WO2000061633A1

WO2000061633A1 - Method for purifying blood coagulation factor viii and blood coagulation factor viii/von willebrand factor complex

Info

Publication number: WO2000061633A1
Application number: PCT/JP2000/002350
Authority: WO
Inventors: Kazuya Hosokawa; Toyoaki Suzuki; Masanori Nagata
Original assignee: Fujimori Kogyo Co., Ltd.; Chisso Corporation
Priority date: 1999-04-12
Filing date: 2000-04-11
Publication date: 2000-10-19
Also published as: AU3676700A; JP2002348300A

Abstract

A method for purifying a blood coagulation factor VIII/Von Willebrand factor complex and blood coagulation factor VIII via a simple step even from a solution containing a large amount of the blood coagulation factor VIII/Von Willebrand factor complex. This method comprises mixing a solution containing the blood coagulation factor VIII/Von Willebrand factor complex with a gel and then separating and removing the gel from the solution.

Description

Description Purification method for blood coagulation factor VIII and blood coagulation factor VIII / von Willebrand factor complex

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.

Among these blood coagulation factors, 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. In addition, since the deficiency or abnormality of the FVIII clotting activity protein is observed in hemophilia A, 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.

As mentioned above, the treatment of hemophilia patients uses anti-hemophilia drugs FVIII or factor IX (prothrombin complex). In some cases, antibodies to the deficiency factor may be raised in patients, and it has been cited as a major cause of viral hepatitis C and AIDS (acquired immunodeficiency syndrome). For this reason, at present, the safety of dry concentrated human blood coagulation factor VIII products heated in liquid form at 60 ° C for 10 hours and dry concentrated human blood coagulation factor VIII products heat-dried at 65 ° C for 95 hours is available. In addition to the use of heat-treated preparations with extremely high potency, blood plasma is used as a raw material, and the virus is inactivated by heat treatment or treatment with an organic solvent / detergent (TNBP / octoxinol 9 treatment), and anti-FVIII A dry concentrated human blood coagulation factor VIII preparation purified and separated using a monoclonal antibody, and disclosed in, for example, Japanese Patent Application Laid-Open No. 7-278,197 (Patent No. 2513993). As a result, recombinant products have been developed in which products produced by genetic recombination techniques have been purified by affinity chromatography using a monoclonal antibody against the C-terminal subunit of factor VIII: C activity, etc. . However, even for preparations in which virus treatment was inactivated by heat treatment or treatment with an organic solvent / surfactant and safety was improved, the risk of viral infections such as non-A non-B hepatitis could not be completely ruled out. In addition, since the heated preparation contains fibrinogen, administration thereof may cause an excessive rise in the concentration of fibrinogen in the blood. On the other hand, when the monoclonal antibody is used for purification and separation, (1) since the protein derived from a heterologous animal is often used in the monoclonal antibody, the protein derived from the heterologous animal is often contained in the preparation. (2) Monoclonal antibodies themselves are extremely expensive in terms of cost, and there is a problem that the final preparation must be expensive.

Therefore, there is a strong need for a technology that can purify FVIII without these problems.However, FVIII clotting protein has a plasma content of about 0.2 g / m1 and other clotting factors. In order to obtain 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.

By the way, 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 ”.

The von Wille brand factor protein, a component of the FVIII / vWF complex, 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. In addition, this 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.

In addition, since 95% or more of the FVIII / vWF complex is occupied by vWF protein, a method of purifying and refining the FVIII / vWF complex and then collecting a FVIII coagulation-active protein was attempted. 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). After further purifying the FVIII / vWF complex by insoluble guinea pig (CIG) or a CNBr-Sepharose 4B column immobilized with anti-IgM rabbit serum IgM fraction), 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-strength ram.The partially purified FVIII / vWF complex is further purified by CNBr- After adsorbing onto a Sepharose 4B-anti-vWF monoclonal antibody column, washing, and then eluting FVIII with a buffer solution containing calcium chloride. However, although 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. . In addition, 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. In order to obtain purified protein equivalent to mg, 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. Therefore, 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. Then, 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.

In addition, 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.

In addition to the above findings, by combining the above two steps, 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. By doing so, FVIII containing almost no impurities We also found that it can be obtained efficiently.

Based on these findings, the present invention has been completed.

That is, the present invention has the following configurations (1) to (10).

(1) After coagulation of a solution containing the complex factor VIII / Phone 'virbrand factor complex with the gel, separation and removal of the gel from the mixture: factor VIII / von Willebrand How to purify factor complexes

(2) The method according to (1), wherein the gel is a dry gel.

