EP0081482A1 - Procede de purification et de concentration du complexe de facteur viii - Google Patents

Procede de purification et de concentration du complexe de facteur viii

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Publication number
EP0081482A1
EP0081482A1 EP19810901787 EP81901787A EP0081482A1 EP 0081482 A1 EP0081482 A1 EP 0081482A1 EP 19810901787 EP19810901787 EP 19810901787 EP 81901787 A EP81901787 A EP 81901787A EP 0081482 A1 EP0081482 A1 EP 0081482A1
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EP
European Patent Office
Prior art keywords
factor viii
solution
glycine
concentration
precipitate
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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
EP19810901787
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German (de)
English (en)
Inventor
E. G. Birger Blombäck
Lars G. Thorell
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Blombaeck E G B
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Blombaeck E G B
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Filing date
Publication date
Application filed by Blombaeck E G B filed Critical Blombaeck E G B
Publication of EP0081482A1 publication Critical patent/EP0081482A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a process in purification and/or concentration of the factor VIII complex. It is started from a preparation prepared in a manner known per se, in which the factor VIII complex is enriched. The preparation is mostly in the form of a precipitate.
  • the factor VIII complex is lacking or its activity is reduced in hemophilia of type A and in v. Willebrand's disease. The symptoms in these diseases are often serious bleedings in joints and muscles and from mucous membranes.
  • the factor VIII complex takes part in the biochemical reactions promoting the coagulation of blood.
  • an insoluble polymer, fibrin is formed from a soluble protein, fibrinogen.
  • the reason for the polymerization or formation of fibrin is an enzymatic change of the fibrinogen molecule, which is caused by the enzyme trombin (factor Ila).
  • This enzyme is formed from protrombin (factor II) under the influence of an enzyme, called factor Xa.
  • Said enzyme is also present as a zymogen in the blood before the coagulation.
  • factor X The zymogen form is called factor X.
  • factor Xa The conversion of factor X to factor Xa also takes place enzymatically by means of an enzyme called factor IXa.
  • factor VIII complex takes part in this reaction (as a co-factor) together with calcium and phospholipide.
  • the most important reactions in the coagulation (clotting) of blood are illustrate Totally a great number of various factors take part here.
  • the factor VIII complex consists of at least two components, one of which is called the factor VIII : C, in which C means that this component is responsible for the coagulation activity of the complex in the reaction chain shown.
  • This component is considered to contain the antigen proved by means of antibodies, which are developed in certain persons suffering from hemophilia and which prevent the coagulation activity of factor VII I : C .
  • the antigen is called F VIII : CAG.
  • the other component has been called factor VIII-RAG or F VHI-related antigen. This antigen is different from the antigen F VIII : CAG.
  • the factor VIII : C and the (antigen) factor VIII : CAG are lacking in hemophilia of type A in a serious form. In this disease there is a normal content of factor VIII: RAG. In v. Willebrand's disease, there Is a lack of factor
  • factor VIII RAG in the blood and a corresponding lack of factor VIII : C.
  • the lack of factor VIII: RAG is almost total and the content of factor VIII : C is about 5 % of the normal content.
  • the activity of the so-called factor VIII : RCF is highly reduced. This activity is an expression of a component in plasma which causes agglutination of trombocytes in the presence of the antibiotic "Ristocetin”. It is now considered that the activity of the factor VIII : RCF is an expression of the component or components in plasma which has (have) the factor Vlll-related antigen (factor VIII : RAG).
