JPS61194035A - Gamma-globulin preparation - Google Patents

Abstract

PURPOSE:To provide the titled heat-treated preparation free from the tendency to increase the amount of polymers and increase the anticomplementary value. CONSTITUTION:An aqueous solution containing gamma-globulin [the content of gamma-glob ulin is preferably 0.1-30% (W/V), and the pH is 4.5-10] is heated at 60 deg.C for 10hr to inactivate the contaminant viruses. The thermal stability of gamma-glob ulin can be improved remarkably by the addition of one or more compounds selected from monosaccharides, disaccharides and sugar alcohols in an amount of 10-100g/100ml. A gamma-globulin preparation having further improved thermal stability is obtained by adding one or more components selected from neutral amino acids (e.g. glycine, alanine, etc.; 1-20g/100g of the above solution), neutral salts (e.g. NaCl, KCl, etc.; 0.1-10g/100ml), surfactants, and organic carboxylic acid salts (e.g. alkali metal salt such as Na salt; 1-30g/100ml).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a T-globulin preparation prepared by heat treatment.

More specifically, a stable T-globulin preparation in which no increase in polymer content or increase in anti-complement value is observed when T-globulin is pasteurized (60°C, 10 hours) with the addition of a stabilizer if desired. Regarding.

[Prior Art] Among immunoglobulins, which are plasma protein components, T-globulin preparations containing IgG as a main component have been widely used for the prevention and treatment of various infectious diseases, but they lack thermal stability. In addition, sterilization by filtration is used because it contains a wide range of antibodies against various types of viruses and bacteria. However, when T-globulin is obtained from plasma protein fractionation, microscopic contaminant viruses such as hepatitis virus and AIDS virus can pass through one pore of the filter, and the resulting T-globulin contains It cannot be 100% denied that they are mixed.

On the other hand, for vaccines that are plasma protein components, a method of heating the vaccine to 60 to 120 degrees Celsius in the presence of a stabilizer has already been used and has been successful.

[The Mano point that the invention attempts to solve]

An object of the present invention is to provide a T-globulin preparation in which viruses that are at risk of contamination are inactivated.

[Means for solving problems]

The present inventors have conducted various studies and found that heat treatment of T-globulin, preferably an aqueous solution thereof, at 60°C for 10 hours is an extremely effective method for inactivating contaminant viruses. Furthermore, it has been found that the thermal stability of r-globulin against heat treatment is significantly increased by adding at least one selected from monosaccharides, disaccharides, and sugar alcohols, and in addition to the above-mentioned stabilizers, it is also an activation method. The inventors have discovered that the thermal stability of γ-globulin can be further enhanced by coexisting at least one selected from neutral amino acids, neutral salts, surfactants, and organic carboxylates, and have completed the present invention.

Furthermore, in the preparation of the present invention, T-globulin can be obtained in a state in which it is dissociated into monomers by heat treatment.

That is, the present invention relates to a heat-treated T-globulin preparation.

In the present invention, it is preferable to heat the T-globulin-containing aqueous solution in the form of a T-globulin-containing aqueous solution. Although it may be an aqueous solution, it is advantageous to heat the aqueous solution at a partial purification or purification stage. The protein (
The content of γ-globulin) is preferably 0.1 to 30% (w/v), and p)I of the aqueous solution is generally p
1 (4.5 to 10, preferably adjusted to pH 6 to 8 with an appropriate buffer).

Regarding the stabilizer added to the aqueous solution containing T-globulin, a single tJ! Examples of m include glucose, mannose, galactose, fructose, etc., and examples of disaccharides include sig sugar, maltose,
Suitable sugar alcohols include lactose and the like, but are not limited to mannitol, sorbitol, xylift, etc. The amount of the stabilizer added is 10% per 100+1 of the γ-globulin aqueous solution. -10
It is 0g.

