JPS5840532B2 - IGA Oyobi IGM Nonetsu Antei Kahou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for thermally stabilizing human plasma or placenta extracts, or IgA and IgM obtained by fractionating these, and specifically relates to a heat treatment for inactivating hepatitis viruses. On this occasion,
The present invention relates to a method for increasing the thermal stability of IgA or IgMJ.

Recently, the onset of serum hepatitis associated with blood transfusion has become a major social problem, and it has been revealed that the cause is a hepatitis virus.

The hepatitis virus was previously called the 2-Australian antigen, but is now called the HB antigen.

However, since it is difficult to inactivate the HB antigen in plasma for transfusion, the concentration of HB antigen is measured in advance using an immunoassay method using anti-HB antigen, and the HB antigen is inactivated at a high concentration.
By excluding plasma containing antigens, some effectiveness in preventing the onset of hepatitis has been achieved, but it is not sufficient.

The problem of hepatitis development is also included in individual human serum protein preparations obtained by fractionating plasma.

However, it has been found that by subjecting albumin preparations to heat treatment at 60°C for 10 hours, the infectivity of hepatitis can be inhibited without altering the albumin. Safe in clinical use.

Since it was discovered that a preparation heat-treated at 60°C for 10 hours was effective in preventing the onset of hepatitis after administration,
This method has been applied to other human serum protein preparations.

A material to which a heat treatment method of 60° C. for 10 hours can be applied must be stable against this treatment.

Therefore, various stabilizers have been discovered to enable this heat treatment.There are substances that cannot withstand heat treatment without a stabilizer, but can make heat treatment possible in the presence of stable machining. .

Amino acids, sugars, and the like are generally used as stabilizers for human serum proteins at physiologically isotonic or lower concentrations.

The inventor previously added amino acids and saccharides to a haptoglobin preparation at physiologically isotonic concentrations of 2, 25%, and 5%, respectively, and heat-treated the mixture at 60°C for 10 hours. It has decreased significantly.

As a result of much research into methods for thermally stabilizing haptoglobin, we found that by adding amino acids or saccharides, especially glycine or mannitol, to a concentration of 15% or more, 6.
Thermal stability against heat treatment at 0°C for 10 hours was significantly increased, making it possible to inactivate the hepatitis virus in the preparation (see Japanese Patent Application No. 128606/1982).

The inventors subsequently discovered that this thermal stabilization method was effective not only for haptoglobin but also for IgA and IgM, and based on this new knowledge, they completed the present invention.

The present invention involves adding neutral amino acids such as glycine, alanine, and valine, monosaccharides such as glucose, mannose, and fructose, and trisaccharides such as sucrose, maltose, and lactose to an aqueous solution of IgA or IgM (human serum immunoglobulin A or IgM). , mannit,
Add sugar alcoholics such as Nrubit, Xylit, etc. singly or in combination to a concentration of 5% or more, and then heat at 60°C.
A heat treatment is performed for 10 hours.

As a result of quantifying the amount of protein remaining in the solution after heating with respect to the protein before heating by one-way immunodiffusion method, it was found that 5 of these stabilizers
% or more, preferably 15 to 20%, a remarkable thermal stabilizing effect was observed.

Thermal stabilizing effects of various stabilizers on IgA before heat treatment
and the residual rate after heat treatment with IgM as 100% (%
) as shown in Table 1.

The thermostabilizing effect of the present invention on IgA and IgM was very clearly effective both in the plasma state, in the solution during the plasma fractionation process, and also in purified IgA and IgM aqueous solutions.

Furthermore, human serum protein preparations can also be obtained from human placenta.

Even when placenta is used as a raw material, amino acids, sugars, and sugar alcohols can be added to the extract or an aqueous solution after an appropriate purification process at a temperature of 5% or more, preferably 15 to 20%. When subjected to heat treatment at ℃ for 10 hours, placenta-derived IgA and IgM were stabilized in the same way as when plasma was used as the material.

After such heat treatment, IgA and IgM
IgA and IgA for medical use, which does not pose a risk of developing hepatitis when administered using a prescribed method to produce a drug.
gM formulation was obtained.

