WO2000061633A1 - Procede de purification du facteur viii de coagulation sanguine et du complexe de facteur viii de coagulation sanguine et de facteur von willebrand - Google Patents

Procede de purification du facteur viii de coagulation sanguine et du complexe de facteur viii de coagulation sanguine et de facteur von willebrand Download PDF

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WO2000061633A1
WO2000061633A1 PCT/JP2000/002350 JP0002350W WO0061633A1 WO 2000061633 A1 WO2000061633 A1 WO 2000061633A1 JP 0002350 W JP0002350 W JP 0002350W WO 0061633 A1 WO0061633 A1 WO 0061633A1
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blood coagulation
fviii
gel
complex
coagulation factor
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PCT/JP2000/002350
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English (en)
Japanese (ja)
Inventor
Kazuya Hosokawa
Toyoaki Suzuki
Masanori Nagata
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Fujimori Kogyo Co., Ltd.
Chisso Corporation
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Application filed by Fujimori Kogyo Co., Ltd., Chisso Corporation filed Critical Fujimori Kogyo Co., Ltd.
Priority to AU36767/00A priority Critical patent/AU3676700A/en
Publication of WO2000061633A1 publication Critical patent/WO2000061633A1/fr

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    • 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)

Definitions

  • the present invention relates to a novel blood coagulation factor VIII and a method for purifying a blood coagulation factor VIII / von Willebrand factor complex. More specifically, the present invention relates to a method for purifying blood coagulation factor VIII and a blood coagulation factor VIII nofon-bilmbrand factor complex with high purity and high yield without reducing its activity. is there. Background art
  • Blood coagulation factors are factors involved in blood coagulation, and are a total of 15 factors, including proteinaceous coagulation factors in 12 types of plasma and 3 types of calcium ions, tissue troponoplastin and phospholipids. Is known to be involved in the blood coagulation reaction.
  • blood coagulation factor VIII (also referred to simply as “FVIII”) functions as a promoter of the blood coagulation reaction, and is an important blood for maintaining a normal hemostasis mechanism. Coagulation is a coagulation factor of the intrinsic system.
  • FVIII is also called anti-hemophilia factor A, anti-hemophilia factor or anti-hemophilia glopurin.
  • Hemophilia A is a sex-chromosomal recessive inheritance and is classified as mild, moderate, or severe, depending on the degree of reduced factor activity.In mild cases, spontaneous bleeding is rare, and it is difficult to stop bleeding during trauma, surgery, or tooth extraction Is noticeable for the first time, In severe or severe cases, spontaneous bleeding is the main symptom, and bleeding occurs in deep tissues due to intrinsic coagulation disorders.Bleeding into joint cavities is characteristic of this disease. The joints contracture and become functionally impaired, as well as intramuscular bleeding, intracranial bleeding, and renal bleeding. Gene therapy or transplantation can be considered as treatment for patients with hemophilia A, but these treatments are currently under clinical research, and have been put into practical use for hemophilia patients. Has not been. For this reason, at present, patients with hemophilia A need to be supplemented with the deficient factor FVIII at the time of bleeding to stop bleeding each time, or to be treated prophylactically when bleeding is expected. Have been.
  • FVIII clotting protein has a plasma content of about 0.2 g / m1 and other clotting factors.
  • purified protein equivalent to mg a large amount of plasma, 20 to 40 liters, is required, and FVIII is denatured during the purification process and becomes fragmented. Then, there was a problem that it was very difficult to purify while maintaining the entire structure.
  • this FVI 11 binds non-covalently to a von Willebrand (von Wile lebrand) factor (herein, also simply referred to as “vWF”) in a blood stream and forms a complex (here, “blood Coagulation factor VIII / von Willebrand factor complex ”or simply“ FVIII / vWF complex ”.
  • vWF von Wile lebrand factor
  • the von Wille brand factor protein acts as a carrier protein for FVIII and adheres platelets to damaged vascular endothelial subcellular tissues exposed during bleeding.
