EP1015020A1

EP1015020A1 - Pharmaceutical substance containing various vitamin k-dependent factors

Info

Publication number: EP1015020A1
Application number: EP98944886A
Authority: EP
Inventors: Yendra Linnau; Peter Turecek; Hans-Peter Schwarz
Original assignee: Baxter AG
Current assignee: Baxter AG
Priority date: 1997-09-19
Filing date: 1998-09-17
Publication date: 2000-07-05
Also published as: AU743102B2; NO20001415D0; WO1999015196A1; CA2304396A1; AU9244998A; BR9812222A; SK4022000A3; HUP0003681A1; JP2001517636A; NO20001415L; ATA159197A; AT409334B

Abstract

The invention relates to a pharmaceutical substance separated from a prothrombin complex, containing at least two different blood factors which are highly purified and vitamin k-dependent.

Description

Pharmaceutical preparation containing vitamin K-dependent factors

The invention relates to a pharmaceutical preparation containing vitamin K-dependent individual factors.

Protein K-dependent proteins are characterized by the fact that they require vitamin K in an essential way for their biosynthesis. For example, prothrombin (factor II), which is formed under the influence of vitamin K antagonists, cannot bind Ca ²⁺ in contrast to normal prothrombin. Normal prothrombin contains γ-carboxyglutamate at the N-terminal end, i.e. a second carboxyl group on the glutamate residue. In the course of the biogenesis of functional prothrombin, the first ten glutamate residues in the amino-terminal region of the protein are carboxylated by a vitamin K-dependent enzyme system to give γ-carboxyglutamate. This γ-carboxyglutamate group is a very strong chelator for calcium ions. ^Prothrombin is bound to phospholipid membranes, which originate from cell membranes, eg from platelets, via these bound Ca ²⁺ ions in order to obtain the correct topology for initiating blood coagulation.

Not only does prothrombin have γ-carboxyglutamate residues, but the coagulation factors VII, IX and X are carboxylated on specific glutamate residues in order to develop a high affinity for calcium ions. However, other proteins involved in the coagulation cascade, such as Protein C, Protein S and Protein Z, also need vitamin K for their biosynthesis.

Vitamin K-dependent individual factors, in particular factors II, VII, IX and X, have similar physico-chemical properties, such as similar molecular weights, pl's, electrophoretic mobility, etc., and are therefore generally referred to as a prothrombin complex (other name : Factor IX complex or PPSB complex). Due to the similar protein characteristics, it is difficult to prepare the factors individually. The production of prothrombin complex preparations with simultaneous isolation of all the factors contained was therefore always preferred in the prior art over the production of blood factor concentrates in the production of pharmaceutical preparations (see Brummelhuis in: Methods of Plasma Protein Fractionation, ed. Curling, 1980, Academic Press, pages 117-128). However, it was also found that the prothrombin complex factors, owing to their different stability or half-life, can never be obtained in a physiological ratio (1 U of the protein in each case) (see Müller et al., Krankenhauspharmazie 13 (11), (1992), 528-531; Köhler et al., Thrombosis Research 60 (1990), pages 63-70).

EP-A-0 700 684 describes a prothrombin complex concentrate together with at least one further component which promotes blood coagulation as an antidote for blood anticoagulants.

There is a risk that prothrombin complex concentrates contain activated coagulation factors, which are mostly active serine proteases, due to their purification from complex protein mixtures. However, patients in particular should not be coagulated, since the development of thrombosis can or is fatal. Even if only traces of these activated coagulation factors, in particular thrombin, are present in such a preparation, proteolytic inactivation of the individual factors occurs, which, depending on the individual stability of the individual factors, can have serious consequences. Overall, prothrombin complex concentrates therefore tend to be unstable and are unsuitable for long storage. These degradation reactions, in particular through thrombin, even occur in the solid state, i.e. drying or lyophilization of the preparations does not lead to reliable storage stability.

Furthermore, prothrombin complex concentrates are extremely inflexible with regard to the relative ratios of the individual factors contained. There are hardly any possibilities to influence these relations in the course of the purification of the prothrombin complex, which is particularly disadvantageous when a prothrombin complex enriched with certain factors is desired. It is therefore an object of the present invention to provide pharmaceutical combination preparations of vitamin K-dependent proteins which are precisely defined or definable in their composition, have high stability, in particular in the case of long-term storage, and high flexibility in variation of their composition.

This object is achieved according to the invention by a pharmaceutical separated prothrombin complex preparation containing at least two chromatographically purified vitamin K-dependent individual factors as active substances. Above all, the preparation according to the invention should contain highly purified factor IX in combination with at least one further highly purified vitamin K-dependent single factor.

