JPH09324001A - Purification method for sodium hyaluronate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a purification method for easily producing a high purity sodium hyaluronate which is low in the content of the pyrogenous substance derived therefrom, and the sodium hyaluronate obtained by using the method. SOLUTION: This method for purification of sodium hyaluronate comprises providing a solution containing sodium hyaluronate dissolved therein or a solution prepd. by passing the solution through a positively charged filter, adding a salt and an organic solvent miscible with water to the solution, to thereby deposit and precipitate sodium hyaluronate, and withdrawing a supernatant liquid therefrom, thereby removing the pyrogenous substance. The sodium hyaluronate is obtained by this purification method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying sodium hyaluronate and sodium hyaluronate obtained by the purification method.

[0002]

2. Description of the Related Art Sodium hyaluronate containing no streptricin and having no hemolytic property (Japanese Patent Publication No. 3-7811)
6), sodium hyaluronate having low chemotactic activity of human polymorphonuclear leukocytes and low cell invasion ability during intraocular administration in rabbits (Japanese Patent Publication No. 6-8323), high-purity hyaluronic acid containing no pyrogenic substance Sodium (JP-A-62-501)
471) and the like are known.

As a method for industrially producing sodium hyaluronate, a method of purifying an extract from a chicken cob or a method of purifying the substance from a fermentation liquor obtained from a strain of the genus Streptococcus is known. As a method for removing a high molecular compound such as an exothermic substance present as an impurity in sodium hyaluronate, a treatment with trichloroacetic acid, chloroform-isoamyl alcohol treatment, an enzyme treatment (Japanese Unexamined Patent Publication (Kokai) No. 6-58242)
0-24194), ion exchange resin treatment (JP-A-63-
12293), adsorption resin treatment (JP-A-2-10320)
3) or alternatively, cetylpyridinium chloride (JP-A-60-133894), a method of selectively precipitating sodium hyaluronate with a large amount of a water-soluble organic solvent, etc. (JP-A-2-268765), 20,000 to 60,000 centistokes. A method of removing a heat-generating substance by passing a solution having a viscosity through a positively charged filter in a solution having a pH of 6 to 10 (JP-A-6-199656) is known.

[0004]

SUMMARY OF THE INVENTION Sodium hyaluronate having physical properties such as no hemolytic property, no chemotaxis, and negative pyrogenicity test has been provided. Complicated operation has been required. A purification method for easily removing a pyrogen from sodium hyaluronate is desired.

An object of the present invention is to provide a purification method for easily obtaining high-purity sodium hyaluronate having a low content of pyrogenic substances, and sodium hyaluronate obtained by the purification method.

[0006]

According to the present invention, a salt and a water-soluble organic solvent are added to a sodium hyaluronate solution containing sodium hyaluronate to precipitate and precipitate sodium hyaluronate, and the supernatant liquid is removed to generate heat. It is possible to provide a method for purifying sodium hyaluronate characterized by removing a volatile substance, and sodium hyaluronate obtained by the purification method.

Further, a sodium hyaluronate solution containing sodium hyaluronate is passed through a positively charged filter to remove a pyrogen by the same method as above, and a method for purifying sodium hyaluronate and a method for purifying the same. The obtained sodium hyaluronate can be provided. Furthermore, sodium hyaluronate obtained by the above method is washed with 50% (v / v) or more of a water-soluble organic solvent, powdered, and then vacuum dried,
Further, it is possible to provide a method for purifying sodium hyaluronate, which is characterized by reducing pyrogenic substances, and sodium hyaluronate obtained by the purification method.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Any sodium hyaluronate that can be used in the present invention can be used regardless of the origin of microorganisms, animals and the like and the molecular weight of sodium hyaluronate. Examples of sodium hyaluronate include those isolated from the vitreous, umbilical cord, synovial fluid, chicken cob, Streptococcus pyogenes , Streptococcus equi , Streptococcus equismilis ,・Disgalactie ( Streptococcus dysgal
actiae ), Streptococcus zooepidemicus ( St
Examples include those produced by microorganisms such as reptococcus zooepidemicus ).