  • Factors that bind platelets to subendothelial tissue during primary hemostasis It functions as a molecule (molecular glue), forms a complex with FVIII through non-covalent bonds, and exists in the circulating blood as a macromolecular glycoprotein that has a stabilizing effect on FVIII.
  • vWF deficiency or abnormality can be attributed to prolonged bleeding time, decreased plasma factor VIII, decreased platelet adhesion and reduced ristocetin aggregation, decreased vascular resistance, transfusion ' It is known to be the cause of von Willebrand disease, which is characterized by an increase in factor activity that is higher than expected.
  • This method includes, for example, (1) first collecting a cryopion in the presence of 3% ethanol and 1% polyethylene glycol (PEG), and removing the supernatant to obtain a cryoprecipitate ( The cryoprecipitate is subjected to heat treatment, glycine-salt precipitation, 3-4% Ficoll 70 precipitation fractionation, etc. to remove fibrinogen, and the FVIII / vWF complex is roughly purified.
  • the complex is subjected to gel filtration chromatography (eg, Sepharose 4B or CL-6B, Ultrogel AcA22 in the presence of a protease inhibitor such as DFP) or affinity chromatography (eg, anti-fibrinogen, anti-cold).
  • gel filtration chromatography eg, Sepharose 4B or CL-6B, Ultrogel AcA22 in the presence of a protease inhibitor such as DFP
  • affinity chromatography eg, anti-fibrinogen, anti-cold
  • the purified FVIII / vWF complex A method in which FVIII and vWF are dissociated by mixing with calcium chloride, and then FVIII appearing in the low molecular weight part is collected by gel filtration; (2) Cryoprecipitate is prepared from fresh plasma in the presence of proteazyme inhibitor A method of adsorbing this to CNBr-Sepharose 4B-anti-vWF monoclonal antibody large force ram, washing and eluting FVIII with a buffer containing calcium chloride; and (3) cryoprecipitate from plasma
  • the FVIII / vWF complex is partially purified by adsorbing and eluting the FVIII / vWF complex, and then added to the polyelectrolyte E5 large-s
  • the method (1) can be used for purification, it is not suitable from an industrial point of view because the target FVIII activity is reduced in the purification stage of the FVIII / vWF complex.
  • the methods (2) and (3) described above are based on the fact that the FVIII coagulation-active protein has a plasma content of about 0.2 g / m1, which is much lower than other coagulation factors, as described above.
  • a large amount of plasma of 20 to 40 liters is required, which requires a correspondingly large volume column, which is not preferable in terms of equipment, and the process is complicated and takes time and effort. There was a disadvantage.
  • an object of the present invention is to provide a method for purifying FVIII / vWF complex and FVIII to a high degree of purity by a simple process even from a solution containing a large volume of FVIII / vWF complex.
  • Another object of the present invention is to provide a method for purifying an FVIII / vWF complex and FVIII to a high degree of purity from a solution containing a large volume of the FVIII / vWF complex in a simple step without reducing the activity of FVIII. Disclosure of the invention that is intended to be provided
  • the present inventors have conducted intensive studies to achieve the above objects, and as a result, when a solution containing the FVIII / vWF complex was mixed with a gel having pores of a specific size, fibrinogen-fibronectin was obtained.
  • Low molecular weight today Small molecules such as protein and water are captured and retained in the gel network, but the FVIII / vWF complex has a large molecular weight and is located outside the gel without entering the gel network.
  • the solution containing the FVIII / vWF complex is mixed with such a gel, and then the gel is separated and removed by filtration or the like, whereby the solution containing the FVIII / vWF complex is concentrated several to several tens of times. And the ability to simultaneously separate and remove low molecular weight proteins such as fibrinogen and fibronectin, and small molecules such as water.
