio of VWF/FVIII<0.15 to:1)applicable for treatment of hemophilia a, but which does not possess the benefits that will be concentrate, enriched VWF, in the treatment of hemophilia and who is not under any circumstances not suitable for the treatment of VWD.

It was found (Siedlecki et al., Blood, vol 88, No. 8, 1996: 2939-2950)that VWF changes its three-dimensional structure in terms of current, providing increased strength of the angular shift (stress angular shift), which ensures the mechanism of adhesion of platelets in vivo. This change in structure leads to the fact that the molecule changes its globular structure towards more than the linear form. In this regard, K.Furuya et al., in his publication (Vox Sanguinis (2006) 91, 119-125) have already pointed out that filtering FVIII concentrate through a pore size of 20 nm should be carried out at high pressure (0.8 bar) in order to provide an acceptable output FVIII, and they suggest that at lower pressures the influence of the speed of the current is not enough to present VWF changed its structure, which makes it difficult to filter. Under these conditions (0.8 bar) they also made good out of the present multimers with a low molecular weight, if you keep in mind that the original content of these multimers to nanofiltration was very low.

The above documents do not disclose the possible use of nanofiltration through pores smaller than 35 nm, such as, for example, 20 nm, for VWF or a complex of VWF/FVIII ratio (>0,4) with the functional content of multimers with high molecular weight, which makes possible their use for the treatment of VWD, or which retains the advantages described for concentrates rich in VWF, in their application for the treatment of hemophilia A. moreover, patent WO 2005040214 clearly says any qualified specialist in the art that such a pore size prevents the passage of VWF with high the molecular weight.

The prior art in which there is a nanofiltration VWF for p is obtaining a therapeutic product, currently limited by nanofiltration through a pore size of 35 nm, described in the patent EP 1632501. This patent discloses a method of obtaining a VWF concentrate with low FVIII content (ratio FVIII.C/VWF:RCo less than 0,06), which includes a step of virus removal by nanofiltration through a filter with a pore size of 35 nm, as indicated in paragraph 23 of the embodiment of the invention in Example 1, paragraph 37. From this patent, we can conclude that the nanofiltration molecules with the size of VWF is impossible for nanofilter having a pore size of less than 35 nm.

As we have seen, at present, no therapeutic concentrate, intended for the treatment of disease von Willebrand, which would pass nanofiltration through a pore size of 20 nm. In addition, the prior art shows that nanofiltration VWF containing multimer with high molecular weight, through a pore size of 20 nm is impossible, as in the case of the molecular complex of FVIII/VWF, if there are multimeric VWF with high molecular weight and the ratio between VWF and FVIII higher than 0,15.

Thus, production of concentrate FVIII/VWF by nanofiltration through a pore size of 20 nm with a ratio between the two components of the macromolecular complex of FVIII and VWF, which is more like a complex, normally present in the plasma of human blood (1 unit VWF each edinica FVIII), and that still has all the advantages of native complex of FVIII/VWF and is suitable for the treatment of VWD and hemophilia a, still represents an unsolved problem.

The method of obtaining VWF or a complex of FVIII/VWF from human blood plasma usually starts with obtaining the fraction cryoprecipitate, which is purified by selective precipitation or, more recently, by using chromatographic techniques, especially using ion-exchange and/or affinity chromatography.

Methods purification of FVIII, which is currently based on immunoaffinity chromatography (using monoclonal antibodies), provide FVIII with very high specific activity, but devoid of functional VWF [K.Furaya et al., Vox Sanguinis (2006) 91, 119-125].

As we have seen, these methods of obtaining VWF or a complex of FVIII/VWF include one or more special stages inactivation or removal of viruses.

In the present invention the activity of VWF is based on the role of VWF as a cofactor for antibiotic Ristocetin (Ristocetin, VWF:RCo) in its ability to induce platelet aggregation (Pharmacopoiea Europea 07/2006:20721). This activity is expressed in international units (International Units, IU VWF:RCo), and its concentration is expressed in IU/ml (IU VWF:RCo/ml).

Under the FVIII activity refers to the coagulating FVIII activity (FVIII:C), which is based on the role of FVII as a cofactor in the activation of FX in the presence of FIXa, calcium ions and phospholipids (Pharmacopoeia Europea 07/2006:20704). This activity is quantitatively determined with a chromogenic substrate and is expressed in international units (International Units, IU FVIII), and its concentration is expressed in IU FVIII/ml (IU FVIII/ml).

