JPH10265407A - Immunoglobulin preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject preparation improved in storage stability by reducing an fine amount of serum albumin contaminating an immunoglobulin fraction. SOLUTION: This preparation reduced in the contamination amount of serum albumin to <=10 μg per 50 mg of immunoglobulin is obtained by preferably dissolving a human immunoglobulin fraction in a proper aqueous solvent (a pH of 4-7 and a low ion strength of 0.0001-0.1 M), subjecting the solution to a contact treatment with an anion exchanger (DEAE groups, etc., bound to an inactive carrier) or an adsorbent (colloidal silicate silica) equilibrated with the aqueous solvent, and subsequently recovering the non-adsorbed fraction. The preparation does not produce inactive foreign matters, even after stored in the solution state at 37 deg.C for 39 days. The preparation is usually intravenously continuously administered at a daily dose of 50-100 mg (as the immunoglobulin) per kg of a body weight for one to several days.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunoglobulin preparation, and more particularly, to an immunoglobulin preparation having excellent storage stability.

[0002]

2. Description of the Related Art Among γ-globulins which are plasma protein components, immunoglobulin preparations containing IgG as a main component, in particular,
Until now, it has been widely used for prevention and treatment of various infectious diseases. By the way, immunoglobulins are unstable in a solution state, and the immunoglobulins called anti-complement activity, as a result of the aggregation of immunoglobulins, that is, the formation of polymers and dimers, especially as a result of denaturation that occurs during the fractionation operation It has been known that the complement fixation ability of the compound increases remarkably, and when administered intravenously to the human body, the serum complement concentration is decreased or serious side effects such as anaphylactic shock are caused. Therefore, immunoglobulins are not liquid preparations, but dry preparations,
In particular, it was formulated in a lyophilized form. However, there is a problem that the dry preparation cannot be easily administered because it has to be dissolved in distilled water for injection or the like at the time of use.

[0003] On the other hand, liquid preparations have the advantage that they do not require the dissolution of distilled water for injection and can be easily administered as compared with dry preparations, but they have the disadvantage that immunoglobulins are inferior in stability as described above. there were. Therefore, development of a liquid composition of intravenous globulin that is stable even in a solution state has been attempted.

For example, Japanese Patent Application Laid-Open No. 63-192724 discloses an intravenous injection which is stable even in a solution state by a combination of low electric conductivity, pH 5.5 ± 0.2 and use of sorbitol as a stabilizer. Liquid compositions of globulin have been proposed. JP-A-58-43914 discloses an immunoglobulin composition containing substantially no immunoglobulin aggregates and having an immune serum globulin monomer concentration of more than about 90%. The ionic strength of the immune serum globulin solution to less than about 0.001,
It is disclosed that the pH is adjusted to 3.5 to 5.0.

Japanese Patent Application Laid-Open No. 63-8340 discloses that immune serum globulin is prepared from a human plasma source by using a cold ethanol fractionation method at a pH equal to or less than about 5.4. The immune serum globulin is then stored at a pH equal to or less than about 4.25 for at least about 3 days to be substantially free of infectious retrovirus, or about 6.8.
A method for the production of immune serum globulin substantially free of acquired virus comprising the step of storing at a temperature of at least 45 ° C. at a pH equal to or less than However, this invention aims at inactivating a retrovirus, and there is no report that the immunoglobulin aggregation was improved in the prepared immunoglobulin preparation.

[0006] Japanese Patent Application Laid-Open No. 7-238036 discloses that
In order to improve the stability, it is disclosed that the acid treatment or storage at room temperature suppresses the aggregation of immunoglobulins, that is, the increase of not only the polymer but also the dimer.

[0007]

As described above, since immunoglobulins are inherently unstable proteins, they are one of the issues most concerned about the stability when a liquid composition is prepared. The present invention solves such a problem, and an object thereof is to provide an immunoglobulin preparation having good storage stability even in a solution state.

[0008] The present inventors have further studied in consideration of such circumstances, and as a result, by further reducing the amount of serum albumin contaminating trace amounts in the immunoglobulin fraction, the storage stability of the immunoglobulin was reduced. Was found to be further improved, and the present invention was completed.

[0009]

SUMMARY OF THE INVENTION The present invention is as follows. (1) An immunoglobulin preparation, wherein the amount of serum albumin contaminant is 10 μg or less per 50 mg of immunoglobulin. (2) An immunoglobulin preparation which does not generate insoluble foreign matter after storage in a solution state at 37 ° C. for 39 days.

