JPS62289523A - Heat treatment of immunoglobulin for intravenous administration - Google Patents

Abstract

PURPOSE:To carry out viral inactivation of the titled globulin, by heating natural type immunoglobulin for intravenous administration in the presence of a specific stabilizer. CONSTITUTION:Natural type immuno(gamma)globulin for intravenous administration, e.g. obtained by fractionating blood plasma proteins, etc., such as IgG, IgA or IgM, is a dry state is heated in the presence of a stabilizer consisting of at least one saccharide selected from sorbitol and saccharose and albumin under condition of <=3% moisture content until contaminating viruses are inactivated to carry out viral inactivation of the natural type immunoglobulin for intravenous administration. The above-mentioned stabilizer is used in an amount to give concentrations of 1-5wt/vol% saccharide and 0.5-5wt/vol% albumin based on a 0.01-2wt/vol% solution of monoclonal immunoglobulin or 2-8wt/vol% solution of polyclonal immunoglobulin.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a heat treatment method for virus inactivation of natural immunoglobulin for intravenous administration.

[Prior art]

Traditionally, 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. )
[The New York Academy-Opmedison (Th
e New York Academy of Med
icine), Rise (5), 341-358 (19
It was adopted based on the report of 55)), 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 proven epidemiologically.

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 activated. It is easy to cause a decrease or disappearance of

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. Experiments using blood coagulation factor II as a model revealed that it was significantly inhibited. However, -m
Even in dry heat treatment, if a stabilizer is not added, the activity of blood protein 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 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 mechanisms of both types overlap with each other, but it has been suggested that they are fundamentally different [Learn, Physical Methods of Stellarization of Macroorganisms, Batteriology] Review (Rahn, Physical
'! Ie-thods of 5terilizati
on of Macroorganisms.

Bact, 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 natural immunoglobulin for intravenous administration.

Another object of the present invention is to provide a virus inactivation heat treatment method for naturally occurring immunoglobulin for intravenous administration, which can maintain good solubility and solubility in water.

[Means for solving problems]

The present inventors have demonstrated that dry heat treatment of natural intravenous immunoglobulin can inactivate viruses without losing the activity of the natural intravenous immunoglobulin, especially in the presence of a stabilizing agent. Dry heat treatment of naturally occurring immunoglobulin for intravenous administration significantly stabilizes the immunoglobulin, and furthermore, the immunoglobulin subjected to dry heat treatment under such conditions has good solubility and solubility in water. They discovered this and completed the present invention.

In the present invention, contaminating winodose is inactivated by drying natural immunoglobulin for intravenous administration in the presence of a stabilizer consisting of albumin and at least one saccharide selected from sorbitol and saccharose. This invention relates to a method for heat treatment of natural immunoglobulin for intravenous administration, which is characterized by heating the natural immunoglobulin for intravenous administration until it is heated, thereby inactivating contaminating viruses and improving the stability and water content of natural immunoglobulin for intravenous administration. Solubility is improved.

The natural immune (gamma) globulin for intravenous administration to be subjected to the heat treatment in the present invention is one that has biological or physiological activity as an immunoglobulin, such as one obtained by fractionating plasma proteins. In addition, its properties are +1+ natural, without any modification or change, and therefore F, which is a fragment of gamma globulin.
Does not include ab, F (ab')z, Fc, etc., (2)
It has various properties such that there is no decrease in antibody titer (at the same time, there is no decrease in antibody spectrum), and (3) anti-complement action (complement fixation) is sufficiently lower than 20 units (CHso value), which is considered safe. refers to something that

The natural immunoglobulin for intravenous administration used in the present invention may be obtained by any method as long as it is in its natural state and has a low anti-complement value, but it can be manufactured using existing equipment. The most efficient method is to use gamma globulin for intramuscular injection, which is already used as a medicine, and to separate the aggregates by acid treatment. However, apart from manufacturing complexity and reduced yield, the method using nonionic surfactants removes gamma globulin aggregates that cause anti-complement effects, and gamma globulin aggregates with low anti-complement
It is preferable to use globulin.

Further, the natural immunoglobulin for intravenous administration of the present invention is exemplified by those derived from humans, horses, or mice, and may be either a polyclonal antibody or a monoclonal antibody (preferably IgG, IgA, or IgM). .

