JPH0899900A - Production of immunoglobulin pharmaceutical preparation for intravenous injection - Google Patents

Abstract

PURPOSE: To provide an effective producing method of immunoglobulin pharmaceutical preparation for intravenous injection having high safety and effectiveness, capable of inactivating contaminant virus, having extremely few contaminant protein and clinically applicable. CONSTITUTION: This method for producing an immunoglobulin for intravenous injection uses a fraction containing immunoglobulin as a starting raw material and includes the following steps: (a) an unabsorbed fraction is recovered by treating the fraction containing the immunoglobulin with an anion exchanger under conditions of pH5-7 and 0.0001-0.1M ion strength; (b) the fraction containing the immunoglobulin is heat-treated under conditions enough to inactivate the contaminant virus; (c) the fraction is treated with 4-12w/v% polyethylene glycol having 1000-10000 molecular weight under conditions of pH4-6, 0.0001-0.1M ion strength and 0-4 deg.C temperature to recover the supernatant; and (d) the supernatant of (c) is treated with 1015w/v% polyethylene glycol under conditions of pH7-9, 0.0001-0.1M ion strength and 0-4 deg.C temperature to recover the precipitate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an intravenous immunoglobulin preparation.

[0002]

2. Description of the Related Art Among immunoglobulins which are plasma protein components, immunoglobulin preparations containing IgG as a main component are
Until now, it has been widely used for the prevention and treatment of infectious diseases.

The inclusion of contaminant viruses such as hepatitis virus in this immunoglobulin preparation has not always been denied. Therefore, as a method for inactivating a contaminating virus, a liquid heat treatment method (see JP-A-61-191622 or the like) or a dry heat treatment method (see JP-A-61-78730 or JP-A-60-270195). Is proposed.

[0004]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The present invention further develops the above-mentioned heat treatment, and aims to improve the yield in the production of an immunoglobulin preparation for intravenous injection, improve productivity, and further remove contaminating proteins. Is. That is, an object of the present invention is to inactivate the contaminating virus, the contaminating protein is extremely small, a clinically applicable preparation, that is, an efficient method for producing an intravenous immunoglobulin preparation having higher safety and efficacy. To provide.

The inventors of the present invention have examined the industrial production method of an immunoglobulin preparation for intravenous injection along this purpose, and have conducted a polyethylene glycol (hereinafter referred to as PEG) fractionation treatment,
The present invention was completed by combining the heat treatment and the anion exchanger treatment and setting the treatment conditions of each step.

[0006]

The gist of the present invention is as set forth in the claims of the invention, and in particular, an intravenous immunoglobulin containing the following treatments starting from a fraction containing an immunoglobulin: The present invention relates to a method for manufacturing a preparation.

(A) pH 5 to 7, ionic strength 0.00
The non-adsorbed fraction is collected by treating with an anion exchanger under the condition of 01 to 0.1M. (B) Heat treatment is carried out under conditions sufficient to inactivate the contaminating virus. (C) pH 4 to 6, ionic strength 0.0001 to 0.1
M, temperature 0-4 ° C, molecular weight 1,000-10,
Treat with 000 PEG 4-12 w / v% and collect the supernatant. (D) The supernatant of (c) was adjusted to pH 7-9 and ionic strength 0.00
Molecular weight of 1,0 under conditions of 01-0.1M and temperature of 0-4 ° C.
Precipitate is recovered by treatment with 10 to 15 w / v% PEG from 00 to 10,000.

(Starting material) As the starting material of the present invention,
A fraction containing immunoglobulin is used, which is derived from human plasma and is not particularly limited as long as it contains the immunoglobulin fraction. Specific examples include a fraction II + III obtained by the Cohn ethanol fractionation, a fraction II, and a paste containing a fraction equivalent to these containing immunoglobulin. The starting material may also include human blood group antibodies, kallikrein, prekallikrein, IgM, IgG polymers and the like.

(Manufacturing Method) The manufacturing method according to the present invention includes the following steps.

Anion exchanger treatment step This step is a step of contact-treating with an anion exchanger to recover the non-adsorbed fraction. This step is performed to remove IgM and IgG polymers.