  • the present inventors have reported that the FVIII / vWF complex has a molecular weight of several million to about 2,000,000, and exists as a very large complex, whereas fipri, which is a protein complex, is present. Focusing on the fact that the molecular weights of nogen and fibronectin are relatively small, ranging from 240,000 to 100,000, compared to the above complex, the solution containing the FVIII / vWF complex can be used in a specific range by utilizing such a difference in molecular weight.
  • the FVIII / vWF complex can be purified efficiently by ultrafiltration or gel filtration chromatography with a membrane containing pores that cut off molecules having a molecular weight of They also discovered that they can be separated with high purity and high yield.
  • the solution containing the FVIII / vWF complex is mixed with a gel having pores of a specific size to concentrate and concentrate the blood FVIII / vWF complex.
  • the partially purified solution containing the FVIII / vWF complex is subjected to ultrafiltration or gel filtration chromatography using a membrane containing pores for cutting off molecules having a specific range of molecular weight.
  • a high purity FVIII / vWF complex free from the complex is obtained, and blood coagulation factor VIII is dissociated from this complex, and further purified using ultrafiltration, gel filtration chromatography, or affinity chromatography.
  • the present invention has the following configurations (1) to (10).
  • a method for purifying blood coagulation factor VIII comprising a step of purifying a mixed solution of blood coagulation factor VIII and von Willebrand factor by ultrafiltration after the dissociation step according to (6).
  • the ultrafiltration membrane used in the ultrafiltration has a cut-off molecular weight of 300,000 to 1
  • a method for purifying blood coagulation factor VI comprising a step of purifying.
  • a blood coagulation factor VIII comprises mixing a solution containing a blood coagulation factor VIII / von Willebrand factor complex with a gel, and then separating and removing the gel from the mixture.
  • a method for purifying a von Willebrand factor complex is provided.
  • the solution containing the FVIII / vWF complex used in the present invention (hereinafter, also simply referred to as “starting solution”) is not particularly limited as long as it contains the FVIII / vWF complex.
  • cryoprecipitate is prepared from plasma by a conventionally known method.
  • a commercially available product such as a cryo preparation or a factor VIII concentrated preparation may be used as it is.
  • the method for preparing a mutant using a gene recombination technique is not particularly limited, and a gene recombination technique known in the art can be used in the same manner.
  • the gel used in the present invention is not particularly limited as long as it has a three-dimensional network structure and has a solvent (solution) retention ability inside.
  • These gels may be produced by a known technique or a commercially available gel may be used as it is.
  • Sephacryl-300 Pharmacia
  • Sepharose 6B gel Pharmacia
  • Sephadex G200 Pharmacia
  • Gels for cell mouth fining such as GCL-2000 (manufactured by Chidso Co., Ltd.) and Cell mouth fins, and xiapatite having a head mouth.
  • the shape of the gel is not particularly limited, and any shape may be used.
  • the size of the gel is not particularly limited, and varies depending on the shape of the gel, the size of the pores, and the like.For example, when the gel is spherical, the diameter is about 0.1 to 100 zm, preferably It is about 50-500 zm.
  • the network structure of the gel is such that impurities such as low molecular weight proteins such as fibrinogen and fibronectin and small molecules such as water are taken in and retained therein, but high molecular weight FVI The II / vWF complex forms pores that are too large to be incorporated.
  • gels usually have an exclusion limit of 300,000 to 2,000,000 proteins, but the molecular weight of the FVI II / vWF complex is several million to 2,000,000. Considering the degree of exclusion, it preferably has a protein exclusion limit of 300,000 to 100,000, more preferably 300,000 to 500,000.
  • the exclusion limit of the gel exceeds 2,000,000, the desired purified product F
  • the VIII / vWF complex is also incorporated into the gel network, which may reduce the purification efficiency of the FVIII / vWF complex.
  • fibrinogen molecular weight: about 330,000
