JPH0761935A - Method for producing intravenous immunoglobulin preparation - Google Patents

Abstract

(57) [Summary] [Structure] Using a fraction containing immunoglobulin as a starting material, under conditions sufficient to inactivate contaminating viruses,
pH 4.5 to 6.5 and heat treatment in a liquid state in the presence of a stabilizer, pH 5.1 to 5.4, ionic strength 0.00
Molecular weight of 1,0 under conditions of 01-0.1M and temperature of 0-4 ° C.
Polyethylene glycol 6 to 10 of 0 to 10,000
The supernatant is recovered by treating with w / v%, and the supernatant is adjusted to pH 7-
10, ionic strength 0.0001-0.1M, temperature 0-4
A method for producing an immunoglobulin preparation for intravenous injection, which comprises a step of treating with polyethylene glycol having a molecular weight of 1,000 to 10,000 at 10 ° C and having a concentration of 10 to 15 w / v% to recover the precipitate under the condition of ° C. [Effects] Immunoglobulins are almost inactivated,
Contaminant virus is inactivated, globulin dimer content is extremely low, solubility is good, anti-complement activity is sufficiently low, and safe and effective immunoglobulin preparations for intravenous injection are efficiently obtained. .

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 (JP-A-61-191622, etc.) or a dry heat treatment method (JP-A-61-178730, JP-A-60-2701).
No. 95) has been proposed.

By developing this heat treatment method and combining it with polyethylene glycol (hereinafter referred to as PEG) fractionation treatment, anion exchanger treatment and the like, a clinically applicable preparation, that is, a highly safe and effective preparation. A method for producing an intravenous immunoglobulin preparation has been established (Japanese Patent Laid-Open No. 63-183539).

[0005]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The present invention aims to further improve the production method of the above-mentioned intravenous immunoglobulin preparation, and to improve the yield, productivity, and further remove contaminating proteins. It is a thing. Therefore, an object of the present invention is to inactivate a contaminating virus, to reduce the amount of contaminating proteins, and to provide a clinically applicable preparation, that is, an efficient method for producing an intravenous immunoglobulin preparation having higher safety and efficacy. To provide.

[0006] 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 heat treatment and the like and setting the treatment conditions of each step.

[0007]

The gist of the present invention is as set forth in the claims of the invention, and in particular, an immunoglobulin for intravenous injection comprising the following treatment using a fraction containing immunoglobulin as a starting material. The present invention relates to a method for manufacturing a preparation.

(A) Heat treatment in a liquid state at a pH of 4.5 to 6.5 and in the presence of a stabilizer under conditions sufficient to inactivate contaminating viruses. (B) pH 5.1 to 5.4, ionic strength 0.0001
~ 0.1M, temperature 0 ~ 4 ℃, molecular weight 1,000
Treat with ~ 10,000 PEG 6-10 w / v% and collect supernatant. (C) The supernatant of (b) is adjusted to pH 7 to 10, ionic strength 0.0
001-0.1M, temperature 0-4 ℃, molecular weight 1,
The precipitate is recovered by treatment with PEG 10-15 w / v% of 000-10,000.

(Starting material) As a 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.

(Production Method) The production method according to the present invention preferably comprises the following treatments. Low Concentration PEG Treatment Step This step is a step of treating the starting material with low concentration PEG and collecting the supernatant. The starting material is suspended in a suitable aqueous solvent. As a solute of an aqueous solvent, for example, sodium chloride,
It may contain sodium phosphate, potassium phosphate, acetic acid, sodium acetate, citric acid, sodium citrate and the like.

This suspension is treated with 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 6 to 10 w / v% (particularly 8 w / v%), pH
5.1-5.4 (especially 5.2-5.3), ionic strength 0.0001-0.1M (especially 0.0001-0.0)
1M). At this time, the protein concentration is preferably 1 to 20 w / v% (in particular, 1 to 5 w / v%). The said process is performed by stirring at 0-4 degreeC normally for about 30 minutes-6 hours. Then, for example, centrifugation (600
The supernatant is collected at 0-8000 rpm for 10-30 minutes).

