JPH11349490A - Albumin preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an albumin preparation having excellent safety, stability, etc., useful for treating hypoalbuminemia, etc., by heat treatment and by extremely reducing an aggregate content and a content of admixture protein such as transferrin, etc. SOLUTION: This albumin preparation is heat-treated and has an aggregate content equal to or lower than a limit of detection measured by a gel filtration analysis method in making 25 (w/v)% albumin solution, <=2 mg/dl transferrin content measured by Mancini method, <=4 mg/dl α1 -acidic glycoprotein content measured by Mancini method and <=6.5 mg/dl haptoglobin content measured by Mancini method. The preparation is obtained by treating a serum albumin- containing aqueous solution with an anion exchanger under conditions of preferably pH 5.1-5.25, with a cation exchanger under conditions of preferably pH 4-8 and then heat-treating the aqueous solution preferably at about 60 deg.C for about 10 hours.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an albumin preparation and a method for producing the same. More specifically, the present invention relates to a serum albumin preparation with reduced aggregate content, contaminant protein content, and the like, and a method for producing the same.

[0002]

2. Description of the Related Art Serum albumin is the most abundant protein in plasma, and has functions such as maintaining osmotic pressure in blood and transporting nutrients and metabolites by binding them. Plays. The above-mentioned preparations containing serum albumin have been used for treatment of hypoalbuminemia, hemorrhagic shock and the like due to albumin loss and reduced albumin synthesis. In the case of albumin preparations, heat treatment in the state of an albumin-containing aqueous solution is generally used in order to inactivate viruses that may be mixed therein. When a commercially available albumin preparation produced by such a method is analyzed by gel filtration analysis, it is known that aggregates are present in the preparation. Since this aggregate (generally referred to as a polymer, hereinafter referred to as a polymer) hardly exists before the above-mentioned heat treatment,
It is considered that albumin was aggregated by the action of heat-unstable contaminating proteins due to the heat treatment. Since commercially available albumin preparations are widely used safely, this polymer is not considered to be particularly harmful to humans. However, since it is a heat-modified product, it is preferable that it is not contained in the preparations as much as possible. In addition, albumin contains contaminant proteins having relatively similar physicochemical properties to albumin, such as transferrin, and it is difficult to efficiently separate the protein by conventional means such as fractionation. However, there is a problem that contaminating proteins remain in the albumin preparation.

The present invention has been made to solve the above-mentioned problems, and as a result of various studies by the present inventors, the content of contaminating proteins such as transferrin can be drastically reduced by highly purifying albumin. It was found that no polymer was detected even when heat treatment was carried out together with the above. That is, an object of the present invention is to provide an albumin preparation having a low polymer content and a low content of contaminating proteins, and a method for producing the same.

[0004]

The albumin preparation of the present invention, which has been made to solve the above-mentioned problems, is a heat-treated albumin preparation, which is a 25 (w / v)% albumin solution. Sometimes, the aggregate content measured by the gel filtration analysis is below the detection limit; the transferrin content measured by the Mancini method is 2 mg / dl or less; the α ₁ -acid glycoprotein content measured by the Mancini method is 4 mg / dl or less; Mancini The haptoglobin content measured by the method is 6.5 mg / dl or less; and the serum albumin-containing aqueous solution is treated with an anion exchanger under conditions of pH 5.1 to 5.25. An albumin preparation obtained by treating with a cation exchanger under conditions of pH 4 to 8 and then heating. . In addition, as an anion exchanger and a cation exchanger used for the treatment of the aqueous albumin solution, a strong anion exchanger and a strong cation exchanger are preferable, respectively.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The origin of albumin, which is the main component and starting material of the preparation of the present invention, is not particularly limited, and specifically originates from mammals such as humans, cows, rabbits and the like. And particularly those derived from humans. As a starting material for preparing albumin, for example, fraction V obtained by cold alcohol fractionation by Kohn, etc. is exemplified.

The albumin preparation of the present invention can be obtained by treating an albumin-containing aqueous solution obtained by dissolving the above-mentioned serum albumin in appropriate purified water with an anion exchanger, treating with a cation exchanger, and then heating. The treatment with the anion exchanger and the cation exchanger yields an aqueous albumin solution from which contaminating proteins having various isoelectric points have been removed. Then, since the purified aqueous albumin solution is subjected to a heat treatment, polymerization caused by contaminating proteins can be suppressed, and an albumin preparation in which the polymer content and the contaminating protein content are low and the virus which may be contaminated is inactivated is obtained. .