(I) Preparation of anion exchanger As the anion exchanger, an anion exchange group bound to an insoluble carrier is used, and as the anion exchange group, a diethylaminoethyl (DEAE) group or a quaternary amino group is used. An ethyl (QAE) group or the like can be used, and agarose, cellulose, dextran, polyacrylamide or the like can be used as the insoluble carrier. The binding is performed by a known method.

(Ii) Treatment method The starting material is dissolved in a suitable aqueous solvent. Aqueous solvent is pH
5 to 7 (preferably pH 5.5 to 6.5), ionic strength 0.0001 to 0.1M (preferably 0.0001 to
0.01M) aqueous solution. As a solute of an aqueous solvent,
For example, sodium chloride, sodium phosphate, potassium phosphate, acetic acid, sodium acetate, citric acid, sodium citrate and the like may be contained. The protein concentration is 1
-15 w / v% (particularly 3-10 w / v%) is preferable. Further, contact treatment is carried out with the anion exchanger equilibrated with the above aqueous solvent. In the treatment, either a batch method or a column method may be used.

For example, in the batch method, 1 ml of an anion exchanger is mixed with about 10 to 100 ml of the solution to be treated, stirred at 0 to 4 ° C. for about 30 minutes to 2 hours, and then centrifuged (6000 to 8000 rpm, (10 to 30 minutes)
And collect the supernatant. Even in the column method, anion exchanger 1
About 10 to 100 ml of the solution to be treated is brought into contact with ml to collect the non-adsorbed fraction.

Heat treatment step This step minimizes the decrease in antibody activity of immunoglobulin, but contaminating viruses such as HB virus,
AIDS virus or the like is a step of heat treatment under conditions in which it is completely inactivated.

The heat treatment is carried out in a dry state having a moisture content of 3% or less (that is, a dry heat treatment) or a solution state, that is, an immunoglobulin aqueous solution state (that is, a liquid heat treatment), and above all, the liquid heat treatment is performed. Is particularly preferable. further,
This heat treatment is preferably performed in the presence of a stabilizer, and as the stabilizer, disaccharides (eg, sucrose,
Maltose) and sugar alcohols (eg, sorbitol, mannitol) are exemplified, and sorbitol is particularly preferable. The amount of the stabilizer added is 0.5 to 5 w / v% (preferably 1 to 3 w / v%) of disaccharides and sugar alcohols in the dry heat treatment, and disaccharides and sugar alcohols in the liquid heat treatment. 10 w / v% or more (preferably 20 to 40 w
/ V%) is preferably exemplified.

The amount of immunoglobulin to be heated is
In the dry heat treatment, the amount of protein is preferably adjusted to 1 to 10 w / v% (preferably 3 to 7 w / v%). In the liquid heat treatment, the amount of protein is preferably adjusted to 0.1 to 30 w / v% (preferably 5 to 20 w / v%). In the case of dry heat treatment, the heat treatment is preferably carried out by adding a stabilizer, followed by sterilization filtration if necessary, and water content of 3% or less, preferably 1% or less, for example, by freeze-drying.
The freeze-drying conditions are 0.5 mmHg vacuum, 20-4
For example, at 0 ° C. for about 24 to 96 hours. Then, for example, 50 to 80 ° C. (preferably about 60 ° C.), 1 to 200
The treatment is performed for a time (preferably about 10 to 100 hours).
In addition, by performing this heat treatment step in an inert gas atmosphere, stability during heating can be further enhanced. Examples of the inert gas include nitrogen gas, argon and helium. In the case of liquid heat treatment, p
H is adjusted to 4.5-6.5, preferably pH 5-6,
For example, the treatment is performed at 50 to 80 ° C. (preferably about 60 ° C.) for 10 minutes to 20 hours (preferably about 10 hours). The ionic strength of the aqueous solution is 0.0001-0.
1M (particularly preferably 0.0001 to 0.01M) is exemplified.

Low concentration polyethylene glycol (PE
G) Treatment step This step is a step of treating with low concentration PEG and collecting the supernatant.

The object to be treated has a molecular weight of 1,000 to 10,0.
Treat with PEG of 00 (preferably about 2,000-6,000) (eg, mix both). The processing condition is P
EG concentration 4 to 12 w / v% (especially 8 to 12 w / v%),
pH 4-6 (especially 4.5-5.5), ionic strength 0.0
001-0.1M (especially 0.0001-0.01M)
And At this time, a protein concentration of 1 to 20 w / v% (especially 5
˜15 w / v%) is preferred. The process is
It is usually carried out by stirring at 0 to 4 ° C. for about 30 minutes to 6 hours. After that, for example, centrifugation (6000 to
8,000 rpm, 10 to 30 minutes) and collect the supernatant.

