JPS6122022A - Method for heat-treating blood plasma protein - Google Patents

Abstract

PURPOSE:To obtain a blood plasma protein having simultaneously improved stability and water-solubility, by heating a blood plasma protein contaminated by viruses in the presence of an amino acid in the dry state until the viruses are inactivated. CONSTITUTION:A blood plasma protein contaminated by viruses, e.g. blood coagulation factor III, fibrinogen, albumin or globulin, in the dry state is heat-treated at 30-100 deg.C, preferably 60 deg.C in the presence of an acidic amino acid, e.g. aspartic acid or glutamic acid, and a basic amino acid, e.g. histidine, lysine or arginine, preferably at 0.5:1-2:1 compounding ratio to stabilize remarkably the blood plasma protein with good water-solubility and dissolution state. The viruses to be an object for inactivation are viruses in fear of contamination for human blood plasma protein, particularly hepatitis viruses, etc. The treatment is carried out in an inert gas atmosphere to improve further the stability.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a heat treatment method for virus inactivation of plasma proteins that are unstable to heat.

[Prior art]

Conventionally, heat treatment in an aqueous solution (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, as described by Murray et al. (The Ne11
York Academy of Medicine+
31 (5), 341-358 (1955)), and has been widely used for many years up to the present day, 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, and plasma proteins that have physiological or biological activity are particularly sensitive to heat and easily undergo thermal denaturation. This can easily lead to a decrease in activity or disappearance.

On the other hand, apart from the liquid heating method, plasma proteins are heated in a dry state that contains no or little moisture (usually water content of 1.5% or less, especially 1% or less) (hereinafter referred to as dry heat treatment). Experiments using the blood coagulation factor as a model revealed that the decrease in activity was significantly suppressed by applying heat treatment compared to the liquid heating method (see Table 1). However, unless a stabilizer is added, plasma protein activity will inevitably decrease, and the solubility and solubility in water will deteriorate.

By the way, the mechanism of action of virus inactivation by heating is that liquid heating is mainly based on the denaturation of the protein components of the virus, whereas dry heat treatment is mainly damaged by oxidation of the lipid components of the virus, resulting in pathogenicity. It is said that the virus inactivation mechanism of the painting method overlaps with each other, but it has been suggested that they are fundamentally different (Rahn, Physical Meth
ods of 5terilization of Ma
croorganisms, Bact, Rev.
9, 1-47 (1945)].

Therefore, the present inventors have searched for stabilizing agents for plasma proteins during dry heat treatment of plasma proteins, and have investigated ways to improve the water solubility of plasma proteins. The present invention was based on the discovery that plasma proteins are significantly stabilized when plasma proteins are subjected to dry heat treatment under such conditions, and that plasma proteins subjected to dry heat treatment under such conditions have good solubility and solubility in water. completed.

[Disclosure of the invention]

That is, the present invention relates to a method for heat treatment of plasma proteins, which comprises heating virus-contaminated plasma proteins in a dry state in the presence of an acidic 7-mino acid and a basic amino acid until the virus is inactivated. This improves the stability and water solubility of plasma proteins at once.

The plasma protein to be heat-treated in the present invention is one obtained by fractionating plasma proteins, or a mixture of two or more thereof, and usually those having biological or physiological activity are the ones to be heat-treated. It is said that Examples of such plasma proteins include blood coagulation factor 1, factor 2, factor X, factor X, factor W, fibrinogen, thrombin, plasminogen, albumin, globulin, and the like.

Examples of acidic amino acids used in the present invention include aspartic acid and glutamic acid, and examples of basic amino acids include histidine, lysine, and arginine.

These may be any of the L-form, 0-form, and DL-form, but preferably the L-form is used. Preferred combinations of acidic amino acids and basic amino acids include, for example, a combination of lysine and glutamic acid (particularly a combination of L-lysine and L-glutamic acid), a combination of lysine and aspartic acid, and the like.

The preferred blending ratio of acidic amino acids and basic amino acids is o, 5:i to 2:1, particularly preferably 1:1.
It is.

Both amino acids are usually 0.01 to 10% by weight of plasma protein as the total amount of acidic amino acids and basic amino acids.
It is sufficient if it exists to some extent. A stabilizing effect and an improvement in water solubility (solution) are observed from about 0.01% by weight, and the stabilizing effect and improvement in water solubility (solution) are saturated at about 10% by weight. A preferable addition amount is about 0.5 to 1% by weight, and this addition amount provides the best balance between stabilizing effect, water solubility (solution state), and formulation.

Both amino acids may be added before or after lyophilization of the plasma protein.