[0007] In the above step, the albumin content in the aqueous solution containing albumin is usually 0.1 to 30%.
(W / V, the same applies hereinafter unless otherwise specified), preferably about 1 to 10%. In the present invention, first, the albumin aqueous solution is subjected to an anion exchanger treatment and purified. By this anion exchanger treatment, contaminant proteins having a lower isoelectric point than albumin such as haptoglobin and α ₁ -acid glycoprotein are removed and purified. As the anion exchanger to be used, any insoluble carrier having an anion exchange group (for example, diethylaminoethyl group or the like) can be used, and more specifically, an anion exchange agent commonly used in this field is used. Body, for example, DEAE-Sepharose, Q-Sepharose (both are registered trademarks, manufactured by Pharmacia), DEAE-Toyopearl, QAE-Toyopearl (both are registered trademarks, manufactured by Tosoh Corporation), A200 Cellulofine (registered trademark, Seikagaku Corporation) Co., Ltd.), anion exchange resin, etc., from the viewpoint of the efficiency of removing contaminating proteins.
It is preferable to use a strong anion exchanger such as Sepharose and QAE-Toyopearl.

The above treatment using an anion exchanger is carried out by bringing an aqueous albumin solution into contact with an anion exchanger. The amount of the anion exchanger used is determined by the content of contaminating proteins in the aqueous solution containing albumin, the amount of the anion exchanger, The anion exchanger is used in an amount of 0.1 to 5 ml, usually about 3 ml, per gram of albumin. This method may be carried out by either a column method or a batch method, but is preferably carried out by a column method from the viewpoint of the efficiency of removing contaminating proteins.

In the case of carrying out by the column method, the above-mentioned aqueous solution of albumin is pH 3-6, preferably pH 5.1-5.
25. The salt concentration is adjusted to about 0.001 to 0.2 M sodium chloride, preferably 0.001 to 0.05 M, and the buffer [for example, 0.02 M sodium acetate (pH
5.1)], and then pass through the anion exchanger column equilibrated in [1], and then develop with the same buffer solution to collect non-adsorbed components. The above operation is preferably performed at a low temperature (usually 10 ° C. or lower) in order to suppress denaturation of albumin.

When the batch method is used, an anion exchanger is added to and brought into contact with an aqueous albumin solution adjusted to the above conditions, mixed at 10 ° C. or less for about 30 minutes to 2 hours, and then centrifuged. This is performed by separating from the anion exchanger by such means as above, and collecting the supernatant.

The aqueous albumin solution purified by the above-described anion exchanger treatment is subjected to a cation exchanger treatment after further pH adjustment, concentration adjustment and the like, if necessary. By this cation exchanger treatment, contaminant proteins such as transferrin having an isoelectric point at a higher pH than albumin or the like are removed and purified. As the cation exchanger to be used, any insoluble carrier having a cation exchange group (for example, sulfo group, carboxyl group, etc.) can be used, and more specifically, a cation exchanger commonly used in this field is used. Examples of the ion exchanger include SP-Sephadex (registered trademark, manufactured by Pharmacia), SP-Toyopearl, and TSKgelSP-5PW (all are registered trademarks, manufactured by Tosoh Corporation). It is preferable to use a strong cation exchanger such as SP-Sephadex and SP-Toyopearl.

The treatment using the cation exchanger is performed by bringing the aqueous albumin solution purified by the anion exchanger treatment into contact with a cation exchanger.
The amount of the cation exchanger used is appropriately adjusted depending on the content of contaminating proteins in the aqueous albumin solution, the exchange capacity of the cation exchanger, and the like.
5 ml, usually about 2 ml, is used. This method may be carried out by either a column method or a batch method, but is preferably carried out by a column method from the viewpoint of the efficiency of removing contaminating proteins.

In the case of carrying out by the column method, the above-mentioned aqueous solution of albumin is pH 4 to about 8, preferably pH 4.5 to 6.
5. More preferably, the pH is adjusted to about 5.25 to 5.5, and the salt concentration is adjusted to about 0.001 to 0.2 M sodium chloride, preferably to about 0.001 to 0.05 M, and the buffer solution [for example, 0.02 M Sodium acetate (pH 5.5)], and then pass through a cation exchanger column, and then develop with the same buffer to collect non-adsorbed components.
The above operation is preferably performed at a low temperature (usually 10 ° C. or lower) in order to suppress denaturation of albumin.