[0010]

Next, the present invention will be described in detail. The mode of the immunoglobulin used in the immunoglobulin preparation of the present invention is not particularly limited, but those prepared by a known method can be used.
For example, a paste prepared from a fraction containing immunoglobulin as a starting material, specifically, fraction II + III, fraction II obtained by corn ethanol fractionation, or a paste of a fraction equivalent thereto containing immunoglobulin And the like. A method in which these fractions are suspended in an aqueous solvent to extract immunoglobulins is exemplified. At this time, an aqueous medium having at least twice the volume, preferably at least 5 volumes of the fraction is used. As the aqueous medium used here, water or an aqueous solution containing sodium chloride, hydrochloric acid, acetic acid, phosphoric acid or a salt thereof is preferable.
Is preferably 4 to 7, and the ionic strength is preferably 0.001 to 0.1M.

The immunoglobulin may be a non-chemically modified type such as a polyethylene glycol-treated type, a pH 4 type, or an ion-exchange resin-treated type; a chemically modified type such as an alkylated type or a sulfonated type; plasmin, pepsin, trypsin, etc. Enzyme-treated immunoglobulins treated with enzymes may be used, and among them, non-chemically modified immunoglobulins are preferably used.

In the present invention, the non-chemically modified immunoglobulin has the following properties. (1) No modification or change has been received as it is, and therefore, immunoglobulin fragments Fab and F (ab ')
₂ Does not include Fc. (2) No decrease in antibody titer and no decrease in antibody spectrum. (3) The anti-complement effect (complement fixability) is sufficiently lower than 20 units (CH50 value) regarded as safe by Japanese standards for biological preparations.

Such a non-chemically modified immunoglobulin may be obtained by any method as long as it is in a natural state and has a low anti-complement titer.

The method of preparing the non-chemically modified immunoglobulin may be performed by a known method. For example, ethanol fractionation, polyethylene glycol fractionation (JP-A-53-475)
No. 15), fractionation by a combination of polyethylene glycol and hydroxyethyl starch (JP-A-51-9).
1321), acid treatment (JP-A-57-32228), anion-exchanger treatment (JP-T-59-50154).
No. 6, JP-A-60-42336), a combination of heat treatment and polyethylene glycol fractionation (JP-A-63-432).
No. 183539), liquid formulation (JP-A-58-43)
914, JP-A-63-197274) and the like.

It is preferable to subject the immunoglobulin obtained by the above method to a heat treatment. For example, as a liquid heating method, a method using a high concentration of sugar or sugar alcohol as a stabilizer (Japanese Patent Application Laid-Open No. 61-191622), a method using a high concentration of sorbitol as a stabilizer, low ionic strength, and acidic pH (JP-A-63-146)
832) and the like. As the drying and heating method, a method using glycine, polyethylene glycol, sodium chloride, mannitol or the like as a stabilizer (JP-A-61-78730), a method using a disaccharide or a sugar alcohol as a stabilizer (particularly, 62-228
024, JP-A-63-283933) and the like.

The origin of the immunoglobulin used in the present invention is not particularly limited, and specific examples include human, mouse, rat and the like, and human is preferable. Specific examples of such immunoglobulins include, for example, polyethylene glycol-treated human immunoglobulins.

The immunoglobulin preparation of the present invention is one from which serum albumin has been removed to some extent, and has a small amount of impurities. Examples of the method for removing serum albumin include the following contact treatment methods using an anion exchanger and contact treatment methods using colloidal silicic acid.

A) Treatment method using an anion exchanger This is a method in which a non-adsorbed fraction is recovered by contact treatment with an anion exchanger. (i) Preparation of anion exchanger The anion exchanger is obtained by bonding an anion exchange group to an inert carrier. Examples of the anion exchange group include a diethylaminoethyl (DEAE) group and a quaternary aminoethyl (QA) group.
E) groups and the like, and agarose, cellulose, dextran, polyacrylamide and the like can be used as the inert carrier. The coupling is performed by a known method.

(Ii) Treatment method The immunoglobulin fraction is dissolved in a suitable aqueous solvent. The aqueous medium preferably has pH 4 to 7 (particularly pH 5 to 6) and low ionic strength (particularly 0.0001 to 0.1 M). Examples of such an aqueous medium include an aqueous sodium chloride solution, distilled water for injection, and an acetate buffer. The prepared immunoglobulin solution has a protein concentration of 1 to 15 watts.
/ V% (particularly 3 to 10 w / v%), pH 4 to 7 (particularly p
H5-6).

