JP2005325132A - Method for producing immunoglobulin preparation for intravenous injection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the efficient production of an immunoglobulin preparation for intravenous injection by inactivating miscellaneous foreign viruses and having extremely low miscellaneous foreign protein content, and high safety and effectiveness. <P>SOLUTION: This immunoglobulin preparation for intravenous injection is produced by using a fraction containing an immunoglobulin as a starting raw material and subjecting the raw material at least to the steps (a), (c) and (d) in this order. (a) A step to treat the material with an anion exchanging material at pH 5-7 and an ionic strength of 0.0001-0.1M and recover a non-adsorbed fraction; (b) a step to inactivate the miscellaneous foreign viruses by heat-treatment; (c) a step to treat the product with a polyethylene glycol having a molecular weight of 1,000-10,000 and a concentration of 4-12 w/v% under a condition of pH 4-6, an ionic strength of 0.0001-0.1M and at a temperature of 0-4°C and recover the supernatant liquid; and (d) a step to treat the supernatant liquid with a polyethylene glycol having a molecular weight of 1,000-10,000 and a concentration of 10-15 w/v% under a condition of pH 7-9, an ionic strength of 0.0001-0.1M and at a temperature of 0-4°C and recover the precipitate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

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

  Among the immunoglobulins that are plasma protein components, in particular, immunoglobulin preparations mainly composed of IgG have been widely used for the prevention and treatment of infectious diseases.

The mixing of contaminating viruses such as hepatitis virus in this immunoglobulin preparation is not necessarily denied. Therefore, a liquid heat treatment method (Patent Document 1) or a dry heat treatment method (Patent Documents 2 and 3) has been proposed as a method for inactivating contaminated viruses.
JP 61-191622 A JP-A-61-78730 Japanese Patent Application No. 60-270195

  An object of the present invention is to further develop the above-mentioned heat treatment, and to improve yield, improve productivity, further remove contaminating proteins, etc. in the production of intravenous immunoglobulin preparations. That is, an object of the present invention is to provide an efficient method for producing a pharmaceutical preparation that can inactivate contaminating virus, has extremely few contaminating proteins, and can be applied clinically, that is, has higher safety and effectiveness. It is to provide.

  The inventors of the present invention examined an industrial production method of an intravenous immunoglobulin preparation along this purpose, and combined polyethylene glycol (hereinafter referred to as PEG) fractionation treatment, heat treatment and anion exchanger treatment, The processing conditions of the process were set and the present invention was completed.

  The gist of the present invention is as described in the claims, and particularly relates to a method for producing an intravenous immunoglobulin preparation including the following treatment using a fraction containing immunoglobulin as a starting material.

(A) A non-adsorbed fraction is collected by treatment with an anion exchanger under conditions of pH 5 to 7 and ionic strength 0.0001 to 0.1M.
(B) Heat treatment under conditions sufficient to inactivate contaminating viruses.
(C) Treatment with PEG 4-12 w / v% with a molecular weight of 1,000-10,000 under conditions of pH 4-6, ionic strength 0.0001-0.1 M, temperature 0-4 ° C. and recovering the supernatant .
(D) The supernatant of (c) was treated with 10 to 15 w / v% of PEG having a molecular weight of 1,000 to 10,000 under conditions of pH 7 to 9, ionic strength 0.0001 to 0.1 M, temperature 0 to 4 ° C. And collect the precipitate.

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

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

I. Anion exchanger treatment step This step is a step of recovering the non-adsorbed fraction by contact treatment with an anion exchanger. 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. As the anion exchange group, a diethylaminoethyl (DEAE) group, a quaternary aminoethyl (QAE) are used. ) Group and the like, and agarose, cellulose, dextran, polyacrylamide, etc. can be used as an insoluble carrier. The coupling | bonding is performed by a well-known method.

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

  For example, in the batch method, 1 ml of 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. Also in the column method, about 10 to 100 ml of the solution to be treated is brought into contact with 1 ml of the anion exchanger, and the non-adsorbed fraction is collected.

II. Heat treatment step This step is a step of heat treatment under conditions in which the decrease in antibody activity of immunoglobulin is minimized, but contaminating viruses such as HB virus and AIDS virus are completely inactivated.

