JP2001270900A - Method for inactivating virus in fibrinogen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inactivating virus by the dry heating of fibrinogen having a fear of virus contamination in which a denaturation rate and/or a devitalization rate of the fibrinogen by heating are/is low, and the solubility and the dissolved condition of the obtained fibrinogen are satisfactory. SOLUTION: This method is to lyophilize an aqueous solution where the fibrinogen, a basic amino acid and sodium chloride are made to coexist and to heat the lyophilized product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for drying a fibrinogen aqueous solution which is likely to be contaminated with a virus and heating the dried fibrinogen aqueous solution to inactivate the virus. More specifically, upon virus inactivation by drying and heating, the rate of fibrinogen denaturation and inactivation is extremely low, fibrinogen after virus inactivation has good solubility in water, and its dissolved state is also stable. And a method for inactivating fibrinogen virus.

[0002]

2. Description of the Related Art Blood products such as human plasma and derivatives thereof may be contaminated with viruses such as AIDS virus, various hepatitis viruses, and human parvovirus B19. Therefore, in the production of therapeutic drugs using these, it is essential to incorporate a step of sufficiently inactivating the virus. As a method for inactivating viruses that may be contaminated in blood products, a heat treatment method in an aqueous solution state (hereinafter, referred to as a liquid heating method) is Mu.
rray et al. (The New York Academy of Medicine, 31
(5), 341-358 (1955)), and since then this method has been widely used as a virus inactivation method for blood products. However, plasma proteins with high heat stability that can withstand this liquid heating method are limited to a very small portion, such as albumin, and many other plasma proteins are unstable to heat, and are denatured or inactivated by this method. High rate.
Therefore, apart from the liquid heating method, a method of inactivating the virus by heat-treating the plasma protein in a dry state (hereinafter referred to as a dry heating method) has been proposed (Japanese Patent Publication No. 58-50054).
8). Although this method can suppress denaturation and deactivation of plasma protein to some extent as compared with the liquid heating method, it is not yet sufficient. Further, a method of adding a certain stabilizer at the time of drying and heating has been attempted, but no satisfactory result has been obtained. Further, there is a problem that the solubility of fibrinogen in water is deteriorated after heating, or the fibrinogen is once dissolved, and becomes unstable due to partial reaggregation with the passage of time.

[0003]

It is said that fibrinogen has a higher risk of viral contamination than other plasma protein preparations. For this reason, it is necessary to treat the virus inactivation more strictly than other blood products. Examples of the virus inactivation method include the above-described liquid heating method, dry heating method, and solvent detergent (SD) method.
As a virus removal method, a virus removal process using a virus removal filter or the like is known. Combining a plurality of these methods is considered to be an effective means for achieving perfect virus inactivation.To do so, it is necessary to minimize the denaturation and inactivation of fibrinogen in each treatment step, and to reduce the fibrinogen in these series of steps. It is important that the solubility is always kept good. It is an object of the present invention to provide a method for inactivating a fibrinogen that is likely to be contaminated with a virus, in which the denaturation and inactivation rates can be kept lower than any of the conventional methods. Still another object is to provide a fibrinogen having good solubility and dissolved state in water even after virus inactivation.

[0004]

