The way to obtain standardized concentrate of human von willebrand factor and the concentrate obtained in this way


(57) Abstract:

Usage: in medicine in obtaining standardized concentrate of human von Willebrand factor. The inventive cryosurgery fraction of plasma pre-treated by aluminum hydroxide, twice successively purified by ion-exchange chromatographies on the macroporous type resin vinyl polymer having a group diethylaminoethyl. This is followed by further purification using affinity chromatography on a column with gelatin-separati shown in the equilibrium buffer solution used as an eluting at the previous stage. The resulting concentrate of von Willebrand factor has high specific activity and high content of multimers with high molecular weight. 2C. and 1 C.p. f-crystals, 2 Il.

The invention relates to a method for production on an industrial scale standardized concentrate of human von Willebrand factor very high purity, having a very high specific activity and high content of multimers with high molecular weight, which is particularly suitable for therapeutic use.

The von Willebrand factor /PV/ beach the ticks S-S, the main element which has a molecular weight close to 260 kilodaltons /KD/. The lowest form of PE in plasma is a dimer with a molecular weight of 440-500 kDa, and most forms are multimer this dimer, the molecular weight of which can reach up to 20 million Daltons. Such a connection of the subcells in multimer can be specific for cells in which it is produced, and the PV is synthesized and polymerized in megakaryocytes and endothelial cells.

This factor plays a major role in hemostasis by two different functions: as an adhesion protein he performs dispersion, adhesion and aggregation of blood platelets /platelets/ on subendothelial vessels and is involved so rapidly zarubezhnie (healing of damaged vessels and, on the other hand, it provides stabilization and mixing factor YIII in the process of circulation.

Congenital insufficiency of PV or structural anomaly of this factor lead to disease Willebrand's disease, which results in bleeding, especially skin and mucous membranes. This disease is very heterogeneous in its clinical expression and poses serious problems in the of mostaza /period blood clotting/ coagulation /period of brain activity and clotting activity of factor YIII/. The disease can be cured in therapy by replacing the derivatives in human plasma, enriched PV /for example, crossdoney fraction of plasma or concentrates of factor YIII, containing enough PV associated with the last/. However, these products are not standardized for the treatment of von Willebrand disease. In addition, alloocimene fraction of blood plasma, in particular cryosurgery faction, not without risk of virus infection, because they often are not effective treatment for viral inactivation. In addition, they lead to excess contaminating proteins that are not needed by the patient and which can cause immunological reactions after repeated injections.

In contrast, the purified factor YIII can be effective treatment for viral inactivation, but the method of its purification was optimized for the treatment of hemophilia A; and not for treatment failure PV.

Thus, methods of obtaining factor YIII, developed recently, such as immunoaffinity or purification by ion exchange, becoming more and more efficient and lead to the production of concentrates, which no longer contain PV in sufficient quantity to be effecctive the treatment of von Willebrand disease, the applicant has developed a new method of purification of PV on an industrial scale, which allows the best way to make cost-effective extraction of the different molecules in the plasma. In particular, it allows the first stage to obtain a concentrate of factor YIII /in accordance with the method described in the application for the European patent 0359593/, and further to carry out the extraction fraction enriched with von Willebrand factor, from the same party cryosurgery faction that allows the best way to make cost-effective use of human plasma. Thus obtained fraction PV then purified by two additional stages by chromatography, resulting concentrate PV very high purity.

The complexity of the molecules of von Willebrand factor makes its getting very difficult. Cleaning methods on a small scale, i.e. from 5 to 2000 ml, in view of analytical radiation have already been described, but these methods could not be adapted for industrial scale /Thorell et al, Jhromb, Res, 35, 1984, 431-450/. Moreover, the principle of maximum profitability cryosurgery faction with the aim of obtaining at the same time factor YIII and the PV has not yet been considered.

The von Willebrand factor was purified is acerina /Berntorp et al, Vox Sang, 56, 1989. 212-217; Winkelman et al, Vox Sang, 57, 1989, 97-103/ and as a result, various methods of chromatographic separation, for example, by use of molecular sieves /Perret et al Haemosfasis, 14, 1984, 289-295 /or by ion exchange /Austen et al Jhromb, Haemostasis, 48, 1982, 46-48; Harrison et al, Jhromb. Res. 50, 1988, 295-304/, however, these methods give relatively small outputs for von Willebrand factor or a small bottle of gel or not at the same time allow the extraction factor YIII and PV, which makes them less attractive for industrial applications.

Austen et al /1982, above/ get concentrate small purity /8 Ed AG/mg protein/ and with relatively low output, is likely to be due to very adverse conditions chromatography was carried out /pH=5,5/.

Harrison et al /1988, above/ use as a matrix of dextran-sulfate-sepharose; this material has only a small holding capacity in relation to PE, such as PE exhibits a specific activity of only 2-4 Units/mg protein.

Moreover, most of these products contain a large fraction denaturirovannykh or inactivated forms along with active multimeric forms, as it shows the ratio of the activity of the cofactor of ristocetin (RCO) / therapeutic use in the treatment of von Willebrand disease. On the contrary, the way of the applicant company allows you to get the concentrate PV; in which the RCO/AG exceeds unity, which is comparable with the same respect for natural PE in plasma.

The present invention relates to a method for producing a concentrate of von Willebrand factor for therapeutic use, and this method allows to obtain standardized parties, each of which comes from very large volumes of plasma /4000 litres or more of which has already been extracted factor concentrate YIII that best makes cost-effective use cryosurgery faction.

In particular, the present invention relates to a method for producing a concentrate of von Willebrand factor, which involves the combination of three successive chromatographic steps, allowing the enrichment multimers with high molecular weight, contributing to the biological activity of FB. The source material is cryosurgery fraction of human plasma, subjected to the classical treatment as a result of adsorption on aluminum hydroxide. This material is then subjected to viral inactivation, for example by treatment with a solvent-detergent before cistilnih chromatographic stages of obtaining VIII of the intermediate product, moreover, the first two stages are ion-exchange chromatography, and the third is affinity chromatography.

Two-stage ion-exchange chromatography is carried out on the same resin of the vinyl polymer grafted groups of DEAE /diethylaminoethyl/, in particular, on columns with resin DEAE-fractogel(M)TSK 650 /Merck/, brought into equilibrium with the buffer solution containing 0.01 M traintravel citrate, 0,11 M sodium chloride, 0.001 M calcium chloride, 0.12 M glycine and 0,016 M lysine, pH 7.

DEAE-Fractogel(M)TSK 650 is a hydrophilic synthetic gel. Carrier is a copolymer of oligoarthritis, glycidylmethacrylate and pentaerythritol-dimethacrylate, which are vaccinated group diethylaminoethyl, such as-O-CH2-CH2N+/C2H5/2HCl, which forms the alkaline aminoalkenes. There is DEAE-Fractogel TSK 650 with particles of two sizes /after rehydration/: S /0,025 0,050 mm/ and type M /0,045 0,090 mm/; both of these types can be used for the present invention.

Cryosurgery fraction of plasma, purified and processed for viral inactivation, is passed through parvo yiruma by increasing the concentration of sodium chloride in a buffer solution of 0.15 to 0.14 M

Allerona thus the fraction enriched with von Willebrand factor, is passed through the second chromatographic column under the same conditions as for the first column. Since the faction introduced in this second column are already exempt from most of the proteins /particularly factor VIII and fibronectin/ who competed for adsorption centers, the adsorption capacity of PV in the second column is significantly higher. After removal of the filtrate and washing the column with buffer solution for equilibrium adsorbed PE eluted by increasing the concentration of sodium chloride in the buffer solution to values of 0.15 to 0.17 M

Due to the high adsorption capacity and efficiency of the resin DEAE-Fractogel with respect to the PV, the latter is extracted with high specific activity (>150 RCo IU/ml), which eliminates the need for an additional stage of concentration by ultrafiltration.

Allerona thus the fraction is subjected to affinity chromatography on a column with gelatin-separate in the same buffer solution for equilibrium that avoids the stage of dialysis or ultrafiltration to izmeneniya gel, associated with gelatinous, is not critical: you can also use gelatin-Metrogel(M)gelatin-Svertex(M)or gelatin-Fractogel(M). Use preferably gelatin-sepharose(M)that binds 5 to 10 mg of fibronectin/ml under the conditions applied by the method in accordance with the invention.

The von Willebrand factor, purified under these conditions, is not associated with the gel and passes into the filtrate; as chromatographic stage in the presence of gelatin does not lead to a significant dilution of PE, it is not necessary to concentrate its /for example, by ultrafiltration/ he may immediately be brought to the condition and liofilizirovanny. The absence of a proteolytic enzyme in the final product ensures its stability during sterilization filtration and freeze-drying, thus avoiding the addition of stabilizing agents.

The concentrate of von Willebrand factor obtained by the method in accordance with the present invention has a degree of purification, exclusively exceeding more than 10,000 times the level relative to the source plasma, and its specific activity is 345 Ed CBA/mg protein/ units AK the second chromatographic stage in cleaning the PV is illustrated in Fig.1.

Improving the quality of the product during successive stages of purification were observed, in particular, depending on the fraction of molecules in the state of multimers with high molecular weight /i.e. molecular forms with high biological activity/ method of electrophoretic analysis.

This analysis shows a progressive enrichment multimers >4 /Fig. 2/ that make up 79% of the PV in the final product compared to 70% in natural plasma, whereas the result of cryosurgery half of them lost. It was unexpectedly found that chromatography on DEAE-fractogel(M)favorable selective retention of multimeric very large and deletes in the filtrate size with abnormal structure /subjected to partial proteolysis/ and with small activity.

Standardized concentrate of PE of great purity with high specific activity and with a high content of multimers with a high molecular weight, obtained by the method in accordance with the present invention, particularly well, therefore, adapted for therapeutic use in the treatment of various forms of von Willebrand disease, which is confirmed will precede the period clotting of blood during bleeding.

Tests in vitro have confirmed the identity of its biochemical and physiological properties with the same properties of natural molecules, in particular, its ability to bind platelets /platelets/ unit for transfusion /perfusion/ and his ability to connect with the endogenous factor VIII.

The high purity of the hub of von Willebrand factor obtained by the method in accordance with the present invention allows also to consider its application for various laboratory needs /for fine structure analysis for radiation functionality, diagnostics and so on/ and to obtain specific antibodies.

The concentrate of the present invention can also be used as a stabilizer in the process of obtaining factor VIII using cells that have been transformed by genetic engineering and during the stages of purification of Factor VIII obtained in this way.

The following example illustrates the implementation of the present invention without limiting, however, its scope.

Example. The source material.

Crossdoney the fraction is obtained on the basis of fresh plasma in the presence of sodium citrate /4%/ or anticoagulative rsma is frozen at a temperature of -60oC, then stored at -35oC. Parties plasma contain from 1800 to 2000 litres and are collected in batches of 4000 litres for each implementation of the method. The purpose of thawing the plasma is placed in a chamber at a temperature of -7oC and for at least 12 hours to ensure a slow and regular heating, then it thawed in the cell thermostatted at a temperature of 0 to 2oC with constant stirring. Cryosurgery fraction (which is around 9 g/l plasma) are recovered by centrifugation in the cold.

After centrifugation extracted cryosurgery faction again transferred into the solution and adsorb on the aluminum hydroxide in order to remove basic impurities, i.e., components of the prothrombin complex /in particular, the factor VII/ Factor XII. Emergent phase is then cooled to a temperature of 15oF /partly removes fibrinogen and fibronectin/.

This processing allows to extract from 80 to 86% of the mixture of Factor VIII von Willebrand Factor from cryosurgery faction. The specific activity of Factor VIII is 0.7 IU 1/mg, a specific activity PE is 0.6 U RCo/mg /cofactor activity of ristocetin/ 1,2 and Ed CBA/mg /activity linking cctor Willebrand's disease, processed under the action of a solvent-detergent, known for its effectiveness against viruses with a lipid envelope /Horowitz et al. Transfusion 1985. 25, 516 522/, which also includes incubation for 8 hours at a temperature of 25oC in the presence of 0.3% tri-n-butylphosphate /TNBP/ 1% Tween 80.

After this processing detects 95% of the activity of factor VIII and von Willebrand measured at the previous stage. Electrophoresis allows you to control what the PV of all time is in a multimeric form.

The method of chromatographic separation.

Purification of von Willebrand factor is a method derived from the method of purification of Fraction VIII, described by the applicant in the application for the European patent 0359593.

First chromatographic stage is carried out on a column with DEAE-Fractogel(M)TSK650 /Merck/. Buffer solution for equilibrium contains traintravel citrate /0.01 M/ calcium chloride/0.001 M/ calcium chloride /0,11 M/ glycine /0.12 M/ L-lysine /0,016 M/. The von Willebrand factor, Factor VIII and fibronectin are retained by the column; contaminating proteins /mainly fibrinogen and IgG/ bind weakly or not associated column, and residues from the treatment for viral inactivation adelayda the flow velocity in the column is equal to 100 cm/hour.

Under these conditions, the implementation of the used column has a holding capacity in relation to PV about 75% /defined in the form of antigen, AG/, and the remainder goes to the filtrate. This holding capacity is 45 Units AG-PV/ ml of gel.

The von Willebrand factor is desorbed from the column by increasing NaCl concentration in the buffer solution to a value of 0.15 M. the Collected fraction PV contains from 30 to 35 initial PV, however, 40% of the last remain adsorbirovannymi with Factor VIII and will soloerotica together with the latter in the second increasing the concentration of buffer solution to a value of 0.25 M and will suicidarse together with the latter.

The fraction containing suirvey from the first column to the PV injected into the second column, identical to the first, under the same conditions after a dilution buffer solution for equilibrium with the aim to bring the ionic strength fraction of the PV (up to the value equivalent to 0.11 M sodium chloride.

As introduced faction already freed from impurities and from Factor VIII, which competed for the binding of the adsorption centers of the first column, then second column observing means is Tate increasing NaCl concentration in the buffer solution to 0.17 M

This second chromatographic stage allows to obtain the ratio of the concentration in 8-10 times higher than the level of the previous stage, which gives the possibility to dispense with additional stages of concentration by ultrafiltration, for example. In the usual variety of measurement systems, the eluate contains the following concentrations or activity PE:

antigen /AG/ 889 IU/ml;

cofactor ristocetin 9719 U/ml;

the ability of binding with collagen /CBA/ 14913 U/ml;

multimer /Z4/ high molecular weight 79%

The ratio of CBA/AG value 1,69 shows that the activity of PV is well preserved. This is in line with a high percentage of /79%/ multiserv with high molecular weight.

Electrophoretic analysis of the eluate from the PV shows the presence of a small contamination of fibronectin and inter-alpha-trypsinogen inhibitor /ITI/.

This second eluate with PV is subjected to a third stage of purification on a column of gelatin-Separate CL4B /pharmacy/ cited in equilibrium with an eluting buffer solution used for the second column, in order to remove fibronectin.

This gel for affinity chromatography has a holding ways redetection quantities (<mg/l) for fractions of PV.

Purified PV is in the filtrate of this last stage and can be directly communicated to the condition and liofilizirovanny:

Electrophoretic analysis no longer detects no pollution;

The content of the PV is 250 IU AG/mg protein.

Its specific activity is 345 Ed CBA/mg protein and 186 220 Ed RDF/mg protein.

General purification efficiency of more than 10 000 times the level of the original plasma.

Obtained after purification PV consists of 65-80% of multimers with high molecular weight, i.e., has a composition comparable to the initial plasma, which is equal to 70% as shown by electrophoretic analysis on agarose-SDS.

The stability of the concentrate was studied in the liquid state at ordinary temperature for 24 hours: there were no signs of proteolysis and no specific activity changes.

The complete absence of thrombogenic activity of the concentrate was controlled using classical tests NAPTT/"non activated partial tromboplastin time"/. The complete absence of thrombin, the RCA and kallikrein was installed.

The final concentrate requires no addition of a stabilizer.

botana solvent-detergent; band 3 fraction, is not associated with DEAE-fractogel; lane 4 1st eluate PV; band 5-2-th eluate PV; lane 6 - fraction, is not associated with the gel on gelatin; lane 7 standards.


IgG immunoglobulins

Alb albumin

Fig.2: Distribution of multimers PV in fractions during purification after electrophoresis on agarose SDS: band 1 natural plasma; lane 2 - cryosurgery fraction; lane 3 cryosurgery fraction-treated solvent-detergent; lane 4 faction is not associated with DEAE-Fractogel; lane 5 1st eluate PV; lane 6 2nd eluate PV; lane 7 - fraction, is not associated with the gel on the gelatin.

1. The way to obtain standardized concentrate of human von Willebrand factor from cryosurgery fraction of plasma pre-treated by aluminum hydroxide, which comprises two successive stages ion-exchange chromatography on macroporous type resin vinyl polymer having a group diethylaminoethyl, characterized in that it further conduct a purification step using affinity chromatography on a column with gelatin-separate, and column shown in the equilibrium buffer solution used as an eluting at prospect is containing von Willebrand factor, collected, poured into individual vials dosage and then lyophilizer.

3. The concentrate of von Willebrand factor obtained by the method according to PP.1 and 2, with the specific activity of the corresponding 345 Eg CBA/mg protein and 186 220 With EgR/mg protein, characterized by respect to the activity measured in units of the NVA, to the amount of antigen that is equal to at least 1.5 and comprising more than 65 80% of multimeric forms high molecular mass.


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