In the present invention, after freeze-drying a naturally occurring intravenous immunoglobulin solution to which a specific stabilizer has been added,
% or less, usually 0.05 to 3%.

The stabilizer used in the present invention is at least one kind of [Q
and albumin are used in combination.

The amount of the stabilizer used is, for example, as follows.

That is, in the case of monoclonal immunoglobulin, 0.01 to 2 W/V% solution thereof, and in the case of polyclonal immunoglobulin, to 2 to 8 W/V% solution thereof, 5W/V%, more preferably 2~
The amount corresponds to a concentration of about 3 W/V%, albumin 0.5 to 5 W/V%, more preferably about 1 to 2 W/V%. This level of addition provides the best balance between stabilizing effect, water solubility, solubility, and formulation.

Natural type immunoglobulin for intravenous administration is usually used as a lyophilized product, but it is preferable to add a stabilizer before the natural type immunoglobulin for intravenous administration is lyophilized.

Furthermore, although the stabilizer may be removed after the drying treatment of the present invention, it is preferable that the stabilizer be incorporated into the immunoglobulin preparation as it is.Thus, the stability of the preparation during lyophilization and storage is improved. be done.

In the present invention, in addition to the above stabilizers, at least one type selected from neutral salts, organic carboxylic acids, and surfactants may be added as a co-stabilizer.

Regarding the co-stabilizer used, examples of neutral salts include halogen salts of alkali metals or alkaline earth metals such as sodium chloride, potassium chloride, and magnesium chloride, and the amount added is equal to that of natural intravenous immunoglobulin. The amount is 0.1 to 10 g per 100 g of the aqueous solution.

The 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), It may be any ring shape. Examples of the hydrocarbon residue include an alkyl group and an aryl group (for example, a phenyl group).

The number of carboxyl groups in the organic carboxylic acid may be plural, but 1 and 2 carboxyl groups are preferable.

Further, the organic carboxylic acid may be substituted with a hydroxyl group. There is no particular limit to the salt in the organic carboxylate as long as it is physiologically acceptable, and preferred salts include alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, etc.). etc.), particularly preferably sodium salts and potassium salts.

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. Mention may be made of salts of acids, especially alkali metal salts (sodium salts, potassium salts).

The preferred number of carbon atoms in such an organic acid is about 3 to 15.

The amount of organic carboxylate added is 100% of the globulin aqueous solution.
- per o, i ~ 3g.

Surfactants include nonionic agents such as alkylphenylpolyoxyethylenes (e.g. Triton® and Nonidet®), anionic agents such as bile salts (e.g. sodium taurocholate); cationic agents such as benzalkonium chloride, and polyhydric alcohols with surface activity such as polymeric copolymers of propylene oxide [Pluronic registered trademark (Pluronic@)
F 68 ), etc., and the addition amount is 0% per 100 μm of natural intravenous immunoglobulin aqueous solution.
．． The amount is preferably about 0.002 to 1 g.

In the present invention, the heat treatment is carried out at a temperature and for a period of time sufficient to inactivate viruses that are likely to contaminate natural immunoglobulin for intravenous administration.

Heating temperature is usually 30 to 100°C, preferably about 60°C
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, and in particular include hepatitis virus and AIDS 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 and heat resistance of naturally occurring immunoglobulin for intravenous administration, and no matter what degree of purification the immunoglobulin is used, the stabilizing effect of the stabilizer,
Consequently, the effects of the treatment of the present invention remain unchanged. Therefore, the heat treatment of the present invention may be performed at any stage of the immunoglobulin purification process.

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%.

[Action/Effect]

According to the present invention, the activity of natural intravenous immunoglobulin, which is a valuable blood product, is not significantly lost.
It is possible to inactivate viruses that are feared to be mixed into pharmaceutical preparations.

Furthermore, the immunoglobulin preparation for intravenous administration that has undergone the treatment of the present invention has excellent solubility in water, and the solubility of water-soluble debris is maintained well.

Therefore, the present invention is extremely useful as an industrial method for producing natural intravenous immunoglobulin preparations.

[Example/Experiment example]

The present invention will be explained below with reference to Examples and Experimental Examples.
The present invention is not limited to these in any way.