(3) Blood coagulation obtained by the method according to (1) or (2) above! /! A method for purifying a blood coagulation factor VI / von-Vilbrand factor complex by performing gel filtration on a solution containing the factor / von-Vilbrand factor complex.

(4) Purifying the solution containing the blood coagulation factor VIII / von Willebrand factor complex obtained by the method according to any one of (1) to (3) by ultrafiltration. How to purify the blood coagulation factor VIII / Fon / Vilbrand factor complex.

(5) The method according to any one of (1) to (4), wherein the exclusion limit of the gel by a protein is in a range of 300,000 to 20,000,000.

(6) Dissociating the blood coagulation factor VIII / von Willebrand factor complex obtained by the method according to any one of the above (1) to (5) into blood coagulation factor VIII and von Willebrand factor A method for purifying blood coagulation factor VIII, comprising the step of:

(7) 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).

(8) The ultrafiltration membrane used in the ultrafiltration has a cut-off molecular weight of 300,000 to 1 The method according to (7), wherein the range is 800,000.

(9) After the dissociation step according to (6) or the ultrafiltration step according to (7), the mixture of blood coagulation factor νπι factor and von Willebrand factor is subjected to affinity chromatography. A method for purifying blood coagulation factor VI, comprising a step of purifying.

(10) The method according to (9), wherein the ligand of the adsorbent used in the affinity chromatography is anhydrothrombin. BEST MODE FOR CARRYING OUT THE INVENTION

According to a first aspect of the present invention, 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. By the above method, large capacity

Even a solution containing the FVIII / vWF complex can be concentrated several times to several tens times in a simple process, and simultaneously separates low-molecular-weight contaminating proteins such as fibrinogen-fibronectin and small molecules such as water. · Can be removed. 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. However, for example, plasma derived from human, preferably normal human [preferably fresh (frozen) plasma], body fluids such as platelets and placenta; cryoprecipitate (cryoprecipitate); fraction I (Fraction I) (8% ethanol precipitation); and a solution containing these mutants which have an amino acid sequence in which at least one amino acid has been deleted, substituted or added by these genetic recombination operations and which maintains factor VIII clotting activity. And so on. At this time, in the present invention, cryoprecipitate (cryoprecipitate) is prepared from plasma by a conventionally known method. Alternatively, 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. For example, 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. However, spherical and fine particles are preferably used in consideration of the packing density in the container and the contact efficiency with the starting solution. Also, 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.

In the present invention, 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. Specifically, 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. At this time, if 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. On the other hand, when the exclusion limit of the gel is less than 300,000, fibrinogen (molecular weight: about 330,000) ゃ 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. In some cases, Nectin is a dimer or tetramer in the S--S bond. In the case where is present, 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. In addition, when fibronectin in the form of a tetramer is present in the low molecular weight protein, 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.

In the present invention, 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. In consideration of these points, 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. When a dry gel is used, 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. Also use 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. )%, More preferably 80 to: LOO (v / v)%, most preferably 90 to: L00 (v / v)%. In this specification, 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. Such a dried gel may be used as it is without equilibrating the gel with a buffer or the like. Alternatively, 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. In the present invention, 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.

In the present invention, 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. For example, 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, pressure filtration, and the like. It is. Of these, pressure filtration and centrifugation are preferably used.

By such a method, 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. In the present specification, 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.

Further, in the present invention, 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. By repeating the above-described operation in this manner, 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. When the above concentration / impurity removal operations are repeated, 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.

According to the second aspect of the present invention, 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.

In the present invention, 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. At this time, 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). Taking into account that, 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. At this time, when the molecular weight cut off of the ultrafiltration membrane exceeds 18 million, 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. On the other hand, if 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. As such an ultrafiltration membrane, 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). In the present specification, 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.

As described above, since fibronectin may be in the form of a dimer or a tetramer due to the S--S bond, the dimer form is present in the low-molecular-weight protein. When fibronectin is present, 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. In addition, if the tetrameric form of fibronectin is present in low molecular weight foreign proteins, 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.

By such a simple ultrafiltration procedure, 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.

In the present invention, 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. By repeatedly performing the above ultrafiltration operation, contaminant proteins such as fibrinogen / fibronectin are further removed, and the target FVI II / vWF complex is purified with higher purity. When the above purification operation is repeatedly performed, 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. .

After the purification step according to the first aspect, 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. For example, Sephacryl-300 (manufactured by Pharmacia) and Sepharose 6B column (manufactured by Pharmacia) Etc. can be used. In this case as well, similarly, when the dimeric form of fibronectin is present in the low molecular weight protein, the exclusion limit of the gel by protein is set at 500,000 to 200,000. In the case where the tetrameric form of fibronectin is present in the low-molecular-weight contaminant protein, 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.

