JP2931319B2 - Method for producing blood coagulation factor VIII - Google Patents

Description

The present invention relates to a method for producing blood coagulation factor VIII. More specifically, the present invention relates to a method for producing high-purity blood coagulation factor VIII with good solubility by removing contaminating proteins contained in blood coagulation factor VIII by gel filtration.

[Problems to be solved by the prior art and the invention]

Blood coagulation factor VIII is formed in the body as a complex with von Willebrand factor as a multimer with a molecular weight of 1 to 20 million, and exists as a macromolecule unique to other plasma proteins.

Blood coagulation factor VIII has been conventionally used to treat bleeding in hemophilia A patients. At present, blood coagulation factor VIII is supplemented to hemophilia A patients by cryoprecipitate or using it as a raw material for PEG fractionation, ethanol fractionation,
A blood coagulation factor VIII concentrate produced by glycine fractionation or the like is used.

However, these preparations are rich in coagulable proteins such as fibrinogen and put a heavy burden on patients. In addition, these preparations are generally prepared in a lyophilized form, and there is a problem that their dissolution is not always easy due to the contained coagulable protein.

In recent years, a production method using a monoclonal antibody has also been developed, but in such preparations, contamination of the monoclonal antibody is feared.

Accordingly, an object of the present invention is to provide a highly pure blood coagulation factor VIII preparation.

Another object of the present invention is to provide a blood coagulation factor VIII preparation having excellent solubility.

[Means for solving the problem]

In view of such circumstances, the present inventors have conducted intensive studies and found that the aqueous solution containing blood coagulation factor VIII was subjected to gel filtration using a water-insoluble porous gel having an exclusion limit molecular weight of 800,000 to 100 million. The present inventors have found that coagulation proteins such as fibrinogen can be efficiently removed without using complicated operations, and that blood coagulation factor VIII having good solubility can be obtained.

That is, in producing the blood coagulation factor VIII, the present invention uses a water-insoluble porous gel of a crosslinked dextran type having an exclusion limit molecular weight of 800,000 to 100 million, and a gel filtration using a solvent containing a calcium salt. The purpose of the present invention is to improve the quality of a conventional factor VIII concentrate by separating blood coagulation factor VIII.

Further, the present invention, in the method for producing blood coagulation factor VIII, characterized in that the aqueous solution containing blood coagulation factor VIII is treated in the following steps, not only to improve the quality of the factor VIII concentrate, The purpose is to improve the recovery and purity of factor VIII.

1) treatment with an aluminum hydroxide gel 2) treatment with a surfactant for virus inactivation 3) glycine fractionation 4) fractionation with sodium chloride, and 5) an exclusion limit molecular weight of 800,000 to 100,000,000, Blood coagulation factor VIII is separated by gel filtration using a cross-linked dextran-based water-insoluble porous gel and a solvent containing a calcium salt.

In the context of the present invention, blood coagulation factor VIII is not particularly limited as long as it is of human origin.

The aqueous solution of blood coagulation factor VIII, which is the starting material of the present invention, is not particularly limited as long as it is an aqueous solution containing blood coagulation factor VIII containing contaminants, and it may be partially purified. Since blood coagulation factor VIII is mainly contained in plasma, it is preferable to use plasma as a starting material, and cryoprecipitate (cold precipitate) [Br
itish Journal of Haematology 21 , 21 (1970) Thrombos
is Diathesis Haemourhagica 11 , 64 (1964)]. Furthermore, partially purified blood coagulation factor VIII is also used. The partial purification method is performed by known means, for example, aluminum hydroxide gel treatment, polyethylene glycol fractionation treatment, ethanol fractionation treatment,
Glycine fractionation treatment, anion exchanger treatment, hydrophobic chromatography treatment, affinity chromatography, fractionation treatment with sodium chloride, and the like (U.S. Pat. JP-A-60-511115 and JP-A-63-108000). Further, Factor VIII which has been subjected to heat treatment or virus inactivation treatment (for example, treatment with a surfactant) can also be used (JP-A-58-74617, JP-A-59-134730, and JP-A-59-134730).
No. 1-222022, No. 60-51116).

In the present invention, it is carried out by purifying blood coagulation factor VIII by gel filtration.

As the carrier for gel filtration, a crosslinked dextran-based water-insoluble porous gel having an exclusion limit molecular weight of 800,000 to 100 million is used. For example, Sephacryl-400, 500, 1000 (manufactured by Pharmacia) and the like can be mentioned.

With a carrier for gel filtration having a lower limit of the exclusion limit molecular weight of less than 800,000, removal of contaminants to be removed such as fibrinogen having a molecular weight of 340,000 and fibronectin having a molecular weight of about 350,000 becomes insufficient. The exclusion limit molecular weight corresponds to the upper limit of the fractionable molecular weight range of the carrier for gel filtration. The gel filtration carrier usable in the present invention can be selected from the description as long as it is a commercially available gel filtration carrier, since the exclusion limit molecular weight can be easily specified from a pamphlet. Also, rather than a commercial product, even a gel filtration carrier prepared uniquely, globular proteins of known molecular weight, such as albumin,
A calibration curve (standard curve) is prepared using globulin, globin, and the like, and the exclusion limit molecular weight is specified from the result, and a gel filtration carrier usable in the present invention can be selected.

Examples of the solvent for pretreatment and development of the carrier include a buffer having a pH of 6 to 8 and an ionic strength of about 1 to 2000 mM. Specifically, 5 to 50 mM Tris buffer (pH 7 to 9), 10 to 100 mM phosphate buffer (pH 6 to 8) and the like are used.