High-concentration polyethylene glycol (PE
G) Treatment step This step is a step of treating the supernatant obtained in the step of with high-concentration PEG and collecting the precipitate.

The above-mentioned supernatant is treated with a molecular weight of 1,000 to 10,000.
Further treatment with 0 (preferably 2,000 to 6,000) PEG (eg, mixing both). The treatment condition is that the PEG concentration is 10 to 15 w / v% (particularly 11 to 13).
w / v%), pH 7-9 (especially 7.5-8.5), ionic strength 0.0001-0.1M (especially 0.0001-)
0.01M). At this time, protein concentration 1 to 20 w / v
% (Particularly 5 to 15 w / v%) is preferable.
The said process is performed by stirring at 0-4 degreeC normally for about 30 minutes-6 hours. Then, for example, centrifugation (6000 to 8000 rpm, 10 to 30 minutes) is performed to collect the precipitate.

In the present invention, in addition to the above treatment, it is preferable to further perform retreatment with an anion exchanger or the following treatment.

Treatment with Immobilized Diamino Compound This step is a step of contacting with the immobilized diamino compound to recover the non-adsorbed fraction. This step is performed to remove prekallikrein or kallikrein.

(I) Preparation of Immobilized Diamino Compound The immobilized diamino compound is a diamino compound immobilized on an insoluble carrier. As the diamino compound, aminobenzamidine, aminobenzaguanidine, lysine, arginine and the like can be used. As the insoluble carrier, agarose, cellulose, dextran, silica gel, glass or the like is used. Immobilization may follow a known method. For example, agarose, cellulose and the like can fix the diamino compound by the CNBr activation method, and silica gel, glass and the like by the oxirane method.

(Ii) Treatment method The treatment object, for example, the precipitate fraction obtained in the above step, has a pH of 5 to 8 (particularly pH 6 to 7) and an ionic strength of 0.01.
Contact treatment with the immobilized diamino compound is performed under the conditions of ˜0.2 M (particularly 0.05 to 0.15 M). In that case, the protein concentration is preferably 1 to 15 w / v% (particularly 3 to 10 w / v%), and both the batch method and the column method are preferably used.

In the batch method, for example, 1 ml of the immobilized diamino compound is mixed with about 10 to 100 ml of the above fraction, and the mixture is mixed at 0 to 10 ° C., preferably 0 to 4 ° C. for 30 minutes to 4 hours, preferably 30 minutes to. After stirring for about 2 hours, centrifuge (6,000 to 8,000 rpm, 10 to 30 minutes)
And collect the supernatant. Also in the column method, about 10 to 100 ml of the above fraction is contacted with 1 ml of the immobilized diamino compound, and the non-adsorbed fraction is collected.

Immobilized human blood group substance treatment step This step is a step of recovering the non-adsorbed fraction by contact treatment with the immobilized human blood group substance. This step is performed to remove human blood group antibodies.

(I) Preparation of immobilized human blood group substance The immobilized human blood group substance is obtained by immobilizing human blood group substance on an insoluble carrier. A known method may be used to prepare the human blood group substance. For example, it can be obtained by subjecting human A, B, AB or O type erythrocytes to hemolysis in a hypotonic solution or ultrasonic treatment, followed by purification by ammonium sulfate fractionation method or PEG fractionation method. As a human blood group substance, a synthetic antigen (sugar chain) can also be used [Human Blood Grou
ps and Carbohydrate Chemistry (1978) Chem. Soc. Rev.
p423-452].

Further, this human blood group substance is said to be effective for inactivating contaminating viruses after being dissolved in physiological saline, for example, about 50 to 70 ° C., preferably about 60 ° C.
At 7 to 13 hours, preferably about 10 hours, or about 8
0 to 130 ° C., preferably 95 to 121 ° C. and about 1 to 40
Heat treatment for minutes, preferably about 2 to 30 minutes. afterwards,
Insoluble matter is removed by centrifugation and dialyzed against distilled water to obtain each human blood group substance. As the insoluble carrier, agarose, cellulose, dextran, silica gel, glass or the like is used. Immobilization may follow a known method. For example, human blood group substances can be immobilized by the CNBr activation method for agarose, cellulose, etc., and by the oxirane method for silica gel, glass, etc.