  • fibronectin molecular weight in monomer form: about 24 It is possible that low-molecular-weight contaminating proteins such as (10,000) will not be incorporated efficiently, that is, it will hinder good purification of the FVIII / vWF complex.
  • Nectin is a dimer or tetramer in the S--S bond.
  • the exclusion limit of the gel by the protein is preferably in the range of 500,000 to 20,000,000, more preferably 500,000 to 100,000,000, and most preferably 500,000 to 5,000,000.
  • the exclusion limit of the gel by the protein is set at 1,000,000 to 20,000,000, more preferably 1,000,000 to 1,000,000. It is preferably in the range of 10,000 to 5,000,000.
  • the amount of the gel used depends on the type and amount of the starting solution to be purified, the type and the shape of the gel, and the like.
  • the solution containing the FVIII / vWF complex can be concentrated, and the amount of the gel such as fibrinogen / fibronectin can be increased. It is not particularly limited as long as it can remove small molecules such as low molecular weight protein and water, but it captures as much foreign protein as possible and retains it in the gel network structure. Preferably it is possible.
  • the gel used in the present invention when added in a dry state by freeze-drying or the like, has a higher water absorption amount than when a wet gel is added, and the gel of the solution It is more preferable in terms of the degree of concentration.
  • a dry gel it is desirable that the network structure can be returned to a wet state, and a gel having a strong bond such as a crosslinked gel is preferable.
  • gel to mix The amount varies depending on the structure of the gel, the size and the retention capacity of the solution, but is usually 20 to 200 (v / v)%, preferably 50 to: L50 (v / v) based on the starting solution.
  • the term “gel in a dry state (dry gel)” is used to mean a gel having a capacity to hold a sufficient amount of liquid inside its network structure, and the water content in the gel The less is the better, but does not necessarily mean that the water content must be zero.
  • a dried gel may be used as it is without equilibrating the gel with a buffer or the like.
  • the above gel is dried in a desiccator containing a desiccant such as silica gel, calcium chloride, phosphorus pentoxide, etc. overnight; passing through dry air; It may be used afterwards.
  • the mixing of the starting solution and the gel is not particularly limited as long as the contact between the starting solution and the gel can be sufficiently achieved.However, in order to complete the contact in a short time, the starting solution and the gel are put into a container and stirred. It is preferable to mix while mixing.
  • a method for separating a gel from a mixture of a starting solution and a gel is the same as a method known in the art.
  • centrifugation usually, 4 to 20, 500 to 5000 xg, 30 minutes, preferably 4 to 8. C, 1,000 to 3,000 xg, 30 minutes
  • suction filtration suction filtration
  • pressure filtration pressure filtration and centrifugation are preferably used.
  • the FVIII / vWF complex can be concentrated in a single operation, usually about 3 to 10 times in volume ratio, and can be reduced to a low molecular weight such as fibrinogen-fibronectin.
  • Small molecules such as sun protein and water are usually removed by 30 to 90% of the total, preferably 70 to 90% of the total.
  • the removal rate of impurities is determined by specific activity and SDS. — On PAGE Therefore, it is a value calculated based on the measured value.
  • the concentration / impurity removal operation may be performed once or repeatedly. When the concentration and impurity removal operations are repeated, it is preferable to repeat the operation about 2 to 5 times.
  • the purity of the FVI II / vWF complex obtained by repeating the operation more than 5 times Although the purity is higher, it is not economical when considering time and labor because the improvement in purity corresponding to the repetition is not much observed. Concentration can be achieved sufficiently.
  • the FVI I I / vWF complex is further concentrated to a small amount, and small proteins such as water and other proteins such as fibrinogen-fibronectin are further removed.
  • the gel used in each operation is washed after each concentration operation to remove miscellaneous proteins so that they can be incorporated into the gel and re-used. Can be used.
