EP1015020A1 - Preparation pharmaceutique contenant divers facteurs tributaires de la vitamine k - Google Patents

Preparation pharmaceutique contenant divers facteurs tributaires de la vitamine k

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Publication number
EP1015020A1
EP1015020A1 EP98944886A EP98944886A EP1015020A1 EP 1015020 A1 EP1015020 A1 EP 1015020A1 EP 98944886 A EP98944886 A EP 98944886A EP 98944886 A EP98944886 A EP 98944886A EP 1015020 A1 EP1015020 A1 EP 1015020A1
Authority
EP
European Patent Office
Prior art keywords
factor
preparation according
protein
preparation
individual factors
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98944886A
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German (de)
English (en)
Inventor
Yendra Linnau
Peter Turecek
Hans-Peter Schwarz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter AG
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Baxter AG
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Publication date
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Publication of EP1015020A1 publication Critical patent/EP1015020A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents

Definitions

  • 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.
  • prothrombin factor II
  • Normal prothrombin contains ⁇ -carboxyglutamate at the N-terminal end, i.e. a second carboxyl group on the glutamate residue.
  • ⁇ -carboxyglutamate 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.
  • prothrombin have ⁇ -carboxyglutamate residues
  • the coagulation factors VII, IX and X are carboxylated on specific glutamate residues in order to develop a high affinity for calcium ions.
  • 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).
  • 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.
  • prothrombin complex concentrates contain activated coagulation factors, which are mostly active serine proteases, due to their purification from complex protein mixtures.
  • activated coagulation factors which are mostly active serine proteases
  • patients in particular should not be coagulated, since the development of thrombosis can or is fatal.
  • proteolytic inactivation of the individual factors occurs, which, depending on the individual stability of the individual factors, can have serious consequences.
  • 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.
  • 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.
  • a pharmaceutical separated prothrombin complex preparation containing at least two chromatographically purified vitamin K-dependent individual factors as active substances.
  • 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.
  • 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.
  • 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.
  • factor VII 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.
  • 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).
  • 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 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • the preparation is therefore free from albumin and / or stabilizers, such as in particular antithrombin III and / or heparin.
  • the preparation according to the invention is also free of phospholipids.
  • 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.
  • 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 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.
  • 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.
  • 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 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
  • 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.
  • 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).
  • 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.
  • Plasmatic factor IX Plasmatic factor IX
  • prothrombin complex 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.
  • factor IX 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.
  • 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 ⁇ g human protein C at two-week intervals by intraperitoneal injection. After six weeks, another 50 ⁇ g 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:
  • 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
  • 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 ⁇ m 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.
  • 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).
  • 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 .
  • 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 ⁇ g 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.
  • 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 ⁇ g / ml protein S. It was free of C4-binding protein.
  • Anti-Protein S monoclonal antibodies were prepared as follows:
  • mice were immunized with 100 ⁇ g of the protein S produced at two-week intervals by intraperitoneal injection. After six weeks, 50 ⁇ g 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).
  • 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 ⁇ g / 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 ⁇ 1
  • 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:
  • Example 5 Pharmaceutical preparation of a partial prothrombin complex:
  • 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.

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  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une préparation pharmaceutique séparée d'un complexe de prothrombine, contenant au moins deux facteurs sanguins individuels, hautement purifiés et tributaires de la vitamine K.
EP98944886A 1997-09-19 1998-09-17 Preparation pharmaceutique contenant divers facteurs tributaires de la vitamine k Withdrawn EP1015020A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT159197 1997-09-19
AT0159197A AT409334B (de) 1997-09-19 1997-09-19 Pharmazeutisches präparat enthaltend vitamin k-abhängige einzelfaktoren
PCT/AT1998/000224 WO1999015196A1 (fr) 1997-09-19 1998-09-17 Preparation pharmaceutique contenant divers facteurs tributaires de la vitamine k

Publications (1)

Publication Number Publication Date
EP1015020A1 true EP1015020A1 (fr) 2000-07-05

Family

ID=3516739

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98944886A Withdrawn EP1015020A1 (fr) 1997-09-19 1998-09-17 Preparation pharmaceutique contenant divers facteurs tributaires de la vitamine k

Country Status (10)

