JPH04352727A - Serum albumin composition having improved heat resistance - Google Patents

Abstract

PURPOSE:To provide a serum albumin composition having improved heat resistance to permit the thermal treatment thereof at high temperatures and enabling the perfect inactivation of various adverse reaction-generating substances such as contaminated viruses and alpha-acid glucoproteins. CONSTITUTION:An albumin composition used as a drug or an additive to the drug and having improved heat resistance is characterized by adding a long chain fatty acid, preferably 16-18C saturated fatty acid to albumin, preferably a natural albumin or genetic recombined human serum albumin usually in a molar ratio of 0.1-10:1, preferably 1-6:1. The albumin composition can be thermally treated at high temperatures above 60 deg.C at which the thermal treatment is impossible by the use of conventional stabilizers. The fatty acid is a substance always existing in living bodies and the amount of the fatty acid is within amounts thereof existing in the living bodies, thereby permitting to provide a blood preparation having extremely high safety and excellent effectiveness on medical treatments.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides a method for improving the stability of albumin, especially its stability against heat, by handling albumin, which is commonly used as a pharmaceutical or pharmaceutical additive. Compositions containing modified albumin are also provided.

0002

[Prior Art] It cannot be denied that serum albumin obtained from plasma fraction components contains contaminant viruses such as hepatitis virus and substances that cause side effects such as α-acid glycoproteins that have immunosuppressive ability. Therefore, before use, serum albumin usually needs to be subjected to heat treatment. Generally, when heat-treating an aqueous solution containing human serum albumin, a stabilizer against denaturation is used that is certified under the Biological Products Standards of the Ministry of Health and Welfare. That is, for 1 g of human serum albumin, 0.16 mmole of N
- sodium acetyltryptophan, or 0.08 mmole each of sodium N-acetyltryptophan and sodium caprylate; 60.
The test was carried out at 0±0.5°C for more than 10 hours.

0003

[Problem to be solved by the invention] The above-mentioned stabilizer can prevent the denaturation of serum albumin, especially human serum albumin, when subjected to heat treatment under the above conditions, but when heated under more severe conditions, For processing, denaturation is difficult to prevent. Furthermore, with these stabilizers, even if the amount added is greater than the above specified amount, it is almost impossible to further improve the heat resistance of human serum albumin.

0004

[Means for Solving the Problems] In view of the above points, the present inventors have discovered that the heat resistance of serum albumin can be improved by incorporating long-chain fatty acids into serum albumin, and have completed the present invention.

More specifically, in order to more completely inactivate substances that cause side effects such as contaminating viruses and α-acid glycoproteins without denaturing serum albumin,
As a result of various studies on methods that enable processing under harsher heating conditions, we found that an aqueous solution containing albumin,
It has been found that the heat resistance of albumin can be improved by adding a long chain fatty acid in a molar ratio of at least 0.1 to albumin. Therefore, the present invention provides for albumin,
Provided is an albumin composition with improved heat resistance, characterized in that it contains a long chain fatty acid in a molar ratio of at least 0.1.

[0006] The albumin used in the present invention particularly refers to serum albumin, which is a serum-derived protein with a molecular weight of about 70,000. The present invention is applicable to all serum albumins such as human, porcine, and bovine, and may be naturally derived or genetically recombinant.

[0007] The long-chain fatty acids are not particularly limited, but those having 16 to 18 carbon atoms are preferred, and include palmitic acid,
Examples include stearic acid, oleic acid, and linoleic acid. Particularly preferred are those whose hydrocarbon residues are completely saturated (hereinafter referred to as saturated fatty acids). That is, in the present invention, palmitic acid and stearic acid, which are saturated fatty acids having 16 to 18 carbon atoms, are preferred. Furthermore, a preferred form of use of long-chain fatty acids is as their salts. The salt is not particularly limited as long as it is physiologically acceptable, but preferably includes alkali metal salts such as sodium salts and potassium salts, and alkaline earth metal salts such as calcium salts, particularly sodium salts and potassium salts. is preferred. The amount of long chain fatty acid used is usually 0.1 to 10 in molar ratio to albumin, preferably 1 to 6.

[0008] These long-chain fatty acids are generally recognized to exist in living organisms, and usually exist in several bound or free forms per albumin molecule. Therefore, there is no concern about toxicity to living organisms. Although the addition of long-chain fatty acids alone can improve the heat resistance of albumin, it is even more effective when used in combination with conventional stabilizers. EXAMPLES The present invention will be explained in more detail by way of Examples below, but these are not intended to limit the present invention in any way.

Example 1 Stearic acid/oleic acid/linoleic acid: 1/1/1 was added to a commercially available human serum albumin (hereinafter abbreviated as HSA) aqueous solution.
0 mole, 0.15 mole, 0.75 mole,
1.40 mole, 2.00 mole, 2.75 mole
e, 5.50 mole, 6.00 mole added, 50
Using eight types of specimens that had been shaken for 1 hour at 70°C,
The degree of denaturation of HSA when heated at six levels of 71°C, 73°C, 75°C, 77°C, and 80°C for 2 minutes each is reported in Clinical Chemical Acta, Volume 26, Page 505 (D.A.
Arvan and A. Ritz, Clin.
chim. Acta, 26, 505 (1969)
According to the method of Alban et al. described in ), the azo dye 2
Evaluation was performed by a dye adsorption method using -(4'-phenylazo)-benzoic acid. The molecular weight of HSA is 66,458
It is. The results are shown in Table 1.

[Table 1] As the amount of long chain fatty acids added increased, the degree of modification of HSA decreased and the heat resistance improved.

Example 2 To an HSA aqueous solution to which sodium N-acetyltryptophan and sodium caprylate were added at a concentration of 4 mM, stearic acid, oleic acid, and linoleic acid, all having the same number of carbon atoms (18) but different degrees of unsaturation, were added. Each sodium salt alone was added at 2 times per mole of albumin.
．． 0 mole was added and shaken at 50°C for 1 hour.
The degree of denaturation of HSA when heated for 2 minutes at standard temperature was measured by the dye adsorption method described above. The results are shown in Table 2.

[Table 2] The degree of modification at temperatures above 75°C is as follows: stearic acid
The order was oleic acid and linoleic acid. That is, it was found that long-chain fatty acids with a lower degree of unsaturation have a lower degree of modification and are more effective in improving heat resistance.

Example 3 Completely saturated sodium salts of myristic acid, palmitic acid, and stearic acid having different carbon numbers were added to an HSA aqueous solution at a concentration of 2.00% per mole of albumin.
The degree of denaturation of HSA was determined by the dye adsorption method when three types of specimens added with mole and shaken at 50°C for 1 hour were heated for 2 minutes at 5 levels of 70°C, 71°C, 73°C, 75°C, and 77°C. It was measured with The results are shown in Table 3.

[Table 3] Although there was almost no difference in the effect of improving heat resistance due to the difference in carbon number, all long chain fatty acids gave good heat resistance.

Example 4 In an HSA aqueous solution, sodium N-acetyltryptophan or sodium caprylate was added to 1 mole of albumin in amounts of 0.05 mole, 1.0 mole, and 5.0 mole.
10.0 mole, 10.0 mole, or 0.5 mole, 1.0 mole of sodium palmitate were added individually and shaken at 50 °C for 1 hour. The degree of denaturation of HSA was measured by a dye adsorption method when heated at 77° C. for 2 minutes at five levels. The results are shown in Table 4.

[Table 4] A specified amount (5.3 m
ole/moleHSA) even if added in a large amount, HS
While the heat resistance of A was hardly improved, a small amount of sodium palmitate significantly improved the heat resistance of HSA.

Example 5 When sodium palmitate was added to an aqueous HSA solution at a rate of 3.0 mole/mole HSA per 1 mole of albumin, the mixture was shaken at 50°C for 1 hour and then heated at 68°C for 20 hours. Almost no denaturation of HSA was observed. I couldn't.

[0014]

[Effects of the Invention] According to the present invention, the heat resistance of serum albumin is improved, and heat treatment can be performed under conditions more severe than before. That is, heat treatment at 60° C. or higher, which is impossible with conventional stabilizers, is also possible. Furthermore, the stabilizer in the present invention is a substance that normally exists in living organisms, and the amount added is within the normal amount, so it is excellent in terms of safety. Therefore, by using serum albumin heat-treated according to the present invention, it is possible to provide a blood product that is medically extremely safe and highly effective.

Claims (6)

[Claims] 1. An albumin composition with improved heat resistance, characterized in that it contains a long chain fatty acid in a molar ratio of at least 0.1 to albumin. 2. The albumin composition according to claim 1, wherein the albumin is naturally derived or recombinant human serum albumin. 3. The albumin composition according to claim 1, wherein the long chain fatty acid is a saturated fatty acid having 16 to 18 carbon atoms. 4. A method for improving the heat resistance of albumin, which comprises adding a long chain fatty acid at a molar ratio of at least 0.1 to albumin to an aqueous solution containing albumin. 5. The method for improving heat resistance of albumin according to claim 4, wherein the albumin is naturally derived or genetically recombinant human serum albumin. 6. The method for improving heat resistance of albumin according to claim 4 or 5, wherein the long chain fatty acid is a saturated fatty acid having 16 to 18 carbon atoms.