Regarding the trapping stabilizer used in the present invention, examples of neutral salts include alkali metal or alkaline earth metal halogen salts such as sodium chloride, potassium chloride, and magnesium chloride, and the amount added is T- The amount is 0.1 to 10 g per 100 ml of globulin aqueous solution.

Examples of neutral amino acids (ie, monoamino monocarboxylic acids) include glycine, alanine, valine, leucine, isoleucine, etc., and the amount added is 1 to 20 g per 100 ml of T-globulin water.

An organic carboxylic acid refers to a hydrocarbon residue substituted with a carboxyl group, and the hydrocarbon residue may be saturated or unsaturated, and may be chain (linear or branched). Examples of the hydrocarbon residue, which may be either cyclic or cyclic, include an alkyl group and an aryl group (for example, a phenyl group). The organic carboxylic acid may have a plurality of carboxyl groups, but 1 and 2 carboxyl groups are preferred.The organic carboxylic acid may also be substituted with a hydroxyl group.As a salt in the organic carboxylate, physiological There is no particular restriction as long as it is acceptable, and preferred are alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, etc.), particularly preferably sodium salts, potassium salts, I can give you salt.

Specific examples of organic carboxylic acid salts include physiologically acceptable salts such as propanoic acid, butanoic acid, pentanoic acid, capric acid, °caproic acid, malonic acid, succinic acid, glutaric acid, adipic acid, citric acid, and mandelic acid. salts, especially alkali metal salts (sodium salts, potassium salts).

It takes! The preferable number of carbon atoms in the l acid is about 3 to 15.

The amount of organic carboxylate added is 1 part of T-globulin aqueous solution.
It is 1 to 30 g per 001.

Surfactants include non-ionic agents such as alkylphenylpolyoxyethylenes (e.g. Triton@ and Non1det'), bile salts (e.g. sodium taurocholate), etc. anionic agents, such as benzalkonium chloride, and cationic agents, such as benzalkonium chloride, and surface-active polyhydric alcohols, such as high molecular weight copolymers of propylene oxide (Pluronic®).
F 68 ), etc., and the amount added is 0.002 to 0.05 g per 1 local of T-globulin aqueous solution.
degree is preferred.

The heat treatment may be carried out at a temperature and time sufficient to inactivate contaminating viruses, for example at 50°C to 130°C, preferably about 60°C, for 25 minutes to 20 hours, preferably 10
Time is done.

In order to examine the heating effect of the present invention, experiments were conducted on the effect of heating on the infectivity of various viruses that are feared to be contained in T-globulin preparations. In this experiment, variola virus, mumps virus, measles virus, vesicular stomatitis virus, titangonia virus, polio virus, coxalaki virus, and echo virus were added to a T-globulin sample, and the mixture was heated at 60°C for 10 hours. process,
The remaining virus infectivity was measured over time, and it was found that the virus had completely lost infectivity after 10 hours, regardless of whether a stabilizer was added or not. This result suggests that the infectivity of viruses other than the virus used can be inactivated if the heat treatment of the present invention is applied.

In the present invention, after the above heat treatment, we conducted not only the appearance and properties but also the quantitative determination of the polymer, the measurement of the anti-complement value, the measurement of the measles antibody titer, and an acute toxicity experiment. It can be said that it is highly effective and highly effective.

The preparation thus obtained is generally in the form of a solution, and if highly purified T-globulin is used as the starting material, it may be used as is, or if a crude product is used, it may be treated according to a known purification method and then treated if necessary. , dialysis, and sterile filtration, the product is dispensed to contain 500 to 10,000 mg of γ-globulin according to the packaging unit. The storage method is not particularly limited as long as high temperatures are avoided, but it is particularly desirable to
It should be stored at 30°C or below, but it may also be a lyophilized preparation if desired.

The T-globulin that has undergone this treatment is administered as it is, or after being subjected to a formulation treatment known per se, for example, diluted or dissolved with distilled water for injection. The dosage for adults is usually 500 to 3,000 doses of T-globulin at a time.
mgl, and for children, 250 to 1500 sgii of T-globulin is used at one time.

