JP2982296B2 - Purification method of aqueous solution containing albumin - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for purifying an albumin-containing aqueous solution, and more particularly, to a method for reducing the content of an aluminum component in an albumin-containing aqueous solution which is a raw material of an albumin preparation. .

[Problems to be Solved by the Prior Art and the Invention] Serum albumin is the most abundant protein in plasma and has functions such as maintaining osmotic pressure in blood, transporting nutrients and metabolites by binding to 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.

The albumin preparation is prepared from the plasma as a raw material through a plurality of purification steps.The present inventors have studied in detail the impurities in the albumin preparation, and found that the filter aid used in the production process of the albumin preparation contains The aluminum content in the final formulation was confirmed to be quite high due to aluminum contamination.

By the way, recently, attention has been paid to the relationship between aluminum and various diseases.For example, severe painful bone diseases frequently occurring in patients undergoing renal dialysis and long-term infusion are caused by aluminum contained in a solution used. It is said that there is.

In the case of dialysis patients with chronic renal failure, accumulation of aluminum in serum is considered to be the cause of dialytic dementia, bone disease, and hypochromic anemia.

Further, attention has been paid to the relationship between aluminum in the brain and Alzheimer's disease (senile dementia).

For this reason, countries in Europe and the United States are moving to regulate the aluminum content in pharmaceutical preparations.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for remarkably reducing the aluminum content in an aqueous solution containing albumin containing aluminum.

Means for Solving the Problems The present inventors have conducted intensive studies in order to reduce the aluminum content in the albumin preparation. As a result, the aluminum-containing aqueous solution containing aluminum was treated with an anion exchanger to obtain aluminum. The inventors have found that the content can be reduced and completed the present invention. That is, the method for purifying an albumin-containing aqueous solution of the present invention is to remove the aluminum mixed therein by treating the plasma-derived (serum) albumin-containing aqueous solution containing aluminum mixed therein with an anion exchanger. The purified aqueous solution containing albumin is prepared into an albumin preparation through processes such as a cation exchanger treatment and a heat treatment.

 Hereinafter, the present invention will be described in detail.

(1) Starting Material The source of albumin, which is the starting material of the method of the present invention, is not particularly limited, and is specifically a mammal, for example, a human,
Examples include those derived from cattle and rabbits, 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.

(2) Treatment of the present invention In the present invention, an albumin-containing aqueous solution in which albumin is dissolved in appropriate purified water is subjected to an anion exchanger treatment for purification.

In the above step, the albumin content in the albumin-containing aqueous solution is generally adjusted to about 0.1 to 30% (W / V, the same applies hereinafter unless otherwise specified), preferably about 1 to 10%.

 The anion exchanger used is an anion exchanger.
Substituents (eg, quaternary alkyl ammonium bases, diethyl
Laminoethyl group etc.)
Can also be used, and more specifically in this area
Conventional anion exchangers, such as DEAE-Sepharose
S , Q-Sepharose (All are Pharmacia
Made), DEAE-Toyopearl , QAE-Toyopearl (Izu
Also manufactured by Tosoh Corporation), A200 Cellulo Fine (Seikagaku Corporation
Aluminum anion exchange resin, etc.
Q-Sepharose, QAE-Toyopa in terms of removal efficiency
It is preferable to use a strong anion exchanger such as a thiol anion.

The treatment using the anion exchanger is performed by bringing the aqueous solution containing albumin into contact with the anion exchanger, and the amount of the anion exchanger used is determined by the aluminum content, the contaminant protein content, and the anion exchange content in the albumin-containing aqueous solution. Although it is appropriately adjusted depending on the exchange capacity of the body, 2 to 5 ml, usually about 3 ml, of an anion exchanger is used per 1 g of albumin.
This method may be performed by either a column method or a batch method, but is preferably performed by a column method from the viewpoint of aluminum removal efficiency.

In the case of performing by the column method, the albumin-containing aqueous solution is pH 3 to about 6, preferably pH 4.5 to 5.5, and the salt concentration is about 0.001 to 0.2 M sodium chloride, preferably 0.001 to 0.2 M.
Pass through an anion exchanger column adjusted to ~ 0.05M and equilibrated with a buffer [eg, 0.02M sodium acetate (pH 5.1)], and then developed with the same buffer to collect non-adsorbed components It is performed by 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 the aqueous solution containing albumin adjusted to the above conditions, and the mixture is 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 means and collecting the supernatant.

(3) Post-treatment The albumin purified by the above anion exchanger treatment
When formulating aqueous solutions containing
After adjusting the concentration and concentration, etc.,
Further purification to remove contaminating proteins.
No. Cation exchanger used as cation exchanger
Insoluble (eg, sulfo group, carboxyl group, etc.)
Any carrier can be used as long as the carrier is more specific.
Typically, cation exchangers commonly used in this field, for example, SP
−Sephadex (Pharmacia), SP-Toyopa
Rule , TSKgelSP-5PW (All manufactured by Tosoh Corporation)
Examples include ion exchange resins and the like, which improve the efficiency of removing contaminating proteins.
Mustards such as SP-Sepharose and SP-Toyopearl
Preferably, an on-exchanger is used.

The treatment using the cation exchanger is performed by bringing the aqueous solution containing albumin 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 contaminant protein content in the albumin-containing aqueous solution, the exchange capacity of the cation exchanger, and the like. . 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.