The sodium hyaluronate solution is 0.00
An aqueous solution containing sodium hyaluronate at a concentration of 1 to 1% (w / v) is preferable, and the water used for dissolving sodium hyaluronate has a heat generating substance of 0.01 EU /
It is preferably less than or equal to ml. The sodium hyaluronate solution was used for sterilization or insoluble matter removal in an amount of 0.2 to 5.0 μm.
You may perform a filtration process using the filter of m.

Further, for the purpose of removing the heat-generating substance during dissolution of sodium hyaluronate, a filtration treatment may be carried out using a filter positively charged in a specific pH range. As the filter, any filter that is positively charged with an aqueous solution having a pH of 3 to 10 can be used. Preferably, a membrane filter can be used.

The filter is preferably a filter made of a copolymer containing polyamide resin such as nylon-66 as one component and having a membrane pore size of 0.20 to 5.0 μm. Specific examples of the filter include a positive pole filter (trade name) manufactured by Nippon Pole Co., Ltd., a profile II plus (trade name), and the like.

When the filtration treatment is carried out using the filter, in order to improve the filterability, the treatment may be carried out by adding a salt or raising the temperature of the hyaluronic acid solution. As the salt, a positive component consisting of an alkali metal ion such as lithium, sodium and potassium, an alkaline earth metal ion such as magnesium and calcium and an aluminum ion as a base component, and a halogen such as chlorine and bromine as an acid component. A salt composed of an ion, an inorganic acid such as sulfuric acid and nitric acid, and a negative component composed of an acid radical of an organic acid such as formic acid and acetic acid can be used, and preferably a sodium salt can be used. These salts can be used alone or in combination.

Specific salts include sodium chloride, sodium sulfate, sodium acetate and the like.
Salt is 0.1 to 30% of the hyaluronic acid solution
(W / v) is preferably added. The liquid temperature of the hyaluronic acid solution can be raised to 80 ° C.

A salt and a water-soluble organic solvent are added to the sodium hyaluronate solution which has been subjected to the filter filtration treatment or the sodium hyaluronate solution which has not been subjected to the treatment. As the salt, the above-mentioned salts can be used alone or in combination. It is preferable to add 1 to 30% (w / v) of salt to the hyaluronic acid solution.

The final concentration of the water-soluble organic solvent is 30 to 90% so that sodium hyaluronate can be precipitated.
It is preferable to add it so as to be (v / v). As the water-soluble organic solvent, any water-soluble organic solvent can be used, and specifically, alcohols such as methanol, ethanol, n-propanol, and isopropanol, ketones such as acetone, and dimethoxyethane. , Ethers such as tetrahydrofuran, dioxane, acetonitrile and the like. These water-soluble organic solvents can be used alone or in combination. Also,
The water-soluble organic solvent used preferably has a low content of exothermic substances.

In order to efficiently remove a heat-generating substance from sodium hyaluronate precipitated and precipitated by adding a water-soluble organic solvent to a sodium hyaluronate solution,
It is preferable to allow the sodium hyaluronate to settle sufficiently, and usually, after adding the water-soluble organic solvent, leave it for 2 hours or more.
Allow sodium hyaluronate to settle. At this time, the precipitated sodium hyaluronate is in a gel-like, semi-transparent state with a small specific volume.

The exothermic substance can be removed by removing the supernatant from the solution in which the sodium hyaluronate is precipitated. Further, the sodium hyaluronate from which the heat-generating substance has been further removed can be obtained from the sodium hyaluronate remaining after removing the supernatant liquid by the following method. 50-100% (v / v) of a water-soluble organic solvent is added to the gel-like sodium hyaluronate from which the supernatant is removed, and the gel is washed. By this operation, the sodium hyaluronate is changed from gel to powder. If necessary, the powdery sodium hyaluronate may further contain 50 to 100% (v /
After adding the water-soluble organic solvent of v), washing and vacuum drying, the content of the exothermic substance is 0.0015 to 0.003E.
U / mg or less of sodium hyaluronate can be obtained.

