JPS6281327A - Heat-treatment of human thrombin preparation - Google Patents

Abstract

PURPOSE:To inactivate viruses without losing the activity of thrombin, by the dry-heat treatment of a human thrombin preparation in the presence of at least one kind of stabilizer selected from amino acid, its salt and sugar. CONSTITUTION:A human thrombin preparation provided usually as a freezedried product is heated at about 60 deg.C and a water-content of <=3%, preferably 0.05-3% for 10-100hr in the presence of at least one kind of stabilizer selected from amino acid, its salt and sugar. Viruses, especially hepatitis viruses are inactivated by this treatment. The amount of the stabilizer is preferably 5-100mg for amino acid or its salt and 50-300mg for sugar based on 5,000 units of thrombin. The stability of the agent can be further improved by the combined use of sodium citrate. Thrombin can be remarkably stabilized and the solubility and liquid nature of thrombin can be improved by the treatment.

Description

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

[Conventional technology]

Conventionally, heat treatment in a water/liquid state (hereinafter referred to as liquid heating method) has been the most reliable method for inactivating viruses that may be contaminated with plasma proteins such as albumin. , Murray et al.
The New York Academy of Medicine
New York Academy of Medicine
ine).

31 F+1.341-358 (1955)), and has been widely used for many years until today, and the virus inactivation effect of the liquid heating method has been epidemiologically proven.

However, only a limited number of plasma proteins, such as albumin, can withstand liquid heating. In particular, plasma proteins with physiological or biological activity are extremely sensitive to heat, easily denatured by heat, and lose their activity. It is likely to deteriorate or disappear.

On the other hand, separate from the liquid heating method, when plasma proteins are heated in a dry state that does not contain or contains little water (hereinafter referred to as dry heat treatment), the activity decreases compared to the liquid heating method. It has been revealed in a patent featuring the blood coagulation factor II that it is significantly inhibited. However, the reality is that even in dry heat treatment, if a stabilizer is not added, the activity of plasma proteins cannot be avoided, and the solubility and solubility in water deteriorates.

By the way, the mechanism of action of virus inactivation by heating is that liquid heating mainly denatures the protein components of the virus, whereas dry heat treatment mainly oxidizes the lipid components of the virus. It is said that the pathogenicity is lost, and although the virus inactivation mechanisms of both types overlap in some parts, it has been suggested that they are fundamentally different [Learn, Physical Methods of Sterilization of Macroorganisms] Logie Levis, (Rahn, Physic
alMethods of 5terilization
of MacroorganismsBact,
Rev.) 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 contaminant viruses without inactivating a thrombin preparation.

[Means for Solving the Problems] The present inventors have discovered that the virus can be inactivated without losing thrombin activity by dry heat treating a thrombin preparation in the presence of a specific stabilizer described below; The present invention was completed by discovering that thrombin is significantly stabilized and that thrombin preparations subjected to dry heat treatment under such conditions have good disintegration and solubility in water.

That is, the present invention provides human thrombin preparations for which there is a risk of virus contamination, in a substantially dry state, in the presence of at least one stabilizer selected from amino acids, their salts, and saccharides, until the virus is inactivated. The present invention relates to a heat treatment method for human thrombin preparations, which involves heating, thereby inactivating contaminating viruses and improving the stability and water solubility of thrombin.

The thrombin preparation to be heat treated in the present invention is
It has biological or physiological activity as thrombin, such as one obtained by fractionating plasma proteins.

Such thrombin preparations include, for example, those that meet the standards prescribed by the Japanese Pharmacopoeia.In the present invention, after freeze-drying the thrombin/8 solution, the moisture content is usually 0.
．． It is carried out by heating under conditions of 0.05 to 3%. At that time, by adding the above-mentioned specific stabilizer, the stability of thrombin is promoted and the solubility and liquid state of thrombin are improved. be done.

Amino acids and salts thereof as stabilizers used in the present invention are not particularly limited, and amino acids include monoaminocarboxylic acids (e.g., glinone, alanine, etc.), acidic amino acids (e.g., glutamic acid, etc.)
, basic amino acids (e.g. lysine, arginine, etc.)
etc.

Examples of amino acid salts include salts of basic amino acids and acidic amino acids among the above amino acids (eg, lysine glutamate, etc.). In addition, as sugars, monosaccharides 8
(e.g. glucose, mannose, mannitol, etc.)
, disaccharides (for example, maltose, sucrose (white sugar), milk vN, etc.), and polysaccharides (for example, starch, dextran, etc.).

The amount of stabilizer to be used is 5 to 500 units when using an amino acid or its salt, preferably 5 to loo+■, per 5000 units of thrombin, and 5 to 500 units when using tJNm. 500LN! , preferably 50-30
It is 0 ■. At this level of addition, the stabilizing effect,
It has the best balance between water cylindrical properties, solubility, and formulation.