  • the gel used in each operation may be the same or different.
  • the solution containing the FVI II / vWF complex is subjected to ultrafiltration or gel filtration chromatography after the concentration removal operation according to the first aspect of the present invention.
  • the present invention provides a method for purifying a blood coagulation factor VIII / Phosphate / Vilbrand factor complex comprising:
  • the ultrafiltration step according to the second aspect of the present invention is hereinafter also referred to as “first ultrafiltration step”.
  • the solution containing the FVI I / vWF complex used in the present invention is the same as defined in the first embodiment.
  • ultrafiltration is effective for the FVI II / vWF complex and other impurities contained in the starting solution, for example, fibrinogen-fibronectin. This is performed using an ultrafiltration membrane that can be separated efficiently.
  • the molecular weight of the FVIII / vWF complex is about several million to about 20 million, whereas the molecular weight of fibrinogen / fibronectin is about 330,000 and about 240,000 (in the form of monomer).
  • the ultrafiltration membrane used for ultrafiltration usually has a cut-off molecular weight of 300,000 to 18,000,000, preferably a cut-off molecular weight of 300,000 to 5,000,000, more preferably A membrane having a molecular weight cut-off of 300,000 to 100,000 is used.
  • the FVIII / vWF complex partially passes through the ultrafiltration membrane together with impurities such as fibrinogen / fibronectin. The yield of the desired FVIII / vWF complex may be reduced.
  • the molecular weight cut-off of the ultrafiltration membrane is less than 300,000, impurities such as fibrinogen / fibronectin cannot pass through the ultrafiltration membrane, resulting in good separation from the FVIII / vWF complex. It may not be done.
  • a commercially available ultrafiltration membrane is used as it is. Examples of commercially available ultrafiltration membranes include BIOMAX lOOOKDa (manufactured by Millipore) and 0.1 micron membrane filter. (Millipore).
  • an ultrafiltration membrane having a molecular weight cut-off of 300,000 to 18,000,000 corresponds to an ultrafiltration membrane having a pore size of about 0.02 to 2 microns.
  • the dimer form is present in the low-molecular-weight protein.
  • the molecular weight cut off of the ultrafiltration membrane used for ultrafiltration is from 500,000 to 18 million, more preferably from 500,000 to 5 million, most preferably It is preferably in the range of 500,000 to 100,000.
  • the molecular weight cut off of the ultrafiltration membrane used for filtration is preferably in the range of 100,000 to 1,800,000, and more preferably in the range of 100,000 to 500,000. Good.
  • low-molecular-weight contaminating proteins such as fibrinogen and fibronectin are usually more than 90% of the total, and preferably more than 99% of the total. Removed at a rate. Ultrafiltration is also preferable because it removes contaminating viruses in addition to low molecular weight proteins such as fibrinogen and fibronectin.
  • the ultrafiltration operation may be performed once or repeatedly, for example, about 2 to 10 times. It is possible to repeat the ultrafiltration operation more than 10 times, but it is more preferable that the number of repetitions is small in order to process in as short a time as possible and prevent loss of components.
  • contaminant proteins such as fibrinogen / fibronectin are further removed, and the target FVI II / vWF complex is purified with higher purity.
  • the ultrafiltration membrane used in each operation may be the same or may be changed to a different one depending on the kind of the foreign matter to be removed. .
  • a purification step by gel filtration chromatography may be performed.
  • the exclusion limit of the gel used at this time is preferably 300,000 to 200,000.
  • Sephacryl-300 manufactured by Pharmacia
  • Sepharose 6B column manufactured by Pharmacia
  • the exclusion limit of the gel by protein is set at 500,000 to 200,000.
  • the exclusion limit of the gel due to the protein is set at 100,000 to 20,000,000. It is preferable to set the range.
  • the purification step according to the first embodiment and the purification step according to the second embodiment have been described independently of each other. However, by using these purification steps in combination, FVIII / vWF with higher purity can be obtained. A complex is obtained.