  • Willebrand's disease has been found to be correlated to the prolonged capillary bleeding time existing in this disease and which is an expression of a defective trombocyte function. This function is normal in hemophilia type A, whereas the coagulation time of the blood is prolonged due to the reduced content of factor VIII : C. In v. Willebrand's disease, the coagulation time is also prolonged as there is also a lack of factor VIII : C in this disease, especially in serious forms thereof. The lack of factor VIII : C in v. Willebrand's disease is considered to be a consequence of the lack of factor VIII : RAG/RCF, which seems to act as carrier molecule for factor VIII : C. In accordance with the existing values, one can illustrate the factor VIII -complex schematically in the following way:
  • a number of processes are known at present for the preparation of plasma concentrates for clinical use, which contain the factor VIII complex or parts thereof.
  • the complex can be precipitated from Cohn's fraction I with alcohol. Additional concentration of the complex can be carried out by extraction of inert protein whith about 1 M glycine solution in cold. Fractionation of plasma with ether or tannic acid has also been used for the preparation of factor VIII concentrates. Concentrates prepared by precipitation in cold of the factor VIII complex have been widely used due to the simplicity of the method. In certain cases, polyethylene glycol has been used for precipitation of the factor VIII complex. Variants of the glycine method, in which either other amino acids than glycine have been used or glycine has been used for precipitation of the factor VIII complex are also known.
  • Concentrates prepared according to the glycine method contain all factors in the factor VIII complex, but the specific activity is low, and therefore great volumes of solution must be injected in the treatment of hemophilia. Preparations with a high specific activity have often been found to lack the factor correcting the prolonged bleeding time in v. Willebrand's disease.
  • Hemophilia A as well as v. Willebrand's disease can be treated with such a concentrate.
  • a specific activity which is 200-300 times greater than that In plasma also permits preparations containing the necessary therapeutic dose in a small volume (5-10 ml) . This makes the treatment easier as the dose can be administered in an injection syringe. This also makes it possible to carry out the treatment for example at home by the sick person himself as is usual in the treatment of diabetes with insulin.
  • the process according to the invention is charactericed in that the resulting preparation of factor VIII complex is dissolved in a glycine solution of at least 1.5 M at a temperature of at least +15°C and a pH of 6.3-7.8, preferably pH 6.8, and that supernatant liquid is recovered as a product or for further working-up.
  • the resulting product (factor VIII complex) contains the antihemophilic factor (factor VIII : C/CAG) and the component (factor VIII-RAG/RCF) which is lacking in v. Willebrand's disease.
  • the starting preparation obtained in a way known per se can be prepared by cryoprecipitation or fraction ation of plasma with alcohol.
  • impurities are then precipitated in a glycine solution at and about room temperature and at a neutral pH.
  • glycine solutions have been used at a low temperature for dissolutiqn of impurities from a precipitated factor VIII complex.
  • Glycine at a concentration of about 2 M has also been used for precipitation of the factor VIII complex from a protein solution at a low temperature.
  • the preparation of factor VIII complex is now treated at a temperature of at least 15°C, preferably at a temperature of 15-37o C, such as 20-35oC and most preferably at about 30 °C.
  • the temperature should not normally exceed 40-45°C.
  • the factor VIII complex remains in solution, while contaminating protein is precipitated.
  • the pH is approximately neutral.
  • the ionic strength of the solution should be between 0.1 and 0.5, calculated as buffer salts.
  • the glycine concentration at the precipitation of impurities can vary between 1.5 M and a saturated solution and is preferably 1.6-2.3 M, such as 1.8-2.2 M, and most preferably about 2.0 M.
  • the supernatant liquid can be worked up in a manner known per se by precipitating the factor VIII complex with organic solvent or polymerisolutions.
  • this factor VIII complex also can be precipitated by the addition of salt.
  • salt is added to the supernatant liquid to a concentration of at least 1.5 M maintaining the glycine concentration of at least 1.5 M to precipitate the factor VIII complex containing product which Is recovered.