In contrast to the prior art, in which the prothrombin complex was always purified as a complex and was not produced by a combination of individual factors and, moreover, in the best case only exists to an intermediate, ie only up to moderate, purity, the present invention a preparation is made available which contains the individual blood factors in highly purified form, which are freed from disturbing impurities, in particular from thrombin activity. In particular, the individual factors to be combined according to the invention are purified from plasma or recombinant cells by chromatographic purification processes, such as ion exchange chromatography, hydrophobic chromatography, affinity chromatography and / or molecular exclusion chromatography. In this way, specific activities of at least 50% of the theoretical purity, preferably at least 70%, in particular at least 90%, up to the theoretical purity for the individual factor can be achieved for most vitamin K-dependent individual factors. Accordingly, it is also preferred to use factors that are substantially free (≤ 5%) of denaturing products.

In the pharmaceutical production of blood coagulation protein preparations, three different degrees of purity are generally distinguished; low, intermediate and high. Table 1

in vivo half-life theoretical purity

Factor II 60 h 0.1 U / mg

Factor VII 2 - 2.5 h 2000 (1667 - 2500) Factor IX 18 - 24 h 250 (200 - 333) Factor X 40 h 118 (100 - 143)

In the determination of factor II activity in particular, there are always falsified results due to the presence of traces of factor Ha, since traces of factor Ha already overlap the concentration determination of factor II in such a way that values are even many times higher than the theoretical Purity can be determined. The factor VII values are therefore somewhat lower than the other factors, since factor VII is a very unstable protein that is converted to factor VIIa very quickly. Therefore, a factor VII preparation that has more than 10% of the theoretical purity is regarded as highly purified.

Since the preparation according to the invention is composed of individual factor preparations which are very precisely defined with regard to their activity or their degree of purity, the ratio of the individual factors to one another can also be set optimally. Problems that arise in the course of further processing of the total protein complex concentrates in the prior art, for example through loss of activity during virus inactivation, but also through processes that occur in the course of the purification process, can also be avoided from the outset, since after the union of the highly purified individual factors for the preparation according to the invention, preferably no further processing steps are carried out.

The preparation according to the invention thus has the overall advantage of being standardizable. This ensures that the respective individual factors are contained in the concentration +/- 10% deviation in the preparation.

Preferred individual factors are selected from the invention the group consisting of factor II, factor VII, factor IX, factor X, protein C, protein S and protein Z. Preferred production methods for highly purified preparations of these proteins can be found, for example, in EP 0 796 623 (factors II and X), the A 594/97 (factor VII), EP 0 496 725 (factor IX), EP 0 533 209 (protein C) and EP 0 406 216 (protein S).

In the preparation according to the invention, at least the factors II, VII, IX and X are preferably combined starting from highly purified individual factors, the highly purified individual factors protein C, protein S and / or protein Z possibly also being added in order to be able to provide a prothrombin complex which is as physiological as possible, ie a prothrombin complex whose composition corresponds to the physiological one - only without the disruptive accompanying proteins that flow from plasma during the course of the prothrombin complex purification, e.g. Thrombin.

The individual factors can be purified from plasma, in particular human plasma, or can be produced recombinantly. Since it is not necessary to separate the structurally and physicochemically very similar factors in the production by recombinant DNA technology, the preparation according to the invention is preferably composed of highly purified recombinant individual factors. The factor can also be produced transgenically; it can be a derivative, in particular a peptide and / or a fragment.

The vitamin K-dependent individual factors Factor II, Factor VII, Factor IX, Factor X, Protein S and C have been cloned and sequenced and their representation is described for example in Falkner et al. , Thrombosis and Haemostasis 68 (2) (1992), pages 119 to 124 for vitamin K-dependent proteins.

According to the invention, the relative values of the highly purified individual factors can be set easily and in any relation to one another in the preparation, for example in that these conditions correspond to the natural conditions in the blood, that is to say approximately one unit of the other per unit of one factor Factor is present. A preferred preparation according to the present invention therefore contains the highly purified individual factors Factor II, Factor VII, Factor IX and Factor X in a relative ratio, based on international units of (0.5 to 2): (0.5 to 2): 0, 5 to 2): (0.5 to 2). If protein C, protein S and / or protein Z are also present in the preparation, these individual factors also preferably have relative ratios of 0.5 to 2.

On the other hand, according to the invention it is also possible to set the individual factors on the basis of their relative stabilities, in particular in relation to their relative half-lives, i.e. that more is provided from a less stable factor and correspondingly less from the stable factor. The intended period of use or effect can also be taken into account, i.e. the longer this period of time, the greater the weighting of these relative values.

Recombinant proteins with a changed half-life, for example, can then also be standardized accordingly. Other factors such as in vivo recovery can be included in a standardization of the factors.