In the above, 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. Considering the fact that the pores of the ultrafiltration membrane are clogged by low-molecular-weight proteins such as urea, 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. According to a third aspect of the present invention, 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.

In the above embodiment, 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.

Further, in the above embodiment, after the dissociation operation of the FVIII / vWF complex into FVII I and vWF is performed in this manner, 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”). In the step of purifying the mixture of blood coagulation factor VIII and von 'Vilbrand factor by ultrafiltration, 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.

After removing the miscellaneous proteins as much as possible by the above-described steps, 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). Through the ultrafiltration membrane. That is, 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. As 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. Is preferentially adsorbed and recovered, so that FVIII with higher purity can be obtained. In this case, 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. For example, 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. Of these, 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. In the affinity chromatography, the ligands may be used alone or in combination of two or more. In addition, 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. Hereinafter, the present invention will be described specifically with reference to examples. 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.

Example 1

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. 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 enabled a 5-fold concentration in a single operation.

Further, the 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.

Subsequently, 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.

After the above ultrafiltration procedure was repeated to concentrate to about 100,000 times, 0.3M calcium chloride was added to this concentrated solution to dissociate the FVIII / vWF complex, and the same as above By ultrafiltration using a 0.1 micron membrane filter (manufactured by Millipore), only FVIII was selectively passed through the membrane to obtain purified FVIII. At this time, the specific activity of the purified FV III was 2,000 u / mg, and the recovery was about 40%. Example 2

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.

Further, this 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.

Subsequently, 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.

After the above procedure was repeated to concentrate to about 1 Q 10,000 times, 0.3 M calcium chloride was added to this concentrate to dissociate the FVIII / vWF complex, and then BIOMAX lOOOKDa By ultrafiltration using (Millipore), only FVIII was selectively passed through the membrane to obtain purified FVIII. At this time, the specific activity of the purified FVIII was 2,200 u / mg, and the recovery was about 30%.

Example 3

FVIII / vWF eluted in the vicinity of the void volume by subjecting 30 ml of the cryo-concentrated solution finally prepared in the same manner as in Example 1 to gel filtration using a Sephacryl-4B (Pharmacia) column (500 ml) to carry out gel filtration The complex was recovered.

Subsequently, 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, ρΗ7.5). The specific activity of the purified FVIII thus obtained was about 2,500 u / mg, and the recovery was about 40%. Industrial applicability

As described above, 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. By following this method, 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. In addition, by such a method, 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.

In particular, 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. By ultrafiltration, 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.

In addition, 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. By purification by chromatography, blood coagulation factor VIII can be separated in a short time and efficiently with almost no impurities.

Claims

The scope of the claims

1. A blood coagulation factor VIII / von-Bilbrand factor complex, which consists of mixing a solution containing the complex with the gel and separating and removing the gel from the mixture. Purification method.

2. The method according to claim 1, wherein the gel is a dry gel.

3. Blood coagulation VI, which is performed by gel filtration chromatography on a solution containing the blood coagulation factor フォン / von Willebrand factor complex obtained by the method according to claim 1 or 2. Factor / Von 'method for purification of the bilant factor complex.

4. Purification by ultrafiltration of a solution containing a blood coagulation factor VIII / von Willebrand factor complex obtained by the method according to any one of claims 1 to 3 Method of purifying blood coagulation factor VIII / von Willebrand factor complex.

5. The method according to any one of claims 1 to 4, wherein the exclusion limit of the gel by the protein is in the range of 300,000 to 20,000,000.

6. The blood coagulation factor VIII / von Willebrand factor complex obtained by the method according to any one of claims 1 to 5 is combined with blood coagulation factor VIII and von 'Vilbrandt. A method for purifying blood coagulation factor VIII, comprising a step of dissociating into factor.

7. A method for purifying blood coagulation factor VIII, comprising: after the dissociation step according to claim 6, purifying a mixed solution of blood coagulation factor VIII and von Willebrand factor by ultrafiltration.

8. The method according to claim 7, wherein the molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration is in the range of 300,000 to 180,000.

9. Dissociation process described in claim 6 or described in claim 7 A method for purifying blood coagulation factor VI, comprising a step of purifying a mixed solution of blood coagulation factor VI and a von Willebrand factor by affinity chromatography after the ultrafiltration step described above.

10. The method according to claim 9, wherein the ligand of the adsorbent used in the affinity mouth chromatography is anhydrothrombin.