MXPA99008941A - Immunotolerant prothrombin complex preparation - Google Patents

Abstract

The invention relates to an immunotolerant prothrombin complex preparation, a method for producing said preparation and the use of the same for producing a medicament.

Description

PREPARATION OF THE IMMUNOTOLERANT PROTROMBIN COMPLEX DESCRIPTION OF THE INVENTION The invention relates to a pharmaceutical composition for the treatment of coagulation problems, especially a factor VIII inhibitor patient. The invention also relates to a process for the preparation of this composition as well as its use. The coagulation of blood is caused by a section of reactions of different proteins or enzymes. Due to lack of blood coagulation factor, fibrin formation from fibrinogen and therefore closure of wounds is avoided; The consequence is hemorrhage. Such a case occurs in the case of hemophilia A. This is the most widespread hemorrhagic disease and is caused by a lack of factor VI11. For the substitution treatment of hemophilia A, gu preparations contain factor VIII. The treatment with these preparations leads in most cases to rapid hemostasis. However, there are also patients in whom only a lack of factor VIII is present, but also an inhibitor directed to factor VIII has been developed. Another group of patients has inhibitors of factor VII, without suffering hemophilia A. Depending on the amount of factor BII inhibitor present, the effect of factor VIII administered by its neutralization is inhibited.

REF .: 31317 For the treatment of factor VIII inhibitor patients, preparations based on plasma fraction are currently offered, which contains a mixture of coagulation factors. The plasma fraction may contain, for example, the factors of the prothrombin complex (factor II, VII, IX and X). A preparation that promotes blood coagulation with deviating activity of the factor VIII inhibitor (FEIBA TI 4, IMMUNO AG) is obtained, for example, according to AT-B 0 368 883 by means of a treatment of the cryosodium. This preparation also contains the coagulation factors II, VII, IX and X. The action of a FEIBA preparation due to its complex composition is multiple. Ariani et al. (Thrombosis Res. 31, 475-488 (1983)) mentions as factor of action factor VII in its activated form. It was determined that after the infusion of a FEIBA preparation a higher Vlla factor content is found in the plasma of hemophiliacs. Likewise, Teitel (in Thrombosis and Haemostasis 66 (5) 559-664 (1991)), describes the role of factor Vlla in prothrombin complex concentrates with a "factor VIII deviating activity". Simultaneously, the principle of effect, factor Xa, is presented in this type of preparations. The prothrombin complex concentrates studied contained factor Vlla, expressed as the factor between the activity of factor il and factor VII antigen of 2.1 and 2.5.