When the batch method is used, a cation exchanger is added to and contacted with an aqueous albumin solution adjusted to the above conditions, mixed at 10 ° C. or less for about 30 minutes to 2 hours, and then centrifuged. This is performed by separating from the cation exchanger by such means as above, and collecting the supernatant.

The amount of contaminating proteins contained in the aqueous albumin solution purified by the anion exchanger treatment and the cation exchanger treatment is below the detection limit of haptoglobin, transferrin and α ₁ -acid glycoprotein.

The aqueous albumin solution in which the content of contaminating proteins has been reduced by the above-mentioned anion exchanger treatment and cation exchanger treatment was adjusted to an appropriate concentration, and was formulated into a desired formulation such as filling in a vial. Thereafter, heat treatment is performed to obtain the albumin preparation of the present invention. The above-mentioned heat treatment inactivates a virus that may be mixed into the albumin preparation, and has an albumin concentration of about 5 to 30%.
Usually, it is performed as an aqueous solution adjusted to about 5 or 20 to 25%, and the heating temperature may be a temperature and time sufficient to inactivate contaminating viruses.
C., preferably at about 60.degree. C., for 5 to 20 hours, preferably for about 10 hours. In the above heat treatment, a stabilizer for albumin, for example, sodium N-acetyltryptophan, sodium caprylate, or the like may be added alone or in combination, if necessary. These albumin stabilizers include albumin 1 contained in the preparation.
It is used in an amount of 20 to 60 mg, preferably about 40 mg per g.

The albumin preparation thus obtained has a polymer content measured by gel filtration analysis below the detection limit, and the transferrin content, α ₁ -acid glycoprotein content and haptoglobin content measured by the Mancini method are below the detection limit. is there. The albumin preparation of the present invention is used in the same dosage and usage as the conventional albumin preparation.

[0018]

The albumin preparation of the present invention is a preparation which is excellent in safety, stability and the like, in which the virus is inactivated by heat treatment, the content of the polymer and the content of contaminating proteins such as transferrin are extremely small.

[0019]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 (1) Preparation of Albumin Aqueous Solution A paste of fraction V (500 g) obtained by Kohn's cold alcohol fractionation was dissolved in 2.0 L of cold sterile distilled water, and the pH was adjusted to 4.6 with acetic acid. , And stirred for about 1 hour. Then, filtration at about -2 ° C (filter:
0.45 μm), and 2.0 L of cold aseptic distilled water was added, and the pH was adjusted to 5.1 with 1N sodium hydroxide to obtain an albumin aqueous solution.

(2) Anion exchanger treatment QAE-Toyopearl (580 ml) was applied to a column (diameter 5c).
mx 18 cm in length), washed thoroughly with 0.5 M sodium chloride, and then 0.02 M sodium acetate (pH
Equilibration was performed in 5.1) to prepare an anion exchanger column. The albumin aqueous solution of the above (1) was passed through the column, and further cooled 0.02 M sodium acetate (pH 5.1,
2 L). Combine the passing solution and the washing solution,
The pH was adjusted to 5.5 with 8M sodium bicarbonate.

(3) Cation exchanger treatment SP-Toyopearl (400 ml) was packed in a column,
After thoroughly washing with 0.5 M sodium chloride, 0.02
Equilibrated with M sodium acetate (pH 5.5) to prepare a cation exchanger column. The aqueous albumin solution obtained in the above (2) was passed through this column, and the column was further washed with 0.02 M sodium acetate (pH 5.5, 1.2 L). After the flow-through solution and the washing solution were combined, the solution was dialyzed and concentrated with Pellicon to prepare A ₂₈₀ = 149 (albumin concentration: 28%).

(4) Heat treatment The aqueous albumin solution obtained in the above (3) is added to the aqueous solution 1
1.2 ml of stabilizer solution per 0 ml (in 100 ml,
5.55 g of N-acetyltryptophan and 3.89 g of sodium caprylate) were added, and the pH was adjusted to 6.85 with 1N sodium hydroxide. Next, after adjusting the albumin concentration to 25%, a predetermined amount was dispensed into vials and heat-treated at 60 ° C. for 10 hours to obtain an albumin preparation.

When the polymer content in the obtained preparation of the present invention was measured by gel filtration analysis, no polymer was detected. For comparison, the polymer content of an albumin preparation (hereinafter referred to as a comparative preparation) prepared in substantially the same manner as in the above-mentioned production method except that the SP-Toyopearl column treatment step was excluded was 2.49 with respect to the albumin content. % By weight, and the polymer content of the albumin preparation of the present invention was remarkably low. The gel filtration analysis was performed under the following conditions.