The implementation of the invention

The present invention discloses a therapeutic factor concentrate of von Willebrand's disease or a complex of factor VIII/factor Von Willebrand's disease, which has passed through the nanofiltration through a sieve with pore size less than the equivalent of 35 nm, in which the resulting product has a ratio of VWF:RCo/FVIII:C, which is greater than or equal to 0.4, which saved the structure of multimeric VWF, including multimer with high molecular weight (greater than 11 bands), and which is suitable for medical compounds for treatment of disease Von Willebrand's disease and hemophilia a, and its production method.

Based on the results of research on nanofiltration VWF or a complex of FVIII/VWF in which VWF stores multimeric structure and contains multimer with high molecular weight, the authors present invention unexpectedly showed that in the case of a solution containing VWF or a complex of FVIII/VWF and calcium ions, can be nanofiltration through nanofilter with a nominal pore size of less than 35 nm, and preferably 20 nm at a maximum pressure less than 0.5 bar and preferably is t 0.2 to 0.4 bar.

The solution, which should be subjected to nanofiltration, has a maximum concentration of 0.6 units of absorbance (AU280), which is equivalent to not more than 12 IU VWF:RCo/ml Protein solution composition in addition to the presence in it of itself VWF and FVIII may include other proteins, such as fibrinogen or fibronectin; the specific activity of the VWF (IU VWF:RCo/mg protein) is equal to or greater than 1, and usually 10 or more. The specific activity of FVIII (IU FVIII/mg protein) in the case of nanofiltration complex FVIII/VWF is also equal to or greater than 1, and usually 10 or more.

The solution, which should be subjected to nanofiltration, may contain calcium chloride) in a concentration of from 0.05 to 0.20 M and at least one basic (alkaline) the amino acid as a stabilizer of protein, preferably a histidine at a concentration of from 20 to 30 mm. The pH value of the solution, which should be subjected to nanofiltration, should be above 5.5 to prevent denaturation.

The ratio of the load (filter), expressed in the biological activity of the protein, which should be subjected to nanofiltration, can reach 50 IU VWF:RCo/cm2area of filter surface, which is equivalent to 0.5 mg of VWF/cm2.

In these conditions, you can filter up to 120 litres of solution per 1 m2area of filter surface with the release of FVIII activity by more than 70%, with the Odom VWF activity more than 60%, with preservation of the multimeric structure of VWF (more than 11 multimers) and with the ratio of VWF:RCo/FVIII, which is at least 80% of this relationship in the source material.

The solution, which should be subjected to nanofiltration, differs in that it relates the activities of VWF:RCo/FVIII is equal to or greater than 0.4, and usually from 1 to 3, and this procedure is equally applicable for solutions without VWF FVIII and solutions for complex FVIII/VWF, because VWF as a multimeric molecule is large in size, is a molecule, which imposes restrictions on the entire process.

Under these conditions, standard filtration rate is a maximum of 30 liters/hour/m2usually from 10 to 20 liters/hour/m2the time filter is 12 hours or less. This makes it possible industrial application, and it is also clear that these parameters can be changed or optimized by adjusting the surface area for nanofiltration, which is a limiting factor for the whole process because of the high rates (nanofilter).

In the preferred embodiment of the pre-nanofiltration using nanofilter with pore size from 35 to 100 nm before nanofiltration is carried out through the pores of size less than 35 nm. The relationship between the square of the first (pre the filter) and the second (< 35 nm) nanofilter is from 1:2 to 1:4.

You can also perform double nanofiltration through a pore size of 20 nm (20 nm + 20 nm), which increases the benefits due to increased product safety nanofiltration.

Thanks to the present invention obtained past nanofiltration VWF or a complex of FVIII/VWF, which makes possible the creation of high-purity medical drug suitable for the treatment of VWD and hemophilia a with VWF activity greater than 100 IU VWF:RCo/ml and having the relationship between the activities of VWF and FVIII 0.4 or more, and in which the multimeric structure of VWF includes multimer with high molecular weight (more than 11 pages).