IgA and I useful for medical purposes according to the present invention
The thermal stability of gM can be significantly increased, making it a safe Ig.
This has the effect that A and IgM preparations can be produced with good yield.

Next, the present invention will be specifically explained with reference to several examples.

Note that the present invention is not limited to the stabilizers and targets shown in these Examples.

Implementation row 1 Dissolve 25M' of glycine in pooled human plasma 11, add O1
Adjust the pH to 8 using normal caustic soda or 0.1N hydrochloric acid.
．． Adjust to 0.

The mixture was filtered for sterilization and heat treated at 60°C for 10 hours.

After heating, the mixture was dialyzed against physiological saline (pH 7y2), and the resulting precipitate was removed by centrifugation to obtain a clear solution.

When IgA was quantified before and after heating by one-way immunodiffusion method, the quantitative values were 175.2'm9/di and 132.81 v/dl, respectively.

Similarly, the quantitative value of IgM is 1091119/dl.
and 6UI19/dl-C-.

Example 2 Mannitol 20Of was used in place of the glycine used in Example 1.
? The same treatment was performed using .

When IgA was quantified before and after heating by one-way immunodiffusion method, the quantitative values were 170.3 m9/dl and 1088 m97 dl, respectively.

Similarly, the quantitative value of IgM is 1161119/dl and 1
031119/di! Q hot.

Example 3 Glucose 250? instead of the glycine used in Example 1 The same treatment was performed using .

The quantitative value of IgA before and after heating was 172.3■/d, respectively.
1 and 139,6 m9/dl.

Similarly, the quantitative values of IgM were 111/dl and 90/dl.
It was l.

Example 4 Water was added to fraction IV30P obtained by fractionating pooled human plasma using Cohn's low-temperature ethanol fractionation method to make a homogeneous suspension.
Centrifuge to remove most of the insoluble material.

Dissolve 60 g of alanine in this solution, adjust the pH to 7.5, and then add water to make a total volume of 300 ml.

After heating this solution at 60° C. for 10 hours, it is dialyzed against a 0.0063M acetate buffer at pH 6.0, and the resulting precipitate is removed by centrifugation to obtain a clear solution.

When the quantitative determination of IgA was carried out using the one-way immunodiffusion method on the liquid before and after heat treatment, the quantitative value was 213 m97d, respectively.
1 and 149 mq/dl.

Similarly, the quantitative value of IgM was 132 m9/dl and 88,
It was 4yny/dl.

Example 5: Add 1 each of glycine and mannitol to pooled human plasma.
A solution dissolved at a concentration of 5% was mixed at a ratio of 1:2, and this solution was heated at 60° C. for 10 hours.

After heating, the mixture was dialyzed against physiological saline, and the resulting precipitate was removed by centrifugation to obtain a clear solution.

When the quantitative determination of IgA in the liquid before and after heating was carried out using a one-way immunodiffusion method, the quantitative values were 72.3 .mu./di and 57.6 .mu./dl, respectively.

Similarly, the quantitative value of IgM is 45■/dl and 34.6■
/dl.

Example 6 Three placentas were frozen and thawed three times, then crushed, and the same weight of physiological saline was added to thoroughly homogenize, followed by centrifugation to separate residual fragrance and obtain a clear liquid.

This liquid 1. Add 200 g of mannitol to e and adjust the pH to 7.2.
After heating at 60° C. for 10 hours.

After heating, the solution is dialyzed against a 0.0063M acetate buffer with a pH of 6.0, and the resulting precipitate is removed to obtain a clear solution.

When the quantitative determination of IgA and IgM was measured using the one-way immunodiffusion method for the liquid before and after heating, the quantitative values for IgA were 261,119/dl and 20.8 m9/dl, respectively.
dl and 16.0■/d for IgM, respectively.
1 and 13.5~/dl.

Claims (1)

[Claims] 1. Human plasma or placenta extract, or a solution containing IgA or IgM obtained by fractionating these, is added to the hepatitis virus in the presence of neutral amino acids, monosaccharides, trisaccharides, and/or sugar alcohols. 1. A method for thermally stabilizing IgA and IgM, which comprises applying heat treatment to inactivate IgA and IgM.