A preparation which is preferred in this regard therefore comprises the individual factors Factor II, Factor VII, Factor IX and Factor X in a relative ratio, based on international units of (0.5 to 2): (5 to 35): (0.5 to 7 ): (0.5 to 5) because the half-lives of prothrombin are 60 hours, of factor VII 2 hours, of factor IX 20 hours and of factor X 40 hours. A preferred preparation with these factors therefore contains the individual factors Factor II, Factor VII, Factor IX, Factor X, Protein C and Protein S in a relative ratio, based on international units of (0.5 to 2): (5 to 35): (0.5 to 7): (0.5 to 5): (1 to 15): (1 to 15).

Since factor II is by far the most stable of these factors and, in its activated form, thrombin also bears the greatest risk of stability, a preparation that does not contains thrombin, preferred. Factor VII is usually considered to be quite unstable and is therefore preferably provided in an increased amount, for example in 10-fold concentration (based on international units) in the preparation according to the invention. A particularly preferred preparation therefore contains individual factor VII and individual factor II in a ratio of greater than 10: 1.

The present preparation preferably provides a prothrombin complex or a partial prothrombin complex composed of highly purified individual factors. In any case, it is preferred that the individual factors in the preparation do not form a complex. This can be demonstrated, for example, by analytical ion exchange chromatography on Q-Sepharose (Pharmacia), the individual factors being able to be eluted discretely when eluted with a salt gradient. In contrast to this are the complexes as they are present in the prothrombinase or in the pro-prothrombinase.

Prothrombinase is an enzyme-substrate complex that forms on a phospholipid surface and enables the activation of prothrombin. By definition, prothrombinase consists of factor II (prothrombin), activated factor X (factor Xa), cofactor V or Va, phospholipids and calcium ions. These factors exist in vivo as a transient complex for activating the prothrombin and formation of the thrombin. A corresponding pro-prothrombinase is defined as a complex of factors which are at least partially modified or activated to form a prothrombinase. The pro-prothrombinase is therefore to be understood as a precursor of the prothrombinase and as a complex in which one or more components are present in their precursors, as zymogens or as proforms and which is formed on the basis of affinities of the components to one another.

For reasons of stability, it is advantageous to avoid any presence of activated coagulation factors in the preparation according to the invention. A preferred embodiment is therefore characterized in that the preparation has no activated coagulation factors, in particular no factor Ha, IXa, Xa and given if Vlla contains.

Preferred preparations according to the invention contain less than 0.1 U factor VIII: C or factor VIII: Ag / mg protein and / or less than 0.1 U factor Ila / unit prothrombin and / or less 0.1 U factor Xa / unit factor X .

In order to keep the excellent stability of the preparation according to the invention as long as possible, it is advantageous to provide the preparation according to the invention in lyophilized form. This makes it possible to store the preparation according to the invention almost indefinitely and still reconstitute it as a lyophilisate to a ready-to-use solution within a short time.

According to a further preferred embodiment, the preparation according to the invention further contains magnesium ions. These ions have a competitive effect on calcium ions and can displace the calcium ions especially in the complete or partial prothrombin complex. This prevents premature thrombin formation in a solution of the preparation according to the invention to an even greater extent and thus stabilizes it to such an extent that it remains stable for many hours even in an aqueous solution.

It has been found that the pharmaceutical preparation according to the present invention can even be provided as a stable infusion solution, especially if it is ensured that it does not contain any free calcium ions. The free calcium ion content can easily be determined by known ion titrations or other analytical methods. For complexing the calcium ions e.g. a pharmaceutically acceptable chelating agent, preferably EDTA, and related substances such as citrate.

The preparation according to the invention preferably also contains antithrombin III in the amounts in which it has hitherto been used as stabilizing agent in prothrombin complex concentrates, optionally together with heparin. Although this measure due to the high degree of purity of the individual factors does not appear to be absolutely necessary, it can be regarded as advantageous for pharmaceutical reasons or also the requirements of pharmacopoeias or other regulations with regard to the prothrombin complex concentrates of the prior art.

In another preferred embodiment, the preparation is therefore free from albumin and / or stabilizers, such as in particular antithrombin III and / or heparin. In particular, the preparation according to the invention is also free of phospholipids.

According to a preferred embodiment, the pharmaceutical preparation according to the invention is treated on the basis of a treatment for virus inactivation of infectious viruses or other infectious agents. This virus inactivation treatment is preferably ensured by two mutually independent virus inactivation or virus depletion methods.

This inactivation treatment is preferably ensured by a tenside and / or heat treatment, for example by a heat treatment in the solid state, in particular a steam treatment according to EP-0 159 311, EP-0 519 901 or EP-0 674 531.

Other treatments for inactivating viruses also include treatment with chemical or chemical / physical methods, e.g. with chaotropic substances according to W094 / 13329, DE 44 34 538 or EP-0 131 740 (solvent) or photo inactivation.

Nanofiltration or antibody-enhanced nanofiltration (see PCT / AT97 / 00075) is also a preferred method for depleting viruses in the context of the present invention.