Therapeutic compositions containing prothrombin prepared according to EP 0 044 343 Bl are suitable for the treatment of coagulation factor inhibitors and contain an activated prothrombin complex, in which the factors are partially activated. The factor Vlla ratio is 8-80 Units / ml. The concentration of factor IX is in the range of 15 to 112 Units / ml. Correspondingly, the content of factor Vlla, in relation to factor IX, is 0.07-5.3 factor units Vlla / Factor IX units. Vinazzer (Thromb.Res.26: 21-29 (1982)) shows the difference of the AUTOPLEX preparation, which is prepared according to EP 0 044 343, and FEIBA. As shown there, AUTOPLEX is characterized by a higher thrombin content (factor lia), measured in NIH units, compared to FEIBA (see table 1 page 24). However, highly purified factor Vlla preparations are also proposed for the therapy of coagulation factor inhibition states (for example in EP 0 082 182-Bl) and Hedner et al. (Haemostasis 19, 335-343 (1989)). One advantage of factor Vlla preparations is their lack of factor VIII. The content of factor VIII in preparations of prothrombin complex or in activated preparations of prothrombin complex such as for example FEIBA in patients with functional inhibitors of factor VIII, has the effect that these inhibitory antibodies are triggered by the administration of factor VIII, whereupon the state of hemophilia by the inhibitor even gets worse temporarily. It has already been determined that for effective haemostasis with preparations of factor VIII inhibitor hemophilia factor Vlla in their effect they outperform prothrombin complex factor preparations (Turecek P. et al., Thromboeis & Haemostasis, 1997, p. 222). A task of the present invention is therefore to present a preparation, which possesses the effectiveness or the efficiency of the prothrombin complex preparations without presenting the undesired immunological side reactions of that type of preparations. The aforementioned task is solved by preparing in accordance with the invention a pharmaceutical preparation of immunotolerant prothrombin complex which contains factors II, IX, X and optionally VII with a reduced content of factor VIII antigen. The content of factor VIII antigen of the preparation according to the invention is preferably less than 10%, especially less than 5%. The content of factor VIII is preferably lower than 0.1 factor VIII antigen: C / FEIBA Unit. In a particularly preferred embodiment, the content of factor VIII antigen is up to less than 0.03 / unit of FEIBA, more preferably less than 0.02 / unit of FEIBA and most preferably less than the determinable limit. The finding that especially in the case of a Subsequent purification of the plasma prothrombin complex factors or a plasma fraction, the content of factor VIII and eventually the content of phospholipids can be reduced in such a way that while the activity of the prothrombin complex factors is kept constant, considered surprising. In particular, the pharmaceutical preparation according to the invention contains at least the factors iXa, Xa and Vlla and has an activity FEIB also reduces the coagulation time of a plasma deficient in factor VIII with a functional inhibitor (see for example AT-B 350 726). A preparation according to the invention can be prepared from plasma or a plasma fraction. The plasma fraction can be prepared from plasma, especially of human origin, by means of chromattraction, precipitation or centrifugation, or the residue of cryoprecipitation can be used. The plasma fraction includes factors dependent on vitamin K as factors of the prothrombin complex, but preferably also contains protein S, C and / or Z. In a particularly preferred embodiment the preparation is free of phospholipids. Preferably the upper limit of the phospholipids obtained is 0. inmol / unit of FEIBA (on the calculation see the examples). Due to this lack of phospholipids, it can be reduced or avoid the formation of unwanted antibodies against factor VIII. In accordance with the present application there is also presented a process for producing said preparation. This procedure covers the following steps: a) obtaining plasma or a plasma fraction containing factors II, IX, X and eventually VII, b) contacting the plasma or the plasma fraction with a carrier material, possibly in the presence of a detergent, such that factor VIII and eventually the phospholipids are separated from factors II, IX, X and possibly VII, c) purify the plasma or plasma fraction, and d) recover a fraction containing the factors II, IX , X and eventually VII. In a preferred variant steps b and c or b, c and d are performed in a single process step. The plasma fraction used is preferably a plasma fraction with an intermediate purity. Under a preparation with intermediate purity should be understood a plasma fraction of such type which is analogous to the definition of intermediate property of factor preparations VIII (see for this for example Ood Clive (ed.), Factor VIII: Purity and prophylaxis, Royal Society of Medicine).

Additional proteins, which are not separated by means of chromatographic purity, may be present in a preparation with intermediate purity. A preparation of common and commercial prothrombin complex factors, such as for example FEIBA S-TIM 4, IMMUNO or activated prothrombin complex, can also be used as starting material. As a corresponding purification process, the methods known in the state of the art can be used, preferably a chromatographic treatment, precipitation or centrifugation is carried out. The residue of cryoprecipitation can also be used as the plasma fraction. The carrier material is a suitable material for chromatography, filtration and / or nanofiltration. In the case of filtration, it is in particular affinity or membrane filtration. If a previously purified material is used, for example a material previously purified for example by means of an anion exchanger, a readsorption in another carrier material, preferably in the same carrier material used for the previous purification, can then be carried out under modified conditions. In a preferred embodiment the carrier material is a carrier material specific for factor VIII, especially a suitable matrix for affinity chromatography. In particular, it is preferred to use a matrix containing F. In a carrier material of that type, factor VIII and, optionally, the phospholipid are adsorbed, while factors II, IX, X and possibly VII do not adhere. The carrier material can also be a non-specific carrier material for factor VIII, for example a weak ion exchanger, for example a ion exchanger DEAE, TMAE or another sufficiently known by the state of the art. Depending on the selected conditions, factor VIII and eventually phospholipids are adsorbed on the carrier material, while factors II, IX, X and eventually VII do not adhere or vice versa. In another preferred embodiment, the carrier material has a higher affinity for the prothrombin complex than for factor VIII. For example, factors II, IX, X and possibly VIII are adsorbed, while factor VIII and possibly phospholipids are eluted. The factor VII can be inactivated selectively and then in that inactive form for example no longer adhering to the carrier material. Such inactivation of factor VIII can be obtained, for example, using, for example, a binder of guelate, by means of degradation, for example by means of serine proteases such as thrombin or an activated protein C, by means of binding of affinity models, such as antibodies or peptides. All the described process variants can also be carried out in the presence of a detergent, especially a non-ionic detergent. Preferably a polyether or a polysorbate, especially Tween or Triton, is used as a detergent. If detergent is used, then in a preferred embodiment it is removed or removed. In another preferred embodiment, a step is provided for the inactivation of pathogens optionally present, especially selected from the group of heat treatment steam treatment, treatment with a solvent and / or treatment with a detergent. The arrangement preparation can also be obtained in general according to one of the procedures described above. It is suitable in particular for the preparation of a medicament, which is suitable for the treatment of patients with factor VIII inhibitor (= haemophilic A), especially for those patients with a titre of inhibitor greater than the Bethesda unit / ml of plasma, preferably greater than 5 units Bethesda / ml plasma. It can also be prepared through the combination of individual factors II, IX and X highly purified as well as eventually VII. Since the biological material, is material that also originates from organisms or body fluids or microorganisms, which can be contaminated with pathogens such as for example infectious molecules or microorganisms or virtue or pyrogens, different procedures have been developed for the inactivation or reduction of pathogens or pyrogens. Such procedures include physical and / or chemical treatments, such as different filtering methods (for example nano-, dia- or ultrafiltration), heat treatment, acid or bleach treatment, detergent treatment and / or organic solvent as well as treatment with UV light or laser light. Also different combinations of these procedures for the inactivation or reduction of pathogens have been proposed multiple times in the state of the art. From EP 0 197 554, for example, a method for depyrogenicizing and inactivating viruses in a biological or pharmaceutical product is known, which encompasses treatment with a viral inactivating agent and depyrogenation agent, such as, for example, an animal substance and / or a solvent in the phase solid, in which the product is adsorbed. By means of this process, the viral inactivating agent or depyrogenation agent is separated from the solid phase, the adsorbed product is washed and finally eluted from the solid phase.