The measurement of the exothermic substance was carried out so that the content of the exothermic substance was 0.00 so that sodium hyaluronate was 2 g / l.
After dissolving in water of 6 EU / ml or less, colorimetric analysis is performed using a Toxicolor system (manufactured by Seikagaku Corporation) according to the attached manual. Hereinafter, examples of the present invention will be described.

[0019]

【Example】

Example 1 25 g of sodium hyaluronate having a pyrogen content of 0.02 EU / mg was dissolved in 10 L of water having a pyrogen content of 0.01 EU / ml or less, and then filtered using a 0.45 μm microfilter, A filtrate was obtained.

58.5 g / l of sodium chloride was added to the filtrate.
Then, ethanol was added so that the concentration would be 50 or 44% (v / v). The solution was allowed to stand for about 5 hours to precipitate and settle sodium hyaluronate, and the supernatant of the solution was extracted to obtain a sodium hyaluronate precipitate. The precipitate was washed with 80% ethanol water and then with 100% ethanol.

20 g of purified sodium hyaluronate was obtained by vacuum-drying the washed product. Table 1 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0022]

[Table 1]

Example 2 25 g of sodium hyaluronate having a pyrogen content of 0.02 EU / mg was dissolved in 10 L of water having a pyrogen content of 0.01 EU / ml or less and then filtered using a 0.45 μm microfilter. Then, a filtrate was obtained. After adding sodium chloride to the filtrate so as to be 58.5 g / l, 50,
Acetone was added to be 44 or 41% (v / v).

By allowing the solution to stand for about 5 hours, sodium hyaluronate was precipitated and precipitated, and the supernatant of the solution was extracted to obtain a sodium hyaluronate precipitate. The precipitate was washed with 80% acetone and then 100% acetone. By vacuum-drying the washed product,
20 g of purified sodium hyaluronate was obtained.

Table 2 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0026]

[Table 2]

Example 3 Streptococcu
s zooepidemicus ) NCTC7023 [J. Gen. Microbiol., 1
5 , 485-491 (1956)] was cultured on Brain Heart Infusion Agar Medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C. for 16 hours to prepare bacterial cells, glucose 1%, peptone 1.5%, yeast extract 0. 0.5%, corn steep liquor 1%, sodium glutamate 0.3%, dipotassium phosphate 0.2
%, Magnesium sulfate 0.05%, sodium thiosulfate 0.1% and calcium carbonate 2% (pH 7.
0) 300 ml was inoculated and cultured with shaking at 37 ° C. for 16 hours.

150 ml of this seed culture was used for glucose 2.
5%, peptone 1.5%, yeast extract 0.5%, corn steep liquor 0.5%, dipotassium phosphate 0.2%, magnesium sulfate 0.005%, sodium thiosulfate 0.1.
5 L containing 3 L of fermentation medium (pH 7.2)
Inoculate a jar fermenter at 37 ° C and aeration of 0.3
The cells were cultured at vvm, pH 7.0 for 26 hours to obtain a hyaluronic acid culture solution.

3 L of the hyaluronic acid culture solution was diluted to 20 L with ion-exchanged water, 200 g of carbon was added, and the mixture was stirred at room temperature for 1 hour and filtered with a Nutsche to obtain 20 L of a carbon-treated solution. The carbon treatment liquid was used as a macroreticular anion exchange resin DIAION HPA-75.
2.5 L (manufactured by Mitsubishi Chemical Co., Ltd.) and 1.25 L of gel-type strongly acidic cation exchange resin SK1B (manufactured by Mitsubishi Chemical Co., Ltd.) were continuously passed at a flow rate of 5 L / h to obtain 20 L of a chromatographic solution.