Next, this immunoglobulin solution is contacted with an anion exchanger equilibrated with the above aqueous medium.
In the treatment, either a batch method or a column method may be used. For example, in the batch method, an anion exchanger 1 m
After mixing with about 10 to 100 ml of the immunoglobulin solution per 1 and stirring at 0 to 4 ° C. for about 30 minutes to 2 hours,
The supernatant is collected by centrifugation (6000 to 8000 rpm, 10 to 30 minutes). In the column method, 10 to 100 ml of the immunoglobulin solution per 1 ml of the anion exchanger
And then contact non-adsorbed fraction.

B) Method by colloidal silicic acid treatment This is a method of contact-treating with colloidal silicic acid to collect a non-adsorbed fraction. (i) Adsorbent Examples of the colloidal silicic acid used as the adsorbent include silica gel, light silicic anhydride, diatomaceous earth, acid clay, bentonite, kaolin, magnesium aluminate silicate, and the like. Preferably, light anhydrous silicic acid (trade name Aerosil (trade name, manufactured by Nippon Aerosil Co., Ltd.), trade name Deripit (trade name, manufactured by Zeta), etc.) is used.

(Ii) Treatment conditions The purified immunoglobulin is dissolved in a suitable aqueous solvent. The aqueous medium preferably has pH 4 to 7 (particularly pH 5 to 6) and low ionic strength (particularly 0.0001 to 0.1 M). Examples of such an aqueous medium include the same aqueous medium as described for the anion exchanger. The prepared immunoglobulin solution has a protein concentration of 1 to 15 watts.
/ V% (particularly 3 to 10 w / v%), pH 4 to 7 (particularly p
H5-6).

The immunoglobulin solution may be a pharmacologically acceptable additive (eg, a carrier, an excipient, a diluent, etc.) usually used for pharmaceuticals, within a range not inconsistent with the object of the present invention.
It may contain stabilizers or pharmaceutically necessary ingredients.

Examples of the stabilizer include monosaccharides such as glucose, disaccharides such as saccharose and maltose, sugar alcohols such as mannitol and sorbitol, neutral salts such as sodium chloride, amino acids such as glycine, polyethylene glycol and polyoxyethylene. -Nonionic surfactants such as polyoxypropylene copolymer (pluronic) and polyoxyethylene sorbitan fatty acid ester (Tween) are exemplified, and it is preferable that about 1 to 10 w / v% is added.

Next, the immunoglobulin solution is contacted with the above adsorbent. The contact conditions include an immunoglobulin concentration of 1 to 100 g / liter (preferably 10 to 1 g / l).
The amount of the adsorbent is 1 to 30 g / liter with respect to (00 g / liter). This treatment can be performed by either a batch method or a column method, but preferably the batch method is used. Conditions for the batch method include, for example, 5 to 25 ° C., 5
Mix and stir for about 1 minute to 1 hour. Thereafter, the supernatant (non-adsorbed fraction) is collected by filtration or centrifugation.

As described above, in the immunoglobulin preparation from which serum albumin has been removed, the amount of serum albumin contaminated per 50 mg of immunoglobulin is 10 μg or less, preferably 5 μg or less.
μg or less, for example, in the case of a 5 w / v% immunoglobulin solution, the amount of serum albumin contaminants is 10 μg / ml or less, preferably 5 μg / ml or less.
It has properties of not more than μg / ml. The immunoglobulin preparations having such properties have better storage stability than conventional ones. For example, even when stored at 37 ° C. for 39 days, the generation of insoluble foreign substances is extremely small. In addition, as a method for quantifying serum albumin in an immunoglobulin preparation, a known method can be used, and for example, an ELISA method, a Mancini method, a turbidimetric method, or the like can be employed.

When the immunoglobulin preparation of the present invention is in the form of a liquid preparation, it can be used as it is or in a suitable solvent (for example,
Diluted with distilled water for injection, physiological saline, glucose solution, etc.), and in the case of a dry preparation, after freeze-drying the immunoglobulin solution, use an appropriate solvent (eg, distilled water for injection, etc.) at the time of use. ) To be used after dissolving.

The route of administration of the immunoglobulin preparation of the present invention is usually injection, particularly preferably intravenous administration. The dosage of the immunoglobulin preparation of the present invention is 50 to 1000 mg / kg of immunoglobulin per kg of body weight,
It is standard to administer intravenously every day for several days, but the dose may be increased or decreased according to symptoms, sex, body weight and the like.