The preparation according to the invention preferably also contains pharmaceutically acceptable buffer substances or stabilizers, for example the substances already provided for prothrombin complex concentrates in pharmacopoeias.

The pharmaceutical preparation according to the invention can of course be used for all previous indications of the prothrombin complex.

The present invention therefore also relates to the use of the preparation according to the invention for the production of a preparation for the treatment of acquired or inherited bleeding disorders, for the treatment of severe bleeding, for the prophylaxis of bleeding, in particular in the presence of inherited bleeding disorders, for substitution therapy and for the treatment of haemophilia B. .

The preparation according to the invention also proves to be indicated for liver dysfunction.

In the case of administration to patients, previous administration regimes can be used for the dosage, although the more precise definition of the preparations according to the invention is advantageous.

The present invention is explained in more detail with reference to the following examples, to which, however, it should not be restricted.

Example:

Example 1: Production of the single factor preparations

1.1. Manufacture of single factor preparations of plastic factor X and plastic factor II:

A lyophilized prothrombin complex factor preparation, which contained factors II, IX, X as well as protein C and protein S, was carried out according to the method of Brummelhuis, HGJ, Preparation of the Prothrombin Complex. In: Methods of Plasma Protein Fractionation, Curling, JM ed., 117-128, Academic Press, New York, (1980), and heat-treated for virus inactivation according to EP 159 311. The lyophilizate was made accordingly (1000 U factor X / g, 1200 U factor II / g) dissolved in distilled water so that it contained 50 000 U factor X / l, and adjusted to pH 7.0. After adding 12% (v / v) TWEEN ^® 80, the mixture was stirred at room temperature for 1 hour. The mixture was then diluted with a 20 mM Tris-HCl buffer, pH 7.0, 1: 5 and the prothrombin complex protein fraction on calcium phosphate (Ca ₃ (P0 ₄ ) ₂ ) in a concentration of 30 g Ca ₃ (P0 ₄ ) ₂ adsorbed per 1 prothrombin complex solution by stirring for one hour at room temperature. The solid phase was then separated by centrifugation, 20 minutes at 5000 rpm, and the precipitate was washed twice with 20 mM Tris-HCl buffer, pH 7.0, containing 10% ammonium sulfate, by resuspension and centrifugation again. A third wash was carried out in an analogous manner with 20 mM Tris-HCl buffer, pH 7.0, containing 150 mM NaCl. The prothrombin complex fraction was eluted with 1 M sodium phosphate solution, pH 7.0, 25 ml of this solution per g of calcium phosphate being stirred at room temperature for 1 hour and then the remaining precipitate being separated off by centrifugation as above. The supernatant was then subjected to ammonium sulfate precipitation with 366 g of ammonium sulfate per 1 h at 4 ° C. with stirring. The precipitate containing the prothrombin complex fraction was centrifuged off as above. The precipitate was 6.0, was added to a 25 mM trisodium citrate buffer containing 100 mM NaCl, 1 mM benzamidine hydrochloride, pH, and on a column filled with Sephadex ^® G-25, at 4 ° C with a linear flow rate of 1 cm / min buffered against 25 mM trisodium citrate dihydrate buffer containing 100 mM NaCl and 1 mM benzamidine hydrochloride, pH 6.0, in order to separate the ammonium sulfate. The UV absorption at 280 nm and the electrical conductivity were measured in the eluate stream. The protein-containing fractions were combined and then subjected to ion exchange chromatography on DEAE-Sepharose FF ^® , from Pharmacia. The fractions were applied to a column (inner diameter: gel bed height = 1: 1.3) with a gel volume of 8.2 1, 0.55 g protein / 1 gel, at a linear flow of 0.36 cm / min. The chromatography was carried out at 22 ° C. Before the proteins were applied, the column had been equilibrated with a 25 mM trisodium citrate dihydrate buffer containing 100 mM NaCl, 1 mM benzamidine hydrochloride, pH 6.0. The protein fractions were eluted in 1

and buffered against a buffer containing 4 g trisodium citrate dihydrate / 1.8 g NaCl / 1, pH 7.0. A factor II preparation prepared in this way had a specific activity of 6.9 U / mg protein. The factor II activity was determined using the 1-step method, based on the thromboplastin time, using a factor II deficiency plasma against the international factor II standard using the reagent combination from IMMUNO, Vienna. Other coagulation factors were traces or no longer detectable in coagulation analyzes (factor VII <0.00002 E / E factor II, factor IX 0.0002 E / E factor II, factor X 0.004 E / E factor II, protein C 0.003 E / E factor II and factor VIII <0.0002 E / E factor II).

As an alternative production method for a highly purified factor II, a method was also used in which factor IX was first separated from a lyophilized prothrombin complex factor preparation by means of hydrophobic chromatography, then factor II was isolated and this was then highly purified by chromatography on hydroxyapatite.