Country Link
EP (1) EP1015020A1 (fr)
JP (1) JP2001517636A (fr)
AT (1) AT409334B (fr)
AU (1) AU743102B2 (fr)
BR (1) BR9812222A (fr)
CA (1) CA2304396A1 (fr)
HU (1) HUP0003681A1 (fr)
NO (1) NO20001415L (fr)
SK (1) SK4022000A3 (fr)
WO (1) WO1999015196A1 (fr)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1458408B1 (fr) 2001-12-21 2009-04-15 Novo Nordisk Health Care AG Composition liquide de polypeptides de facteur vii
EP1485121A4 (fr) * 2002-03-08 2007-11-07 Lilly Co Eli Formulations de proteines c activees
CN1671410B (zh) 2002-06-21 2010-05-12 诺和诺德医疗保健公司 因子ⅶ多肽的稳定化固体组合物
US7897734B2 (en) 2003-03-26 2011-03-01 Novo Nordisk Healthcare Ag Method for the production of proteins
RU2364609C2 (ru) 2003-05-23 2009-08-20 Ново Нордиск Хелт Кэр Аг Стабилизация белка в растворе
EP1641487B1 (fr) 2003-06-25 2012-02-29 Novo Nordisk Health Care AG Composition liquide de polypeptides du facteur vii
JP5653572B2 (ja) 2003-08-14 2015-01-14 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト 第ｖｉｉ因子ポリペプチドの液状水性医薬組成物
CN1917861B (zh) 2003-12-19 2012-03-21 诺和诺德医疗保健公司 因子vii多肽的稳定化固体组合物
US20080260858A1 (en) * 2005-02-16 2008-10-23 The Board Of Trustees Of The University Of Illnois Universal Procoagulant
EP1869082B1 (fr) 2005-03-04 2011-04-13 The Board Of Trustees Of The University Of Illinois Modulateur de coagulation et de cascades fibrinolytiques
WO2009046194A2 (fr) 2007-10-05 2009-04-09 The Board Of Trustees Of The University Of Illinois Produit d'étanchéité de fibrine
US20100297257A1 (en) 2007-11-09 2010-11-25 National Institutes Of Health (Nih), U.S. Dept. Of Health And Human Services (Dhhs) Anticoagulant antagonist and hemophillia procoagulant
WO2011073235A1 (fr) 2009-12-18 2011-06-23 Csl Ltd. Procédé de purification de polypeptides
CN103221061A (zh) * 2010-10-06 2013-07-24 米迪缪尼有限公司 用于治疗止血障碍的因子ii以及纤维蛋白原
DK3579857T3 (da) * 2017-02-09 2022-06-07 Csl Behring Gmbh Et blodkoagulationsfaktorerstatningsprodukt til anvendelse ved behandling eller profylakse af blødninger

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Publication number Priority date Publication date Assignee Title
GB8729822D0 (en) * 1987-12-22 1988-02-03 Central Blood Lab Authority Chemical process
IT1262899B (it) * 1992-03-27 1996-07-22 Sclavo Spa Processo per l'isolamento di fattore ix, fattore x e fattore ii altamente purificati dal complesso protrombinico o dal plasma umano
DE4342132C1 (de) * 1993-12-10 1994-11-03 Octapharma Ag Verfahren zur Herstellung virusinaktivierte Vitamin K abhängige Plasmakomponenten sowie Protein C und Protein S enthaltender Mittel durch Membran-Chromatographie
DE4430205A1 (de) * 1994-08-26 1996-02-29 Behringwerke Ag Zusammensetzungen, die als Gegenmittel für Blut-Antikoagulanzien geeignet sind und deren Verwendung
DK0796623T3 (da) * 1996-03-20 2005-08-01 Baxter Ag Farmaceutisk præparat til behandling af blodkoagulationslidelser

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9915196A1 *

Also Published As

Publication number Publication date
AU9244998A (en) 1999-04-12
HUP0003681A1 (hu) 2001-02-28
SK4022000A3 (en) 2000-09-12
BR9812222A (pt) 2000-07-18
NO20001415L (no) 2000-05-19
ATA159197A (de) 2001-12-15
JP2001517636A (ja) 2001-10-09
AU743102B2 (en) 2002-01-17
CA2304396A1 (fr) 1999-04-01
WO1999015196A1 (fr) 1999-04-01
NO20001415D0 (no) 2000-03-17
AT409334B (de) 2002-07-25

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