KR950010321B1 - Method of heating treatment of human thrombin preparation - Google Patents

Abstract

No content.

Description

Heat treatment method of human thrombin preparation

The present invention relates to a heat treatment method for inactivating a virus in a human thrombin formulation.

Conventionally, in plasma proteins such as albumin, as the most reliable method of inactivating a virus that is concerned about incorporation, a heat treatment method in an aqueous solution state (hereinafter referred to as a liquid phase heating method) is described in Murray et al., The New York Academy of Medicine, 31 (5), 341 to 358 (1055), which has been widely used for a long time to date, and mechanically heated by virus. The deactivation effect of has been demonstrated.

However, the resistance to liquid heating like albumin is extremely limited among plasma proteins, and particularly, plasma proteins having physiological or biological activity are very sensitive to heat, and thus are prone to heat denaturation and are easily deteriorated and lost.

On the other hand, unlike the liquid phase heating method, if plasma proteins are heat-treated (hereinafter referred to as dry heat treatment) in a dry state that contains little or no moisture, the decrease in activity is significantly suppressed as compared to the liquid phase heating method. This has been demonstrated in experiments that model blood coagulation agents. However, in general, even in the dry heat treatment, it is a preliminary fact that the addition of a stabilizer does not inevitably reduce the activity of plasma proteins, and the solubility and dissolution in water deteriorate.

However, the mechanism of action of virus inactivation by heating is mainly based on denaturation of the protein component of the virus in liquid phase heating, whereas in dry heat treatment, the virus is mainly pathogenic due to the damage of the virus by oxidizing the lipid component of the virus. The two mechanisms of viral inactivation are suggested to be fundamentally different, although overlapping with each other, although these mechanisms are overlapping with one another. [Rahn, Physical Methods of Sterilization] of Macroorganisms Bact. Rev.) 9, 1-47 (1945)]

It is an object of the present invention to provide a heat treatment method which inactivates the incorporated virus without losing the activity of the thrombin formulation.

The present inventors can dry heat the thrombin preparation in the presence of a specific stabilizer described below, thereby inactivating the virus without losing the activity of thrombin, and also significantly stabilizing thrombin, as well as performing the dry heat treatment under such preconditioning. The thrombin formulation carried out was found to have good solubility and dissolution in water, thus completing the present invention.

That is, the present invention is characterized in that a human thrombin preparation of concern for incorporation of the virus is heated in a dry state until the virus is inactivated in the presence of at least one stabilizer selected from amino acids, salts and sugars thereof. The heat treatment method of the human thrombin preparation, which inactivates the entrained virus and improves the stability and solubility of thrombin.

In the present invention, the thrombin preparation to be subjected to heat treatment has a biological activity or physiological activity as thrombin, that is, one obtained by fractionating plasma proteins.

Such a thrombin formulation is suitable for the specification prescribed | regulated by the Japanese pharmacy room, for example.

The present invention is usually carried out by lyophilizing a thrombin solution (pH 5 to 9: but not limited thereto), and then heating under conditions of a moisture content of 0.05 to 3%. The addition of certain stabilizers promotes stabilization of thrombin and also improves the solubility and liquidity of thrombin.

The amino acids used as stabilizers in the present invention and salts thereof are not particularly limited, but amino acids include monoamino carboxylic acids (e.g. glycine, alinine, etc.), acidic amino acids (e.g., glutamic acid, aspartic acid, etc.) and Basic amino acids (eg, lysine, arginine, histidine, and the like). Examples of amino acid salts include salts of basic and acidic amino acids in the amino acids (for example, lysine-glutamate). Examples of the sugars include monosaccharides (eg, glocose and mannose), disaccharides (eg, maltose, sucrose (lactose), lactose and the like), polysaccharides (eg, starch and dextran, etc.). And sugar alcohols (eg, mannitol and sorbitol, and the like).

The amount of the stabilizer is based on 5,000 units of thrombin, and is 5 to 500 mg, preferably 5 to 100 mg when using an amino acid or a salt thereof, and 5 to 500 mg, preferably 50 to 100 when using a saccharide. 300 mg. At this level, the balance between the stabilizing effect, water solubility, dissolution state and formulation is best.

In addition, by using together the sodium citrate conventionally used as a stabilizer of a plasma protein fraction preparation, the stability in the liquid phase by the buffering action can be ensured. The amount of addition is usually 10 to 300 mg, preferably 30 to 150 mg per 5,000 units of thrombin.