The prothrombin complex factor preparation was dissolved as described above and incubated with detergent for 1 h at room temperature. A Factor II, IX and X was subsequently by ion exchange chromatography on DEAE-Sepharose FF ^®, Fa. Pharmacia, containing fraction isolated. From this then the Factor IX-containing fraction was purified by interaction with Butyl-Toyopearl ^®, Fa. Toso Haas, removed. The adsorption supernatant was then purified on Phenyl-Sepharose High Performance ^®, Fa. Pharmacia through a further hydrophobic interaction, with about 4 grams of protein / 1 gel could be adsorbed. The protein fraction was adsorbed in a column with an internal diameter: gel bed height = 1: 1.9 ratio at a linear flow of 0.25 cm / min, then by washing with 20 mM Tris-HCl, 3 M NaCl, pH 7.4. the inert protein was removed and finally the fraction II containing fraction II, which desorbed from the gel with decreasing conductivity at 1.9 M NaCl, was isolated by stepwise elution. The fraction containing factor II was then adsorbed directly onto Ceramic-Hydroxyapatit ^{® from} Biorad. This was done on a column with a

1 1 1

exhibited, while the factor VIIa content was below one unit / ml. Accordingly, a highly purified factor VIII preparation could be obtained without a substantial activation of factor VII. The chromatography yield was over 50%.

1.3. Plasmatic factor IX:

150 ml of prothrombin complex were pre-cleaned using dextran sulfate (Miletich et al., Analytical Biochemistry 105, 304 (1980)). 20 ml of eluate (912 IU factor IX, 160 IU factor X) was applied to a column with 20 ml of an agarose polymer with octyl groups (Octyl-Sepharose-CL-4B (Pharmacia, Sweden)) at a flow rate of 360 ml / h plotted. The column was previously equilibrated with 80 ml of buffer A. After washing the loaded gel with 120 ml of buffer A, the factor IX-containing fraction was eluted with 80 ml of a 250 mmol NaCl solution.

The yield of factor IX was 54% of the initial activity. The specific activity was 186 IU. Factor IX / mg protein. Factor X was only present in traces.

1.4. Obtaining plastic protein C

High-purity Protein C was obtained from a crude Protein C fraction that was made from commercially available prothrombin complex concentrate. The purification was carried out by affinity chromatography using monoclonal antibodies. Anti-Protein C monoclonal antibodies were prepared as follows:

BALB / C mice were immunized with 100 µg human protein C at two-week intervals by intraperitoneal injection. After six weeks, another 50 μg of human protein C were injected and the fusion was carried out three days later. The myeloma cell line (P3-X-63-AG8-653, 1.5 x 10 ⁷ cells) was mixed with 1.7 x 10 ⁸ spleen cells from a mouse, and the fusion according to the modified Koehler & Milstein method using PEG 1500 carried out (Koehler G., Milstein C., Nature 256 (1975), 495-497). Positive clones, tested using an ELISA, were subcloned twice. Ascites production was carried out by injection of 5 x 10 ⁶ hybridoma cells per BALB / C mouse two weeks after Pristan treatment.

The immunoglobulin was purified from ascites by ammonium sulfate precipitation and subsequent chromatography using QAE-Sephadex and then chromatography on Sephadex G200. To reduce the risk of transmitting murine viruses, the antibody was subjected to a virus inactivation step before immobilization. The monoclonal antibodies against protein C thus obtained were coupled to CNBR-Sepharose 4B (Pharmacia). The following buffers were used to purify Protein C using affinity chromatography:

As adsorption buffer: 20 mM Tris, 2 mM EDTA, 0.25 M NaCl and 5 mM benzamidine; the following was used as washing buffer: 20 mM Tris, 1 M NaCl, 2 mM

Benzamidine, 2mM EDTA; the pH was 7.4; the following was used as elution buffer: 3 M NaSCN, 20 mM Tris, 1 M

NaCl, 0.5mM benzamidine, 2mM EDTA.

Another suitable monoclonal antibody for the purification of protein C is described in US Pat. No. 5,336,610. This antibody binds to the activation peptide of protein C and is therefore suitable for selectively purifying the zymogen protein C from the activated form.

In detail: The prothrombin complex concentrate was dissolved in the adsorption buffer, about 10 g of the prothrombin complex concentrate being used for a 20 ml monoclonal antibody column. The dissolved prothrombin complex concentrate was then filtered, centrifuged at 20,000 rpm for 15 minutes and sterile filtered through a 0.8 μm filter. The sterile-filtered and dissolved prothrombin complex concentrate was applied to the column at a flow rate of 10 ml / h. The column was then washed protein-free with the washing buffer and finally the bound protein C with the elu tion buffer eluted at a flow rate of 5 ml / h and the fractions collected. The eluted protein C was dialyzed against a buffer (0.2 M Tris, 0.15 M glycine and 1 mM EDTA, pH 8.3). The protein C content was determined antigenically by means of the Laurell method and in terms of activity after Protac activation.