  The heat treatment is performed in a dry state (ie, dry heat treatment) with a moisture content of 3% or less, or in a solution state, that is, in an immunoglobulin aqueous solution state (ie, liquid heat treatment). . Further, this heat treatment is preferably carried out in the presence of a stabilizer, and as this stabilizer, in both cases of the dry heat treatment and the liquid heat treatment, disaccharides (eg, saccharose, maltose), sugar alcohols are used. (Examples include sorbitol and mannitol), and sorbitol is particularly preferable. The amount of stabilizer added is 0.5 to 5 w / v% (preferably 1 to 3 w / v%) for disaccharides and sugar alcohols in dry heat treatment, and disaccharide and sugar alcohol for liquid heat treatment. The use of 10 w / v% or more (preferably 20 to 40 w / v%) is preferably exemplified.

  The amount of immunoglobulin to be heated is preferably adjusted to 1 to 10 w / v% (preferably 3 to 7 w / v%) as the amount of protein in the dry heat treatment. In the liquid heat treatment, the protein amount is preferably adjusted to 0.1 to 30 w / v% (preferably 5 to 20 w / v%). In the case of a dry heat treatment, the heat treatment is preferably performed after sterilization filtration as necessary after adding a stabilizer, and the water content is 3% or less, preferably 1% or less, for example, by freeze drying. The lyophilization condition is exemplified by about 24 to 96 hours at 20 to 40 ° C. under a vacuum of 0.5 mmHg. Next, for example, the treatment is performed at 50 to 80 ° C. (preferably about 60 ° C.) for 1 to 200 hours (preferably about 10 to 100 hours). In addition, the heat treatment process can be performed in an inert gas atmosphere to further improve the stability during heating. Examples of the inert gas include nitrogen gas, argon, helium and the like. In the case of liquid heat treatment, the pH of the aqueous solution is adjusted to 4.5 to 6.5, preferably pH 5 to 6, for example, 50 to 80 ° C. (preferably about 60 ° C.) for 10 minutes to 20 hours (preferably 10 Processed about hours). Examples of the ionic strength of the aqueous solution include 0.0001 to 0.1M (particularly preferably 0.0001 to 0.01M).

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

  The object to be treated is treated with PEG having a molecular weight of 1,000 to 10,000 (preferably about 2,000 to 6,000) (for example, both are mixed). The treatment conditions are PEG concentration 4 to 12 w / v% (especially 8 to 12 w / v%), pH 4 to 6 (especially 4.5 to 5.5), ionic strength 0.0001 to 0.1 M (especially 0.8. 0001-0.01M). At this time, the protein concentration is preferably 1 to 20 w / v% (particularly 5 to 15 w / v%). The said process is performed by stirring at about 0-4 degreeC normally for about 30 minutes-6 hours. Thereafter, for example, the supernatant is recovered by centrifugation (6000 to 8000 rpm, 10 to 30 minutes).

IV. High concentration polyethylene glycol (PEG) treatment step This step is a step in which the supernatant obtained in step III is treated with high concentration PEG to recover the precipitate.

  The supernatant is further treated with PEG having a molecular weight of 1,000 to 10,000 (preferably 2,000 to 6,000) (for example, both are mixed). The treatment conditions were PEG concentration of 10-15 w / v% (particularly 11-13 w / v%), pH 7-9 (particularly 7.5-8.5), ionic strength 0.0001-0.1 M (particularly 0 .0001-0.01M). At this time, the protein concentration is preferably 1 to 20 w / v% (particularly 5 to 15 w / v%). The said process is performed by stirring at about 0-4 degreeC normally for about 30 minutes-6 hours. Then, for example, the precipitate is collected by centrifugation (6000 to 8000 rpm, 10 to 30 minutes).

  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.

V. Treatment with Immobilized Diamino Compound This step is a step of collecting a non-adsorbed fraction by contact treatment with an immobilized diamino compound. This step is performed to remove prekallikrein or kallikrein.

(I) Preparation of immobilized diamino compound The immobilized diamino compound is obtained by immobilizing a diamino compound 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 and the like are used. Immobilization may be in accordance with a known method. For example, diamino compounds can be immobilized by, for example, CNBr activation method for agarose, cellulose and the like, and oxirane method for silica gel, glass and the like.

(Ii) Treatment method The precipitate to be treated, for example, the precipitate fraction obtained in the above step IV, has a pH of 5 to 8 (particularly pH 6 to 7) and an ionic strength of 0.01 to 0.2 M (particularly 0.05 to 0.00). 15M) is contacted with the immobilized diamino compound. At that time, 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.

  For example, in the batch method, about 10 to 100 ml of the above fraction is mixed with 1 ml of the immobilized diamino compound, and 0 to 10 ° C., preferably 0 to 4 ° C. for 30 minutes to 4 hours, preferably about 30 minutes to 2 hours. After stirring, the supernatant is recovered by centrifugation (6,000-8,000 rpm, 10-30 minutes). Also in the column method, about 10 to 100 ml of the fraction is brought into contact with 1 ml of the immobilized diamino compound, and the non-adsorbed fraction is collected.

VI. Immobilized human blood group substance treatment process This process is a process for 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 a human blood group substance on an insoluble carrier. The human blood group substance can be prepared by a known method. For example, human A, B, AB or O type red blood cells are hemolyzed or sonicated in a hypotonic solution and then purified by an ammonium sulfate fractionation method or a PEG fractionation method. Synthetic antigens (sugar chains) can also be used as human blood group substances (see Human Blood Groups and Carbohydrate Chemistry (1978) Chem. Soc. Rev. p423-452).

  Furthermore, this human blood group substance is effective for inactivating the virus which is contaminated after being dissolved in physiological saline, for example, about 50 to 70 ° C., preferably about 60 ° C. Is heated at about 80-130 ° C, preferably 95-121 ° C for about 1-40 minutes, preferably about 2-30 minutes. Thereafter, the 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 and the like are used. Immobilization may be in accordance with a known method. For example, human blood group substances can be immobilized by CNBr activation method for agarose, cellulose and the like, and oxirane method for silica gel, glass and the like.

(Ii) Treatment method The treatment object, for example, the non-adsorbed fraction of the step V is pH 5-8 (particularly pH 6-7) and ionic strength 0.01-0.2M (particularly 0.05-0.15M). A contact treatment is then performed with the immobilized human blood group substance equilibrated with the aqueous solvent. At that time, the protein concentration is preferably 1 to 15 w / v% (particularly 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 0 to 10 ° C., preferably 0 to 4 ° C., for 30 minutes to 4 hours, preferably 30 minutes. After stirring for about 2 hours, the supernatant is collected by centrifugation (6,000-8,000 rpm, 10-30 minutes). Also in the column method, about 10 to 100 ml of the solution to be treated is brought into contact with 1 ml of the immobilized human blood group substance, and the non-adsorbed fraction is collected.

(Preparation of liquid formulation)
The obtained immunoglobulin composition is dissolved in a suitable solvent (for example, distilled water for injection) so as to be 1 to 10 w / v% (preferably 3 to 7 w / v%), and further a stabilizer, for example, Disaccharides (sucrose, maltose, etc.), sugar alcohols (sorbitol, mannitol, etc.), particularly preferably sorbitol 1-20 w / v% (preferably 2-10 w / v%), pH 5-6 (preferably pH of 5.3 to 5.7, particularly preferably about 5.5), low conductivity, preferably 1 mmho or less (especially 0.6 mmho or less, both converted to 8 ° C.) in a means known per se. After adjustment, sterilization filtration, dispensing, etc. are performed based on normal formulation technology. Thus, an intravenously administrable immunoglobulin preparation is prepared.

  In the present invention, since an intravenous globulin preparation was produced by the method as described above, no IgG polymer was detected in the obtained preparation, and the yield of IgG was improved during the production process.

  In the preparation obtained by the present invention, the immunoglobulin is hardly inactivated, and since the heat treatment is performed, the contaminating virus is also inactivated, the solubility is good, and the anti-complement activity is sufficiently low, Further, in a preferred embodiment, it has the property that it does not contain impurities such as prekallikrein, kallikrein, and anti-human blood group substance antibody. It is a safe formulation that can be passed.

  In addition, the preparation obtained by the present invention 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.

  Examples will be given to describe the present invention in more detail, but the present invention is not limited to these examples.

Example 1
After adding 3 liters of water to 1 kg of corn fraction II + III and extracting at pH 4, DEAE-Sephadex was added to this solution (1 ml per 50 ml solution), and contact treatment was carried out under conditions of pH 6.0 and 4 ° C. The supernatant (IgG solution) was recovered by centrifugation (7000 rpm, about 20 minutes). Further, 50 g of sorbitol was added to 100 ml to adjust the pH to 5.5, followed by heat treatment at 60 ° C. for 10 hours. After the 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 resulting supernatant was adjusted to pH 8.8 using 1N sodium hydroxide, PEG # 4000 was added to a final concentration of 15 w / v%, and centrifuged at 2 ° C., and IgG was added to the precipitate fraction. A fraction was obtained. The IgG fraction was dialyzed against distilled water, 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 intravenous immunoglobulin liquid preparation.