  • the order in which the purification step according to the first aspect and the purification step according to the second aspect are used in combination is not particularly limited, and may be performed in any order. Thus, the starting solution can be concentrated to a small volume and a small amount of sample can be used in the next purification step.If cryoprecipitate is used directly in the purification step according to the second embodiment, a large amount of fibrinogen / fibronectin can be obtained.
  • the purification process according to the first aspect is first performed, and then the purification is performed according to the second aspect. It is preferable to purify the FVIII / vWF complex by performing the step.
  • the purification step according to the first aspect as described above, or a combination of the purification step according to the first aspect and the purification step according to the second aspect preferably purification according to the first aspect Of the blood coagulation factor VIII comprising dissociating the FVIII / vWF complex purified by the combination of the step and the purification step according to the second embodiment into blood coagulation factor VIII and von Willebrand factor.
  • a purification method is provided.
  • a conventionally known method can be applied to the method of dissociating the FVIII / vWF complex into FVIII and vWF in the same manner. Specifically, 0. 25 ⁇ 1 M, preferably a method of dissociating FVIII / vWF complex in FVIII and vWF in the presence of 0. 25-0. 5 M calcium chloride (C a C l 2) Is raised I can do it.
  • the mixed solution of FVIII and vWF after dissociation is further subjected to ultrafiltration to further remove FVIII. It is preferable to perform a purification step (hereinafter, also referred to as a “second ultrafiltration step”).
  • the molecular weight of FVIII is 250,000 to 300,000 and fibrinogen (molecular weight: about 340,000) and fibronectin (The molecular weight in the form of a monomer is approximately 240,000), so that the dissociation step is performed by combining the purification step according to the first embodiment and the purification step according to the second embodiment. It is preferably carried out after removing contaminant proteins such as gender fibronectin as much as possible.
  • the FVIII / vWF complex is dissociated into FVIII and vWF to substantially reduce FVIII (molecular weight of 250,000 to 300,000) and vWF (molecular weight of about A second ultrafiltration process is performed on this mixed solution to obtain only a pure FVIII (molecular weight of 250,000 to 300,000).
  • FVIII molecular weight of 250,000 to 300,000
  • vWF molecular weight of about
  • the FVIII / vWF complex is purified to a high degree of purity by passing through the purification step according to the first aspect and / or the purification step according to the second aspect, so that the solution containing the purified FVIII / vWF complex FVIII purified by the second ultrafiltration step is also obtained in high purity and high yield.
  • the ultrafiltration membrane used in the second ultrafiltration step the same one as described in the first ultrafiltration step can be used.
  • the ultrafiltration membrane used in performing the ultrafiltration step may be the same or different. Further, after performing the dissociation operation of the FVIII / vWF complex into FVIII and vWF, or after the second ultrafiltration step, purification by affinity chromatography is further performed to obtain FVIII.
  • the ligand of the adsorbent used in affinity mouth chromatography is not particularly limited, and a known ligand can be used in the same manner.
  • an anti-FVIII monoclonal having affinity for FV III can be used.
  • examples include a null antibody and anthrom- thrombin, which is a thrombin derivative that has a binding ability to FVIII but has lost serine protease activity.
  • anhydrothrombin is particularly preferably used as a ligand for the adsorbent, in consideration of the purification efficiency of the objective FVIII and the risk of contamination with a protein derived from a heterologous animal.
  • the ligands may be used alone or in combination of two or more.