The protein C eluate obtained was prepared in the following manner for a pharmaceutically administrable preparation:

The eluate was first subjected to an ultrafiltration and a diafiltration step. A buffer with a pH of 7.4, which contained 150 mM NaCl and 15 mM trisodium citrate .2H ₂ O, was used for the diafiltration. The filtrate obtained was freeze-dried and virus inactivated by steaming for one hour at 80 ° C. ± 5 ° C. and 1375 ± 35 mbar.

The lyophilized virus-inactivated material was then dissolved in a sterile isotonic NaCl solution and any antibodies or serum amyloid P present were removed by means of ion exchange chromatography on Q-Sepharose. The purified solution was concentrated through a further ultrafiltration and diafiltration step. Thereafter, 10 albumin, 150 mM NaCl and 15 mM trisodium citrate were added to the solution obtained. The pH of the solution was 7.5. Mouse immunoglobulin and factors II, VII, IX and X could not be detected. The solution was then sterile filtered, filled and lyophilized. The specific activity was 14 units of protein C / mg. An amidolytic test was used as the activity test, the protein C being activated by means of Protac (Pentapharm).

1.5. Purification of plasma protein S

Human protein S was produced from factor IX concentrate (prothrombin complex STIM-3 IMMUNO AG Vienna) using QAE-Sephadex and Blue Sepharose CL-6B chromatography (Pharmacia) in the following way: The lyophilized concentrate (100 g) was dissolved in 200 ml of sterile, ion-free water and against a buffer consisting of 0.01 M 2- (N-morpholineethanesulfonic acid), pH 6.0; 0.18 M NaCl, 10 mM EDTA, 2 mM benzamidine HCl and 0.02% NaN ₃ (start buffer), dialyzed. The dialyzed material was then applied to a QAE-Sephadex column (8 x 19 cm) and equilibrated with the buffer mentioned. 1.5 l buffer (starting buffer) was used as the washing solution.

Protein S was eluted at 110 ml / h with a linear NaCl gradient consisting of 1.2 l of start buffer and 1.2 l of a further buffer, which differs from the first buffer by adding 0.5 M NaCl. The Protein S fractions were examined by Fast Flow SDS Page (Pharmacia) and antigen determination (Laurell) for Protein S, and fractions containing Protein S were pooled and finally dialyzed against a buffer. This buffer contained 50 mM TRIS-HC1, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM benzamidine-HCl and 0.2% NaN ₃ . After dialysis, the Protein S pool was applied to a Blue Sepharose column CL-6B (2.5 cm x 10.5 cm) and equilibrated with the start buffer.

The washing was carried out at a rate of 15 ml / h with 500 ml of starting buffer. The protein S could thus be eluted in the "void volume" while prothrombin adsorbed on the column. The protein S-rich fractions were again determined using the SDS-Page Fast Flow System (Pharmacia) and according to Laurell (Scand. J. Clin. Invest. (Suppl.) 29 (1977) 21 (Suppl. 124)).

The protein S produced in this way had the characteristic morphology for protein S on a reduced SDS page, namely two close bands (doublet) with a molecular weight of approximately 86,000 and 76,000. The protein concentration was determined spectrophotometrically using an extinction coefficient of 0.1 at 280 nm for human protein S, and was confirmed by the LOWRY method (Lowry 0., Rosebrough N., Farr AL, Randall R., Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265). The pre-purified protein S thus produced was used to produce sheep antiserum against protein S by making four immunization injections, 100 μg of protein S being administered subcutaneously with Freund's adjuvant in the first two injections and in the following booster- gene with incomplete adjuvant. After further boosters, the antiserum was tested by double immunodiffusion and showed a precipitation with purified protein S and with normal plasma.

The IgG fraction from 450 ml of antiserum was obtained by alcohol precipitation and subsequent adsorption on Sephadex A 50 in TRIS-HCl buffer, pH 6.8. The supernatant from 450 ml of antiserum contained 1.14 g of anti-protein S-IgG. The IgG fraction was coupled to 450 ml Sepharose CL-4B using 5.7 mg protein / ml Sepharose. The coupling efficiency was 76%. The AntiProtein S column was equilibrated with glycine-HCl, pH 3, and adsorption buffer, pH 7.5.

The adsorption buffer consisted of 20 mM TRIS, 2 mM EDTA, 0.25 M NaCl, 2 mM benzamidine, 0.02% Tween ^® 20, and 0.02% NaN _3, pH 7.4. The washing buffer solution had the following concentrations: 20 mM TRIS, 2 mM EDTA, 1.0 M NaCl, 0, 5 mM benzamidine, 0.01% Tween ^® 20; 0.02% NaN ₃ , pH 7.4.