The treatment solution was adjusted to pH 7.0 with sodium hydroxide.
After adjusting to, 200 g of carbon was added, and the mixture was stirred at room temperature for 1 hour. After stirring, the mixture was filtered using a Nutsche filter, and the obtained filtrate was filtered using a 0.45 μm microfilter to obtain 20 L of a filtrate. The content of the pyrogen in the filtrate is
It was 0.04 EU / mg per sodium hyaluronate.

To the filtrate, 1.2 kg of sodium chloride was added, and 14 L of acetone was added. By allowing it to stand for about 5 hours, sodium hyaluronate will precipitate and settle,
The supernatant of the solution was extracted to obtain a sodium hyaluronate precipitate. After washing the precipitate with 80% acetone,
It was washed with 100% acetone. The washed product was vacuum dried to give 11.5 g of purified sodium hyaluronate.
I got it.

Table 3 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0033]

[Table 3]

Example 4 25 g of sodium hyaluronate having a pyrogenic content of 0.02 EU / mg, which was derived from a chicken cob, was mixed with a pyrogen content of 0.01E.
After dissolving in 10 L of water of U / ml or less, the mixture was filtered using a 0.45 μm microfilter to obtain a filtrate. Sodium chloride was added to the filtrate at 58.5 g / l, and then acetone was added at 41% (v / v).

By allowing the solution to stand for about 5 hours, sodium hyaluronate was precipitated and precipitated, and the supernatant of the solution was taken out to obtain a sodium hyaluronate precipitate. The precipitate was washed with 80% acetone and then 100% acetone. By vacuum-drying the washed product,
20 g of purified sodium hyaluronate was obtained.

Table 4 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0037]

[Table 4]

Example 5 25 g of sodium hyaluronate having a pyrogen content of 0.02 EU / mg and a molecular weight of 2.3 million, and a pyrogen content of 0.
It was dissolved in 10 L of water of 01 EU / ml or less. Sodium chloride was added to the solution such that the concentration was 10 g / l. p
Microfilters that are positively charged in H3-10
[Absolute filtration accuracy 0.45 μm, Posidyne filter (trade name) manufactured by Nippon Pall Co., Ltd.] is used and the filtration temperature is 25 ° C.
Then, the hyaluronic acid solution was filtered to obtain a filtrate.

58.5 g / l of sodium chloride was added to the filtrate.
Then, ethanol was added so as to be 44% (v / v). The solution was allowed to stand for about 5 hours to precipitate and settle sodium hyaluronate, and the supernatant of the solution was extracted to obtain a sodium hyaluronate precipitate. After washing the precipitate with 80% ethanol water, 1
It was washed with 00% ethanol. By vacuum-drying the washed product, 20 g of purified sodium hyaluronate was obtained.

Table 5 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0041]

[Table 5]

Example 6 50 g of sodium hyaluronate having a pyrogen content of 0.02 EU / mg and a molecular weight of 600,000 was added to a pyrogen content of 0.0
It was dissolved in 10 L of water at 1 EU / ml or less. Sodium chloride was added to the solution such that the concentration was 10 g / l. pH
Membrane filter that is positively charged in 3-10
[Absolute filtration accuracy 0.45 μm, Posidyne filter (trade name) manufactured by Nippon Pall Co., Ltd.] is used and the filtration temperature is 25 ° C.
Then, the hyaluronic acid solution was filtered to obtain a filtrate.

58.5 g / l of sodium chloride was added to the filtrate.
Then, ethanol was added so as to be 44% (v / v). The solution was allowed to stand for about 5 hours to precipitate and settle sodium hyaluronate, and the supernatant of the solution was extracted to obtain a sodium hyaluronate precipitate. After washing the precipitate with 80% ethanol water, 1
It was washed with 00% ethanol.