The present inventors have conducted various studies to solve the above-mentioned problems. As a result, during freeze-drying and subsequent heat treatment of an aqueous fibrinogen solution, basic amino acids, particularly lysine and / or arginine, were obtained. When coexisting with sodium chloride, fibrinogen exhibits extremely high stability against freeze-drying and long-term heat treatment, and fibrinogen whose virus has been inactivated by heat treatment has poor solubility and dissolution in water. The inventors have found that the present invention is very good, and have further studied and completed the present invention. That is, the present invention provides (1) a method for inactivating fibrinogen, which comprises heating an aqueous solution containing fibrinogen, basic amino acid, and sodium chloride which may be contaminated with virus, and then heating until the virus is inactivated.
(2) the virus inactivation method according to (1), wherein the basic amino acid is lysine or arginine; (3) the virus inactivation method according to (1), wherein the basic amino acid is lysine; (4) the basic amino acid Is the lysine and arginine, (5) the aqueous solution is fibrinogen and 4 to 40 w of fibrinogen with respect to fibrinogen.
/ W% of a basic amino acid and 5 to 25 w / w% of sodium chloride, wherein the virus inactivation method according to the above (1), (6) the aqueous solution is 2 to 20 w / w with respect to fibrinogen and fibrinogen. w% lysine and 5%
(1) containing ２５25 w / w% sodium chloride
The virus inactivation method as described in (7), wherein the aqueous solution is fibrinogen and 2-20 w / w% lysine, 2-20 w / w% arginine and 5-25 w / w with respect to fibrinogen.
the virus inactivation method according to the above (1) containing w% sodium chloride; (8) heating at 50 to 80 ° C. and 24 to 19;
The virus inactivation method according to the above (1), which is performed for 2 hours, (9)
The virus inactivation method according to the above (1), wherein the dried fibrinogen has a water content of 3 w / w% or less;
0) fibrinogen in which the virus has been inactivated by the virus inactivation method according to any of (1) to (9),
It is.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The aqueous fibrinogen solution to be inactivated in the virus of the present invention is prepared by, for example, dissolving corn fraction I or cryopaste derived from human plasma in an aqueous sodium citrate solution, and using a filter aid as necessary. It is preferably a purified fibrinogen obtained by filtration, purification with ethanol having different concentrations, virus removal treatment with a virus removal filter, and the like.
Although this fibrinogen has a low possibility of virus contamination due to the above-mentioned special filtration and purification methods, its presence cannot be completely ruled out. In the present invention, a basic amino acid and sodium chloride are added to an aqueous fibrinogen solution, and if necessary, a virus removal treatment by a virus removal filter or ultrafiltration concentration is performed. Lyophilize. Basic amino acids include lysine, arginine and the like. The addition ratio of the basic amino acid to fibrinogen is 4 to 40 w / w%, preferably 6 to 35 w / w%. In particular, when lysine and arginine are used, the content of each is preferably 2 to 20 w / w%, preferably 3 to 17.5 w / w%. The addition rate of sodium chloride to fibrinogen is 5 to 25 w /
w%, preferably 8 to 18 w / w%. The basic amino acid and the salt may be added at any stage before heating, but it is preferably after purification with ethanol having different concentrations and before treatment with a virus removal filter.

More specifically, the concentration of purified fibrinogen in the solution is 9-11 w / v%, the concentration of basic amino acid is 0.7-3.5 w / v%, and the concentration of sodium chloride is 0.7-w / v%. It is preferable to prepare it to be 1.6 w / v%.
The aqueous solution containing fibrinogen, basic amino acid and sodium chloride is dried so that the water content is 3 w / w% or less, preferably 1.5 w / w% or less. Drying conditions are desirably low-temperature drying, particularly freeze-drying. The shape of the composition obtained by drying is powdery, massive,
There is a scaly shape, but any of them may be used. The dried fibrinogen thus obtained is heated until the virus is inactivated. The method of heating may be any method, and examples include infrared irradiation, sand bath, water bath, and heating with an oven. The atmosphere at the time of heating is usually air at atmospheric pressure. However, if necessary, reduced-pressure air, nitrogen, or other inert gas may be used. Heating temperature is usually 50 ~
80 ° C, preferably 55-75 ° C, more preferably 6 ° C
0-70 ° C. Heating time is usually 24-192 hours,
Preferably from 48 to 168 hours, more preferably from 72 to 168 hours.
144 hours.

[0007] The dried fibrinogen thus heated completely inactivates contaminating viruses, and the biological activity of fibrinogen before heating, that is, the blood coagulation ability is almost maintained even after heating, and the remaining activity remains. The rate is over 98%. In addition, the obtained virus-inactivated fibrinogen, particularly those containing lysine or lysine and arginine as basic amino acids, are easily soluble in water, so that a very high concentration aqueous solution of fibrinogen such as 8 w / v% or more can be prepared, The dissolved state after dissolution is also stable.

[0008]

The present invention will be specifically described below with reference to examples and test examples. Example 1 1.6 kg of corn fraction I derived from human plasma
A solution obtained by dissolving in 33 liters of a 5 mM aqueous sodium citrate solution (pH 7.0) (absorbance of A280 = about 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 adding an aqueous solution of citric acid M, and sodium chloride was further added to a concentration of 0.9 w / v%. Next, the liquid was cooled to 0 ° C., and 24 kg of 25 v / v% ethanol was added so that the ethanol concentration became 7.5 v / v%. The mixture was allowed to stand still for 1 hour, and the resulting precipitate was again subjected to 55 mM containing 0.9% w / v sodium chloride.
It was dissolved in 22 liters of aqueous sodium citrate solution (pH 7.0). Thereafter, 36 kg of 15 v / v% ethanol was added so that the ethanol concentration became 2 v / v%, and the mixture was allowed to stand for 10 minutes. Then, the precipitate was removed, and the ethanol concentration was changed to 8 v / v%.
9.9 kg of 25 v / v% ethanol so as to be v%
After addition, 0.6 kg of an 8 v / v% ethanol precipitate of purified fibrinogen was obtained. 0.6 kg of the above 8 v / v% ethanol precipitate was added to 1.2 w / v% lysine and 0.9 w / v
v. 22% of a fibrinogen solution (5.4 mg / ml) obtained by dissolving with 21 liters of a solution containing sodium chloride (35 times the amount of the precipitate) at 1.2 μm and 0.2 μl.
After successively filtering through a 2μ filter, 0.8 kgf / c
m with a primary pressure of virus removal filter (Planova 35
N, manufactured by Asahi Kasei Corporation). The obtained virus-removed filter-passed solution was concentrated using an ultrafilter to obtain 9%.
A 3 mg / ml purified fibrinogen solution was obtained. This solution was divided into two parts, and the freeze-dried powders obtained by freeze-drying (all water contents were 1 w / w%) were heat-treated at 65 ° C. for 120 hours to obtain samples (1-1) and ( 1-
2).