From EP 0 131 740 it is known to treat a composition containing proteins in a solution with organic solvents such as di- or trialguylphosphate, optionally in the presence of a detergent (solvent / detergent treatment), it being possible to obtain virus-free protein compositions. that contain lipids. A thermal treatment is known from AT-PS 402151, in which a preparation which is in an aqueous solution is added with a surfactant at a concentration of at least 1% by weight before heating it. Another method for the reduction or suppression of undesired activities in biological or pharmaceutical products is known from ep 0 083 999. This is based on prolonged contact with a solution or suspension of an amphiphile with non-denaturing effect. The depyrogenated product is treated with an ion exchanger to remove the amphiphile. A disadvantage of many of those methods known in the state of the art is that there frequently occur loses of activity in the labile proteins obtained in the compositions to be treated, for example in the blood proteins. Especially when performing a chromatographic purification step, a relatively extensive inactivation of the proteins occurs. A breakdown of proteins can also lead to activation. For example, it is known that factor VII in the case of a Chromatographic purification due to autocatalytic phenomena, it is easily activated in factor Vlla undesirable because it is very labile. Another disadvantage is the high expenditure in time and apparatus of many procedures, which greatly reduce their practicability and therefore make their use frequently technically unsuitable to a great extent. In the context of the present invention, a careful process for proteins, in particular for labile blood proteins, must be used for the effective inactivation of pathogens in biological materials, which can be used at a technically superior level and which is economically viable. Especially for the procedure to inactivate pathogens, disintegration and possible activation of sensitive proteins must be continuously avoided. In this method for inactivating pathogens, especially viruses, in a biological material this material is incubated with a chemical agent, the incubation being carried out in the presence of an eluotropic salt corresponding to a NaCl concentration of at least 200 mmol / 1, preferably when minus 300 mmol / 1. The inactivation of pathogens in the solution against the treatment of an adsorbent offers some advantages. Thus, for example, it is more possible to practice such a procedure in a homogeneous one-phase system and also the validation of the inactivation step. It also seems that better access to pathogens in a relatively homogeneous phase increases the efficiency of the process step. The biological material preferably contains a human protein and is in particular plasma or a plasma fraction or originates from a cell culture. Preferably the biological material contains a blood factor, such as factor XII, XI, VIII, V, von Willebrand factor or fibrinogen, especially a protein derived from vitamin like factor II, factor VII, factor IX, factor X, protein C , protein S or protein Z. The proteins may be present as individual factors, preferably in purified form, or in a complex mixture. In a particularly preferred embodiment, the biological material contains at least one factor of the prothrombin complex and is in particular a fraction containing a prothrombin complex or a material containing factor VII, it is derived, for example, after the cryoprecipitation of the residue plasma corresponding (cryosidual). The preparation according to the invention is preferably one with FEIB activity (Factor eight Inhibitor Byoassing Activity - deviating activity of the factor eight inhibitor), also a preparation which is suitable for the treatment of patients with factor VIII inhibitor.