  • anhydrothrombin which is particularly preferably used as a ligand in the present invention can be prepared by a known method, for example, WO99 / 20655%, Ashton, RW et al., "Preparation and characterization of anhydrothrombin". , Biochemistry (1995), Vo. 34, No. 19, p. 6454-6463.
  • the degree of purification of the FVIII / vWF complex and FVIII was calculated in the same manner as described in the embodiment of the present invention.
  • cryoprecipitate Ten liters of frozen citrate plasma was thawed at 4 ° C to obtain cryoprecipitate. Next, the cryoprecipitate was separated from plasma by centrifugation at 4 ° C (3,000 xg, 30 minutes), and 1 liter of cryoprecipitate was separated. It was dissolved in a phosphate buffer (PH 7.0) to obtain a cryo-dissolved solution. To this lysate was added 90% (v / v) of dried Sephacry 300 (Pharmacia), and the mixture was stirred at 20 ° C for 30 minutes. Was isolated.
  • PH 7.0 phosphate buffer
  • cryo-concentrated solution was subjected to gel filtration with a Shepharose 6B (Pharmacia) column (500 ml) to recover an FVI II / vWF complex eluted near the void volume.
  • the thus obtained peak recovered solution having FVIII activity is subjected to ultrafiltration with a 0.1 micron membrane filter Yuichi (manufactured by Millipore) to obtain a small amount of filtrate contained in the recovered solution. Impurities such as Plinogen-Fibronectin were removed.
  • Dried Shepharose 6B (manufactured by Pharmacia) was added to the cryo-lysate solution prepared in the same manner as in Example 1 so as to have a concentration of 90% (v / v).
  • the supernatant was separated by filtration.
  • the same operation as described above was repeated again for about 200 ml of the obtained supernatant to obtain about 40 ml of a cryo-concentrated solution (a concentrated solution containing the FVIII / vWF complex). This As a result, a 5-fold concentration was possible with a single operation.
  • crys- tal concentrate was subjected to gel filtration using a Sephacry 400 (manufactured by Pharmacia) column (500 ml) to recover an FVI II / vWF complex eluted near the void volume.
  • the thus obtained peak recovery solution having FVIII activity is subjected to ultrafiltration using a 0.1-micron membrane filter (manufactured by Millipore) to obtain a small amount of fibrinogen contained in the recovery solution. (4) Impurities such as fibronectin were removed.
  • the thus-obtained peak recovery solution having FVIII activity was applied to a 20 m1 immobilized column of anhydrothrombin immobilized with a buffer (50 mM Tris-HCl, 0.15 M NaCl, pH 7.5). 5 mg / m1), completely elute the non-adsorbed peaks with the same buffer, and use the dissociation solution (50 mM Tris-HCl, O.IM NaCl, 0.25 M CaCl 2 . After dissociation and elution of FVIII, FVIII was eluted with an eluate (50 mM Tris-HCI, 0.15 M NaCl, 0.15 M, benzamidine, ⁇ 7.5). The specific activity of the purified FVIII thus obtained was about 2,500 u / mg, and the recovery was about 40%. Industrial applicability
  • the method for purifying the blood coagulation factor VIII / von-Bilbrand factor complex of the present invention comprises the steps of: mixing a solution and a gel containing the blood coagulation factor VIII / von-Bilbrand factor complex; It consists of separating and removing the gel from the solution.
  • the solution containing the FVIII / vWF complex can be concentrated to a very small volume in a simple step, so that the purification operation in the next step can be performed without requiring large equipment.
  • foreign proteins such as fibrinogen and fibronectin contained in the solution containing the FVIII / vWF complex can be efficiently separated and removed by simple steps.
  • a solution and a gel containing the blood coagulation factor VIII z von Willebrand factor complex are mixed, and the solution is concentrated by separating and removing the gel from the solution.
  • a solution containing a large volume of FVIII / vWF complex can be concentrated to a small volume in a simple step, and the FVIII / vWF complex can be concentrated in a simple step and reduce the activity of FVIII. It is possible to purify to high purity without reduction.
  • the FVIII / vWF complex purified to high purity as described above is dissociated into blood coagulation factor VIII and von's Vilbrand factor, and the dissociated solution is subjected to ultrafiltration and / or affinity.
  • blood coagulation factor VIII can be separated in a short time and efficiently with almost no impurities.

