JP2001335509A - Method for removing virus from solution containing fibrinogen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an industrially advantageous method for removing viruses in which the filtration efficiency is improved for removing the viruses from a fibrinogen solution having fear of viral contamination. SOLUTION: A basic amino acid and sodium chloride are coexisted in the fibrinogen solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for removing a virus from a solution containing fibrinogen which may contain viruses by using a virus removal membrane, the method comprising the step of containing a basic amino acid or a salt thereof and sodium chloride in the solution. The present invention relates to the provision of a method for removing viruses more efficiently.

[0002]

BACKGROUND ART It cannot be denied that blood products such as human plasma and derivatives thereof such as fibrinogen preparations may be contaminated with viruses such as AIDS virus, various hepatitis viruses and human parvovirus B19. Therefore, in the production of drugs using these, it is essential to incorporate a step of sufficiently inactivating and / or removing viruses. As a method of inactivating viruses that may be contaminated in blood products, heat treatment in an aqueous solution state (hereinafter referred to as liquid heating) is described by Murray et al. (The New York Acad).
emy of Medicine, 31 (5) 341-358 (195)
5), and since then this method has been widely adopted as a virus inactivation method for blood products. On the other hand, as a method for removing a virus, JP-A-2-167232 describes a method in which a virus intentionally added to a blood coagulation factor VIII preparation is removed by filtration with a porous hollow fiber filter made of regenerated cellulose. . Sekiguchi et al. Reported a method of introducing a virus removal step using a hollow fiber filter for virus removal during the immunoglobulin preparation manufacturing process (Japanese Journal of Trans.
fusion Medicine, 34 (6) 615-617 (198)
8). However, the virus removal method using these virus removal filters has problems such as poor filterability when the protein is fibrinogen, a decrease in yield and an increase in the amount of membrane used, and a problem in industrial productivity. This is a major issue. Also, among plasma proteins, fibrinogen is said to have a higher risk of viral contamination than other protein preparations. For this reason, virus inactivation and removal must be performed more strictly than other protein preparations. Examples of the virus inactivation method include a liquid heating method, a drying heating method, a solvent detergent (SD) method, and the virus removal method includes a virus removal method using a virus removal filter. It is also known that the virus can be removed by the ethanol precipitation method and the column chromatography method.

[0003]

The combination of a plurality of the various methods described above is considered to be an effective means for perfecting the inactivation and removal of the virus. For that purpose, fibrinogen is used in each treatment step. It is industrially important to minimize the decrease in the yield of methane and to efficiently process the phenol. An object of the present invention is to provide a virus removal step for a fibrinogen solution that is at risk of virus contamination,
An object of the present invention is to provide a safe fibrinogen preparation by removing viruses more efficiently and industrially advantageously.

[0004]

Means for Solving the Problems The present inventors have conducted various studies to solve the above-mentioned problems, and as a result, when treating a solution containing fibrinogen with a virus-removing membrane, a basic amino acid or salts thereof, In particular, when lysine and / or arginine and sodium chloride are coexisted, fibrinogen exhibits high stability and excellent filterability, and fibrinogen from which virus has been removed by a virus removal membrane treatment is subjected to various membrane filtration steps to be performed thereafter. And found that they exhibited excellent filterability, and further studied to complete the present invention. That is, the present invention provides (1) a method for removing a virus from a solution containing fibrinogen which may be mixed with a virus using a virus removal membrane, wherein the solution containing fibrinogen contains a basic amino acid or a salt thereof and sodium chloride. (2) the virus removal method according to the above (1), wherein the basic amino acid is lysine or arginine; and (3) the concentration of the basic amino acid or the salt thereof in the solution containing fibrinogen. 0.01 to 10.
0 w / v%, and the concentration of sodium chloride is 0.01-1.
The virus removal method according to the above (1), wherein the concentration of the basic amino acid or the salt thereof in the solution containing fibrinogen is 0.25 to 3.0 w / w%;
/ V%, and the concentration of sodium chloride is 0.25 to 3.0.
w / v%, the method for removing viruses according to the above (1).

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The fibrinogen to be virus-removed according to the present invention is obtained by dissolving corn fraction I or cryopaste derived from human plasma in an aqueous sodium citrate solution and, if necessary, using a filter aid for filtration and concentration. Those obtained by purification with ethanol are preferred. In the present invention, a solution containing fibrinogen is added with a basic amino acid or a salt thereof and sodium chloride, and subjected to various pre-filtrations, followed by a virus removal membrane treatment. Examples of the basic amino acid or salts thereof include lysine, lysine hydrochloride, arginine, arginine hydrochloride and the like. The amount of the basic amino acid or its salt is usually from 0.01 to 1 in a solution containing fibrinogen.
0.0 w / v%, preferably 0.25 to 3.0 w / v
v%. The amount of sodium chloride added is usually 0.01
To 10.0 w / v%, preferably 0.25 to 3.0 w / v.
w / v%. The basic amino acid may be added at any stage before the virus removal membrane treatment, but is preferably added before the preliminary filtration treatment.

[0006] The solution containing fibrinogen thus prepared exhibits unprecedentedly high filterability and fibrinogen recovery. Furthermore, a solution containing fibrinogen to which a basic amino acid or a salt thereof and sodium chloride are added has high membrane filtration properties.For example, even when an ultrafiltration membrane is used, it is stably concentrated to an unprecedentedly high concentration. This makes it possible to obtain an unprecedentedly high recovery, and it is possible to continuously introduce various filtration steps in the step after the virus removal membrane treatment.

[0007]

The present invention will be described below in more detail with reference to Examples, Comparative Examples and Test Examples. Example 1 1.6 kg of corn fraction I derived from human plasma
A solution obtained by dissolving 33 liters of a 5 mM aqueous sodium citrate solution (pH 7.0) (A280 absorbance = 1
To 4), 3.8 kg of barium sulfate was added, and 90 ° C. was added at 25 ° C.
After stirring for minutes, insolubles were removed. 55 m in the obtained liquid
The pH was adjusted to 6.4 by addition of an aqueous solution of citric acid M, and sodium chloride was further added to a concentration of 0.9 w / v%. Then, the solution was cooled to 0 ° C., and ethanol was added so that the ethanol concentration became 7.5 v / v%. The precipitate obtained by centrifugation is subjected to a 55 mM sodium citrate aqueous solution (pH 6.4) containing 0.9% w / v sodium chloride.
Dissolved in 22 liters. After that, the ethanol concentration becomes 2 v
/ V%, and ethanol was added to remove the precipitate. Then, ethanol was added so that the ethanol concentration became 8 v / v%, and a purified fibrinogen fraction was obtained as a precipitate. The fibrinogen fraction precipitate 0.6 k
g, dissolved in 6.4 liters of a solution containing 0.2% w / v arginine and 0.25% sodium chloride.
m and 0.1 μm filters, sequentially.
The virus was passed through a virus removal filter (Planova 35N, manufactured by Asahi Kasei Corporation) at a primary pressure of 8 kgf / cm2. The amount of fibrinogen in the filtrate was measured, and the yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by multiplying the obtained value by volume and the value obtained by multiplying the amount of fibrinogen before filtration by volume. The results are shown in [Table 1].

Example 2 8 of purified fibrinogen obtained in the same manner as in Example 1
0.6 kg of the v / v% ethanol precipitate is reduced to 0.04 w / v%
The fibrinogen solution obtained by dissolving with 6.4 liters of a solution containing arginine and 0.05% sodium chloride was 10 μm, 1.2 μm, 0.5 μm and 0.1 μm.
0.8kgf / cm2
Virus removal filter (Planova 35N,
(Made by Asahi Kasei Corporation). The yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by measuring the amount of fibrinogen in the filtrate and the obtained value multiplied by the volume and the value obtained by multiplying the amount of fibrinogen before filtration by the volume. The results are shown in [Table 1].

Example 3 8 of purified fibrinogen obtained in the same manner as in Example 1
0.6 kg of the v / v% ethanol precipitate was added to 1.2 w / v%
Solution 1 containing arginine and 0.9% sodium chloride
The fibrinogen solution obtained by dissolving with 2 liters is sequentially filtered through a 10 μm, 1.2 μm, 0.5 μm and 0.1 μm filter, and then a virus removal filter (Planova 35N, Asahi Kasei Corporation) at a primary pressure of 0.8 kgf / cm 2. (Manufactured by Co., Ltd.). The amount of fibrinogen obtained in the filtrate was measured, and the yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by multiplying the obtained value by the volume and the value obtained by multiplying the amount of fibrinogen before filtration by the volume. The results are shown in [Table 1].

Example 4 8 of purified fibrinogen obtained in the same manner as in Example 1
A solution 21 containing 0.6 kg of v / v% ethanol precipitate containing 1.2% arginine hydrochloride and 0.9% sodium chloride
1 liter of the resulting fibrinogen solution
After sequentially filtering through filters of 0 μm, 1.2 μm, 0.5 μm and 0.1 μm, the mixture was passed through a virus removal filter (Planova 35N, manufactured by Asahi Kasei Corporation) at a primary pressure of 0.8 kgf / cm 2. The yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by measuring the amount of fibrinogen in the filtrate and the obtained value multiplied by the volume and the value obtained by multiplying the amount of fibrinogen before filtration by the volume. Further, the area of the virus removal filter required when all the solution was passed was calculated. The results are shown in [Table 1] and [Table 2].

Comparative Example 1 Purified fibrinogen 8 obtained in the same manner as in Example 1
A fibrinogen solution obtained by dissolving 0.6 kg of a v / v% ethanol precipitate with 6.4 liters of a 0.25% sodium chloride solution was used to obtain 10 μm, 1.2 μm, and 0.5 μm
And sequentially filtered through a 0.1 μm filter,
The solution was passed through a virus removal filter (Planova 35N, manufactured by Asahi Kasei Corporation) at a primary pressure of kgf / cm2. The yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by measuring the amount of fibrinogen in the filtrate and the obtained value multiplied by the volume and the value obtained by multiplying the amount of fibrinogen before filtration by the volume. The results are shown in [Table 1].

Comparative Example 2 The purified fibrinogen 8 obtained in the same manner as in Example 1
A fibrinogen solution obtained by dissolving 0.6 kg of a v / v% ethanol precipitate in 6.4 liters of a 0.20% arginine solution was sequentially filtered through a 10 μm, 1.2 μm, 0.5 μm and 0.1 μm filter. 0.8 kg later
It was passed through a virus removal filter (Planova 35N, manufactured by Asahi Kasei Corporation) at a primary pressure of f / cm2. The yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by measuring the amount of fibrinogen in the filtrate and the obtained value multiplied by the volume and the value obtained by multiplying the amount of fibrinogen before filtration by the volume. The results are shown in [Table 1].

Example 5 1.6 kg of corn fraction I derived from human plasma was
A solution obtained by dissolving 33 liters of a 5 mM aqueous sodium citrate solution (pH 7.0) (A280 absorbance = 1
To 4), 3.8 kg of barium sulfate was added, and 90 ° C. was added at 25 ° C.
After stirring for minutes, insolubles were removed. 55 m in the obtained liquid
The pH was adjusted to 6.4 by addition of an aqueous solution of citric acid M, and sodium chloride was further added to a concentration of 0.9 w / v%. Then, the solution was cooled to 0 ° C,
Ethanol was added at 7.5 v / v%. The precipitate formed after centrifugation was washed with a 55 mM aqueous sodium citrate solution (pH 6.4) containing 0.9% w / v sodium chloride.
Dissolved in 2 liters. Thereafter, the ethanol concentration was 2 v /
After adding and separating ethanol to remove the precipitate, the ethanol was added so that the ethanol concentration became 8 v / v% to obtain a purified fibrinogen fraction. 0.6 kg of an 8 v / v% ethanol precipitate of purified fibrinogen obtained in the same manner as in Example 1 was added to 1.2 w /
22 liters of a fibrinogen solution obtained by dissolving with 21 liters of a solution containing v% lysine hydrochloride and 0.9% sodium chloride was sequentially filtered through 10 μm, 1.2 μm, 0.5 μm and 0.1 μm filters. 0.8k
It was passed through a virus removal filter (Planova 35N, manufactured by Asahi Kasei Corporation) at a primary pressure of gf / cm2. The yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by measuring the amount of fibrinogen in the filtrate and the obtained value multiplied by the volume and the value obtained by multiplying the amount of fibrinogen before filtration by the volume. In addition, the area of the virus removal filter required when all of the solution was passed was calculated. The results are shown in [Table 2].

Example 6 The purified fibrinogen 8 obtained in the same manner as in Example 1
22 liters of a fibrinogen solution obtained by dissolving 0.6 kg of a v / v% ethanol precipitate in 21 liters of a solution containing 1.2% w / v lysine hydrochloride, 1.2% arginine hydrochloride and 0.9% sodium chloride. 10 μm, 1.2
After sequentially filtering through a filter of μm, 0.5 μm and 0.1 μm, the mixture was passed through a virus removal filter (Planova 35N, manufactured by Asahi Kasei Corporation) at a primary pressure of 0.8 kgf / cm 2. The yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by measuring the amount of fibrinogen in the filtrate and the obtained value multiplied by the volume and the value obtained by multiplying the amount of fibrinogen before filtration by the volume. Further, the area of the virus removal filter required when all the solution was passed was calculated. The results are shown in [Table 2].