Sample: The preparation of the present invention and the comparative preparation were
Dilute 50-fold with the following buffer, and filter (filter:
20 μl of the solution (0.45 μm) was injected. Column: TSKgelG3000SW (Tosoh Corporation)
Was used (diameter 7.8 mm × length 30 cm). Buffer: 0.1 M KH ₂ PO ₄ /0.3 M NaCl
(PH 6.9) Flow rate: 1 ml / min Detection wavelength; λ = 280 nm Apparatus; Waters HPLC system

Further, alpha ₁ in the present invention the formulation obtained and Comparative Composition - acid glycoprotein (alpha ₁ -AG), performed haptoglobin (Hp) and the measurement of transferrin (Tf) content, the results are given in Table 1 Show. The contaminant protein content was measured by a one-way immunodiffusion method (Mancini method: Mancini method).
iG. Et al., Immunochemistry, 2nd.
Vol. 3, No. 3, pp. 235-254, 1965). In this case, the anti-α ₁ -acid glycoprotein serum, anti-haptoglobin serum and anti-transferrin serum used were prepared by a conventional method using rabbits as immunized animals.
Using a one-piece immunodiffusion gel prepared from these antisera, a standard curve of the area of the settling ring for each corresponding contaminating protein is shown in FIGS. As shown in Table 1, the albumin preparation of the present invention contains α ₁ -AG, Hp and Tp.
f was below the detection limit, indicating that the content of contaminating proteins was extremely low. 1 to 3, the detection limit of α ₁ -AG is 4 mg.
/ Dl, the detection limit for Hp is 6.5 mg / dl, and the detection limit for Tf is 2 mg / dl.

[0026]

[Table 1] In the above Table 1, <DL indicates below the detection limit.

Example 2 In Example 1, the pH was adjusted with 0.8 M sodium bicarbonate in the anion exchanger treatment step (2).
An albumin preparation was prepared in the same manner as in Example 1 except that the pH was adjusted to 5.25 and the pH of the cation exchanger treatment step (3) was adjusted to 5.25. About the obtained albumin preparation, the polymer content and the contaminating protein content were measured in the same manner as in Example 1. As a result, the polymer was below the detection limit, and the Hp, α ₁ -AG and Tf contents were below the detection limit.

[Brief description of the drawings]

FIG. 1 is a graph showing a standard curve obtained by a one-way immunodiffusion method for α ₁ -acid glycoprotein.

FIG. 2 is a graph showing a standard curve obtained by a one-way immunodiffusion method for haptoglobin.

FIG. 3 is a graph showing a standard curve obtained by a one-way immunodiffusion method for transferrin.

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[Procedure amendment]

[Submission date] February 10, 1999

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction contents]

In the case of carrying out by the column method, the above-mentioned aqueous albumin solution is pH 3-6, preferably pH 4.5-5.
5, more preferably about pH 5.1 to 5.25, and a salt concentration of about 0.001 to 0.2 M sodium chloride, preferably about 0.1 to 0.2 M.
The solution is adjusted to 001 to 0.05M, passed through an anion exchanger column equilibrated with a buffer [for example, 0.02M sodium acetate (pH 5.1)], and then developed with the same buffer to collect non-adsorbed components. This is done by: The above operation is preferably performed at a low temperature (usually 10 ° C. or lower) in order to suppress denaturation of albumin.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Shinji Tomioka 4-12-1 Otsukacho, Takatsuki-shi, Osaka Yoshitomi Pharmaceutical Yodogawa Plant Co., Ltd. (72) Inventor Kazumasa Yokoyama 2-7-2, Terauchi, Toyonaka-shi, Osaka

Claims (2)

[Claims] 1. In a heat-treated albumin preparation, when the preparation is a 25% (w / v)% albumin solution, the aggregate content measured by gel filtration analysis is below the detection limit; measured by the Mancini method albumin which is a; acidic glycoprotein content 4 mg / dl or less - Mancini method was measured by alpha _1; that; haptoglobin content determined by Mancini method 6.5 mg / dl or less transferrin content of 2 mg / dl or less was Formulation. 2. An aqueous solution containing serum albumin having a pH of 5.1.
After treatment with an anion exchanger under conditions of ~ 5.25,
The albumin preparation according to claim 1, which is obtained by treating with a cation exchanger under the conditions of 4 to 8, followed by heat treatment.