Thrombin formulations are commonly used as lyophilized products, but it is preferred to add stabilizers prior to the lyophilization of plasma proteins. The stabilizer is also useful as a stabilizer during lyophilization.

In addition, although the stabilizer may be removed after the dry heat treatment of the present invention, it is preferable to mix the stabilizer as it is in the thrombin formulation.

In the heat treatment, the heating temperature is usually 30 to 100 ° C., preferably about 60 ° C., and the heating time is usually 10 minutes to 200 hours, preferably about 10 to 100 hours.

The virus to be inactivated as the heat treatment of the present invention is a virus which is concerned about incorporation into human plasma proteins, in particular hepatitis virus.

In addition, when the heat treatment of the present invention is carried out in an inert gas atmosphere, the stability upon heating can be further improved. Examples of inert gases include nitrogen, argon and helium.

The heat treatment of the present invention can be carried out at any stage of the purification process of the thrombin formulation, such as powder bulk or final formulation.

In the dry heat treatment of the present invention, the dry heat state is substantially anhydrous, and a state in which water is as low as possible is preferable. The water content is usually 3% or less, preferably 1% or less, and generally about 0.05 to 3%.

Since the present invention can inactivate viruses that may be incorporated into the preparation without losing the activity of thrombin, a valuable blood preparation, the heat treatment method of the present invention is advantageous as an industrial preparation of plasma protein preparations. .

EXAMPLE

The present invention will be described based on the following experimental examples and examples, but the present invention is not limited thereto.

Example 1

Barium chloride adsorption and DEAE-Sepadex column chromatography from plasma of normal persons [Bajaj, S. P., et al., J. Biol. Chem.) 248, 7729 (1973), purify prothrombin, add thrombo plastin prepared from the human placenta, human plasma and calcium chloride solution to the prothrombin, change it to thrombin, and change thrombin (thrombin activity per mg of protein). 10 units). Zothrombin was purified by SP-Sephadex column chromatography [(Lundblad, RL, Biochemistry), 10, 2501 (1971)], concentrated purified thrombin and then 100 mM citric acid containing 7.5% D-mannitol Dialysis against buffer (pH 7.0) prepares thrombin solution (3, 500 units / ml, 500 units of thrombin activity per mg of protein). The thrombin solution is sterile filtered and then lyophilized by dispensing 2 ml into a 15 ml vial bottle. The thrombin activity after lyophilization is 6,800 u / V and the humidity is 0.07%. The lyophilized thrombin formulations were heat-treated at 60 ° C. for 72 hours and tested for solubility, thrombin activity, cellulose acetate membrane electrophoresis and gel filtration compared to thrombin formulations prior to heat treatment. No thrombin formulation was stable under heating conditions according to the invention.

Experimental Example 1

The thrombin preparation containing thrubin activity (Thrombin activity 6, 000) was carried out on the purified thrombin (550 units of thrombin activity per mg of protein) prepared according to Example 1, and was subjected to operations such as concentration, dialysis and lyophilization. Unit / bottle). The thrombin formulation is heat treated at 60 ° C. for 72 hours and then tested for thrombin activity. The results are shown in Table 1.

Experimental results have shown that the use of stabilizers is extremely improved compared to the case where no stabilizers are added.

TABLE 1

Effect of stabilizer on heating thrombin formulation (500 units / bottle) (60 ° C., 72 h, sodium citrate 55 mg / bottle)

Example 2

To the thrombin solution prepared in Example 1 (finished with sterile filtration), a virus suspension of phosphate buffer (pH 7.1) is added, mixed uniformly, then distributed in small portions into glass bottles and lyophilized. After lyophilization, the mixture is sealed at normal pressure using nitrogen gas, and is immersed in a 60 ° C. warm bath and heated. It took 10 to 15 minutes for the temperature in the bottle to reach 60 ° C., so the heating time was actually extended by 30 minutes each.

Infectivity of each virus was measured by the Plaque method.

The results are shown in Table 2.

TABLE 2

a) Plaque forming unit / ml <50, <500 detection limit or less.

Claims (2)

The human thrombin preparations concerned about the incorporation of viruses are dried at a humidity of 3% or less; It is heated for 10 minutes to 200 hours at a temperature in the range of 30 ° to 100 ° C. in the presence of a stabilizer selected from amino acids, salts and sugars thereof. The method according to claim 1, which is heated under the conditions of using sodium citrate as a stabilizer.