High Concentration PEG Treatment Step This step is a step of treating the supernatant obtained in the step of with high concentration PEG and collecting the precipitate. Molecular weight of the above supernatant is 1,
000-10,000 (preferably 2,000-6,000)
0) PEG is further processed (for example, both are mixed). The processing conditions are PEG concentration of 10 to 15 w / v%
(Especially 11 to 13 w / v%), pH 7 to 10 (especially 8
~ 9), ionic strength 0.0001-0.1M (especially,
0.0001 to 0.01M). At this time, the protein concentration is preferably 1 to 20 w / v% (in particular, 1 to 5 w / v%). The treatment is usually at 0 to 4 ° C. for about 30 minutes
It is performed by stirring for about 6 hours. Then, for example, centrifugation (6000-8000 rpm, 10-30
For 1 minute) to collect the precipitate.

Anion exchanger treatment step In this step, the precipitate fraction obtained in the step of is dissolved in an aqueous solvent, or after the treatment of the step described below, contact treatment with an anion exchanger is performed to collect a non-adsorbed fraction. It is a process to do. This process is
This is done to remove IgM, IgG polymers.

(I) Preparation of anion exchanger The anion exchanger is one in which an anion exchange group is bound to an insoluble carrier. The anion exchange group is a diethylaminoethyl (DEAE) group or a quaternary aminoethyl group. (QA
The group E) and the like can be used as the insoluble carrier such as agarose, cellulose, dextran and polyacrylamide. The binding is performed by a known method.

(Ii) The precipitation fraction obtained in the step of the treatment method is dissolved in a suitable aqueous solvent. The aqueous solvent has a pH of 5.8 to 6.2 (preferably pH).
6.0) and an ionic strength of 0.0001 to 0.1M. A solute similar to that in the step of may be included. The protein concentration is 1 to 15 w / v% (particularly 3 to 10)
w / v%) is preferred. 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 a 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. The book process can be omitted if desired. Further, when the liquid heat treatment is performed, it is preferable to perform the anion exchanger treatment after the treatment of the immobilized human blood group substance.

Treatment with immobilized diamino compound In this step, the precipitate fraction obtained in step or the non-adsorbed fraction obtained in step is contact-treated with the immobilized diamino compound to recover the non-adsorbed fraction. It is a process to do. 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, aminobenzguanidine, 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 non-adsorbed fraction of the above step is adjusted to pH 5-8.
(In particular, pH 6 to 7), ionic strength 0.0001 to 0.
Contact treatment with the immobilized diamino compound is performed under the condition of 1 M (particularly 0.0001 to 0.01 M). At that time, protein concentration 1
It is preferably ˜15 w / v% (particularly 3 to 10 w / v%), and both batch method and 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. The book process can be omitted if desired.

Immobilized human blood group substance treatment step In this step, the precipitation fraction of step or the non-adsorbed fraction of or the non-adsorbed fraction of It is a process of collecting. 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 a 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 step, is adjusted to pH 5-8.
(Especially pH 6 to 7), ionic strength 0.0001 to 0.1
Contact treatment with the immobilized human blood group substance equilibrated with the above aqueous solvent is performed under the condition of M (particularly 0.0001 to 0.01 M). At that time, a protein concentration of 1 to 15 w / v% (especially 3 to
10 w / v%), and the batch method,
Either of the column methods 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. Even in the column method, the solution to be treated 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.

Heat treatment step This step minimizes the decrease in immunoglobulin antibody activity in the presence of a stabilizer at a desired stage, but completely eliminates contaminating viruses such as HB virus and AIDS virus. This is a step of heat treatment under a condition of activation.
The heat treatment is performed in a solution state, that is, an immunoglobulin aqueous solution state (that is, a liquid heat treatment). Suitable stabilizers include disaccharides (eg, saccharose, maltose), sugar alcohols (eg, sorbitol, mannitol), and more preferably sorbitol. A suitable amount of the stabilizer added is preferably 10 w / v% or more (preferably 10 to 50 w / v%) of disaccharides, sugar alcohols and the like.