By vacuum-drying the washed product, 40 g of purified sodium hyaluronate was obtained. Table 6 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0045]

[Table 6]

Example 7 Streptococcu
s zooepidemicus ) NCTC7023 [J. Gen. Microbiol., 1
5 , 485-491 (1956)] was cultured on Brain Heart Infusion Agar Medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C. for 16 hours to prepare bacterial cells, glucose 1%, peptone 1.5%, yeast extract 0. 0.5%, corn steep liquor 1%, sodium glutamate 0.3%, dipotassium phosphate 0.2
%, Magnesium sulfate 0.05%, sodium thiosulfate 0.1% and calcium carbonate 2% (pH 7.
0) 300 ml was inoculated and cultured with shaking at 37 ° C. for 16 hours.

150 ml of this seed culture was added with glucose 2.
5%, peptone 1.5%, yeast extract 0.5%, corn steep liquor 0.5%, dipotassium phosphate 0.2%,
Magnesium sulfate 0.005%, sodium thiosulfate 0.0.
3 L of fermentation medium (pH 7.2) consisting of 1% was added 5
Inoculate L-jar jar fermenter at 37 ° C and aeration of 0.
Culturing was carried out at 3 vvm, pH 7.0 for 26 hours to obtain a hyaluronic acid culture solution.

3 L of the hyaluronic acid culture solution was diluted to 20 L with ion-exchanged water, 200 g of carbon was added, and the mixture was stirred at room temperature for 1 hour and filtered with a Nutsche to obtain 20 L of a carbon-treated solution. The carbon treatment liquid was used as a macroreticular anion exchange resin DIAION HPA-75.
2.5 L (manufactured by Mitsubishi Chemical Co., Ltd.) and 1.25 L of gel-type strongly acidic cation exchange resin SK1B (manufactured by Mitsubishi Chemical Co., Ltd.) were continuously passed at a flow rate of 5 L / h to obtain 20 L of a chromatographic solution.

The treated solution was adjusted to pH 7.0 with sodium hydroxide.
After adjusting to, 200 g of carbon was added, the mixture was stirred at room temperature for 1 hour, and filtered using a Nutsche to obtain a filtrate. Sodium chloride was added to the solution such that the concentration was 10 g / l. Using a membrane filter that is positively charged at pH 3 to 10 [absolute filtration accuracy 0.45 μm, Posidyne filter (trade name) manufactured by Nippon Pall Co., Ltd.], filtration temperature 2
The hyaluronic acid solution was filtered at 5 ° C. to obtain 18 L of filtrate.

The pyrogen content of the filtrate was 0.01 EU / mg per sodium hyaluronate. To the filtrate was added 1.1 kg of sodium chloride, and 14 L of ethanol was added. By allowing the mixture to stand for about 5 hours, sodium hyaluronate was precipitated and precipitated, and the supernatant of the solution was extracted to obtain a sodium hyaluronate precipitate.

After washing the precipitate with 80% ethanol, 1
It was washed with 00% ethanol. The washed product was vacuum dried to obtain 10.0 g of purified sodium hyaluronate.
I got it. The molecular weight of the sodium hyaluronate was 2.2 million when analyzed by the limiting viscosity method. Table 7 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0052]

[Table 7]

Example 8 50 g of sodium hyaluronate having a pyrogenic substance content of 0.02 EU / mg and a molecular weight of 1,000,000 was dissolved in 10 L of water having a pyrogenic substance content of 0.01 EU / ml or less.
Sodium chloride was added to the solution such that the concentration was 10 g / l.

Membrane filter positively charged at pH 3 to 10 [Absolute filtration accuracy 0.45 μm, Nippon Pole
Using a Posodyne filter (trade name) manufactured by Co., Ltd., at a filtration temperature of 25 ° C., the hyaluronic acid solution is filtered.
A filtrate was obtained. Sodium chloride was added to the filtrate at 58.5 g / l
Then, acetone was added so as to be 41% (v / v).

By allowing the solution to stand for about 5 hours, sodium hyaluronate was precipitated and precipitated, and the supernatant of the solution was extracted to obtain a sodium hyaluronate precipitate. The precipitate was washed with 80% acetone and then 100% acetone. By vacuum-drying the washed product,
40 g of purified sodium hyaluronate was obtained.