[0029]

EXAMPLES The present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto. Example 1 Fraction II + III obtained by Mr. Kohn's cold alcohol fractionation method
Was dissolved in 10 liters of 0.6% sodium chloride, adjusted to pH 3.8 with 1N hydrochloric acid, and stirred at 4 ° C. for 60 minutes to perform an acid treatment. While adding and dissolving 500 g of polyethylene glycol having an average molecular weight of 4,000 to this solution, 1N-sodium hydroxide was added thereto to gradually raise the pH, and finally adjusted to pH 5.0. Immediately thereafter, a clear supernatant was obtained by removing the precipitate by centrifugation. 700 g of polyethylene glycol having an average molecular weight of 4000 was added to the supernatant, adjusted to pH 8.0 with 1N-sodium hydroxide with gentle stirring, and the precipitated immunoglobulin was collected by centrifugation. The immunoglobulin was dissolved, for example, in saline or a solution of 0.6% sodium chloride and 2% mannitol in a 0.02 M acetate buffer. Immunoglobulin 5w / v
% Solution was passed through a filter (manufactured by Zeta Delipit Cuno) carrying anhydrous silica to collect a non-adsorbed fraction. Furthermore, by performing sterilization filtration and freeze-drying, an intravenous immunoglobulin preparation that can be used clinically was obtained. The amount of albumin contaminating in the 5 w / v% solution of the immunoglobulin preparation thus obtained was 10 μg / ml or less as determined by the Mancini method.

Example 2 Corn fraction obtained from human plasma by the cold ethanol method
10 liters of water was added to 1 kg of II + III to extract IgG, 50 g of sorbitol was added per 100 ml of the supernatant, the pH was adjusted to 5.5, and the mixture was heated at 60 ° C. for 10 hours. After adjusting the pH to 5.5, the solution was diluted three times with cold water for injection, and polyethylene glycol (average molecular weight: 4000) was added to a final concentration of 8%, followed by centrifugation at 2 ° C. The supernatant was obtained. This supernatant is
After adjusting the pH to 8.8 by adding N-sodium hydroxide, polyethylene glycol (average molecular weight: 4000) was added to a final concentration of 12%, and centrifuged at 2 ° C. to precipitate the precipitated IgG fraction. I got This IgG fraction was dissolved in water for injection, and D was equilibrated with water for injection.
Add EAE-Sephadex (2 per 50 ml of solution)
ml) and subjected to a contact treatment at 0 to 4 ° C. for about 1 hour, followed by filtration to remove DEAE-Sephadex and collect a filtrate (IgG solution). The IgG solution was adjusted to a 5 w / v% IgG solution with water for injection, the pH of the solution was adjusted to about 5.5 with sodium acetate, and sorbitol was added to a final concentration of 5%. This aqueous solution (conductivity: about 1 mmho) was passed through a filter carrying anhydrous silica in the same manner as in Example 1 to collect a non-adsorbed fraction. The solution was further sterilized and filtered to obtain a liquid immunoglobulin preparation for intravenous injection. 5 w / v% Ig thus obtained
Albumin contamination in the G solution was determined by the Mancini method and found to be 5 μg / ml.

EXPERIMENTAL EXAMPLE 1 The immunoglobulin preparation (5 w / v% solution) of the present invention was treated with colloidal silicic acid and was not treated with serum albumin in the immunoglobulin solution. The degree of formation of insoluble foreign matter after storage at 37 ° C. for 39 days was compared. The treatment with colloidal silicic acid was in accordance with the method described in Examples. Serum albumin was quantified by the Mancini method. The insoluble foreign matter was visually observed. Table 1 shows the results.

[0032]

[Table 1]

From the above results, it can be seen that the immunoglobulin preparation treated with colloidal silicic acid has an extremely small amount of serum albumin, and no insoluble foreign matter is observed even after storage at 37 ° C. for 39 days.

[0034]

As is apparent from the above description, the immunoglobulin preparation of the present invention has a small amount of contaminating serum albumin, and therefore has less storage of insoluble foreign substances even in a solution state than conventional ones. Good stability is obtained.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Yahiro Uemura 2-25-1, Shodai Otani, Hirakata-shi, Osaka Inside Midori Cross Research Institute, Ltd.

Claims (2)

[Claims] 1. An immunoglobulin preparation characterized in that the amount of serum albumin contaminant is 10 μg or less per 50 mg of immunoglobulin. 2. An immunoglobulin preparation which does not generate insoluble foreign matter after storage in a solution state at 37 ° C. for 39 days.