The elution buffer was composed as the washing buffer, with the modification that 0.05% Tween ^® 20 and additionally 243.3 g NaSCN, pH 7.4 (a 3 M thiocyanate solution) were used.

The dialysis buffer solution contained 20 mM Tris, 0.15 mM glycine, 1 mM EDTA, 2 mM benzamidine, pH 8.3.

For further purification of the protein S fraction, prepared from prothrombin complex concentrate, 100 g of the fraction were dissolved in 1 l adsorption buffer and dialyzed overnight against an adsorption buffer solution. After application of the sample, the column was washed with washing buffer, approx. 5 l, protein-free, followed by elution with 3 M NaSCN in the elution buffer solution. The eluate was dialyzed immediately until SCN was below the detection limit; the eluate had a concentration of 500 μg / ml protein S. It was free of C4-binding protein.

Anti-Protein S monoclonal antibodies were prepared as follows:

BALB / C mice were immunized with 100 μg of the protein S produced at two-week intervals by intraperitoneal injection. After six weeks, 50 μg of the human protein S were injected again and the fusion was carried out three days later. The myeloma cell line (P3-X-63-AG8-653, 1.5 x 10 ⁷ cells) was mixed with 1.7 x 10 ⁸ spleen cells from a mouse, and the fusion according to the modified Koehler & Milstein method using PEG 1500 carried out (Köhler G., Milstein C, Nature 256 (1975) 495-497).

Positive clones, tested using an ELISA, were subcloned twice. Ascites production was carried out by injection of 5 x 10 ⁶ hybridoma cells per BALB / C mouse two weeks after Pristan treatment.

The immunoglobulin was purified from ascites by ammonium sulfate precipitation and subsequent chromatography using QAE-Sephadex and then chromatography on Sephadex G200.

The IgG fraction obtained from ascites and pre-purified on protein A-Sepharose was coupled to Sepharose CL-4B. The affinity-chromatographic purification of protein S, which was obtained from prothrombin complex concentrate, was carried out under the conditions described for polyclonal protein S antibodies. The concentration of the protein S eluate was 600 μg / ml. It was free of C4 binding protein.

The protein S preparations, highly purified by the method of polyclonal or monoclonal affinity chromatography, were subjected to an SDS page (gradient gel 8 to 12%) and they can be determined using Coomassie staining (Laemmli UK: Cleavage of structural proteins during the assembly of the head ε 1

Example 3: Pharmaceutical preparation of the prothrombin complex:

As described in the previous example, a highly purified prothrombin complex, containing the factors prothrombin, factor VII, factor IX, and factor X, but without protein C and protein S, was prepared and sterile filled. According to international recommendations, 10,000 international units of heparin / 1 were added to the solution before bottling.

Example 4: Pharmaceutical preparation of the vitamin K-dependent proteins for permanent substitution and maintenance of constant plasma levels:

After substitution with a vitamin K-dependent protein concentrate, in which the plasma levels of the factors of the prothrombin complex are to be kept as constant as possible, the different half-lives of the individual factors make it necessary to carry out the substitution rates of these proteins in an non-equivalent ratio. Accordingly, a pharmaceutical preparation was described as described above, which contained prothrombin, factor VII, factor IX, factor X, protein C and protein S in a ratio of 1: 30: 3: 1.5: 6: 6.

Example 5: Pharmaceutical preparation of a partial prothrombin complex:

The different in vivo half-lives of the factors of the prothrombin complex, in particular due to the long half-life of prothrombin of 2-5 days compared to the short half-life of factor VII of 4 to 7 h and of factor IX of less than 24 h, results in permanent substitution with prothrombin complex preparations which, after setting a normal plasma concentration of 1 unit factor VIII / ml, plasma concentration after 24 h has already decreased again below the concentration required for functioning hemostasis of at least 20%, while, for example, the prothrombin plasma level is still increasing 1

Chromogenic substrates S-2238, S-2222, S-2444, S-2366 and S-2251 from Chromogenix were used to determine the content of activated prothrombin complex factors. The determinations were carried out using the buffer conditions specified by the manufacturer. The activated factors thrombin, factor VIIa, factor IXa, factor Xa, activated protein C, which developed proteolytic activities against the peptide substrates used, could be determined by the chromogenic substrates. After evaluating the photometric analysis of the chromogenic conversion of the substrate, it was possible to determine that for all substrates the content in the preparations described above, containing vitamin K, proteins or selected factors of the prothrombin complex, were in each case below 10 mU / ml.

Claims

Patent claims:

1. Pharmaceutical separated prothrombin complex preparation, characterized in that it consists of at least 2 chromatographically purified vitamin K-dependent individual factors as active substances.