When performing the column method, the albumin-containing aqueous solution is about pH 4 to 8, preferably pH 4.5 to 6.0, more preferably pH 5.5, and the salt concentration is about 0.001 to 0.2 M sodium chloride, preferably about 0.001 to 0.2 M. Adjusted to 0.05M, passed through a cation exchanger column equilibrated with a buffer [eg, 0.02M sodium acetate (pH 5.5)], then developed with the same buffer to collect non-adsorbed components. Done. The above operation is performed at a low temperature (usually 10 ° C) to suppress denaturation of albumin.
The following is preferred.

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

The albumin-containing aqueous solution in which the aluminum content and contaminant protein content have been reduced by the above anion exchanger treatment and cation exchanger treatment is adjusted to an appropriate concentration, for example,
After being formulated into a desired formulation such as filling into a vial, it is subjected to a heat treatment to obtain an albumin formulation. The above heat treatment inactivates a virus that may be mixed into the albumin preparation, and is performed as an aqueous solution adjusted to an albumin concentration of about 5 to 30%, usually about 5 or 20 to 25%. The reaction may be carried out at a temperature and for a time sufficient to inactivate contaminating viruses, for example, at 50 to 70 ° C., preferably about 60 ° C., for 5 to 20 hours, preferably 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 may be contained in an amount of 20 to 60 per gram of albumin contained in the preparation.
mg, preferably about 40 mg.

The albumin preparation thus obtained has a low aluminum content of less than about 200 ppb (by atomic absorption spectroscopy).

[Effects of the Invention] According to the method of the present invention, the aluminum content in an albumin-containing aqueous solution mixed with aluminum can be significantly reduced. Therefore, the aluminum content in the albumin preparation, which is the final product, is kept to a low level of about 200 ppb or less, and a more stable preparation can be provided.

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

Example (1) Preparation of Albumin-Containing Aqueous Solution The fraction V paste (500 g) obtained by Kohn's cold alcohol fractionation was dissolved in cold sterile distilled water 2.0, and the pH was adjusted to 4.6 with acetic acid. And stirred for about 1 hour.
Then, the mixture was filtered using a filter filled with a filter aid at about −2 ° C. (filter: 0.45 μm), further added with cold sterile distilled water 2.0, and adjusted to pH 5.1 with 1N sodium hydroxide. An albumin-containing aqueous solution was obtained.

(2) Anion exchanger treatment QAE-Toyopearl (580 ml) was columned (diameter 5 cm x length 1)
8 cm), washed thoroughly with 0.5 M sodium chloride, and equilibrated with 0.02 M sodium acetate (pH 5.1) to prepare an anion exchanger column. The albumin-containing aqueous solution of the above (1) was passed through this column, and further washed with cold 0.02 M sodium acetate (pH 5.1, 2). The flow-through solution and the washing solution were combined, and the pH was adjusted to 5.5 with 0.8 M sodium hydrogen carbonate.

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

(4) Heat treatment To the aqueous solution containing albumin obtained in the above (3), 1.2 ml of a stabilizer solution per 10 ml of the aqueous solution (5.55 g of N-acetyltryptophan and 3.89 g of sodium caprylate in 100 ml)
Was added and the pH was adjusted to 6.85 with 1N sodium hydroxide, followed by sterilization filtration. Then, the albumin concentration was 25
%, And a predetermined amount was dispensed into vials and heat-treated at 60 ° C. for 10 hours to obtain an albumin preparation.

The content of aluminum in the obtained albumin preparation was measured by an atomic absorption method and found to be 70 ppb.

Experimental Examples In order to investigate the effect of anion exchangers, the system without the anion exchanger treatment (exchanger-untreated system), the weak anion exchanger treatment system (DEAE treatment system) and the strong anion exchanger treatment system ( The efficiency of aluminum removal in the QAE treatment system was compared.
Operations other than the anion exchanger treatment were performed according to the examples. The aluminum content in the obtained albumin preparation was measured by an atomic absorption method. The results are shown in Table 1.

As is clear from Table 1, the aluminum content can be reduced by treating with an anion exchanger, and a remarkable reduction in the aluminum content was observed, particularly when a strong anion exchanger was used.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Koichi Furuta 4-12-1, Otsuka-cho, Takatsuki-shi, Osaka Inside the Midori-Cross Yodogawa Plant Co., Ltd. No. 1 Inside the Midori Cross Central Research Institute Co., Ltd. (72) Inventor Kazumasa Yokoyama 2-1-1180 Shodai Otani, Hirakata City, Osaka Prefecture 1 Within the Midori Cross Central Research Institute Co., Ltd. (56) References A) JP-A-2-191226 (JP, A) JP-A-4-506349 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/765 C07K 1/18 BIOSIS ( DIALOG) WPI (DIALOG) MEDLINE (DIALOG)

Claims (2)

(57) [Claims] 1. A method for purifying an albumin-containing aqueous solution, comprising removing a mixed aluminum by treating a plasma-derived albumin-containing aqueous solution containing aluminum with a strong anion exchanger. 2. The method for purifying an albumin-containing aqueous solution according to claim 1, wherein the aluminum content in the plasma-derived albumin-containing aqueous solution obtained by the strong anion exchanger treatment is 700 ppb or less.