Further, although both amino acids may be removed after the dry heat treatment of the present invention, it is preferable that they be blended as they are in the plasma protein preparation.

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

The virus to be inactivated by the heat treatment of the present invention is a virus that is likely to contaminate human plasma proteins.
Especially hepatitis virus.

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.

Furthermore, there is a poor correlation between the degree of purification of plasma protein and its heat resistance, and the stabilizing effect of both amino acids remains unchanged no matter what degree of purification plasma protein is used.

Therefore, the heat treatment of the present invention may be performed at any stage of the plasma protein purification process.

The drying state in the drying process of the present invention is substantially anhydrous, preferably with as little moisture as possible. The moisture content is usually 1.5% or less, preferably 1.
% or less.

The present invention is useful as an industrial method for producing plasma protein preparations because it is possible to inactivate viruses that are likely to be mixed into the preparation without significantly losing the activity of plasma proteins, which are valuable blood products.

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

Experimental Example 1 A blood coagulation factor ■ solution and a freeze-dried powder of the solution were each heat-treated at 60°C for 20 hours in a hot bath, and changes in the activity of factor ■ in each were examined. The results are shown in Table 1. Indicated.

Table 1 Experimental Example 2 The stabilizer listed in Table 2 was added to the blood coagulation factor ① solution and then freeze-dried, or added after freeze-drying, and then the drying bed was placed in an autoclave at 121°C. 20
Heat-treated for 1 minute to determine the residual rate of factor Ⅰ activity.
sty was determined by APTT measurement using a one-stage method. Also,
Each drying bed after heat treatment was dissolved in distilled water, and its solubility and solution state (clarity) were visually observed. The results are shown in Table 2.

In addition, the residual rate in Table 2 is based on the activity before dry heat treatment as 100%.
This is from when.

(Leaving space below) From the above results in Table 2, it is understood that plasma protein stabilization and solubility can be improved only by dry heat treatment in combination with acidic 1″ acid, basic amino acid and basic amino acid. It will be.

Example 1 After adding and dissolving 10 ml of 0.02 M citrate/salt buffer (pH 6,8) to 1 g of purified factor X paste, 0.05 g of L-lysine and 0.0 g of L-glutamic acid were added.
Add 5 g, dissolve, homogenize, sterilize and filter, 11
A series of operations such as dispensing each sample into glass ampoules, freeze-drying, and explosion were performed to obtain a dried product of factor Ⅰ. After this, the dried ampoule was heated in a water bath at 60° C. for 72 hours. After heating, 1111 l of distilled water was added to each ampoule to dissolve it, and the solution state was observed and factor Ⅰ activity was measured. As a result, the group to which the L-lysine/L-glutamic acid mixture was added showed better solubility than the group to which it was not added, and almost 100% of the activity was maintained. Furthermore, the ratio of ■R:Ag/■:C was 2.88, indicating that there was almost no denaturation due to heat treatment.

Example 2 0.02 M citrate/salt buffer (pH 6,8) was added to 1 g of purified factor To the thus obtained dry powder, 0.05 g of L-lysine and 0.0 g of L-glutamic acid were added aseptically.
．． 05g was added and mixed uniformly. This dried sample was heated in a water bath at 60° C. for 72 hours. The same operation was performed for the L-lysine and L-glutamic acid-free group. As a result, as in Example 1, the L-lysine/L-glutamic acid mixture addition group showed better dissolution than the non-addition group, and almost 100% activity was maintained.

Example 3 After adding 1% of a mixture of L-lysine and L-glutamic acid (1:1) to the liquid bulk of the factor II complex, it was freeze-dried and subjected to an experiment under the same heat treatment conditions as in Example 1. I did it. On the other hand, after previously freeze-drying the liquid bulk of the factor
: 1% amount of 1) was added and heat treated in the same manner. Also,
The same operation was performed for the L-lysine and L-glutamic acid-free group. As a result, in the non-additive group, the activity decreased by about 20% compared to the value before heat treatment, while in the two groups with the addition of the mixture, the activity decreased by less than 7% in both groups. No difference was observed between the groups added after drying.

Example 4 A mixture of L-lysine and L-glutamic acid (1:
1) was added in an amount of 0.5%, and dry heat treatment was performed in the same manner as in Example 1. As a result, dry heat-treated preparations were obtained in which the potency was maintained at 90% or more and the solubility and solubility were extremely good.

Claims (1)

[Claims] A method for heat treatment of plasma proteins, which comprises heating virus-contaminated plasma proteins in a dry state in the presence of acidic amino acids and basic amino acids until viruses are inactivated.