Table 8 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0057]

[Table 8]

Example 9 10 g of sodium hyaluronate having a content of exothermic substance of 0.1 EU / mg and a molecular weight of 2.3 million, and a content of exothermic substance of 0.0
It was dissolved in 10 L of water at 1 EU / ml or less. Sodium chloride was added to the solution such that the concentration was 10 g / l. pH
The hyaluronic acid solution was subjected to a filtration treatment at a filtration temperature of 25 ° C. using a microfilter that is positively charged in 3 to 10 [absolute filtration accuracy: 0.2 μm; Posidyne filter (trade name) manufactured by Nippon Pall Co., Ltd.]. Then, a filtrate was obtained.

58.5 g / l of sodium chloride was added to the filtrate.
Then, ethanol was added so as to be 44% (v / v). The solution was allowed to stand for about 5 hours to precipitate and settle sodium hyaluronate, and the supernatant of the solution was extracted to obtain a sodium hyaluronate precipitate. After washing the precipitate with 80% ethanol water, 1
It was washed with 00% ethanol. By vacuum-drying the washed product, 2 g of purified sodium hyaluronate was obtained.

Table 9 shows the content of pyrogenic substances in the obtained sodium hyaluronate.

[0061]

[Table 9]

[0062]

Industrial Applicability According to the present invention, it is possible to provide a purification method for easily obtaining high-purity sodium hyaluronate having a low content of pyrogenic substances, and sodium hyaluronate obtained by the purification method.

Claims (19)

[Claims] 1. A heat-generating substance is removed by adding a salt and a water-soluble organic solvent to a sodium hyaluronate solution containing sodium hyaluronate to precipitate sodium hyaluronate and removing the supernatant liquid. Purification method of sodium hyaluronate. 2. The sodium hyaluronate contained in the sodium hyaluronate solution is 0.001-1% (W /
The purification method according to claim 1, which is V). 3. The sodium hyaluronate is sodium hyaluronate derived from a chicken cob or a microorganism.
Purification method as described. 4. The purification method according to claim 3, wherein the microorganism is a microorganism belonging to the genus Streptococcus. 5. A microorganism belonging to the genus Streptococcus is Streptococcus zooepidemics NCTC7.
The purification method according to claim 4, which is 023. 6. The purification method according to claim 1, wherein the addition of the salt is 1 to 30% (W / V). 7. The purification method according to claim 1 or 6, wherein the salt is a sodium salt. 8. The purification method according to claim 7, wherein the sodium salt is a salt selected from sodium chloride, sodium sulfate and sodium acetate. 9. Addition of a water-soluble organic solvent is 30 to 45%.
The purification method according to claim 1, which is (V / V). 10. The purification method according to claim 1, wherein the water-soluble organic solvent is methanol, ethanol or acetone. 11. The sodium hyaluronate solution containing sodium hyaluronate is a sodium hyaluronate solution that has been treated by passing through a positively charged microfilter in a solution having a pH of 3 to 10. Purification method of sodium hyaluronate. 12. The purification method according to claim 11, wherein the positively charged microfilter has a pore size of 0.20 to 5.0 μm. 13. The purification method according to claim 11, wherein the sodium hyaluronate solution contains a salt. 14. The addition of salt is 0.1 to 30% (W / V).
The purification method according to claim 17, which is 15. The salt of claim 17, wherein the salt is a sodium salt.
Or the purification method according to item 18. 16. The purification method according to claim 19, wherein the sodium salt is a salt selected from sodium chloride, sodium sulfate and sodium acetate. 17. The sodium hyaluronate obtained by the purification method according to claim 1 has 50 parts by weight.
A method for purifying sodium hyaluronate, which comprises adding 100% (V / V) of a water-soluble organic solvent, washing sodium hyaluronate, and vacuum drying. 18. Sodium hyaluronate obtained by the purification method according to any one of claims 1 to 21. 19. Sodium hyaluronate having a pyrogen content of 0.003 endotoxin unit / mg or less.