The amount of immunoglobulin to be heated is
0.1 to 30 w / v% as protein (preferably 5 to 2)
It is preferably adjusted to 0 w / v%). The liquid heat treatment adjusts the pH of the aqueous solution to 4.5 to 6.5, preferably 5 to 5.
The temperature is adjusted to 6, and the treatment is performed at 50 to 70 ° C. (preferably about 60 ° C.) for 10 minutes to 20 hours (preferably about 10 hours). The ionic strength of the aqueous solution is 0.0
001-0.1M (particularly preferably 0.0001-0.
01M) is exemplified. The liquid heat treatment step is preferably performed on the starting material or after the step. In addition, when performing after the step of, it is more preferable to perform the treatments of and again from the viewpoint of removing impurities.

(Preparation of liquid preparation) The obtained immunoglobulin composition is adjusted to an aqueous solution by a conventional method so as to have a concentration of 1 to 10 w / v% (preferably 3 to 7 w / v%), and a stabilizer, for example, , Disaccharides (sucrose, maltose, etc.), sugar alcohols (sorbitol, mannitol, etc.), more preferably sorbitol 1-20 w / v%
(Preferably 2 to 10 w / v%), pH 5 to 6 (preferably pH 5.3 to 5.7, particularly preferably about 5.
5), low conductivity, preferably adjusted to 1 mmho or less (particularly 0.6 mmho or less, both converted to 8 ° C.) by means known per se, and then based on ordinary formulation technology, Perform sterilization filtration and dispensing. Thus, an intravenously administrable immunoglobulin preparation is prepared.

[0029]

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 according to the present invention has almost no immunoglobulin inactivated, does not contain contaminants such as anti-human blood group antibody, and is heat-treated so that contaminant viruses are also inactivated. It is a safe preparation that has properties such as good solubility and sufficiently low anti-complementary activity, and can pass the Japanese biologics standards (hereinafter referred to as raw standards) issued in 1985.

The preparation obtained by the present invention is dissolved in a suitable solvent (for example, distilled water for injection) at the time of use,
It is used for the prevention or treatment of infectious diseases by intravenous administration, infusion and the like. That is, the present invention is useful as an industrial method for producing an intravenous immunoglobulin preparation.

[0032]

EXAMPLES AND EXPERIMENTAL EXAMPLES Examples and experimental examples are given to explain the present invention in more detail, but the present invention is not limited to these.

(Experimental Example 1) Low-concentration PEG fractionation treatment at each pH In Example 1, the supernatant collection step by 8% PEG fractionation treatment was carried out at each pH, and the IgG yield, presence / absence of IgG polymer, Supernatant turbidity was measured. The IgG yield was calculated after measuring the amount of IgG by an immunoturbidimetric method (Dinaquant aIgG, manufactured by Boehringer Mannheim) using an anti-IgG antiserum. The IgG polymer is subjected to gel filtration by HPLC (carrier is TSKgel G3000SW _XL , manufactured by Tosoh Corporation, eluent is 50 mM acetate buffer containing 0.3% sodium chloride, pH.
6.7). The supernatant turbidity was visually observed.
The results are shown in Table 1.

[0034]

[Table 1]

When the pH is set to a low value (pH 5.1
~ 5.3, sample 2-4), the yield of IgG was increased compared to the conventional method (pH 5.0, sample 5), and the supernatant was clear, and IgG polymer was detected by HPLC analysis. Was not done. In Sample 1 in which the pH was adjusted to 5.0, the centrifugal supernatant became cloudy and the IgG polymer was
Detected by analysis.

(Experimental Example 2) DEAE treatment at each pH In Example 1, the pH at the time of DEAE treatment was compared between 5.5 (conventional method) and 6.0 (invention) to compare the IgG yield, The sedimentation line was measured by the immunoelectrophoresis method. Further, in the immunoelectrophoresis method, the presence of contaminant proteins other than IgG can be confirmed by the shade of the abnormal sedimentation line. The immunoelectrophoresis method was as follows.