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Abstract

L'invention concerne un procédé de purification du facteur VIII de coagulation sanguine et du complexe de facteur VIII de coagulation sanguine et de facteur Von Willebrand en une seule étape à partir d'une solution contenant une quantité importante du complexe de facteur VIII de coagulation sanguine et de facteur de Von Willebrand. Ce procédé consiste à mélanger une solution contenant ce complexe avec un gel, puis à séparer et retirer le gel de la solution.
PCT/JP2000/002350 1999-04-12 2000-04-11 Procede de purification du facteur viii de coagulation sanguine et du complexe de facteur viii de coagulation sanguine et de facteur von willebrand WO2000061633A1 (fr)

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Application Number Priority Date Filing Date Title
AU36767/00A AU3676700A (en) 1999-04-12 2000-04-11 Method for purifying blood coagulation factor viii and blood coagulation factor viii/von willebrand factor complex

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JP10458799A JP2002348300A (ja) 1999-04-12 1999-04-12 血液凝固第ｖｉｉｉ因子および血液凝固第ｖｉｉｉ因子／フォン・ビルブラント因子複合体の精製方法
JP11/104587 1999-04-12

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PCT/JP2000/002350 WO2000061633A1 (fr) 1999-04-12 2000-04-11 Procede de purification du facteur viii de coagulation sanguine et du complexe de facteur viii de coagulation sanguine et de facteur von willebrand

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WO (1) WO2000061633A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111592591A (zh) * 2020-06-17 2020-08-28 博雅生物制药集团股份有限公司 一种人血管性血友病因子/人凝血因子ⅷ复合物的制备方法及产物和应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3205665A1 (fr) * 2016-02-11 2017-08-16 Octapharma AG Procédé de séparation de facteur viii à partir de produits sanguins

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4455301A (en) * 1980-02-26 1984-06-19 Cutter Laboratories, Inc. Antihemophilic factor concentrate
JPS59167519A (ja) * 1983-03-15 1984-09-21 Nippon Sekijiyuujishiya 不活化トロンビンゲルにより血漿タンパク質混合液からフイブリノ−ゲンを除去する方法
EP0245875A2 (fr) * 1986-05-15 1987-11-19 Green Cross Corporation Méthode pour la purification du facteur VIII
JPH0236199A (ja) * 1988-07-22 1990-02-06 Asahi Medical Co Ltd 血液凝固因子の精製方法および精製用吸着材
JPH02255698A (ja) * 1989-03-29 1990-10-16 Green Cross Corp:The 血液凝固第８因子の製造法
WO1991007438A1 (fr) * 1989-11-09 1991-05-30 Novo Nordisk A/S Procede d'isolation du facteur viii
EP0882789A2 (fr) * 1997-06-05 1998-12-09 Fujimori Kogyo Co., Ltd. Procédé de synthese d'anhydrothrombine

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4455301A (en) * 1980-02-26 1984-06-19 Cutter Laboratories, Inc. Antihemophilic factor concentrate
JPS59167519A (ja) * 1983-03-15 1984-09-21 Nippon Sekijiyuujishiya 不活化トロンビンゲルにより血漿タンパク質混合液からフイブリノ−ゲンを除去する方法
EP0245875A2 (fr) * 1986-05-15 1987-11-19 Green Cross Corporation Méthode pour la purification du facteur VIII
JPH0236199A (ja) * 1988-07-22 1990-02-06 Asahi Medical Co Ltd 血液凝固因子の精製方法および精製用吸着材
JPH02255698A (ja) * 1989-03-29 1990-10-16 Green Cross Corp:The 血液凝固第８因子の製造法
WO1991007438A1 (fr) * 1989-11-09 1991-05-30 Novo Nordisk A/S Procede d'isolation du facteur viii
EP0882789A2 (fr) * 1997-06-05 1998-12-09 Fujimori Kogyo Co., Ltd. Procédé de synthese d'anhydrothrombine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KAERSGAARD, P. ET. AL.: "Isolation of the factor VIII-von Willebr and factor complex directly from plasma by gel filtration.", J. CHROMATOGR. B. BIOMED. SCI. APPL., vol. 715, no. 2, 1998, pages 357 - 367, XP002928075 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111592591A (zh) * 2020-06-17 2020-08-28 博雅生物制药集团股份有限公司 一种人血管性血友病因子/人凝血因子ⅷ复合物的制备方法及产物和应用

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AU3676700A (en) 2000-11-14

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