(Ii) Treatment method The treatment target, for example, the non-adsorbed fraction of the above process, is adjusted to pH
(Especially pH 6-7), ionic strength 0.01-0.2M
Contact treatment with the immobilized human blood group substance equilibrated with the above aqueous solvent is carried out under the condition of (particularly 0.05 to 0.15 M).
At that time, a protein concentration of 1 to 15 w / v% (especially 3 to 10 w
/ V%), and either the batch method or the column method may be used.

For example, in the batch method, 1 ml of immobilized human blood group substance is mixed with about 10 to 100 ml of the solution to be treated, and the mixture is mixed at 0 to 10 ° C., preferably 0 to 4 ° C. for 30 minutes.
After stirring for about 4 minutes, preferably about 30 minutes to 2 hours, centrifugation (6,000 to 8,000 rpm, 10
30 minutes) and collect the supernatant. Also in the column method, the solution to be treated is 10 to 10 ml per 1 ml of the immobilized human blood group substance.
About 0 ml is contacted and the non-adsorbed fraction is collected.

(Preparation of Liquid Preparation) The obtained immunoglobulin composition is used in an amount of 1 to 10 w / v% (preferably 3 to 7 w / v).
%) Suitable solvent (eg distilled water for injection)
And a stabilizer such as a disaccharide (sucrose,
Maltose etc.), sugar alcohols (sorbitol, mannitol etc.), particularly preferably sorbitol 1-20 w /
v% (preferably 2 to 10 w / v%) is added to adjust the pH.
5 to 6 (preferably pH 5.3 to 5.7, particularly preferably about 5.5), low conductivity, preferably conductivity 1 mmho.
After being adjusted by a means known per se so as to be the following (particularly, 0.6 mmho or less, both converted at 8 ° C.), sterilization filtration, dispensing, etc. are performed based on ordinary formulation technology. Thus, an intravenously administrable immunoglobulin preparation is prepared.

[0032]

In the present invention, since the globulin preparation for intravenous injection was produced by the above method, IgG polymer was not detected in the obtained preparation, and the yield of IgG in the production process was increased. Has improved.

The preparation obtained by the present invention has almost no immunoglobulin inactivated, and since it is subjected to heat treatment, it also inactivates contaminant viruses, has good solubility, and has sufficient anti-complement activity. In a preferred embodiment, it has a property of not containing impurities such as prekallikrein, kallikrein, anti-human blood group antibody, etc.
Japanese biologics standards issued in fiscal year 0 (hereinafter, raw standards)
It is a safe formulation that can pass.

The preparation obtained by the present invention is used for the prevention or treatment of infectious diseases by intravenous administration, infusion, etc. That is, the present invention is useful as an industrial method for producing an intravenous immunoglobulin preparation.

[0035]

EXAMPLES Examples will be given to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1 To 1 kg of Cohn fraction II + III was added 3 liters of water, and after extraction at pH 4, DEAE-Sephadex was added to the solution (5
The solution (1 ml per 0 ml solution) was subjected to contact treatment under the conditions of pH 6.0 and 4 ° C., and after the treatment, centrifugation (7,000 rpm, about 20 minutes) was performed to collect a supernatant (IgG solution). 100 more
Add 50 g of sorbitol to ml and adjust the pH to 5.5.
After adjusting to, the mixture was heat treated at 60 ° C. for 10 hours. After completion of heating, the pH was adjusted to 5.5, PEG # 4000 was added so that the final concentration was 8 w / v%, and the mixture was centrifuged at 2 ° C. The obtained supernatant was adjusted to pH 8.8 with 1N-sodium hydroxide, PEG # 4000 was added to a final concentration of 15 w / v%, and the mixture was centrifuged at 2 ° C. to precipitate IgG in the precipitated fraction. Fractions were obtained. The IgG fraction was dialyzed against distilled water and adjusted to a 5 w / v% IgG solution,
The pH of the solution was adjusted to about 5.5 with sodium acetate, and sorbitol was added to a final concentration of 5 w / v%. This aqueous solution (conductivity of about 1 mmho) was sterilized and filtered to obtain an immunoglobulin liquid preparation for intravenous injection.