Information confirming the possibility of carrying out the invention

From commercially available nanofilters in the Examples of the present invention is applied nanofilter brand Planova® company Asahi Kasei Corporation, Japan, which are made from regenerated cellulose and have a pore size of about 35±2 nm in the case of Planova 35N and 19±1 nm in the case of Planova 20N. Filters of this type can be used for different filtering options, as for the "dead-end" filtration, when the entire solution is forced through the filter (dead end filtration mode)and running the filter, when the solution is pumped over the filter (tangential filtration mode). In the Examples below, when using a Planova filters were used options, the ant dead-end nanofiltration, however, for the purposes of the present invention is also suitable flow-variant filtering, and you can use other brands of nanofilters and compositions that are well-known qualified specialists in this field of technology. In the manufacturer's instructions detail how to assemble the device for filtering to nanofiltration and conduct testing to ensure that relevant to the present invention nanofilter are intact.

Example 1. Preparation of the source material

Before nanofiltration of the solution of a complex of FVIII/VWF from human blood plasma can be obtained, for example, from solubilizing cryoprecipitate by precipitation with polyethylene glycol and subsequent purification by affinity chromatography, as described in patent EP 0411810. Last nanofiltration of the solution can be further purified to obtain a product with high purity, for example, by precipitation of glycine, as described in patent EP 0639203. An alternative to this, FVIII or VWF or both of these factors can be obtained by biosynthesis using recombinant DNA technology in transgenic cells or animals [W.I. Wood et al., Nature (1984) 312: 330-337]; ['toole, JJ. et al., Nature (1984) 312: 342-347].

Example 2. Nanofiltration complex FVIII/VWF

Was conducted consistent filtrazione filters Planova 35N area of 0.12 m 2and Planova 20N area of 0.3 m214.7 liters of a solution of partially purified complex of FVIII/VWF with a specific activity of 10.4 IU FVIII/AUNmand the concentration of VWF: RCo 5,69 IU/ml, which is equivalent to 0,235 AUNmin the presence of 25 mm histidine and 0.14 M calcium at pH 6,77 and at a temperature of 20±5°C. Filtration was carried out at the constant speed of the current is approximately 14 l/h/m2maintaining the pressure difference between 0.20 to 0.30 bar in the case of filter Planova 20N during the filtration of the entire solution, which was completed for 3.3 hours. Productivity per unit area per unit time was 3.6 IU FVIII/cm2/h 8,2 IU VWF RCo/cm2/h (ratio VWF: RCo/FVIII: C=2,3) and 9.8 IU VWF: Ag/cm2/hour. Exit activity was 94% for FVIII and 95% for VWF RCo.

Example 3. Characteristics of the complex of FVIII/VWF after nanofiltration through Planova 20N

According to the method described in Example 2 was analyzed 7 different samples of the source material. As starting material there was used a complex of FVIII/VWF with a specific activity of about or more than 10 IU FVIII/AUNmand the protein concentration of 0.3±0,2 AUNmin the presence of 25 mm histidine and 0.14 M calcium at pH 6.8±0.2 and a temperature of 20±5°C. was conducted clearing through a pore size of 0.1 μm and subsequent nanofiltration through filters Planova 35N and Planova 20N. Filtration was conducted at a constant ck is grow current between 10 and 20 liters/h/m 2. Working pressure on the filter Planova 20N was maintained between 0.2 and 0.4 bar during the filter in all cases.

The results obtained (table 1) show that nanofiltration complex FVIII/VWF through a pore size of 20 nm under these conditions has no effect on the activity and purity (specific activity) of the received nanofiltration.

A comparative analysis of relations VWF:RCo/FVIII and VWF:RCo/VWF:Ag (table 1)obtained for the material before and after nanofiltration, allows us to conclude that in these conditions it is possible to carry out filtering of VWF present in the concentrate FVIII/VWF, through a sieve with a pore size of 20 nm without changing the functionality of VWF as a cofactor of Ristocetin.

The average values for 7 samples ± standard deviation (SD)

Example 4. Multimeric structure of VWF detected by electrophoresis shows the preservation of the original structure, including multimer with high molecular weight, containing more than 11 pages.

The figure 1 shows the multimeric structure of VWF in different samples of the final product before and after stage nanofiltration. In the original photos of the samples that pass nanofiltration, we can count at least 16 of bands in each lane of the gel.

Example 5. The influence of pressure difference on the nanofiltration of complex FVII/VWF.

Partially purified solution FVIII/VWF in which the specific activity of FVIII was approximately 10 IU/AUNmand with a concentration in the range of 0.1-0.3 AUNmwas filtered through a filter Planova 35N under the conditions described in Examples 2 and 3. The nanofiltration was carried out at high pressure (0.8 bar) and at low pressure (0.3 bar), the results obtained are presented in table 2.

Table 2
Planova 35NFilter conditionsPressure (bar)The protein concentration in the sample (ODNm)The flow speed % relative to the initial velocity of the current
After 1 hourAfter 3 hours
High pressure0,17910,67,9
0,80,27114,6the concentration is(1)
Low pressure (n=5)0,187±0,039 of 98.2±7,388,6+3,5
0,3of 0.133-0,236to 92.1-110,884,5-92,5
(1)It is impossible to determine, because the filtering was aborted due to contamination and clogging of nanofilter.

Similarly the solution of a complex of FVIII/VWF, pre-filtered through a filter Planova 35N, was filtered through the filter Planova 20N according to the conditions described in example 2. The nanofiltration was carried out at high pressure (0.8 bar) and at low pressure (0.3 bar), and the following results were obtained.

Table 3
Planova 20NFilter conditionsPressure (bar)The protein concentration in the sample (ODNm)The current velocity relative to the initial velocity current(%)
After 1 hourAfter 3 hours
High pressure0,80,14640the concentration is (1)
Low pressure (n=4)0,199±0,0586,5±9,9to 75.7±4,5
0,30,139-0,25475,7-98,472,1-82,1
(1)It is impossible to determine, because the filtering was aborted due to contamination and clogging of nanofilter.

In the case of filtration through the filter Planova 35N at high pressure, there was a sharp decrease in the rate of current: within one hour of filtration was observed speed reduction current to 10.6% and 14.6% of the initial velocity of the current, and in one case to 7.9% from the initial velocity of the current through the 3 hours of filtration. In the case of filtration through the filter Planova 35N when the differential pressure of 0.3 bar flow rate was 88.6 per cent even after 3 hours nanofiltration.

In the case of filtration through the filter Planova 20N solution of a complex of FVIII/VWF, the last pre-filtered through a filter Planova 35N, as the source material with a pressure difference of 0.8 bar the flow rate was 40% after one hour nanofiltration, and after 3 hours, filtration was stopped due to contamination and clogging of nanofilter. In the case of filtration through the filter Planova 20N with a pressure difference of 0, bar the flow rate was 75.7% even after 3 hours nanofiltration.

From the above examples it is obvious that maintaining a low pressure in the filtration process when the pore size of 35 nm or less allows you to avoid the sudden speed reduction current. Consequently, in conditions of low pressure was not observed clogging the pores of the filter membranes because of their dirt molecules with high molecular weight that are present in the solution FVIII/VWF, such as multimeric forms of VWF, reaching the size 20000000 Yes.

Example 6. The effect of the concentration of the source material on the nanofiltration of a complex of FVIII/VWF

Was held nanofiltration different solutions of partially purified complex of FVIII/VWF with a specific activity of FVIII about 10 IU FVIII/AUNmand VWF activity against FVIII activity (VWF:RCo:FVIII) about 2 concentrations in the range of 0.1 to 0.65 AUNmthrough the filters of Planova 20N with a pressure difference of about 0.5 bar in the same manner and under the same conditions described in Examples 1 and 2 (except in concentration).

The results obtained are presented in table 4.

2,19
Table 4
Planova 20NUsed protein concentration (ODNm)The ratio of the velocity of the current is the initial velocity of the current (%) Used relationsPerformance (activity, filtered through a unit area of the 20N filter per unit of time)(2)The protein yield (ODNm) (%)
After 1 hourAfter 3 hours(AUNm/c m2filter 20N)IU FVIII/ cm2/hIU FVW:RCo/ cm2/h
0,10680,771,41,772,65,277,6
0,17063,630,51,392,04,187,6
0,31460,7332,85a 3.97,882,5
0,34352,9323,26,588,6
0,64816,2the concentration is(1)1,160,91,846,8
(1)Concentration: it is impossible to determine, because the filtering was aborted due to contamination and clogging of nanofilter.
(2)Values are calculated based on the number of protein, filtered for 6 hours, in all cases, the specific activity was 10 IU FVIII/ AUNmthe attitude FVW:RCo/FVIII was 2, and the protein yield was calculated in each case.

Observed during nanofiltration decrease the speed of the current is directly proportional to the concentration of coating material. Thus, the observed within one hour of the current velocity was 80,7%, 63,6%, 60,7%, 52,9% and 16.2% for solutions with initial concentration 0,106 AU, 0,17 AU, 0,314 AU, 0,343 AU and 0,648 AU, respectively. In terms of speed current observed after 3 hours nanofiltration, we can say that the decrease in the rate of current below approximately 30.5% of the initial speed of the current may be due to the fact that the nanofiltration was carried out at this pressure, which is the top item is published by third parties for pressure filtration process.

The values of optimal performance (7,8 IU of VWF:RCo/cm2per hour, which is equivalent to 46.8 IU VWF:RCo/cm2) and protein yield (see table) were obtained using an initial concentration close to that of 0.3 AU. When used, the maximum initial concentration (0,648 AU)from the start of filtration was observed contamination and clogging of nanofilter, and the performance and yield of protein was significantly decreased to values below 2 IU VWF:RCo/cm2/hours and 46.8% of the total yield of protein, which makes impossible the carrying out of nanofiltration at this concentration.

From the given data one can conclude that the range of concentrations for nanofiltration of the solution FVIII/VWF with a specific activity of approximately 10 IU FVIII/AUNmis ≤0,6 AU, equivalent to about ≤6 IU/ml FVIII and ≤12 IU/ml VWF:RCo.

Example 7. The influence of calcium concentration on the nanofiltration of a complex of FVIII/VWF

Was held filtering different solutions of the complex of FVIII/VWF, mixed with albumin and having a specific activity of more than 10 FVIII/AU280and FVIII activity about 3 IU/ml, which is approximately equivalent to 4 IU/ml VWF:RCo, through filters with a pore size of 20 nm in the presence of 0.1 M arginine, 25 mm histidine and 0.05 mm calcium at pH 7,3±0,1. Filtration was carried out at a pressure difference on the filter Planova 20N approximately 0.5 bar. Table 5 shows the most important parameters, recip is installed when testing two different samples of the product.

Table 5
The composition of the used samplePerformance (IU/cm2/hour)Output (%)
ODNmFVIII (IU/ml)FVIIIVWF:RCoThe FVIII activity
0,1432,771,62,0(1)43,3
0,1882,961,62,0(1)46,6
(1)Values are calculated based on IU FVIII, filtered per unit time through a unit surface, taking the ratio of VWF:RCo/FVIII) is equal to 1.24.

These data show that the almost complete absence of calcium (0.05 mm) and its replacement by arginine (0.1 M) resulted in a significant decrease in performance, up to the value of 1.6 IU FVIII/cm2/h and 2.0 IU VWF:RCo/cm2/h in both cases, which is significantly less than the values which were obtained in Example 2 (3,6 IU FVIII/cm /h and 8.2 IU VWF:RCo/cm2per hour), although the activity of FVIII in the source material was the same.

Similarly, the output of FVIII activity observed in the two tests, falls to 43.3% and 46,6%, respectively. However, even in these conditions, nanofiltration macromolecular complex of FVIII and VWF provided the ratio of activities of VWF and FVIII, close to the ratio observed in nature (1:1).

Example 8. Getting nanofiltration complex FVIII/VWF in industrial scale

Using partially purified solution of a complex of FVIII/VWF obtained from more than 3,000 liters of blood plasma and has a specific activity of 15.6 IU FVIII/AU280has been filtered in several stages through the filter with a nominal pore size of 35 nm (Planova 35N) with an area of 4 m2and through two filters with a nominal pore size of 20 nm (Planova 20N) with an area of 4 m2in the presence of 25 mm histidine and 0.14 M calcium at pH to 6.80. Filtration was carried out at a constant speed current of about 107 l/h, maintaining the pressure difference on the filter Planova 20N between 0.20 and 0.35 bar, with a total load of (a solution of the product + subsequent flushing) 120,2 kg/m2filter Planova 20N. The total activity deposited on a unit area, was 8.9 IU FVIII/cm2and 19.1 IU VWF:RCo/cm2. Exit activity after nanofiltration was 70.4% for FVIII and 77.3% for VWF:RCo. Taking into account subsequent stage is th washing and concentrating the obtained nanofiltration product, the observed release of activity amounted to 97.5% for FVIII and 86,8% for VWF:RCo.

Subsequent precipitation with sodium chloride and glycine (according to the patent EP 0639203) provides obtaining nanofiltration concentrate FVIII/VWF with high purity.

Received nanofiltration concentrate FVIII/VWF with high purity was stabilized and brought to the desired activity, and then poured the bottles.

The relative content of VWF in relation to the content of VWF, expressed as the ratio of VWF:RCo/FVIII, during the cleaning process, shown below in table 6.