Test method: - Since turbidity is a problem in appearance, 0.0.
,,. The absorbance at nm was measured.

The polymer was quantified by high performance liquid chromatography.

The anti-complement titer was measured using the method of Capant and Mayer (Experimental Immunochemistry).
ntal Klunochemistry L 225
．． (1961)) and the method of Nishioka and Kaida [Biochemistry of Immunology, 103. Based on the method of 1972 (Kyoritsu Shuppan)]. That is, the number of units reduced by adding the sample from 100 units of complement was measured, and the unit of reduction was expressed as the anti-complement value.

Measles antibody titer is hemagglutination
Test (Hemagglutination Inhi)
It is measured using the international unit (bitiontest) method.
ILI/15kg).

Example 1 An experiment was conducted to confirm the stabilizing effect of the present invention. For the experiment, 5% γ-globulin containing about 30% polymer was used.
The concentration was adjusted accordingly. After adding various stabilizers (the amount added is specified in the table), heat treatment was performed at 60°C for 10 hours.
The turbidity of the solution (0,0,...·nm), the quantification of the polymer, and the anti-complement value were examined. As a result, the heat stability of T-globulin was increased by adding the stabilizer (Table 1).

In addition, a decrease in the polymer content, especially 21, was confirmed.

Example 2 Glucose was added to a T-globulin solution containing about 20% of a polymer at each fI concentration, and the T-globulin concentration was adjusted to 5%.Nikki 60 was heat-treated and 0.0
, 5anlI (iii, I combined quantification, anti-complement titer, measles antibody titer, etc.) were measured.

In a system to which glucose is added, as the amount added increases, γ-
Globulin stability has increased. In the system to which 100 g glucose was added, no clouding occurred at all even after heating at 60° C. for 10 hours, and no decrease in measles antibody titer was observed. Furthermore, the dimer value decreased to only 109 A, and the anti-complement value also decreased to 19 units (Table 2).

Example 3 Glucose is a stabilizer, and co-stabilizers are a neutral amino acid (glycine), a neutral salt (sodium chloride), an organic carboxylate (sodium citrate), a surfactant (Bluronic F1a), etc. was added, and the stability of T-globulin in heat treatment at 60°C was investigated. As in Example 1, a T-globulin solution containing about 15% of the polymer was used.

The amount of glucose added was 75 g per 100 ml of T-globulin aqueous solution, and 5.8% sodium chloride, 5% glycine, 10% sodium citrate, 0.01% Pluronic F6, and 5.8% sodium chloride were added.
% and Plurony/ReF1a 0.01%. Each group containing both auxiliary stabilizing chlorine was subjected to heat treatment at 60°C. The results are shown in Table 3, and it was possible to further reduce the polymer and anti-complement value by adding a co-stabilizer.

Example 4 An acute toxicity experiment was conducted as a safety test.

Samples A, B, C, D, E, and F, which were heat-treated at 60°C for 10 hours in Example 3, were thoroughly dialyzed with sterile physiological saline, and a total amount of 0.5 per mouse was taken from the tail vein of each mouse.
ml and 1.0+ol were administered to each group of 5 animals,
Although observed for 7 days, no abnormality was observed.

(Margin below) Procedural amendment (1 button) 1985? Month 1st

Claims (5)

[Claims] (1) Heat-treated γ-globulin preparation. (2) The preparation according to claim (1), which is heat-treated in the presence of a stabilizer. (3) The preparation according to claim (2), wherein the stabilizer is at least one selected from monosaccharides, disaccharides, and sugar alcohols. (4) The preparation according to claim (1), wherein the heat treatment is at 60°C for 10 hours. (5) In addition to the above stabilizers, neutral amino acids, neutral salts,
The preparation according to claim (1), wherein at least one co-stabilizer selected from organic carboxylates having 3 to 10 carbon atoms and surfactants is added.