Example 2 3 liters of water was added to 1 kg of Cohn fraction II + III and extracted at pH 4 and DEAE-Sephadex was added to this solution (2
The solution (1 ml per 0 ml solution) was subjected to contact treatment under the conditions of pH 6.0 and 4 ° C., and after the treatment, centrifugation (7,000 rpm, about 20 minutes) was performed to collect a supernatant (IgG solution). 100 more
Add 50 g of sorbitol to ml and adjust the pH to 5.5.
After adjusting to, the mixture was heat treated at 60 ° C. for 10 hours. After completion of heating, the pH was adjusted to 5.3 to 5.4, and then PEG # 4
000 to give a final concentration of 12 w / v%, 2
Centrifugation was performed at ° C. The resulting supernatant was adjusted to pH 8.8 with 1N sodium hydroxide, and then PEG # 4000.
Was added to a final concentration of 15 w / v%, and the mixture was centrifuged at 2 ° C. to obtain an IgG fraction as a precipitate fraction. This IgG
The fraction was dissolved in distilled water, DEAE-Sephadex was added (1 ml per 33 ml solution), contact treatment was performed under the conditions of pH 6.0 and 4 ° C., and after treatment, centrifugation (7,000 rpm, about 20 minutes) was performed. The supernatant (IgG solution) was collected. This I
The gG fraction was dialyzed against distilled water and adjusted to a 5 w / v% IgG solution, the pH of the solution was adjusted to about 5.5 with sodium acetate, and sorbitol was added to a final concentration of 5 w / v%. This aqueous solution (conductivity of about 1 mmho) was sterilized and filtered to obtain an immunoglobulin liquid preparation for intravenous injection.

The preparations obtained in Examples 1 and 2 above essentially contained only IgG monomer and had an anti-complementary value of 10-15.
It was around CH ₅₀ and passed the raw standard for intravenous globulin.

Experimental Example 1 In the above-mentioned Example 1, a method in which the pretreatment with the anion exchanger was not carried out before the heat treatment was carried out by the conventional method 1 (JP-A-63-1).
No. 83539), and the globulins in the preparation thus obtained are quantified by an immunoturbidimetric method,
The recovery rate of globulin was calculated. Similarly, the recovery rate of globulin in Example 1 was obtained. As a result, the recovery rate in Example 1 was 146% when the recovery rate in Conventional Method 1 was 100%.

Experimental Example 2 In the above-mentioned Example 2, the method in which the pretreatment with the anion exchanger was not carried out before the heat treatment was carried out by the conventional method 2 (JP-A-63-1).
No. 83539), and the globulins in the preparation thus obtained are quantified by an immunoturbidimetric method,
The recovery rate of globulin was calculated. Similarly, the recovery rate of globulin in Example 2 was obtained. As a result, the recovery rate in Example 2 was 154% when the recovery rate in Conventional Method 2 was 100%.

Experimental Example 3 In the liquid immunoglobulin preparation for intravenous injection obtained in Example 2 above, contaminant proteins were quantified by immunodiffusion method and IgG polymer was quantified by HPLC, and the properties of the preparation were analyzed. The results shown in Table 1 were obtained.

[0042]

[Table 1]

Claims (1)

[Claims] 1. A method for producing an immunoglobulin preparation for intravenous injection, which comprises the following steps using an immunoglobulin-containing fraction as a starting material: (a) pH 5 to 7, ionic strength 0.0001 to 0.1
Under the conditions of M, it is treated with an anion exchanger and the non-adsorbed fraction is collected. (B) Heat treatment is carried out under conditions sufficient to inactivate the contaminating virus. (C) pH 4 to 6, ionic strength 0.0001 to 0.1
M, temperature 0-4 ° C, molecular weight 1,000-10,
000 polyethylene glycol 4-12 w / v% to treat and collect the supernatant. (D) The supernatant of (c) was adjusted to pH 7-9 and ionic strength 0.00
Molecular weight of 1,0 under conditions of 01-0.1M and temperature of 0-4 ° C.
Polyethylene glycol of 10 to 10,000
Treat with 5 w / v% to recover the precipitate.