The material originating from a cell culture is preferably a material containing recombinantly prepared blood factors, including intrinsic and intrinsic coagulation factors, fibrinolysis, thrombolysis or its inhibitors, in particular vitamin K-dependent blood factors. common cells for the expression of recombinant proteins are preferred, preferably mammalian cells, such as Vero, CHO or BHK cells. The corresponding proteins can be directly subjected from the crude cell extract to the process according to the invention for the inactivation of the pathogens eventually present, however it can also be a previously purified cell fraction. The gum agent is for example a detergent (amphiphile, surfactant which preferably is contained in an amount of at least 1%, more preferably more than 5%, most preferably 10%, however other chemical agents according to the invention may be used, especially those of which already They are known to have a virucidal, bactericidal or depyrogenicidal effect, or mixtures of the most diverse guiding agents, although the selection is limited because the nature of the biological material must not be impaired in an essential way. a guímica substance that contains more than 50% of biological activity of the material, in relation to the activity before the incubation, preferably at least 70%, especially more than 85%. The term biological activity content means that the proteins contained in the biological material can exert their natural inherent function or different functions. This biological activity can be determined and indicated depending on the type of the protein, for example by means of a standardized chromogen analysis or by means of the determination of antigens. Eventually the chemical agent is removed after incubation. Detergent is generally understood as a synthetic, organic, surfactant substance. Preferably, a non-ionic detergent is used in the process according to the invention. Nonionic surfactants such as polyether, especially alkylphenol polyglycol ether, are among other products of the ethoxylation of fatty acids, fatty acid amides, fatty amines, fatty alcohols, amino-oxides, fatty acid esters of polyalcohols and sucrose esters. . Such a surfactant acts on the proteins in a non-denaturing manner and is preferably selected from the group of polysorbats and Triton. As polysorbate, for example Tween ™ is used. When detergents are used as guimic agents then in a preferred embodiment they are used without the addition of other agents, especially without the addition of toxic organic substances or solvents such as TNBP for example. This minimizes the risk of contamination. The biological material according to the process according to the invention is incubated with a gum agent. Incubation means contacting the biological material with a solution, suspension or emulsion of a guímico agent for a pathogen or pyrogen optionally present for inactivation, for a sufficiently long period of time at a certain temperature. Contacting can be done simply by letting the mixture stand for a defined period of time. The incubation is carried out according to the present invention in the presence of an eluotropic salt. Under the term "eleutropic salt" it should be understood from here onwards salt mixed with a chemical agent or salt in a complex composition, with the property of releasing and / or expelling the adsorbed substances, solid adsorbents or impregnated with liquid or gelatinous Preference is given here to the case of eluotropic salts of a desorption agent, such as is used in chromatographic processes. The substances adsorbed inter alia is sufficiently soluble in the presence of the eluotropic salt that is, preferably conditions are selected, which do not cause the precipitation of biological material. The type and concentration of the salt or composition is usually selected according to the selected adsorbent. The eluant effect of a salt depends, for example, on the polarity of the solvent, for example in the ethanol-acetone-methanol-water sequence. The adsorbent may be a solid phase, especially a suitable matrix for ion exchange chromatography. Other additives, such as, for example, other salts, may also be contained in the composition containing the eluotropic salt. Preferably here the composition is an aqueous composition with a pH value in the range between 6.0 and 8.0, preferably around 7.0. In a preferred embodiment sodium chloride is used as an eluotropic salt, but also other alkali metal salts or also alkaline earth metal salts, including CaCl 2. As eluotropic salts, so-called chaotropic salts, such as for example urea, rhodanide or guanidine, can also be used. The concentration of the salt is at least > 200 mmol / 1, preferably > 300 mmol / 1. The upper limit of the concentration used depends in particular on the solubility of the salt in question and for the case of NaCl is for example about 2 mol / 1. Chaotropic substances, such as for example urea, can be used up to a concentration of 8 mol / 1.

The incubation of the biological material with the guímico agent is carried out for the inactivation of the pathogens eventually present, during a sufficiently long period of time, preferably during a period of time between 10 minutes and 10 hours, most preferred between 1 and 5 hours. The period of time required for the method according to the invention can be determined in a previous study by means of model viruses such as HIV, Sindbis, FSM.E or hepatitis viruses. Also the selection of the temperature influences the period of time to use. In the process according to the invention, it is most preferably incubated at room temperature, for example in a temperature range between 15 and 45 ° C, especially between 20 and 30 ° C. In the process according to the invention the biological material is preferably adsorbed on a solid carrier, purified and the incubation performed directly after the elution of the purified material. The elusion and incubation can be performed consecutively, but also simultaneously. According to another preferred embodiment, the incubation of a biological material is carried out after a chromatographic purification, whereby the eluate was further processed, for example by means of centrifugation, filtration or other physical methods.

The solid carrier in the most preferred form or material suitable for chromatography, especially a material suitable for ion exchange chromatography, hydrophobic chromatography or affinity chromatography. For example, materials such as Sepharose®, Superdex, Sephadex®, Spherodex®, Toyoperarl® or inorganic materials with hydroxylapatit are used. As an ion exchanger, anion exchange materials can be used, such as DEAE-Sephacel®, DEAE-Sephadex®, DEAE-Sepharose®, CL6B, DEAE-Sepharose® Fast Flow, QAE-Sephadex®, Q-Sepharose® Fast Flow, Q -Sepharose® High Performance, DEA-Tris-Acryl, DEAE-Spherodex®, Q-Hyper-D (obtainable from Sepracor), DEAE-Toyopearl®, QAE-Toyopearl®, Fractogel® EMD-TMAE or gold fractogel material. Examples of hydrophobic chromatography materials which may be mentioned are, for example, butyl-Sepharose® ™, octyl-Sepharose®, phenyl-Sepharose®, Fractogel®TSK-butyl, t-butyl-HIC support or Toyoparl® butyl TSK-gel. The biological material can be directly adsorbed from a complex mixture in a carrier and purified to the inactivation step, however other steps for the purification of the material can precede it or follow it, with other steps of chromatographic purification being preferred within the framework of the present invention. Through the procedure according to the invention the pathogens are inactivated. Pathogens are understood to be fragments of, for example, viruses, especially also the isolated genome or its fragments. Pathogens can be lipid-coated pathogens, such as hepatitis B virus or non-lipid-enveloped pathogens such as hepatitis A virus. Virus inactivation procedures are nowadays listed as effective when after the use of the procedure In a sample of a biological material that was mixed with a high dose of a test virus, for example the IH virus or the Sinbis as a model virus, none of the viruses could be demonstrated in the sample and the virus concentration was thus reduced by below the determinable limits. The determination and quantification of nucleic acids can be carried out, for example, by means of a PCT method, as described in AT-PS 401 062 or by means of direct titration. As a measure of the inactivation, the so-called reduction factor is known, which is calculated after an addition of the test virus of the decimal logarithm of the quotient of the initial and final concentration. Based on the European guidelines EC III-8115/89-EN of the commission of European communities, the so-called total reduction factor is known. It is calculated from the sum of the reduction factors of subsequent individual inactivation measures. Another step is also preferably carried out independent for the inactivation or reduction of pathogens. All the methods known in the state of the art, to minimize the risk of infection, come into consideration here. Especially as an additional step for the inactivation or reduction, a filtering and / or a heat treatment is carried out. As a filtrate, a nanofiltration is preferably carried out. A preferred heat treatment is carried out in the solid biological material, for example in a lyophilisate with a controlled water content, for example a water content between 5 and 8% and a temperature between 50 and 80 ° C, as described in EP -0 159 311. In a preferred embodiment, a 2-stage treatment with a detergent as a chemical agent is envisaged. Thus a detergent is used in a first step in an amount of at least 1%, preferably at least 5%, most preferably at least 10%. In a second step, another detergent is used in an amount of at least 10%, preferably at least 12%, most preferably at least 14%. The detergent used can be the same in both stages, but different detergents can also be used. Generally, by means of the combination of steps for the inactivation of viruses, the risk of a viral infection is greatly reduced or excluded after the administration of the corresponding preparation.

According to the present invention, a chromatographically purified preparation is also prepared, which contains a blood factor activatable autodynamically with a fraction of activated blood factor less than 50%, in relation to the content of activated and non-activated blood factor, preferably less than 40% , more preferably less than 30%, still more preferably less than 20%, still more preferably less than 10% and most preferably less than 1% and a detergent content. In particular, the preparation is a preparation containing a prothrombin complex with a factor Vlla activity of less than 50%, based on the content of activated and non-activated factor VII, preferably less than 10%, most preferably less than 1%. . The detergent content of the preparation according to the invention is in a pharmaceutically acceptable amount, preferably between 1% and the determinable limit of the detergent. Under the term autodinamically activatable blood factor, according to the present invention, a blood factor must be understood that is activatable autocatalytically by means of surface contact or by means of processes, such as for example chromatographic processes. Especially that type of blood factor is one selected from the group consisting of factor VII, factor XII, factor XI and pre-kallikrein.

In another preferred embodiment, the preparation is free of serine protease inhibitors, such as for example thrombin inhibitors or cofactors such as, for example, heparin. In a special embodiment, the absence of this type of substance is already shown during a chromatographic process. Therefore, the present invention also relates to the corresponding preparations obtainable by means of the process according to the invention. Other additives may also be contained in the preparation according to the invention, for example stabilizing substances such as amino acids. The following examples should clarify the present invention in detail, without limiting it to them. Example 1: Determination of factor VIII The determination of the factor VIII antigen in FEIBA was carried out according to the method of Moritz B. al. (Thromb. Haemost, 1997, supplement: 31). In this determination by means of a monoclonal antibody directed against the light chain of the molecule as capture antibody and by means of a monoclonal antibody also against the light chain of the factor VIII molecule, but against another epitope, as detection antibody, The presence of factor VIII is shown selectively with other plasma proteins in FEIBA.

Example 2: Determination of phospholipid Organically bound phosphate is extracted from the lyophilized powder of the FEIBA fraction by means of the method of Folch J. et al. (J.Biol.Chem. 1957, 226: 497-509) by means of a mixture of solvents consisting of chloroform, methanol in a ratio of 2 volumetric parts of chloroform to 1 volumetric part of methanol. The extract obtained containing the entire phospholipid fraction was subsequently conducted in teflon containers and the organic solvent evaporated under a stream of nitrogen. After the addition of a buffer (20 mM Tris HCl, 150 mM NaCl, pH 7.4) and Oxisolv reagent (Merck), the teflon containers were sealed and digested for 5 hours at 160 °. The phosphate released by means of the digestion process was determined quantitatively by means of photometry as a molybdate complex according to the method of Ames B.N. (Methods an Enzymlogy 1966, 8: 115-118). Example 3: Preparation of an affinity carrier binding to a factor VII A CHO cell clone that produces the von factor Recombinant Willebrand is prepared as described in FEBS.Lett. 1994; 351: 345-348. By means of transfection with a coding vector, the human furin cDNA (van den Ouweland et al., Nucleic Acids Res. 1990; 18: 664) took the cell family to the co-expression of human furin. That type of clones Stable cells are fermented to a large extent in perfusion reactors on microcarriers (Blüml et al., in. Spier RE, Griffith JB, Berthold W, eds, Animal cell technology, Oxford, London: Butterworth-Heinemann 1994: 267-269). The purification was carried out by means of a 2-step chromatographic procedure according to Thromb.Haemost. nineteen ninety five; 73: 1160. The desorbed fraction was obtained by elution with common salt and buffer was changed by means of gel filtration on Sephadex G25 (Firma Pharmacia) in a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5. Then the preparation by means of ultraconcentration on an Amicon YM30 membrane (cut: 30,000 D) was concentrated at a protein concentration of 3 mg / ml. The concentration of von Willebrand factor in this preparation consisted of 60 Units vWF antigen / mg protein. The preparation of the recombinant von Willebrand factor was diluted to 1.5 mg / ml with a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5. A previously activated gel suitable for affinity chromatography (Actigel, ALD-Superflow, Signature Sterofene), was pre-washed excessively with a buffer containing 20 mM tris-HCl, 150 mM NaCl, pH 7.5. One part by volume of the previously washed gel was mixed with 1.1 volume parts of the protein solution to be immobilized and then 0.15 parts were added. of a solution of 0.1 M cyanoborohydride (NaCNBH3) in 0.1 M phosphate buffer, pH 7.0. The gel was suspended in the buffer by means of stirring and by subsequent stirring it was incubated for 16 hours at room temperature. The gel was then washed in a sintering funnel with 10 times the volume of a buffer containing 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and washed with 5 times the volume of a buffer, containing 20 mM Tris-HCl, 2 M NaCl, pH 7.5. Then it was equilibrated again with 5 volumetric parts of the buffer 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 and the gel was transferred to a chromatographic column with a dimensional diameter at the height of the gel bed of 1: 4. By determining the concentration of protein in the separate solutions of the sinter funnel of the incubation residue of the von Willebrand factor solution and the affinity gel as well as the washing solutions, a higher coupling rate could be determined. 