Example 1 After adding 101 kg of 0.001 M sodium chloride solution to the corn fraction + 1 + I [1] and adjusting the pH to 5.0, polyethylene glycol (P E G #4000) was added.
was added to a final concentration of 8%, and centrifuged at 2°C.

The obtained supernatant was adjusted to pH 8 using IN-sodium hydroxide.
After setting it to 0, the final concentration of PEG #4000 was 12%.
Centrifuge at 2°C to remove IgGII.
ii minutes were collected.

This IgG fraction is 0.6% sodium chloride? Using 8 liquids, dissolve p)I so that the IgG concentration is 7%.
．． Adjusted to 5.

Henzamidine Sepharose column (registered trademark, manufactured by Pharmacia) prepared separately with 100% of this IgG solution
5I1) 7 and human blood group substance Formyl Cephalofine Column 3- to adsorb and remove human blood group antibodies. Blood type antibodies are <1:3 due to adsorption in this step.
2) to (1:2).

To this unadsorbed fraction, human albumin was added at IW/V% and saccharose was added at 2W per IgG5W/V% solution.
/V% was added, filtered for sterilization, and freeze-dried.

After freeze-drying, heat treatment was performed at 60° C. for 72 hours to obtain a natural immunoglobulin preparation for intravenous injection.

This preparation substantially contains only IgG monomer, has a sufficiently low amount of human blood group antibodies, and has an anti-complement value of 10 to 15 CH. It was quite strange.

It also has high solubility and has passed the biological preparation standard as an intravenous immunoglobulin.

Experimental Example 1 (Type of stabilizer) The Fr-If CIgG fraction obtained from normal human blood protein according to Cohn's cold alcohol fractionation method was used as a raw material and treated according to Example 1 to obtain a natural A type of immunoglobulin for intravenous administration was obtained, and each stabilizer listed in Table 1 was added to the IgG.
Each fraction was added to a 5W/■% solution, and the pH was adjusted to 6.3.
After adjusting to ~6.5, it was freeze-dried. The IgG powder is 6
After heat treatment at 5℃ for 96 hours, ? It was tested for 8-lysis, solubility, diphtheria antitoxin titer, measles antibody titer, and anti-complement titer. The results are shown in Table 1. The test method followed the "Biological Products Standards."

As a result, particularly effective amounts of the stabilizer added by the dry heat treatment of the present invention are 1 to 5 W/V% for sugars and 0.5 to 5% for albumin.
It was found that the combined use of 5W/V% is suitable.

Experimental Example 2 To a 5W/V% aqueous solution of the preparation (IgG) prepared according to Example 1, 2W/V% saccharose and IW/V% albumin were added, and to this was added 0.05M phosphate buffer. Various virus suspensions (pH 7.1) were added and mixed uniformly, followed by freeze-drying.

After freeze-drying, return to normal pressure with nitrogen gas and seal tightly.
It was immersed in a 0°C warm bath and heated. In addition, it takes 10 to 15 minutes for the temperature in the bottle to reach 60℃, so it actually takes 3
The heating time was extended to 0 minutes.

The infectivity of each virus is determined by plaque forming (plaque forming).
que forming) method.

The results are shown in Table 2.

(Margin below)

Claims (6)

[Claims] (1) Heating natural immunoglobulin for intravenous administration in a dry state in the presence of a stabilizer consisting of albumin and at least one type of saccharide selected from sorbitol and saccharose until contaminating viruses are inactivated. A method for heat treatment of natural immunoglobulin for intravenous administration, characterized by: (2) The method according to claim (1), which comprises heating under conditions where the humidity content is 3% or less. (3) The method according to claim (1), wherein the natural immunoglobulin for intravenous administration is derived from humans. (4) The method according to claim (3), wherein the human-derived immunoglobulin is a polyclonal or monoclonal antibody. (5) The natural intravenous immunoglobulin is IgG,
The method according to claim (1), characterized in that it is IgA or IgM. (6) The concentration of the stabilizer added to natural intravenous immunoglobulin is 0.01 compared to monoclonal immunoglobulin.
The amount is equivalent to a concentration of 1 to 5 W/V% for sugars and 0.5 to 5% W/V for albumin for a ~2 W/V% solution or a 2 to 8 W/V% solution of polyclonal immunoglobulin. A method according to claim (1), characterized in that: