JPH04234326A - Albumin preparation and production thereof - Google Patents

Abstract

PURPOSE:To obtain serum albumin preparation having extremely reduced albumin aggregate content and contaminant protein content and production thereof. CONSTITUTION:A process wherein an aqueous solution containing albumin is treated with an anion exchanger and a cation exchanger to remove contaminant protein and the aqueous solution containing albumin thus purified is heat- treated. The prepared albumin preparation is a preparation substantially containing neither albumin aggregate nor transferin and having high safety.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention 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 albumin aggregate content and contaminant protein content, and a method for producing the same.

0002

BACKGROUND OF THE INVENTION Serum albumin is the most abundant protein in plasma, and performs functions such as maintaining osmotic pressure in the blood and binding and transporting nutrients and metabolites. The above serum albumin-containing preparations may cause hypoalbuminemia due to albumin loss and decreased albumin synthesis.
It is used to treat conditions such as hemorrhagic shock. Albumin preparations are generally subjected to heat treatment in the form of albumin-containing aqueous solutions in order to inactivate viruses that may be contaminated therein.

0003

Problems to be Solved by the Invention When commercially available albumin preparations produced by such a method are analyzed by gel filtration analysis, it is known that albumin aggregates are present in the preparations. This aggregate (usually referred to as a polymer, henceforth referred to as a polymer) is hardly present before the heat treatment described above, so it is likely that albumin aggregates due to the action of heat-labile contaminant proteins during the heat treatment. considered to be a thing. Since commercially available albumin preparations are widely used safely, this polymer is not thought to be particularly harmful to the human body, but since it is a heat-denatured product, it is preferable to contain it in the preparation as little as possible.

[0004]Albumin also contains contaminant proteins such as transferrin, which have relatively similar physicochemical properties to albumin, and cannot be efficiently separated by conventional means such as fractionation. There is a problem that contaminant proteins remain in the albumin preparation. The present invention was created to solve the above problems, and an object of the present invention is to provide an albumin preparation with a low polymer content and a low content of contaminant proteins, and a method for producing the same.

[0005]

[Means for Solving the Problems] As a result of various studies conducted by the present inventors in order to solve the above problems, the present invention has been achieved by highly purifying albumin to drastically reduce the content of contaminant proteins such as transferrin. This was completed after discovering that no polymer was detected even after heat treatment. That is, the albumin preparation of the present invention is a preparation substantially free of polymers and a preparation substantially free of transferrin.

[0006] Furthermore, the method for producing the albumin preparation of the present invention includes:
This method is characterized in that the serum albumin-containing aqueous solution is treated with an anion exchanger, then treated with a cation exchanger, and then heated. In the above production method, the anion exchanger and cation exchanger used for treating the albumin-containing aqueous solution are preferably a strong anion exchanger and a strong cation exchanger, respectively.

[0007] There is no particular restriction on the origin of albumin, which is the main component of the preparation according to the present invention and the starting material for the production method according to the present invention.
Examples include those derived from cows, rabbits, etc., and those derived from humans are particularly used. Examples of starting materials for preparing albumin include, for example, Cohn's fraction V obtained by cold alcohol fractionation.

The albumin preparation of the present invention can be obtained by treating an albumin-containing aqueous solution prepared by dissolving the above-mentioned serum albumin in suitable purified water with an anion exchanger, followed by treatment with a cation exchanger, and then heat treatment. In the above step, the albumin content in the albumin-containing aqueous solution is usually adjusted to about 0.1 to 30% (W/V, the same applies hereinafter unless otherwise specified), preferably about 1 to 10%.

In the present invention, first, an albumin-containing aqueous solution is purified by subjecting it to an anion exchanger treatment. By this anion exchanger treatment, contaminant proteins such as haptoglobin and α1-acid glycoprotein, which have an isoelectric point lower than that of albumin, are removed and purified. As the anion exchanger used, any insoluble carrier having an anion exchange group (e.g., diethylaminoethyl group, quaternary ammonium group, etc.) can be used. Anion exchangers commonly used in (manufactured by Seikagaku Kogyo Co., Ltd.), anion exchange resins, etc., and it is preferable to use strong anion exchangers such as Q-Sepharose and QAE-Toyopearl in view of the efficiency of removing contaminant proteins.

[0010] The above treatment using an anion exchanger is carried out by bringing an albumin-containing aqueous solution into contact with the anion exchanger, and the amount of anion exchanger used depends on the content of contaminant proteins in the albumin-containing aqueous solution and the anion exchanger. The amount of anion exchanger used is 0.1 to 5 ml, usually about 3 ml, per 1 g of albumin, although it is adjusted as appropriate depending on the exchange capacity of the body. This method may be carried out by either a column method or a batch method, but it is preferably carried out by a column method from the viewpoint of efficiency in removing contaminant proteins.

When using the column method, the albumin-containing aqueous solution is adjusted to a pH of about 3 to 6, preferably about 4.5 to 6.
5.5, the salt concentration is about 0.001 to 0.2 M sodium chloride, preferably 0.001 to 0.05
M, and passed through an anion exchange column equilibrated with a buffer [e.g., 0.02M sodium acetate (pH 5.1)], and then developed with the same buffer to collect the unadsorbed content. It will be done. The above operation is preferably carried out at a low temperature (usually 10° C. or lower) in order to suppress denaturation of albumin. In addition, when performing the batch method, an anion exchanger is added and brought into contact with the albumin-containing aqueous solution adjusted to the above conditions, mixed at 10°C or less for about 30 minutes to 2 hours, and then subjected to centrifugation etc. This is carried out by separating the anion exchanger by a means and collecting the supernatant.

[0012] The albumin-containing aqueous solution purified by the above anion exchanger treatment is subjected to pH adjustment and
After adjusting the concentration, etc., it is further purified by cation exchanger treatment. Through this cation exchanger treatment, contaminant proteins such as transferrin, which has an isoelectric point at a higher pH than albumin, are removed and purified. Any insoluble carrier having a cation exchange group (e.g., sulfo group, carboxy group, etc.) can be used as the cation exchanger, and more specifically, cation exchangers commonly used in this field can be used. Ion exchangers, such as SP-Sephadex (trade name, manufactured by Pharmacia), SP-Toyopearl,
TSKgelSP-5PW (trade name, both manufactured by Tosoh Corporation), cation exchange resin, etc. are exemplified, and from the point of view of contaminant protein removal efficiency, it is recommended to use strong cation exchangers such as SP-Sephadex and SP-Toyopearl. is preferred.

[0013] The treatment using the cation exchanger described above is carried out by bringing the albumin-containing aqueous solution purified by the anion exchanger treatment into contact with the cation exchanger. The amount of the cation exchanger used is adjusted appropriately depending on the content of contaminant proteins in the albumin-containing aqueous solution, the exchange capacity of the cation exchanger, etc., but 0.1 to 5 ml of the cation exchanger is used per 1 g of albumin, usually about 2 ml. be done. This method may be carried out by either a column method or a batch method, but it is preferably carried out by a column method from the viewpoint of efficiency in removing contaminant proteins.

When using the column method, the albumin-containing aqueous solution has a pH of about 4 to 8, preferably about 4.5 to 8.
6.5, more preferably pH 5.5 to 6.0, the salt concentration is adjusted to about 0.001 to 0.2 M sodium chloride, preferably 0.001 to 0.05 M, and a buffer solution [e.g. 0.02M sodium acetate (pH 5.5)
It is carried out by passing through a cation exchanger column equilibrated with ] and then developing with the same buffer to collect the non-adsorbed content. The above operation is preferably carried out at a low temperature (usually 10° C. or lower) in order to suppress denaturation of albumin. In addition, when performing the batch method, add a cation exchanger to the albumin-containing aqueous solution adjusted to the above conditions, bring it into contact, mix at 10°C or less for about 30 minutes to 2 hours, and then perform centrifugation etc. This is carried out by separating the cation exchanger by a means and collecting the supernatant.

[0015] The albumin-containing aqueous solution purified by such anion exchanger treatment and cation exchanger treatment has an extremely low content of contaminant proteins, and the contents of transferrin, haptoglobin, and α1-acid glycoprotein are below the detection limit, and are substantially It does not contain these contaminant proteins.

The albumin-containing aqueous solution whose contaminant protein content has been reduced by the above-mentioned anion exchanger treatment and cation exchanger treatment is adjusted to an appropriate concentration and formulated into a desired pharmaceutical form, for example, by filling it into a vial. After that, the albumin preparation of the present invention is obtained by heat treatment. The above heat treatment is to inactivate viruses that may be mixed into albumin preparations, and is performed as an aqueous solution with an albumin concentration of about 5 to 30%, usually about 5 or 20 to 25%, and the heating temperature is , it may be carried out at a temperature and for a time sufficient to inactivate the contaminating virus, for example, 50 to 7
It is carried out at 0°C, preferably about 60°C, for 5 to 20 hours, preferably about 10 hours. In addition, during the above-mentioned heat treatment, an albumin stabilizer such as sodium N-acetyltryptophan, sodium caprylate, etc. may be added alone or in combination, if necessary. These albumin stabilizers include albumin 1 contained in the formulation.
It is used in an amount of 20 to 60 mg, preferably about 40 mg per g.

The albumin preparation thus obtained is substantially free of polymers (for example, the polymer content relative to albumin is less than 0.63% by weight as measured by gel filtration analysis) and substantially free of transferrin. (e.g. less than 0.008% transferrin content relative to albumin as measured by one-way immunodiffusion). The albumin preparation of the present invention is used in the same dosage and usage as conventional albumin preparations.

[0018]

EFFECTS OF THE INVENTION The albumin preparation of the present invention has viruses inactivated by heat treatment, has extremely low polymer content and contaminant protein content such as transferrin, and is excellent in safety and stability. In addition, the method for producing the albumin preparation of the present invention involves heating an albumin-containing aqueous solution from which contaminant proteins with various isoelectric points have been removed by treatment with an anion exchanger and a cation exchanger. An albumin preparation can be obtained in which the resulting polymerization can be suppressed, the polymer content and the contaminant protein content are low, and viruses that are likely to be contaminated are inactivated.

[0019]

[Examples] Hereinafter, examples will be given to explain the present invention in more detail, but the present invention is not limited to these examples in any way.

Example 1 (a) Preparation of albumin-containing aqueous solution Fraction V paste (500 g) obtained by Kohn's cold alcohol fractionation was dissolved in 2.0 liters of cold sterile distilled water and diluted with acetic acid. After adjusting the pH to 4.6, approx.
Stir for hours. Next, the mixture was filtered at about -2°C (filter: 0.45 μm), 2.0 liters of cold sterile distilled water was added, and the pH was adjusted to 5.1 with 1N sodium hydroxide to obtain an albumin-containing aqueous solution.

(b) Anion exchanger-treated QAE-Toyopearl (580 ml) was placed in a column (diameter 5 cm x length 18 cm).
m), washed thoroughly with 0.5M sodium chloride, and equilibrated with 0.02M sodium acetate (pH 5.1) to prepare an anion exchange column. The albumin-containing aqueous solution of (a) above was passed through this column, and further cooled to 0.
．． Washed with 02M sodium acetate (pH 5.1, 2 liters). The passing liquid and washing liquid were combined, and the pH was adjusted to 5.5 with 0.8M sodium hydrogen carbonate.

(c) Fill a column with cation exchanger-treated SP-Toyopearl (400 ml), thoroughly wash with 0.5 M sodium chloride, equilibrate with 0.02 M sodium acetate (pH 5.5), and cation exchanger treatment. An ion exchanger column was prepared. The albumin-containing aqueous solution obtained in (b) above was passed through this column, and 0.02M sodium acetate (
pH 5.5, 1.2 liters). After combining the passing liquid and the washing liquid, it is dialyzed and concentrated using Pellicon, and A2
80 = 149 (albumin concentration: 28%).

(d) Heat treatment To the albumin-containing aqueous solution obtained in the above (c), 1.2 ml of a stabilizer solution (100 ml
(containing 5.55 g of N-acetyltryptophan and 3.89 g of sodium caprylate) was added thereto, the pH was adjusted to 6.85 with 1N sodium hydroxide, and the mixture was filtered for sterilization. Next, after adjusting the albumin concentration to 25%, a predetermined amount was dispensed into a vial and heat-treated at 60°C for 10 hours to obtain an albumin preparation (hereinafter referred to as the preparation of the present invention).
I got it. Further, as a comparative example, an albumin preparation (hereinafter referred to as a comparative preparation) was prepared in substantially the same manner as the above manufacturing method except that the SP-Toyopearl column treatment step was omitted.

(i) Measurement of polymer content in albumin preparations The polymer contents in the obtained preparations of the present invention and comparative preparations were measured by gel filtration analysis. Note that the gel filtration analysis was conducted under the following conditions. (a) Sample: The preparation of the present invention and the comparative preparation were diluted 50 times with the following buffer and filtered (filter: 0.45
20 μl of the solution (μm) was injected. (b) Column: Column packed with TSKgelG3000SW (manufactured by Tosoh Corporation) (diameter 7.8 mm x length 30 cm)
m) was used. (c) Buffer: 0.1M KH2 PO4 /0.
3M NaCl (pH 6.9) (d) Flow rate: 1 ml/min (e) Detection wavelength: λ = 280 nm (f) Equipment: Waters HPLC system

002
5] The measurement results are shown in FIG. 1 (preparation of the present invention) and FIG. 2 (comparative preparation). As is clear from FIG. 2, in the comparative formulation, a clear polymer peak was detected, and the polymer content relative to albumin was 2.6% by weight. On the other hand, as shown in FIG. 1, in the preparation of the present invention, no polymer peak was detected, indicating that the drug was substantially free of polymer. In FIG. 1, the content of albumin dimer relative to albumin is 0.63% by weight, indicating that this gel filtration analysis method can detect this amount of impurities. Therefore, since no polymer peak was detected in the formulation of the present invention, it was revealed that the polymer content in the formulation of the present invention was less than 0.63% by weight based on albumin.

(ii) Measurement of contaminant proteins in albumin preparations α1-acid glycoprotein (
α1-AG), haptoglobin (Hp) and transferrin (Tf) contents were measured. The results are shown in Table 1. In addition, the measurement of contaminant proteins is carried out using the one-way immunodiffusion method (Manc
ini method: Mancini G. et al., Immunoch
emistry, Volume 2, No. 3, No. 235-254
Page, 1965). At this time, the anti-α1
- Acidic glycoprotein serum, anti-haptoglobin serum, and anti-transferrin serum were prepared by conventional methods using rabbits as the immunized animals. Standard curves of sedimentation ring area for contaminant proteins corresponding to each gel using one-dimensional immunodiffusion gels prepared from these antisera are shown in FIGS. 3 to 5. As is clear from Figures 3 to 5, the detection limit for α1-AG is 4 mg/dl, and the detection limit for Hp is 6.5 mg/dl.
dl, and the detection limit of Tf is 2 mg/dl. As shown in Table 1 below, the albumin preparation of the present invention has α
It was found that 1-AG, Hp, and Tf were all below the detection limit, and the content of contaminant proteins was extremely low and was substantially absent. As mentioned above, the detection limit of Tf is 2 mg/dl, and the albumin content of the formulation of the present invention is 25%, so the Tf content of the formulation of the present invention is less than 0.008% by weight based on albumin. It became clear that.

[0027]

[Table 1]

Example 2 In Example 1, the pH adjustment with 0.8M sodium bicarbonate in the anion exchanger treatment step (b) was carried out to pH 5.
．． An albumin preparation was prepared in the same manner as in Example 1, except that the pH was adjusted to 25 and the pH in the cation exchanger treatment step (c) was adjusted to 5.25. Regarding the obtained albumin preparation, the polymer content and the contaminant protein content were measured in the same manner as in Example 1. The results showed that the polymer peak was not detected and was substantially absent.
Furthermore, the contents of Hp, α1-AG, and Tf were also below the detection limit, and it was found that they were not substantially contained.

Reference Example In the preparation of the present invention preparations and comparative preparations shown in Examples 1 and 2 above, the albumin recovery rates after anion exchanger treatment, cation exchanger treatment and concentration treatment are shown in Table 2. It was as shown in Note that the albumin recovery rate was measured by absorbance at 280 nm.

[0030]

[Table 2]

As shown in Table 2 above, the recovery rate of albumin after anion exchanger treatment, cation exchanger treatment and concentration treatment is high, especially when the cation exchanger treatment is performed at pH
5.5, the recovery rate was found to be significantly higher.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of gel filtration analysis of the formulation of the present invention.

FIG. 2 is a diagram showing the results of gel filtration analysis of comparative formulations.

FIG. 3 is a diagram showing a standard curve for α1-acid glycoprotein measurement by one-way immunodiffusion method.

FIG. 4 is a diagram showing a standard curve for haptoglobin measurement by one-way immunodiffusion method.

FIG. 5 is a diagram showing a standard curve for transferrin measurement by one-way immunodiffusion method.

Claims (3)

[Claims] 1. A serum albumin preparation, the albumin preparation being substantially free of albumin aggregates. 2. A serum albumin preparation, which is characterized in that it does not substantially contain transferrin. 3. A method for producing an albumin preparation, which comprises treating an aqueous serum albumin-containing solution with an anion exchanger, followed by treatment with a cation exchanger, and then heat treatment.