Example 7 8 of purified fibrinogen obtained in the same manner as in Example 1
A solution 21 containing 0.6 kg of v / v% ethanol precipitate containing 1.2% w / v lysine hydrochloride and 0.9% sodium chloride.
Fibrinogen solution 22 obtained by dissolving in 1 liter
One liter is 10 μm, 1.2 μm, 0.5 μm and 0.1 μm.
The solution was sequentially filtered through a 1 μm filter. After adding arginine hydrochloride to the mixture at a concentration of 1.2 w / v%,
It was passed through a virus removal filter (Planova 35N, manufactured by Asahi Kasei Corporation) at a primary pressure of gf / cm2. The yield of fibrinogen after filtration relative to that before filtration was calculated from the value obtained by measuring the amount of fibrinogen in the filtrate and the obtained value multiplied by the volume and the value obtained by multiplying the amount of fibrinogen before filtration by the volume. Further, the area of the virus removal filter required when all the solution was passed was calculated. The results are shown in [Table 2].

Test Example 1 Measurement of Fibrinogen Concentration The Laki method and the Blomback method were combined to measure the fibrinogen concentration in the samples obtained in Comparative Example 1 and Examples 1 to 7 before and after filtration. That is, each sample was diluted to obtain a fibrinogen concentration of 3 to 5 mg / ml.
This was used as a sample solution. 0.1 ml of 0.5 M phosphate buffer (pH 7.0) is added to 2.0 ml of the sample solution,
1.25 ml of a 0.2 M potassium chloride solution and 0.25 ml of purified water were added. Then 250 NIH UNITS
A 0.1 ml / ml thrombin (physiological saline) solution was added and mixed, and the mixture was allowed to stand at 37 ° C. for 1 hour to form an aggregate. This aggregate is collected by filtration, washed twice with purified water, and
After adding to a 20 ml volumetric flask containing 2.0 ml of sodium hydroxide solution and heating to 60 ° C. to dissolve, make up to exactly 20 ml with purified water, measure the absorbance at 278 nm and 325 nm, and determine the respective values as A278 and A278. A325. From the obtained values, the fibrinogen concentration (mg / ml) in each sample was calculated according to the following formula.

(Equation 1) (The value 16.17 is the molecular extinction coefficient of a 1% fibrinogen solution.)

[0017]

[Table 1] As shown in Table 1, the high-concentration fibrinogen solution of Example 1 to which arginine and sodium chloride were added was the same as the solution of Comparative Example 1 to which only the same concentration of sodium chloride was added under the same conditions, and only arginine was added. The fibrinogen yield after filtration using the virus removal membrane was improved several times or more as compared with the solution of Comparative Example 2 described above. The arginine concentration was set to 1.2%, and the sodium chloride concentration was set to 0.9%.
The yield after filtration using the virus removal membrane was 100% even when the concentration of fibrinogen was 9%. From this, it became clear that a high filtration yield of fibrinogen can be obtained by adding arginine and sodium chloride.

[0018]

[Table 2] As is clear from the results in Table 2, it was proved that not only the addition of arginine of Example 4 but also the addition of lysine of Example 5 alone significantly increased the yield. Further, it is clear from Example 6 that both the amino acids are mixed and added to significantly reduce the membrane area to be used, indicating that the invention is industrially extremely useful. In addition, as is clear from the results of Example 7, the results show that all of the steps of adding amino acids show high filterability before the virus removal membrane treatment.

[0019]

According to the present invention, in a method for removing a virus from a fibrinogen solution in which a virus may be mixed using a virus removal membrane, the virus can be removed more efficiently. That is, since the fibrinogen solution has a high membrane filtration property according to the present method, the virus removal membrane treatment proceeds extremely smoothly, and various filtration steps can be advantageously advanced also in the steps after the treatment.

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Claims (4)

[Claims] (1) A method for removing a virus from a solution containing fibrinogen which may contain a virus by using a virus removal membrane, wherein the solution containing fibrinogen contains a basic amino acid or a salt thereof and sodium chloride. Characterized virus removal method. 2. The method according to claim 1, wherein the basic amino acid is lysine or arginine. 3. The concentration of a basic amino acid or a salt thereof in a solution containing fibrinogen is from 0.01 to 10.0 w / w.
v%, and the concentration of sodium chloride is 0.01 to 10.0.
The virus removal method according to claim 1, wherein the amount is w / v%. 4. The concentration of a basic amino acid or a salt thereof in a solution containing fibrinogen is from 0.25 to 3.0 w / v.
%, And the concentration of sodium chloride is 0.25 to 3.0 w /
The virus removal method according to claim 1, which is v%.