Example 2 Four kinds of 8 v / v% ethanol precipitates (0.6 kg) obtained in the same manner as in Example 1 using corn fraction I of different lots were subjected to the concentration shown in [Table 1]. 21 liters of arginine and sodium chloride solution (35
(2 times the volume) to obtain 21.5 liters of a 5.1 to 5.5 mg / ml fibrinogen solution. Each solution was added to 1.2
μ and 0.2μ filter sequentially, after 0.8
After passing through a virus removal filter (Planova 35N, manufactured by Asahi Kasei Corporation) at a primary pressure of kgf / cm, the solution was concentrated by an ultrafilter to obtain 85, 93, 99 and 10 respectively.
A 1 mg / ml purified fibrinogen solution was prepared. The freeze-dried powders obtained by freeze-drying these samples (each having a water content of 1 w / w%) were subjected to heat treatment at 65 ° C. for 120 hours to obtain samples (2-1) to (2-4). .

Comparative Example 1 8v of purified fibrinogen obtained in the same manner as in Example 1
/ V% ethanol precipitate with respect to fibrinogen
Dissolved in an aqueous solution containing only 2.9 w / w% lysine, only 11.8 w / w% arginine, and only 9.68 w / w% sodium chloride to remove 9.3 w / w fibrinogen.
/ V% was prepared, but the stability after dissolution was low,
No further experiments were performed due to the difficulty in filtering through 1.2 μ and 0.2 μ filters.

Test Example 1 Measurement of Fibrinogen Concentration and Purity The concentration of fibrinogen contained in the samples obtained in Examples 1 and 2 was measured by combining the Laki method and the Blomback method. That is, the sample No. (1-1)
~ (2-4) to reduce the fibrinogen concentration to 3-5 m
g / ml, and this was used as a sample solution. Sample liquid 2.0
0.1 M of 0.5 M phosphate buffer (pH 7.0) per ml
1, 1.25 ml of 0.2 M potassium chloride solution and 0.25 ml of purified water were added. Then 250 NIH UN
0.1% ITS / ml thrombin (physiological saline) solution
Then, the mixture was added and mixed, and allowed to stand at 37 ° C. for 1 hour to form a coagulated mass. This coagulated mass is collected by filtration and washed twice with purified water,
20 containing 2.0 ml of 2.5 M sodium hydroxide solution
Add to a volumetric volumetric flask, dissolve by heating to 60 ° C,
Make exactly 20 ml with purified water, 287 nm and 325 n
m were measured and the respective values were taken as A278 and A3.
25. From the obtained values, the concentration (mg / ml) of fibrinogen in each sample was calculated according to the following formula.

(Equation 1) In addition, the residual liquid from which the coagulated mass has been removed (hereinafter, referred to as residual liquid).
And 2.0 ml of sample solution in 0.5 M phosphate buffer (pH
7.0) and 278 n of each of the solutions (hereinafter referred to as control solutions) obtained by adding 0.1 ml and allowing to stand at 37 ° C. for 1 hour.
The absorbance at m and 325 nm was measured, the values from the residual solution were designated as B278 and B325, and the values from the control solution were
278 and C325. From the obtained values, the purity of fibrinogen in the sample was calculated according to the following formula.

(Equation 2) The above results are shown in [Table 1].

[0012]

[Table 1] As is clear from Table 1, the samples of Examples 1 and 2 obtained by the method of the present invention both have a purity of fibrinogen after heat treatment at 65 ° C. for 120 hours, that is, a solidification ability that is equal to that before heating. There was little change, which proved that fibrinogen was very heat-stable.
In addition, all of these samples could be dissolved in water for injection in the Japanese Pharmacopoeia to prepare a fibrinogen solution of about 8% w / v, and the solutions were stable.