The solvent contains a calcium salt. The effect of stabilizing blood coagulation factor VIII is exerted by the presence of the calcium salt.

As a sample, 5 to 10% of the column capacity is desirable. By recovering the fraction containing the blood coagulation VIII fraction in this way, purification and production can be performed. Preferably, a fraction having a molecular weight of 1,000,000 or more is collected.

The obtained blood coagulation factor VIII can be further purified using a known purification method (salinization of factor VIII using glycine and salt [Thorell et al., Thromb, Res., 35, 431).
-450, 1984] and a method using aminohexyl-sepharose (Austen, Br. J. Haematol, 43, 669-674, 197).
9]).

In formulating, for example, addition of a stabilizer, addition of a compounding agent, sterilization filtration, dispensing subdivision, freeze-drying and the like can be performed.

The lyophilized preparation thus obtained is dissolved in distilled water for injection at the time of use and administered by injection or the like.

The dose of blood coagulation factor VIII obtained by the production method of the present invention may be in accordance with conventional preparations.

〔Example〕

Example 1 Cryoprecipitate obtained by freezing and thawing normal human / plasma was extracted and dissolved 5-fold with a 20 mM Tris-10 mM citrate buffer (pH 7.0) as a starting material. Was added to 1/10 volume of the charged weight of cryoprecipitate and stirred for 30 minutes. Then 6 g of bentonite
, And stirred for 1 hour, followed by centrifugation at 4000 rpm for 30 minutes to obtain a supernatant, followed by treatment with a surfactant for virus inactivation (to a concentration of 0.3% Tri-n-butylphosphate, 1% Tween 80). , And stirred at 30 ° C for 6 hours).
This was subjected to glycine fractionation. The conditions for glycine fractionation were
Glycine was added to this solution at a rate of 150 g / 30 ° C.,
After stirring for 1 hour, centrifugation (4000 rpm, 30 minutes, 30 ° C.) was performed to obtain a supernatant, and sodium chloride was added to the supernatant (87 g /).
After stirring for one hour, the Factor VIII fraction was salted out to obtain a centrifugal precipitate (4000 rpm, 30 minutes, 30 ° C.).

Thereafter, the salted-out precipitate was dissolved in a 20 mM Tris buffer (pH 7.0) to adjust the absorbance at 280 nm to 25 to 30, and then used as a sample to be injected into a gel filtration column. The column packing is made of Pharmacia
Using Sephacryl S-400 HR, a factor VIII-active fraction discharged from the column was collected at an injection amount of 5 to 7% of the column bed capacity. The conditions are as follows: Injected sample: Salting-out precipitation solution Column: Sephacryl S-400HR (1.5 cm ID × 90 cm bed vol.cal 60 ml) Flow rate: 0.3 ml / min. Solvent: 20 mM Tris (HCl ), 10mM CaCl ₂ , 1M NaCl buffer,
pH 7.0 As a result, blood coagulation factor VIII was observed in the column void fraction as shown in FIG. 1, and contaminating proteins were highly separated.

Examples 2 to 6 In the same manner as in Example 1, blood coagulation by gel filtration was performed.
The degree of purification of factor VIII was determined by activity and recovery of contaminating protein. The conditions are as follows.

 Injection sample: Salting-out precipitation solution Column: Sephacryl S-400HR (1.5 cm ID × 90 cm bed vol.ca 160 ml) The same solvent as in Example 1 was used.

In Examples 5 and 6, Sephacryl S-400 was used.
An HR column size (5 cm ID × 90 cm bed cal 700 ml) was used.

Blood coagulation factor VIII activity and protein content were measured before gel filtration (applied) and after fractionation (void) above the exclusion limit molecular weight after gel filtration. The results are shown in Table 1.

In the table, "A280" indicates the absorbance at 280 nm of the aqueous protein solution, the specific activity of "F.VIII / A280" is "F.VIII / m
g ".

〔The invention's effect〕

As is clear from the examples, the treatment of the present invention has the effect of efficiently purifying blood coagulation factor VIII and providing a preparation having practically very low solubility of contaminating proteins.

Therefore, the method of the present invention is considered to be extremely useful for preparing a medical preparation of blood coagulation factor VIII.

[Brief description of the drawings]

FIG. 1 shows the development pattern of blood coagulation factor VIII by gel filtration of the present invention.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Toshiharu Honkubota 2-1,180-1, Odani Otani, Hirakata-shi, Osaka 1 Inside the Midori Cross Research Institute, Inc. No. 1 Inside Midori Cross Central Research Institute Co., Ltd. (72) Inventor Kazumasa Yokoyama 2-1-1180 Shodai Otani, Hirakata City, Osaka Prefecture 1 Inside Midori Cross Central Research Institute Co., Ltd. (56) References JP-A-52-57310 (JP, A)

Claims (2)

(57) [Claims] An aqueous solution containing blood coagulation factor VIII is subjected to gel filtration using a crosslinked dextran-based water-insoluble porous gel having an exclusion limit molecular weight of 800,000 to 100 million and a calcium salt-containing solvent. A method for producing blood coagulation factor VIII, comprising separating blood coagulation factor VIII. 2. The method for producing blood coagulation factor VIII according to claim 1, wherein the aqueous solution containing blood coagulation factor VIII is treated in the following steps: 1) treatment with aluminum hydroxide gel 2) virus Treatment with a surfactant for inactivation 3) Glycine fractionation 4) Fractionation with sodium chloride, and 5) Use of a crosslinked dextran-based water-insoluble porous gel having an exclusion limit molecular weight of 800,000 to 100,000,000 The blood coagulation factor VIII is separated by gel filtration using a solvent containing a calcium salt.