JPS6289628A - Heat treatment of human fibrinogen pharmaceutical - Google Patents

Abstract

PURPOSE:To attain the titled safe pharmaceutical of high quality, by dry heat- treating a human fibrinogen pharmaceutical in the presence of a disaccharide or sugar alcohol which is a stabilizer to inactivate hepatitis virus, etc., without deteriorating the fibrinogen pharmaceutical. CONSTITUTION:A human fibrinogen pharmaceutical is dried to 0.05-3%, preferably <=1% moisture content and heat-treated in the presence of a disaccharide, e.g. white sugar or maltose, or sugar alcohol, e.g. D-sorbitol or D-mannitol, as a stabilizer. The above-mentioned stabilizer in an amount of 1-2g based on 1g human fibrinogen is preferably added. Furthermore, sodium citrate which is a conventional stabilizer is preferably used together. The heating is preferably carried out at about 60 deg.C for 65-90hr. Viruses can be inactivated and water solubility and solution properties of the resultant pharmaceutical can be improved without deteriorating the human fibrinogen.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a heat treatment method for virus inactivation of human fibrinogen preparations.

[Conventional technology]

Regarding plasma proteins such as albumin, as the most reliable method to inactivate viruses that may be contaminated, a report by Hurray et al.
academy of Mttdicina), 3
1 (5), 341-358 (1955)), and based on this report, a heat treatment method in an aqueous solution state (hereinafter referred to as liquid heating method) has been carried out. This liquid heating method has been widely used for many years, and its virus inactivation effect has been epidemiologically proven. However, only a limited number of plasma proteins, such as albumin, can withstand liquid heating, and plasma proteins with physiological or biological activities are particularly sensitive to heat.
This tends to cause thermal denaturation, leading to a decrease or loss of 1 activity.

On the other hand, when plasma proteins are heated in a dry state with no or little moisture content (hereinafter referred to as dry heat treatment), the decline in their activity is significantly suppressed compared to liquid heating. ζ Plasma coagulation tube■ This was revealed through an experiment using a model of a child. However, in general, even in the dry heat treatment method, the fact is that unless a stabilizer is added, the activity of plasma proteins inevitably decreases, and the solubility and solubility in water deteriorates.

By the way, the mechanism of action of virus inactivation by heating is that the liquid heating method is mainly based on the denaturation of the protein components of the virus, whereas the dry heat treatment method is mainly based on the oxidation of the lipid components of the virus, causing damage to the virus. It is said that the pathogenicity is lost when the virus is exposed to the virus. Rahn suggests that although the virus inactivation mechanisms of both types overlap, they are fundamentally different.
s of Sterilization of Macr
oorganisms Bact, Rttv, ).

9.1-47 (1945)).

[Problem that the invention seeks to solve]

An object of the present invention is to provide a heat treatment method that inactivates contaminating viruses without altering fibrinogen preparations.

[Means for solving problems]

The authors researched stabilizers for dry heat treatment of human fibrinogen preparations and found that disaccharides and sugar alcohols were specifically effective as stabilizers for this dry heat treatment. Based on this, the present invention was completed.

In the present invention, human fibrinogen preparations that are likely to be contaminated with viruses are brought into a substantially dry state and heated in the presence of a disaccharide or sugar alcohol as a stabilizer until the virus is inactivated. The present invention uses a disaccharide or sugar alcohol as a stabilizer in the dry heat treatment method of human fibrinogen preparations, thereby making it possible to significantly stabilize fibrinogen and inactivate viruses without losing its activity. It was also possible to improve the water solubility and solubility of the obtained human fibrinogen preparation.

The human fibrinogen preparation in the present invention has physiological or biological activity as fibrinogen, is obtained by fractionating plasma proteins, and is compliant with biological product standards. The present invention freeze-dries a solution of a human fibrinogen preparation to a substantially anhydrous dry state, usually to a moisture content of 0.05 to 3%, preferably 1% or less,
Heat the human fibrinogen preparation under these conditions.

Among the stabilizers in the present invention, disaccharides include white sugar and maltose, and sugar alcohols include D-sorbitol and D-! It is ninitol.

50% of these stabilizers per 1 ton of human fibrinogen.
Add at a rate of 0 ml-39, preferably 1-2 t. In the amount added within this range, the stabilizer effect, water solubility, solubility, and formulation are best balanced. Sodium citrate has traditionally been used as a stabilizer for plasma protein fraction preparations. In the present invention, it is also desirable to use sodium citrate as a stabilizing agent because stability in liquid form can be ensured by the buffering effect of sodium citrate. The amount added is 100-1000#1t1 preferably 300-s to 1t human fibrinogen.
O o my. Human fibrinogen preparations are usually used as lyophilized products. In the present invention, it is preferable to add a stabilizer before this freeze-drying process. Although the stabilizer may be removed after the freeze-drying process, it is preferable to leave it in the formulation.

The heating temperature in the present invention is usually 30 to 100°C, preferably about ω°C, and the heating time is 10 minutes to 100°C at 100°C.
time, usually 40-100 hours at ω°C, preferably 6-100 hours
(a) It is time. Viruses to be inactivated in the present invention are viruses that are likely to be mixed into human plasma proteins, particularly hepatitis viruses.