Table 6
Change activity (IU VWF:RCo/IU FVIII) in the course of the cleaning process
Nanofiltration product (n=1)Nanofiltration product (n=6)
Source material1,71,57±0,19
The intermediate concentrate1,91,71±0,11
Highly purified product1,51,14±0,25
Put up highly purified product 1,51,24±0,19

These results show that the application of nanofiltration in the cleaning process of the complex of FVIII/VWF does not impact significantly on the subsequent purification step, which leads to the production of high-purity product. Thus, this shows that the selected conditions nanofiltration concentrate FVIII/VWF through a membrane with pore size of 20 nm do not change for the worse the content of multimeric forms of VWF with high molecular weight, since such a change, as you might expect, has had a negative impact on subsequent treatment with precipitation.

Last nanofiltration concentrate FVIII/VWF has such relative concentration of VWF, which is sufficient for its use as a therapeutic product for the treatment of VWD, and the content of FVIII, which also makes it possible its use for the treatment of hemophilia And providing additional benefits from the presence of VWF (natural stabilizer FVIII) in amounts close to those found in nature, which gives, as noted above, additional benefits in the treatment of hemophilia A.

Although the present invention is disclosed with its preferred embodiments and illustrative examples, it is necessary to understand that on the basis of disclosed material liners is qualified specialist in the art may make many new options in the embodiment of the present invention, which will remain within the framework of the present invention, given the content items of the following claims and the equivalents of these items.

1. A method of obtaining a concentrate of factor a Background of Villebranda or complex factor a Background of Villebranda/factor VIII from human blood plasma or recombinant nature, including
a) preparation of a solution of factor a Background of Villebranda or complex factor a Background of Villebranda/factor VIII, which contains VWF in concentrations up to 12 IU VWF:RCo/ml and is related factor a Background of Villebranda/factor VIII, equal to 0.4 or more;
b) nanofiltration of the solution prepared according to (a) through a filter with pore size less than 35 nm at a pressure less than or equal to 0.5 bar and in the presence of from 0.05 to 0.2 M calcium ions.

2. The method according to claim 1, characterized in that obtained after nanofiltration factor a Background of Villebranda saves a multimeric structure, which includes multimer with a number of bands 11 or more.

3. The method according to claim 1, characterized in that the output of the factor a Background of Villebranda after nanofiltration is 60% or more.

4. The method according to claim 1, characterized in that the yield of factor VIII after nanofiltration is 70% or more.

5. The method of claim 1, characterized in that the applied load on the filter to 50 IU of factor a Background of Villebranda on 1 cm2area of filter surface.

6. The method according to claim 1, great for the present, however, what is the maximum concentration of the solution, which is filtered, is 0.6 AU (OD280).

7. The method according to claim 1, wherein the nanofiltration is carried out at a pressure of from 0.2 to 0.4 bar.

8. The method according to claim 1, characterized in that the standard rate of current when nanofiltration is from 10 to 20 l/h/m2.

9. The method according to claim 1, wherein the nanofiltration is carried out using a filter with a pore size of less than 35 nm.

10. The method according to claim 1, wherein the nanofiltration is carried out using a filter with a pore size of 19±1 nm.

11. The concentrate obtained according to the method according to any one of claims 1 to 10, for the manufacture of medical devices for the treatment of hemophilia a or disease Von Willebrand's disease.



 

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41 cl, 12 tbl, 8 ex

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24 cl, 2 tbl, 14 ex

FIELD: medicine.

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Vaccine preparation // 2409386

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly concerns a vaccine preparation which can be used for controlling viral and other diseases of infectious nature. A reaction product of cationic oligomer and antigen determinant in an equimolar amount are used as an antigen polypeptide.

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2 dwg, 1 tbl, 1 ex

FIELD: chemistry.

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FIELD: chemistry.

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EFFECT: low consumption of time, funds and energy.

12 cl, 5 ex, 1 tbl

FIELD: chemistry; biochemistry.

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1 dwg, 10 ex

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FIELD: medicine, pharmaceutics.