2. Preparation according to claim 1, characterized in that the individual factors are selected from the group factor II, factor VII, factor IX, factor X, protein C, protein S and protein Z.

3. Preparation according to claim 1 or 2, characterized in that it contains at least the factors II, VII, IX and X.

4. Preparation according to one of claims 1 to 3, characterized in that it contains the individual factors protein C and protein S.

5. Preparation according to one of claims 1 to 4, characterized in that at least one of the individual factors is a recombinant factor, a transgenic factor, a derivative, in particular a peptide, and / or a fragment.

6. Preparation according to one of claims 1 to 5, characterized in that it comprises the individual factors contained in a ratio which corresponds essentially to the ratio of these factors in the blood.

7. Preparation according to one of claims 1 to 6, characterized in that it contains the individual factors Factor II, Factor VII, Factor IX and Factor X in a Relatiwerhaltnis-, based on international units, from (0.5 to 2): (0 , 5 to 2): (0.5 to 2): (0.5 to 2).

8. Preparation according to one of claims 1 to 7, characterized in that it contains the individual factors Factor II, Factor VII, Factor IX, Factor X, Protein C and Protein S in a Relativver- Ratio, based on international units, from (0.5 to 2): (0.5 to 2): (0.5 to 2): (0.5 to 2): (0.5 to 2): (0 , 5 to 2).

9. Preparation according to one of claims 1 to 5, characterized in that it comprises the individual factors contained in a ratio which corresponds to the relative half-lives of the individual factors.

10. A preparation according to claim 9, characterized in that it contains the individual factors factor II, factor VII, factor IX and factor X in a relative ratio, based on international units, of (0.5 to 2): (5 to 35): ( 0.5 to 7): (0.5 to 5).

11. Preparation according to one of claims 9 or 10, characterized in that it contains the individual factors factor II, factor VII, factor IX, factor X, protein C and protein S in a relative ratio, based on international units, of (0.5 to 2):

(5 to 35): (0.5 to 7): (0.5 to 5): (1 to 15): (1 to 15).

12. A preparation according to claim 1, 2, 4, 5, 6 or 9, characterized in that it comprises factor VII and factor II in a ratio greater than 10: 1.

13. Preparation according to one of claims 1 to 12, characterized in that the individual factors in the preparation do not form a complex.

14. Preparation according to one of claims 1 to 13, characterized in that it comprises a partial prothrombin complex.

15. Preparation according to one of claims 1 to 14, characterized in that it contains no activated coagulation factor selected from Ha, IXa, Xa and optionally Vlla.

16. A preparation according to any one of claims 1 to 15, characterized in that it contains less than 0.1 U factor VIII: C or factor VIII: Ag / mg protein.

17. Preparation according to one of claims 1 to 16, characterized in that it contains less than 0.1 U factor Ila / U prothrombin.

18. Preparation according to one of claims 1 to 17, characterized in that it contains less than 0.1 E factor Xa / E factor X.

19. Preparation according to one of claims 1 to 18, characterized in that it is in lyophilized form.

20. Preparation according to one of claims 1 to 19, characterized in that it contains magnesium ions.

21. Preparation according to one of claims 1 to 20, characterized in that it contains no free calcium ions.

22. A preparation according to claim 21, characterized in that it contains a chelating agent for complexing free calcium ions.

23. Preparation according to one of claims 1 to 22, characterized in that it further contains antithrombin III in stabilizing amounts, optionally together with heparin.

24. Preparation according to one of claims 1 to 23, characterized in that it is free from infectious viruses due to a virus inactivation or virus depletion treatment.

25. A preparation according to claim 24, characterized in that the freedom from infectious viruses is ensured by two mutually independent inactivation or depletion methods for viruses.

26. Preparation according to one of claims 1 to 25, characterized in that it further comprises pharmaceutically acceptable buffer substances or stabilizers.

27. A preparation according to any one of claims 1 to 26, characterized records that it consists of highly purified vitamin K-dependent individual factors that are virus inactivated.

28. Preparation according to one of claims 1 to 27, characterized in that it comprises the single factor factor X in its α-shape and / or _/ S-shape.

29. Diagnostic preparation containing a preparation according to one of claims 1 to 28.

30. Use of a preparation according to one of claims 1 to 28 for the manufacture of a preparation for the treatment of acquired or inherited blood coagulation disorders.

31. Use of a preparation according to one of claims 1 to 28 for the manufacture of a preparation for the treatment of severe bleeding.

32. Use of a preparation according to one of claims 1 to 28 for the manufacture of a preparation for the prophylaxis of bleeding, in particular in the presence of inherited blood coagulation disorders.

33. Use of a preparation according to one of claims 1 to 28 for the preparation of a preparation for substitution therapy.

34. Use of a preparation according to one of claims 1 to 28 for the preparation of a preparation for the treatment of hemophilia B.