The heat treatment of the present invention is performed in a vacuum or an inert gas atmosphere. Examples of inert gas include nitrogen gas, argon,
Use helium. Note that there is a poor correlation between the degree of purification and heat resistance of human fibrinogen preparations, and the stabilizing effect of the stabilizer does not change no matter what degree of purification human fibrinogen preparations are used. Therefore, in the present invention, the heat treatment may be performed either in the powder bulk process or in the final formulation process during the manufacturing process of human fibrinogen preparations.

〔Effect of the invention〕

According to the present invention, viruses that may be contaminated can be inactivated without altering human fibrinogen, which is a valuable plasma preparation, and the water solubility and solubility of the obtained human fibrinogen preparation can also be improved. Therefore, it is effective in producing safe and high-quality human fibrinogen preparations with high yield.

〔Example〕

A paste of human fibrinogen obtained from normal human plasma by Cohn's ethanol fractionation method or glycine-ethanol fractionation method was mixed with 55rsld containing 4.5% white sugar so that the fibrinogen concentration was 3 W/v%. Citric acid solution C
pH 6,8). After confirming that the pH of this person's fibrinogen preparation solution was 6.8,
35 m in a OfpLI-capacity vial! Dispense into portions and freeze-dry. The human fibrinogen preparation after freeze-drying contains 1 t/bottle of human fibrinogen, 1.6 f/bottle of white sugar, and 588 m of sodium citrate? / bottle, the humidity content was 0.8%. Freeze-dried human fibrinogen preparation (a)
It was heat-treated at ℃ for 72 hours.

The amount of human fibrinogen in the obtained human fibrinogen preparation,
Solubility, cellulose acetate membrane electrophoresis, and gel filtration were all tested, and when compared with those before heat treatment, no significant differences were observed, indicating that the human fibrinogen preparation was stable under these heating conditions.

[Experimental Example 1] Various stabilizers listed in Table 1 were added to the human fibrinogen paste obtained according to Example 1, and a lyophilized human fibrinogen preparation (human fibrinogen amount 1 t/bottle, 50"' distilled The pH after dissolution with water was adjusted to 6.0 to 7.4).After heating each individual's fibrinogen preparation at ω°C for 72 hours, the amount of remaining human fibrinogen in each individual was measured.
The percentage of human fibrinogen amount before heat treatment was calculated.

A reagent for measuring human fibrinogen manufactured by Dito, Inc., USA was used for the measurement, and the measurement method was according to the instruction manual. The measurement results are shown in Table 1, and those using disaccharide sucrose and sugar alcohol D-mannitol as stabilizers are as follows:
The amount of residual fibrinogen was much higher than that using no additives or monosaccharides such as fructose and glucose, and disaccharides and sugar alcohols were found to be specific and effective.

Furthermore, the stabilizing effect of these stabilizers was further increased by using sodium citrate in combination.

(Margin below) Table 1 Comparison of stabilizing effects [Experimental example, B] Human fibrinogen preparation solution prepared in Example 1 (human fibrinogen amount IP/bottle, sucrose 1600 m?/bottle, sodium citrate 588-?/bottle) bottle), 0.05Af! j
A virus suspension in acid buffer pH 7.1 was added and mixed uniformly, followed by freeze-drying in a vial. After that, the pressure was returned to normal with nitrogen gas, the cap was sealed, and the cap was immersed in a hot bath at ω°C to heat it.
After 72 hours, virus infectivity was examined. Note that since it takes 10 to 15 minutes for the temperature inside the bottle to reach ω°C, the heating time was actually extended for 30 minutes in each case. The infectivity of each virus is determined by Plaque 7 Ohming (Plaqug
(forming) method and expressed in units of plaque/7 oming per ls+j. The results are shown in Table 2, and the infectivity of each virus disappeared after n hours.

(Left below) Table 2 Virus Infectious Patent Applicant Agent Midori Juji Co., Ltd.
Patent attorney Ike 1) Mikio et al. Proceedings Amendment 3, Relationship with the person making the amendment Applicant Moxibustion Co., Ltd. Midori Juji 4, Agent No. 5 Fuji, 1-2-8 Nishihonmachi, Nishi-ku, Osaka Inside the new building
(6383) Patent Attorney Mikio Ikeda; 5. Date of order or notification Initiated 1920, Month, Day 6, Subject of amendment, /〆=\ Detailed explanation of the invention column in the specification Mi\ -1I ゝ - 1, -'.

7. Contents of the amendment (1) "Factor" on page 2, line 19 of the specification is corrected to "factor", and "qualitative" on page 5, line 2 of the same specification is corrected to "buy" to "J". .

(2) r40-100 J t-r40 on page 6, line 7
~15o” Correct.

(3) Corrected "adjustment" on the 2nd line of page 11 to "preparation", and rpH7, 1) on the 5th line of page 11) J t r (
p) 17.1] Correction to J

Claims (3)

[Claims] (1) People who are at risk of virus contamination People who are characterized by keeping the fibrinogen preparation in a substantially dry state and heating it in the presence of a disaccharide or sugar alcohol as a stabilizer until the virus is inactivated. Heat treatment method for fibrinogen preparations. (2) The method for heat treatment of a human fibrinogen preparation according to claim 1, wherein the human fibrinogen preparation is heated under conditions of a humidity content of 3% or less. (3) The heat treatment method for human fibrinogen preparations according to claim 1 or 2, which uses sodium citrate as a stabilizer.