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2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: in formula (1) A is a nitrogen atom or CH; when A is a nitrogen atom, B is NR9 (where R9 is a C1-10alkyl group), when A is CH, B is a sulphur atom, R1 is a phenyl group (where the phenyl group is substituted with one or more substitutes selected from a group consisting of halogen atoms, C1-10alkyl group and C1-10alkoxy groups (where C1-10alkyl groups and C1-10alkoxy groups are not substituted of substituted with one or more halogen atoms)); L1 is a bond; X is OH; R2 is a C1-6alkyl group; L2 is a bond; L3 is NH; L4 is a bond or NH; Y is an oxygen atom or sulphur atom; R3 is a thienyl group (where the thienyl group is substituted with CONR29R30 (where R29 is hydrogen or a C1-10alkyl group, and R30 is an amino group (where the amino group is not substituted or substituted with a pyridyl group), mono- or di-C1-10alkylamino group, N-methylpiperzinyl group, piperidine group, morpholine group or C1-10alkyl group (C1-10alkyl group is substituted with one or more substitutes selected from a group consisting of a carboxyl group, carbamoyl groups, pyrroldinyl groups, tetrahydrofuryl groups or morpholine groups) or R29 and R30 together denote -(CH2)m3-G-(CH2)m4- (where G is CR31R32 (where R31 is a hydrogen atom and R32 is a C1-10alkylcarbonylamino group or pyrrolidinyl group) and each of m3 and m4 is independently equal to an integer from 0 to 5 provided that m3+m4 equals 3, 4 or 5), or NR29R30 as a whole denotes a piperidine group or pyrrolidinyl group (where the piperidine group or pyrrolidinyl group is substituted with two substitutes independently selected from a group consisting of: hydroxyl groups and C1-10alkoxy groups) or 2-(4-oxopyrridin-1(4H)-yl)acetyl group), phenyl group (where the phenyl group is substituted with one substitute selected from a group consisting of C1-10alkyl groups, C1-10alkylcarbonyl groups and C1-10alkylaminocarbonyl groups, (where C1-10alkyl group, C1-10alkylcarbonyl group and C1-10alkylaminocarbonyl group are substituted with one or two substitutes selected from a group consisting of hydroxyl groups, carboxyl groups and carbamoyl groups)), phenyl group (where the phenyl group is substituted with one C1-10alkylaminocarbonyl group or one halogen atom), dihydrobenzo[1,4]dioxine group or benzo[1,4]oxazine group. The invention also relates to a medicinal agent containing the disclosed compound as an active ingredient and to a thromopoeitin receptor activator which is a formula (1) compound.

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8 cl, 11 tbl, 128 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology. The invention describes a method of producing a modified vitamin K dependent polypeptide which involves modification of activation peptide which includes at least part of an amino acid sequence of another vitamin K dependent polypeptide and where the said part is at least 3 adjacent amino acids of activation peptide FVII, or 5 adjacent amino acids of activation peptide FX, or 5 adjacent amino acids of activation peptide FIX provided that QSFNDFTR peptide is excluded, or 8 adjacent amino acids of activation prothrombin peptide, or an extension on adjacent amino acids of the activation peptide of the second vitamin K dependent polypeptide which has length at least equal to 15% of the total length of the amino acid sequence of the activation peptide of the second vitamin K dependent polypeptide, in which the second vitamin K dependent polypeptide is selected from a group consisting of protein Z, GAS6 and protein S, and where the second vitamin K dependent polypeptide has longer half-life in plasma. The invention discloses a modified vitamin K dependent polypeptide obtained using the described method and having coagulation activity.

EFFECT: invention enables production of modified vitamin K dependent polypeptides with longer half-life in plasma.

40 cl, 2 dwg, 4 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns methods of treating tracheal, bronchial or alveolar haemorrhage or haemoptysis. Substance of the invention involves intratracheal, intrabronchial or intraalveolar introduction to a person of a blood-coagulation factor which is activated factor VII.

EFFECT: advantage of the invention consists in application of the blood-coagulation factor active at the earlier stages of the coagulation cascade.