90% of the protein used. Example 4: Preparation of prothrombin complex free of factor VIII by contact with an affinity gel. { at this time considered by the applicant as the best way to carry out the invention): The pharmaceutical preparation FEIBA S-TIM4 IMMUNO (1000 units) was reconstituted with 2 ml of distilled water. After the complete dissolution of the dry powder by freezing, the solution had a concentration of active substance of 5 units per ml. 10 of that solution were contacted with 100 mg of the immobilized von Willebrand factor as previously described and incubated for 1 hour at room temperature under slight agitation. Then immobilized and removed by means of filtering through a sintering funnel. The FEIBA solution obtained in the filtrate showed an activity of 49 FEIBA units / ml. By means of the binding of factor VIII contained in the preparation and immobilized von Willebrand factor, the de facto antigen VIII in this preparation was found below the determinable limit. Example 5: Preparation of activated prothrombin complex libr of factor VIII by means of readsorption in a nonspecific carrier 15 mg of DEAE-Sephadex® A-50, Pharmacia Signature, were incubated at room temperature with 1 ml of a solution of 30 g / 1 NaCl in water for its swelling. The gel is then separated from the upper residue by means of centrifugation. Subsequently, five washes were carried out with the same one of absorber (9 g / 1 Na2HP04.2H20, 7 g / 1 NaCl, pH 7.0) and two other washes with a buffer (7 g / 1 Na2.2 citrate). ,, 0 7 g / 1 NaCl) also by means of resuspension and centrifugation. 30 ml of freshly frozen human citrate plasma was thawed at 0- + 4 ° C and the cryoprecipitate was separated by centrifugation at + 2 ° C. The resulting "cryosoduode" was incubated with the washed DEAE-Sephadex®, whereby FEIBA was generated and together with the factors of the prothrombin complex, factor VIII and an inert protein was adsorbed on the gel. Then the inert coadsorbed protein was separated from the DEAE gel by washing with a buffer (9 g / 1 Na2HP04.2H20, 7 g / 1 NaCl). The gel-protein complex moistened with buffer was now suspended with 1.5 ml of a solution of 150 mg / ml TWEEN®-80 and 30 mg / ml NaCl 1 hour at 26 ° C. By means of the treatment with the solution of greater ionic strength the protein was desorbed together with the factors of the prothrombin complex factor VIII. The suspension was then diluted by the addition of 6.5 ml of water and readsormed for 1 hour at room temperature, the prothrombin complex fraction readsorbing again. Simultaneously only a reduced part of the factor VIII contained in the protein fraction was readsorbed in the gel. The gel / protein complex was washed five times with 1 ml of a solution of 7 g / 1 NaCl in detergent-free water. For the elution the gel was treated with 0.7 ml of a solution of 30 g / 1 NaCl in water with stirring. The eluate was dialyzed against distilled water, frozen and lyophilized. After reconstitution of the lyophilisate, the FEIB activity was determined according to AT 350 726. In addition, the factor antigen content was determined VIII. As a control, a FEIBA preparation prepared in a conventional manner was used, as described in AT 350 726. The preparation obtained according to the described procedure had a content of factor VIII reduced by a factor of 10 in comparison with the control. By means of the contact with the detergent the pathogens eventually present were also inactivated, especially the viruses enveloped in lipids. Example 6: Preparation of the free prothrombin complex of factor VIII and free of phospholipids by means of readsorption in a non-specific carrier. 15 mg of DEAE-Sephadex®-50, from Pharmacia, were incubated for 15 minutes at room temperature with 1 ml of a solution of 30 g / 1 NaCl in water until it was swollen. The gel was then separated by centrifugation of the swelling residue. Subsequently, 5 washes of the gel were performed with 1 ml of buffer (9 g / 1 Na2HP04.2H20, 7 g / 1 NaCl, pH 7.0) and another two washes with a buffer (7 g / 1 citrate Na3.2H20, 7 g / 1 NaCl) also by means of resuspension and centrifugation. 30 ml of freshly frozen human citrate plasma was thawed at 0- + 4 ° C and the cryoprecipitate was separated by centrifugation at + 2 ° C. The resulting "cryosoduode" was incubated with the washed DEAE-Sephadex®, whereby FEIBA was generated and together with the prothrombin complex factors, the factor VIII, phospholipids and an inert protein were adsorbed on the gel. Then the inert coadsorbed protein was separated from the DEAE gel by washing with a buffer (9 g / 1 Na2HP04.2H20, 7 g / 1 NaCl). The gel-protein complex moistened with buffer is now suspended in 1.5 ml of a solution of 1 mg / ml TWEEN®-80 and 30 mg / ml of NaCl for 1 hour at room temperature, where the protein fraction and impurities nonspecifically adsorbed were desorbed. The gel was then separated by filtration. The protein solution, by means of the subsequent addition of TWEEN®-80, was brought to a detergent concentration of 150 mg / ml and then incubated for 1 hour at 40 ° C with stirring. It was then diluted by the addition of 6.5 ml of water and readsorbed in a previously prepared and freshly washed DEAE-Sephadex® A-50 gel, where again the prothrombin complex fraction was readsorbed. Simultaneously, a small part of the factor VIII contained in the protein fraction was ligated to the gel. Subsequently, by means of five washes with 1 ml of a solution of 7 g / 1 NaCl in water, the detergent was removed. Here also the present phospholipid, which was solubilized by contact with the amphiphile, was eliminated. For the elution the gel was treated with 0.7 ml of a solution of 30 g / 1 NaCl in water with stirring. The eluate is dialyzed against distilled water, frozen and lyophilized. After reconstitution of the lyophilisate, the FEIB activity was determined according to AT 350 726. In addition, the content of de facto VIII antigen and phospholipid was determined. A preparation of FEIB prepared in a conventional manner, obtained in the manner described in AT 350 726, served as control. The preparation obtained according to the described procedure had a lower factor VII content compared to the control and was free of phospholipids. . The results of the analysis are summarized in Table I. Table 1 FEIBA: CONTENT OF FVIII / FOSFOLIPIDOS