Test Example 2 Measurement of Virus Infectivity Using a microplate, porcine parvovirus infectivity was measured on ESK (Embryonic Swine Kidney) cells cultured at 37 ° C. in a 5% carbon dioxide gas culture solution. The sample was dissolved in water for injection in the Japanese Pharmacopoeia. The virus infectivity was measured by confirming the cytopathic effect of porcine parvovirus on ESK cells. That is, a sample containing a virus was serially diluted 10-fold with an ESK cell culture medium and inoculated to ESK cells. Continued to observe the 7 days of culture and cytopathic effects with carbon dioxide gas incubator (CPE) was calculated viral infectivity titer _(TCID 50 / ml). The virus inactivation effect is calculated by the following formula using LRV (Log Reduction).
Value).

(Equation 3)

Test Example 3 Inactivation experiment of swine parvovirus (PPV) by heating dried fibrinogen A small amount of model experiment by adding 1 volume of swine parvovirus solution to 9 volumes of purified fibrinogen solution before freeze-drying prepared in Example 1 Was carried out. That is, the virus-containing sample (virus infectivity titer 10 ^6.25 ) was freeze-dried, and then subjected to heat treatment, and the virus infectivity titer and LRV in the sample were calculated according to the aforementioned virus infectivity titer measurement method. The results are shown in [Table 2].

[0015]

[Table 2] As is clear from Table 2, fibrinogen gives an LRV of 1.0 by freeze-drying, and then is subjected to a heat treatment of 96 hours for 3.0 and a heat treatment of 3.0 for 120 hours.
An LRV of 5 was obtained. From this, it was confirmed that the virus inactivation effect was equal to or higher than that of the liquid heat treatment of albumin at 60 ° C. for 10 hours, which was already confirmed not to cause disease.

Example 3 Arginine was added to the sample obtained in Example 1 to give 9.3 w / v% of fibrinogen, 1.2 w / v% of lysine,
Arginine 1.2% w / v and sodium chloride 0.9
A purified fibrinogen solution containing w / v% was prepared. This solution was freeze-dried to obtain a powder (water content: 1.0%).
(w / w%) at 65 ° C. for 120 hours. The purity of fibrinogen was 95.2% before heating and 9 after heating.
It was 5.0%. Further, 1.25 g of the powder after heating was dissolved in 8 ml of water for injection in the Japanese Pharmacopoeia in a test tube, and when the mixture was continuously stirred at 25 ° C., it was completely dissolved in about 30 minutes to form a clear liquid, and the solution was stable. Was.

[0017]

According to the present invention, contaminating viruses can be inactivated with almost no denaturation or inactivation of fibrinogen. The obtained virus-inactivated fibrinogen can be used as a high-concentration solution of fibrinogen at 8 w / v% or more, and the dissolved state after dissolution is stable.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Kenji Kaneko 3-1, Shinizumi, Narita City, Chiba Prefecture Nippon Pharmaceutical Co., Ltd. Narita Factory F-term (reference) 4B065 AA95X BD08 BD13 BD22 BD33 BD39 CA44 CA56 4C084 AA02 AA06 BA44 DC11 NA06 ZA542 ZC022 4H045 AA10 AA30 BA10 CA40 DA65 EA29 GA01 GA05 GA10

Claims (10)

[Claims] 1. A method for inactivating fibrinogen virus, comprising drying an aqueous solution containing fibrinogen, basic amino acid and sodium chloride which may be contaminated with virus, and heating until the virus is inactivated. 2. The method according to claim 1, wherein the basic amino acid is lysine or arginine. 3. The method according to claim 1, wherein the basic amino acid is lysine. 4. The method according to claim 1, wherein the basic amino acids are lysine and arginine. 5. An aqueous solution comprising fibrinogen, 4-40% w / w of basic amino acid and 5-5% w / w fibrinogen.
The method for inactivating a virus according to claim 1, wherein the method comprises 25 w / w% of sodium chloride. 6. An aqueous solution comprising fibrinogen, 2-20 w / w% lysine and 5-25 w / w% fibrinogen.
2. The method for inactivating virus according to claim 1, wherein the method comprises 1 / w% of sodium chloride. 7. An aqueous solution comprising fibrinogen and 2-20 w / w% lysine, 2-20 w / w relative to fibrinogen.
The virus inactivation method according to claim 1, comprising 5% arginine and 5 to 25% w / w sodium chloride. 8. The method according to claim 1, wherein the heating is performed at 50 to 80 ° C. for 24 to 192 hours. 9. The dried fibrinogen having a water content of 3% w / w.
The method for inactivating a virus according to claim 1, which is as follows. 10. A fibrinogen whose virus has been inactivated by the virus inactivation method according to any one of claims 1 to 9.