16 cl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula (1), their tautomers and pharmaceutically acceptable salts. The disclosed compounds have thromobopoietin receptor agonist properties. In formula (1) , A is a nitrogen atom or CH, when A is a nitrogen atom, B is NR9 (where R9 is a C1-10 alkyl group), and when A is CH, B is a sulphur atom, R1 is a phenyl group (the phenyl group is substituted with one or more substitutes selected from a group consisting of halogen atoms, C1-10 alkyl groups and C1-10 alkoxy groups (C1-10 alkyl groups and C1-10 alkoxy groups are unsubstituted or substituted with one or more halogen atoms)), L1 is bond, X is OH, R2 is a C1-10 alkyl group, L2 is a bond, L3 is NH, L4 is a bond or NH, Y is a sulphur atom, and when L4 is a bond, R3 is a piperidinyl group, a piperazinyl group (the piperidinyl group and the piperazinyl group are substituted with substitutes selected from a group containing C1-10 alkoxycarbonyl groups, carboxyl group, hydroxyl groups, di-C1-10 alkylaminocarbonyl groups, C1-10 alkylaminocarbonyl groups and C1-10 alkyl groups (C1-10 alkylaminocarbonyl groups and C1-10 alkyl groups are substituted with a substitute selected from a group containing pyridyl groups, hydroxyl groups and carboxyl groups)), or when L4 is NH, R3 is a C1-10 alkyl group (C1-10 alkyl group is substituted with a substitute selected from a group containing C1-10 alkoxy groups, C1-10 alkoxycarbonyl groups or carboxyl groups).

EFFECT: obtaining a thrombopoietin receptor activator which is a formula (1) compound and a medicinal agent which contains the disclosed compound as an active ingredient.

10 cl, 3 tbl, 47 ex

Haemostatic sponge // 2385726

FIELD: medicine.

SUBSTANCE: invention relates to medicine, particularly to haemostatic sponges for local haemostasis. The offered haemostatic sponge represents lyophilised 1% collagen in ammonium carbonate with pH 8.5-8.9 which contains feracryl in the following mixing ratio, %: collagen 98.0-99.2, feracryl - 0.8-2.0. The sponge can be additionally textured in vaporised aldehydes and can additionally contain to 3% of antiseptics and 10% of broad-spectrum antibiotics.

EFFECT: invention provides production of the haemostatic sponge exhibiting high haemostatic activity.

3 cl, 5 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to chemical-pharmaceutical industry. The material for haemostasis contains dialdehyde cellulose of the oxidation level 6.5±0.33% - 1 g, gelatinol 60±3 mg - (0.75 ml), ε-aminocapronic acid 50±0.25 mg, lysozyme 5±0.25 mg, water 5.75 ml. The method for making the textile material expressing haemostatic action consists that ε-aminocapronic acid, gelatinol and lysozyme are successively dissolved in the distilled water at room temperature. After said components are dissolved completely, the prepared solution is placed into dialdehyde cellulose solution of the oxidation level 6.5%±0.33% in the form of a cloth and kept for 2 hours. Then the cloth is wrung out, air-dried to residual humidity no more than 10% with cutting out napkins of weight approximately 1 g and dimensions 7.5×5.0 cm, then sealed in polyethylene bags and sterilised by gamma irradiation in a dose 25 kGy. Besides for haemostasis of irregularly shaped deep stab, missile and shell fragment wounds, the prepared material is ground in a rotor impact mill to lint condition (cotton wool).

EFFECT: making the effective styptic (haemostatic) product of partially oxidised cellulose.

1 tbl, 5 ex, 3 cl

FIELD: medicine.

SUBSTANCE: present invention concerns medicine, specifically a powder delivery system containing a composition which contains a gelatinous or collagen powder with minimal average particle size 10 mcm. Usually, said gelatinous or collagen powder are presented in dry form, i.e. no liquid components and/or propellants are added to the composition. Besides the invention refers to the advanced powder delivery system which contains a protective structure, such as edge enclosure nearby a hole of the delivery system. The invention refers to the gelatine or collagen compositions used for haemostasis, and also to the powder delivery system including the ready-to-use gelatinous or collagen powder in the dry form. Additionally, the powder delivery system can contain a dry agent incompatible with humidity and/or water.

EFFECT: invention provides fast and more effective haemostasis.

34 cl, 10 dwg, 8 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to production of versions of the Gla domain of human factor VII or human factor VIIa, and can be used in medicine. Amino acid sequence of the FVII or FVIIa version is obtained, which differs on 1 to 15 amino acid residues with amino acid sequence of the human factor VII (hFVII) or human factor VIIa (hFVIIa), in which a negatively charged amino acid residue is introduced by substitution in position 36. Obtained variants of FVII or FVIIa are used in a composition for treating mammals with diseases or disorders, where blood clotting is desirable.

EFFECT: invention allows for producing versions of FVII or FVIIa with high clotting activity and/or high activation of factor X, compared to natural form of hFVIIa.

42 cl, 3 dwg, 5 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.

EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.

29 cl, 5 dwg, 6 tbl, 7 ex

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

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