Example 2
After adding 3 liters of water to 1 kg of corn fraction II + III and extracting at pH 4, DEAE-Sephadex was added to this solution (1 ml per 20 ml solution), and contact treatment was carried out under conditions of pH 6.0 and 4 ° C. The supernatant (IgG solution) was recovered by centrifugation (7000 rpm, about 20 minutes). Further, 50 g of sorbitol was added to 100 ml and the pH was adjusted to 5.5, followed by heat treatment at 60 ° C. for 10 hours. After the heating, the pH was adjusted to 5.3 to 5.4, PEG # 4000 was added so that the final concentration was 12 w / v%, and the mixture was centrifuged at 2 ° C. The resulting supernatant was adjusted to pH 8.8 using 1N sodium hydroxide, PEG # 4000 was added to a final concentration of 15 w / v%, and centrifuged at 2 ° C., and IgG was added to the precipitate fraction. A fraction was obtained. Dissolve this IgG fraction in distilled water, add DEAE-Sephadex (1 ml per 33 ml solution), contact treatment under conditions of pH 6.0 and 4 ° C., and centrifuge after treatment (7000 rpm, about 20 minutes) The supernatant (IgG solution) was recovered. The IgG fraction was dialyzed against distilled water, 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 intravenous immunoglobulin liquid preparation.

The preparations obtained in Examples 1 and 2 substantially contain only an IgG monomer, have an anti-complement value of about 10 to 15 CH ₅₀ , and passed the raw standard as an intravenous globulin.

Experimental example 1
In Example 1 above, the method in which the pretreatment with the anion exchanger is not performed before the heat treatment is the conventional method 1 (the method described in JP-A-63-183539), and the globulin in the preparation obtained thereby Was quantified by immunoturbidimetry to determine the recovery rate of globulin. Similarly, the recovery rate of globulin in Example 1 was determined. As a result, when the recovery rate in the conventional method 1 was 100%, the recovery rate in Example 1 was 146%.

Experimental example 2
In Example 2 above, the method in which the pretreatment with the anion exchanger is not performed before the heat treatment is the conventional method 2 (the method described in JP-A-63-183539), and the globulin in the preparation obtained thereby Was quantified by immunoturbidimetry to determine the recovery rate of globulin. Similarly, the recovery rate of globulin in Example 2 was determined. As a result, when the recovery rate in the conventional method 2 was 100%, the recovery rate in Example 2 was 154%.

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

Claims (2)

Intravenous immunoglobulin preparation characterized by comprising the following steps starting from a fraction containing immunoglobulin, wherein at least the steps (a), (c) and (d) are processed in this order: Manufacturing method:
(A) A non-adsorbed fraction is collected by treatment with an anion exchanger under conditions of pH 5 to 7 and ionic strength 0.0001 to 0.1M.
(B) Heat treatment under conditions sufficient to inactivate contaminating viruses.
(C) Under the conditions of pH 4-6, ionic strength 0.0001-0.1 M, temperature 0-4 ° C., treatment with polyethylene glycol 4-12 w / v% with a molecular weight of 1,000-10,000, and the supernatant to recover.
(D) The supernatant of (c) is 10 to 15 w / v% polyethylene glycol having a molecular weight of 1,000 to 10,000 under the conditions of pH 7 to 9, ionic strength 0.0001 to 0.1 M, temperature 0 to 4 ° C. To recover the precipitate. Intravenous immunoglobulin characterized by comprising the following steps starting from a fraction containing immunoglobulin and further performing treatment in the order of (a), (b), (c) and (d) Preparation method of the preparation:
(A) A non-adsorbed fraction is collected by treatment with an anion exchanger under conditions of pH 5 to 7 and ionic strength 0.0001 to 0.1M.
(B) Heat treatment under conditions sufficient to inactivate contaminating viruses.
(C) Under the conditions of pH 4-6, ionic strength 0.0001-0.1 M, temperature 0-4 ° C., treatment with polyethylene glycol 4-12 w / v% with a molecular weight of 1,000-10,000, and the supernatant to recover.
(D) The supernatant of (c) is 10 to 15 w / v% polyethylene glycol having a molecular weight of 1,000 to 10,000 under the conditions of pH 7 to 9, ionic strength 0.0001 to 0.1 M, temperature 0 to 4 ° C. To recover the precipitate.