  • the salt used should be non-toxic and should be able to provide the necessary concentration of at least 1.5 M, e.g. 1.8 M, preferably about 2.0 M or more, e.g. 2.2 M. It is possible to use a saturated salt solu tion.
  • the glycine concentration can be the same as in the dissolving step.
  • the temperature can be as low as the freezing point of the supernatant liquid up to as high temperatures as e.g. 50°C or 60 °C. Temperatures below
  • salts that can be used are NaCl, KCl, CaCl 2 , MgCl 2 , NH 4 Cl, (NH 4 ) 2 SO 4 , Na,SO 4 , K 2 SO 4 , Na 3 PO 4 , Na 2 HPO 4 , NaH 2 PO 4 and citrates of sodium, potassium and ammonium; thus alkaline metal salts, such as sodium and potassium salts and ammonium salts, are preferred.
  • Fig. 1 shows protein and activity in the supernatant liquid and the specific activity as a function of the molarity of the glycine solution.
  • Fig. 2 shows the activity in supernatant liquid and in precipitate and protein in the supernatant liquid as a function of the temperature.
  • Figs. 3 and 4 the same quantities are shown as a function of pH and ionic strength, respectively.
  • Fig. 5 shows the activity in vivo of the factor VIII concentrate in a patient suffering from severe v. Willebrand's disease, and also the inverted value of the bleeding time;
  • Fig. 6 is a diagram Illustrating the yield of factor VIII in the supernatant liquid and the precipitate versus the salt concentration.
  • Example 1 Blood is collected in citrate-phosphate-dex trose-adenine solution in an amount of 50 ml of citrate solution per 430 ml of blood. Blood cells are removed by centrifuging and plasma is sucked off. The plasma is fractionated with alcohol according to Cohn's fractionation method. Fraction I is washed in cold with glycine solution. The remaining precipitate, called fraction 1-0, contains factor VIII complex and is dissolved in sodium citrate solution (pH 6.8) of 0.055 M to a protein concentration of about 2 %. 8 parts of a buffer with a pH of 6.8 and containing varying amounts of glycine, 0.125 M NaCl and 0.025 M imidazole are added to 4 parts of this solution at room temperature.
  • Example 2 Blood and plasma are prepared in the way described in example 1.
  • a counterpart of fraction I-0 is prepared by freezing of the factor VIII complex by adding polyethylene glycol 4000 in an amount of 1 % to the plasma. The mixture is frozen at -70°C. At a slow thawing to +4°C a precipitate called cryoprecipitate remains when all ice has melted. This precipitate contains between 60 and 90 % of the factor VIII complex of the plasma. The precipitate is dissolved in 0.055 M sodium citrate solution (pH 6.8) to a protein content of about 3 % .
  • the separation of the factor VIII complex from inert protein is highly dependent on the temperature of the glycine-protein mixture. At temperatures above 15 °C, the main part of the factor VIII complex is found in the supernatant liquid, while at lower temperatures, the main part is in the precipitate.
  • Example 5 The cryoprecipitate (see example 2) is dissolved in a 0.055 M citrate solution (pH 6.8) to a protein content of about 3 %. 8 parts of buffer containing 3 M glycine, 0.125 M NaCl and 0.025 M imidazole, the pH of which has been adjusted to 6.3: 6.8; 7.3 and 7.8, respectively, are added dropwise to 4 parts of this solution. After stirring for 15 min at 30°C, the precipitate formed is removed by centrifugation and dissolved in citrate solution (pH 6.8). The supernatant liquid and the precipitate are analyzed according to example 1.
  • Example 4 10 parts of glycine-imidazole solution with a varying content of co-men salt were added dropwise to 5 parts of the cryoprecipitate (see examples 2 and 3).
  • the composition of the different additional solutions is as follows:
  • Example 5 Fraction I-0 is dissolved in citrate solution (see example 1) to a protein content of about 2 % . 10 parts of 3 M glycine containing 0.125 M NaCl and 0.025 M imidazole, pH 6.8, are added to 5 parts of the resulting solution. After stirring at 20°C the precipitate (F I) is removed by centrifugation. The supernatant liquid (upper liquid I) is cooled to 4°C. Aqueous polyethylene glycol 6000 of 30 % is then added to a final concentration of 10 % . After stirring for 30 min at 4°C, the precipitate (F II) is removed from supernatant liquid (upper liquid II) by centrifuging.
  • Example 6 10 parts of solution containing 3 M glycine, 0.125 M NaCl and 0.025 M imidazole at pH 6.8 are added to 5 parts of solution of the cryoprecipitate (see example 4) at 20°C. After stirring for 10 min the pre cipitate (F I) formed is removed by centrifuging. The supernatant liquid ( Upper liquid I) is cooled to
  • Example 7 Cryoprecipitate (see example 2) is dissolved in 0.055 M citrate solution, pH 6.8, to a concentration of 3 % . 3.3 parts of a solution containing 2.6 M glycine and 0,025 M imidazole, pH 7.3, are added to 1 part of this solution. The temperature of the mixture is simultaneously brought to 7°C. After the addition, the mixture is brought to equilibrium under stirring at 7°C for 15 min. A precipitate (F I) is formed and removed by centrifugation. The supernatant liquid (Upper liquid I) is saved for analysis.
  • the precipitate is dissolved in 0.055 M citrate solution, pH 6.8, at 30°C to about the same volume as the original cryoprecipitate, after which 3.3 volumes of a solution containing 2.6 M glycine, 0.3 M NaCl and 0.025 M imidazole, pH 6.8, are added. Stirring is carried out at 30°C for 20 min.
  • the precipitate (F II) formed is removed by centrifugation.
  • the supernatant liquid (Upper liquid II) is adjusted to pH 7.5 with a weak sodium hydroxide solution after cooling to 4 °C.
  • An aqueous solution of 30 % polyethylene glycol 6000 is then added dropwise to a concentration of 6.5 % .
  • the mixture is stirred at 4°C for 30 min, after which the precipitate formed is remo ⁇ 'ed by centrifuging.
  • the precipitate (F III) is dissolved in 0.055 M sodium citrate solution containing 0.055 M imidazole, pH 7.40.
  • the supernatant liquid (Upper liquid II) is saved for analysis. All fractions are analyzed in the way described in example 1, and the results appear from the following table 3.
  • Example 8 In the previous examples, the analyses of the factor VIII complex have only comprised factor VIII : C. In the following table 4 analyses of a factor VIII complex is shown, which has been isolated substantially in the way described in example 7.
  • the factor VIII : C can be determined according to Nilsson et al: Acta Med. Scan. 159, 35 (1953) or according to Savidge et al : Thromb. Res. 16 , 355 (1979).
  • the factor VIII : CAG can be determined according to Lazarchick and Hoyer, J. Clin. Invest. 62 , 1048 (1978).
  • the complex contains the same concentration of coagulation factor VIII : C as of factor VIII : CAG.
  • the content of factor VIII : RAG is somewhat higher and the content of factor VIII : RCF (lacking in v. Willebrand's disease) is somewhat lower than of factor VIII : C/CAG.
  • a solution consisting of 2.6 M glycine, 0.3 M NaCl and 25 mM tris is thereafter added at a temperature of 20-30°C to a final concentration of 2 M glycine. Stirring is carried out for 20 min. The precipitate formed is separated by centrifugation. The supernatant is cooled to 4°C, after which solid sodium chloride is added to a final concentration of 2 M. Stirring is carried out for 30 min. after which the precipitate formed is separated by centrifugation. This precipitate is dissolved in 0,055 M sodium citrate solution at a pH of 7.30.
  • the inverted value of the bleeding time is also plotted versus the period of time (hours) elapsed after injection to this patient.
  • the Iwy's method [(Nilsson, Magnusson, Borchgrevink, Thr. Diat. Hamorrh, 10, 223
  • Example 10 A supernatant liquid is prepared from a cryoprecipitate as in example 9. This supernatant is cooled to 4°C and divided in three aliquots; thereafter, solid sodium chlorid is added to each aliquot to a final concentration of 1.0, 1.5 and 2.0 M, respectively. Each aliquot is then treated in the following way: stirring is carried out for 30 minutes, after which the precipitation formed is separated by centrifugation.
  • the precipitation is dissolved in 0.055 M sodium citrate solution at a pH of 7.30.
  • the yield of factor VIII is plotted versus the concentration of NaCl in the diagram of fig.6.
  • the line 0 II and the line F II show the yield, based on the cryoprecipitate, of factor vIII in the supernatant liquid and in the precipitation, respectively.
  • Example 11 is repeated with the exception that room temperature is used Instead of 4 °C during the precipitation with sodium chloride.
  • the following table gives the result from this example; thus the volume and the content of the factor VIII : C is given in units as well as percentage in the cryoprecipitate, the supernatant liquid from the solution process with 2 M glycine solution and in the salt precipitate from the precipitation with 2 M sodium chloride.

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  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Un procede de purification et/ou de concentration du complexe de facteur VIII, commence a partir d'une preparation du complexe de facteur VIII obtenu et concentre d'une maniere connue, generalement sous la forme d'un precipite, tel qu'un cryoprecipite ou une fraction de Cohn 1-0. Cette preparation est dissoute dans une solution de glycine d'au moins 1,5 M a une temperature d'au moins + 15 C et un pH compris entre 6,3 et 7,8 et un liquide surnageant est recupere en tant que produit ou pour un traitement ulterieur, en particulier une precipitation du complexe de facteur VIII a partir du liquide surnageant avec une solution d'un sel de concentration superieure a 1,5 M maintenant la concentration de glycine a au moins 1,5 M.
EP19810901787 1981-06-18 1981-06-18 Procede de purification et de concentration du complexe de facteur viii Withdrawn EP0081482A1 (fr)

Applications Claiming Priority (1)

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PCT/SE1981/000183 WO1982004395A1 (fr) 1981-06-18 1981-06-18 Procede de purification et de concentration du complexe de facteur viii

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EP0081482A1 true EP0081482A1 (fr) 1983-06-22

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Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT391808B (de) * 1986-11-03 1990-12-10 Immuno Ag Verfahren zur herstellung einer faktor viii (ahf)-haeltigen fraktion
AT406867B (de) 1997-02-27 2000-10-25 Immuno Ag Verfahren zur gewinnung von hochreinem vwf oder faktor viii/vwf-komplex

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3631018A (en) * 1970-05-01 1971-12-28 Baxter Laboratories Inc Production of stable high-potency human ahf using polyethylene glycol and glycine to fractionate a cryoprecipitate of ahf concentrate
US3920625A (en) * 1973-06-19 1975-11-18 Kabi Ab Isolation of coagulation factors from biological material using cross linked sulfated, sulfonated carbohydrates
DE2459291C3 (de) * 1974-12-14 1981-06-04 Biotest-Serum-Institut Gmbh, 6000 Frankfurt Verfahren zur Herstellung eines antihämophiles Globulin A enthaltenden Konzentrats neben einem Prothrombin- Komplex und einer Lösung von lagerstabilen Serumproteinen
US4069216A (en) * 1975-06-16 1978-01-17 Edward Shanbrom, Inc. Simplified methods for preparation of very high purity Factor VIII concentrate
CA1074698A (fr) * 1977-12-19 1980-04-01 Gail A. Rock Methode pour augmenter la production de facteur antihemophilique viii dans le sang et le plasma
SE443293B (sv) * 1978-01-25 1986-02-24 Blombaeck E G B Blodfraktionsframstellning
DE2848529A1 (de) * 1978-11-09 1980-05-29 Behringwerke Ag Verfahren zur herstellung des kaelteunloeslichen globulins und dieses enthaltende arzneimittel
DE2916711A1 (de) * 1979-04-25 1980-11-06 Behringwerke Ag Blutgerinnungsfaktoren und verfahren zu ihrer herstellung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8204395A1 *

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Publication number Publication date
WO1982004395A1 (fr) 1982-12-23

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