At this time, by using sodium citrate, which has conventionally been used as a stabilizer for plasma protein fraction preparations, stability in liquid form can be ensured due to its buffering action. The amount added is usually 10 to 300 μl, preferably 30 to 1 μl per 5000 units of thrombin.
It is 50■.

Thrombin preparations are usually used as lyophilized products, but it is preferable to add a stabilizer before lyophilizing plasma proteins. The stabilizer is also useful as a stabilizer during freeze-drying.

Furthermore, although the stabilizer may be removed after the dry heat treatment of the present invention, it is preferable to include it in the thrombin preparation as it is.

The heating temperature in the heat treatment is usually 30 to 100"C1
Preferably, the temperature is about 60°C, and the heating time is usually about 10 minutes to 200 hours, preferably about 10 to 100 hours.

Viruses to be inactivated by the heat treatment of the present invention are viruses that are likely to contaminate human plasma proteins, particularly hepatitis viruses.

Furthermore, by performing the heat treatment of the present invention under an inert gas atmosphere, stability during heating can be further improved.

Examples of the inert gas include nitrogen gas, argon, and helium.

The heat treatment of the present invention may be performed at any stage of the thrombin preparation purification process, such as in the bulk powder or final formulation.

The dry state in the dry heat treatment of the present invention is substantially anhydrous, preferably with as little moisture as possible. The water content is usually 3% or less, preferably 1% or less, and usually about 0.05 to 3%.

According to the present invention, it is possible to inactivate viruses that may be mixed into the preparation without significantly losing the activity of thrombin, which is a valuable blood type agent, and it is useful as an industrial manufacturing method for plasma protein preparations. .

(Examples) The present invention will be explained below using experimental examples and examples, but the present invention is not limited by these in any way.

Example 1 From normal human plasma, barium chloride adsorption method and DEAE-Sephadex column chromatography method [Bajaj, P. et al., Journal of Biological Chemistry (Bajaj, S.P.).

et al,, J, old o1. Chem,) 248
．． 7729 (1973)], and to this prothrombin, thromboplastin prepared from human placenta, human plasma, and calcium chloride solution were added, and thrombin was converted to produce crude thrombin (1 mg of rhino1-thrombin activity 10 units). I got it. This crude thrombin was purified by SP-Sephadex column chromatography [Landbrand, R.L. 9. Biochemistry (Lundblad+ R,L,, [lio
chemistry)+, 2501 (1971)
), and after concentrating the purified tron, 7.
Dialyzed against 100mM citrate buffer (pH 7.0) containing 5% D-mannitol, thrombin solution/& (3
500 maki/f(1). Thrombin activity (500 units per protein) was prepared. After the thrombin solution was sterilized and filtered, 2 ml portions were dispensed into 15I1) 7-volume vials and freeze-dried. Thrombin activity after lyophilization is 6800 u/V, humidity content is 0.07
%Met. This freeze-dried thrombin preparation
The product was heat-treated at ℃ for 72 hours and tested for solubility, thrombin activity, cellulose acetate membrane electrophoresis, and gel filtration while comparing with the thrombin preparation before heat treatment. As a result, no significant changes were observed even after heat treatment. The thrombin preparation was found to be stable under these heating conditions.

Experimental Example 1 Purified thrombin (550 units of thrombin activity per protein) prepared according to Example 1 was purified by concentration, dialysis,
Thrombin preparation (thrombin activity 6000 units/bottle) containing the amount of stabilizer listed in Table 1 by freeze-drying or other operations
was prepared. The thrombin preparation was heat treated at 60° C. for 72 hours and then tested for thrombin activity. The results are shown in Table 1.

As a result, it was found that the use of a stabilizer significantly improved stability compared to the case without the addition.

(Space below) Experimental Example 2 Virus-suspending FA? of phosphate buffered saline (pH 7.1) to the thrombin solution prepared in Example 1 (sterilized and filtered)? f
1, mix it evenly, and divide it into glass bottles.
It was dispensed and freeze-dried. After the freeze-drying was completed, the pressure was returned to normal using nitrogen gas, the tube was sealed tightly, and the tube was immersed in a hot bath at 60.degree. C. and heated. In addition, please wait 10-15 minutes until the temperature inside the bottle reaches 60℃.
Since the heating time was actually 30 minutes, the heating time was extended for 30 minutes.

The infectivity of each virus was measured by the plaque method.

The results are shown in Table 2.

(Margin below) Procedural amendment stamp button January 5, 1985

Claims (3)

[Claims] (1) Persons at risk of virus contamination The thrombin preparation should be heated in a substantially dry state in the presence of at least one stabilizer selected from amino acids, their salts, and sugars until the virus is inactivated. A method for heat treatment of a human thrombin preparation, characterized by: (2) The method according to claim (1), characterized in that the heating is performed under conditions where the humidity content is 3% or less. (3) Claim No. 1 (1) characterized in that heating is performed under conditions in which sodium citrate is also used as a stabilizer.
) or the method described in paragraph (2).