Preparation of agar plate Agarose was dissolved in veronal buffer solution to a concentration of 1%, and 5.5 ml of the solution was dispensed into test tubes and poured on a glass plate (50 x 75 mm) on a horizontal table. It is. After the gel was solidified by leaving it at room temperature, a groove for pouring antiserum and a hole for inserting a sample were formed on an agar plate on a plan view prepared in advance. Preparation of samples and antisera Samples were prepared at 5% concentration (70A ₂₈₀ ). Bromphenol blue (hereinafter referred to as BPB) was added to plasma to be used as an index of migration distance. Electrophoresis The agar plate was placed in an electrophoresis tank filled with veronal buffer solution on both sides, and 5 μl of the sample was added to the sample hole, and then a bridge of the agar plate and the buffer solution was made using No. 2 filter paper. When electricity is applied at a rate of 1 to 2 mA per cm of agar plate and the position of albumin colored with BPB is 1.5 cm from the anode side (about 1 hour)
Electrophoresis was completed. Addition of antiserum Antiserum 60 was added to the groove previously prepared after electrophoresis.
μl was added. The next day, the sedimentation line was observed. The results are shown in Table 2.
The IgG yield was expressed with the amount of IgG before the DEAE treatment as 100%.

[0038]

[Table 2]

In sample 6 treated with pH 5.5 during DEAE treatment, an abnormal sedimentation line substance was detected.
As a result of confirmation by an electrophoresis method, samples 7 and 8 treated with pH 6.0 at the time of treatment were found to have the abnormal sedimentation line substances and transferrin removed to below the detection limit. Also, samples 7 and 8 treated at pH 6.0
The IgG yield of was improved compared to sample 9 (conventional method).

Example 1 1 kg of corn fraction II + III paste was suspended in 10 l of distilled water to adjust the pH to 5.5 and then centrifuged to collect the supernatant, and sorbitol was added to 100 ml of the supernatant. 50 g (final concentration 33 w / v%) was added, and the mixture was heated at 60 ° C. for 10 hours. After heating, pH
Was adjusted to 5.3, and PEG # 4000 was added to a final concentration of 8
%, 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 12%, and the mixture was centrifuged at 2 ° C. to precipitate the IgG fraction. Got After adjusting this solution to pH 6, DEAE-
Add Sephadex (1 ml per 50 ml solution), 0
Contact treatment under conditions of ~ 4 ° C for about 1 hour, centrifugation after treatment (7,000 rpm, about 20 minutes), supernatant (IgG solution)
Was recovered. The IgG solution was adjusted to a 5% IgG solution with distilled water, and the pH of the solution was adjusted to about 5.5 with sodium acetate.
Further, sorbitol was added to a final concentration of 5%. This aqueous solution (conductivity of about 1 mmho) was sterilized and filtered to obtain an immunoglobulin liquid preparation for intravenous injection.

This preparation contained substantially only IgG monomer and had an anti-complementary value of about 10 to 15 CH ₅₀ , and passed the raw standard as an intravenous globulin.

Claims (2)

[Claims] 1. A method for producing an immunoglobulin preparation for intravenous injection, which comprises a process comprising the steps of starting from a fraction containing immunoglobulin as a starting material: (a) inactivating contaminating viruses Under sufficient conditions, the liquid is heat-treated at pH 4.5 to 6.5 and in the presence of a stabilizer. (B) pH 5.1 to 5.4, ionic strength 0.0001
~ 0.1M, temperature 0 ~ 4 ℃, molecular weight 1,000
~ 10,000 polyethylene glycol 6-10w /
Treat with v% and collect the supernatant. (C) The supernatant of (b) is adjusted to pH 7 to 10, ionic strength 0.0
001-0.1M, temperature 0-4 ℃, molecular weight 1,
000 to 10,000 polyethylene glycol 10
Treat with 15 w / v% to recover the precipitate. 2. The precipitate obtained in step (c) is dissolved in an aqueous solvent to have a pH of 5.8 to 6.2 and an ionic strength of 0.0001 to
The method for producing an immunoglobulin preparation for intravenous injection according to claim 1, comprising a step of recovering a non-adsorbed fraction by treating with an anion exchanger under a condition of 0.1M.