Claims (20)

1. CLAIMS l.- Immunotolerant pharmaceutical preparation of prothrombin complex containing factors II, IX, X and possibly VII, characterized in that it has a content of factor VIII antigen less than 0.1 factor VIII: C antigen / FEIBA unit.
2. 2. - Pharmaceutical preparation according to claim 1, characterized in that it is prepared from plasma or a plasma fraction.
3. 3. - Pharmaceutical preparation according to claim 1 or 2, characterized in that the preparation contains at least one of the factors IXa, Xa and Vlla and exhibits FEIB activity.
4. 4. Pharmaceutical preparation according to one of claims 1 3, characterized by being free of phospholipids.
5. 5. - Process for producing a preparation according to one of claims 1 to 4, characterized in that it consists of the following steps: a) obtaining plasma or a plasma fraction containing the factors II, IX, X and eventually VII, b ) contacting the plasma or plasma fraction with a carrier material, possibly in the presence of a detergent, such that factor VIII and eventually the phospholipids are separated from factors II, IX, X and optionally VII, c) purify the plasma or plasma fraction, and d) recover a fraction containing the factors II, IX, X and eventually VII.
6. 6. Method according to claim 5, characterized in that steps b and c are performed as a single procedure step.
7. 7. Method according to claim 5 or 6, characterized in that the carrier material is a material suitable for chromatography, filtration and / or nanofiltration.
8. 8. - Process according to one of claims 5 to 7, characterized in that the carrier material is a specific carrier material for factor VIII, especially a suitable matrix for affinity chromatography.
9. 9. - Method according to claim 8, characterized in that the matrix is a matrix containing vWF.
10. 10. - Process according to one of claims 5 to 9, characterized by the factor VIII and possibly the phospholipids are adsorbed on the carrier material.
11. 11. Process according to one of claims 5 to 7, characterized in that the carrier material has a higher affinity for the prothrombin complex than for factor VIII and is in particular a weaker anion exchanger.
12. 12. - Process according to claim 11, characterized in that factors II, IX, X and possibly VII are adsorbed while the factor VIII and the eventual phospholipids are eluted.
13. 13. Process according to claim 11, characterized by factors II, IX and X and eventually VII are not bound to the carrier material.
14. 14. Method according to one of claims 5 to 13, characterized in that the detergent is a non-ionic detergent, especially a polyether.
15. 15. Method according to one of claims 5 to 14, characterized in that the detergent is removed after contact with the plasma or the plasma fraction.
16. 16. - Method according to one of claims 5 to 15, characterized in that the plasma fraction is prepared by means of chromatography, precipitation or centrifugation, or is the cryoprecipitation residue.
17. 17. Method according to one of claims 5 to 15, characterized in that the plasma fraction has at least an intermediate purity.
18. 18. Method according to one of claims 5 to 17, characterized in that at least one step is carried out for the inactivation or reduction of viruses or constituent parts of viruses, especially selected from the group of thermal treatment, steam treatment, treatment with a solvent and / or treatment with a detergent and nanofiltration.
19. 19. - Preparation according to claim 1 characterized in that it can be obtained with a method according to claim 5.
20. 20. Use of a preparation according to claim 1 for the preparation of a medicament for the treatment of patients with inhibitor of factor VIII (= hemophilia A), especially of those patients with a concentration of inhibitor greater than 1 unit Bethesda / my plasma, preferably greater than 5 units Bethesda / ml plasma.