JP2938880B2 - Purification method of hyaluronic acid - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for separating and purifying hyaluronic acid from a hyaluronic acid-containing liquid. Hyaluronic acid has been used as a pharmaceutical in ophthalmology, orthopedic surgery, dermatology, etc. in addition to cosmetic moisturizers.

[Conventional technology and issues to be solved]

Conventionally, hyaluronic acid has been produced by extraction from animal tissues, for example, chicken limbo, etc. on an industrial scale.However, as a contaminant, chondroitin sulfate is mixed in, or hyaluronidase contained in the tissue is used. Those which are easily reduced in molecular weight and therefore purified with high purity by using a polymer have a high cost.

In order to solve these problems, production of hyaluronic acid by a fermentation method has recently been performed. It has long been known that hyaluronic acid is produced by a group of bacteria of the genus Streptococcus, and there are many reports (JBWoolcock, Journal of General Microbiology 85 37).
2-375 1976).

Hyaluronic acid produced by the fermentation method is produced with a constant raw material and a constant method as compared with the extraction method, and therefore, the quality of the product is kept constant, so that the industrial utility value is large. .

However, a method for obtaining a high-purity product by separating and removing high-molecular compounds as impurities in a fermentation solution has been studied.

For example, the present inventors have also previously proposed a method for purifying hyaluronic acid using alumina to remove exothermic substances, proteins, and the like (Japanese Patent Application No. 63-144728).

In the conventional purification treatment methods including the method using alumina, the purification treatment is performed in the state of an aqueous solution of hyaluronic acid. When such a method is used, pyrogens, proteins, and the like are not sufficiently removed due to a difference in raw materials, and a stable removal effect is often not obtained.
There remains a problem in practical use, and development of a method for stably obtaining a high-quality product that can be used as a pharmaceutical product with good reproducibility has been awaited.

An object of the present invention is to provide a simple and high-quality method for separating and purifying hyaluronic acid with good reproducibility.

[Means for solving the problem]

The present invention provides a purification method in which impurities such as pyrogens, proteins, nucleic acids, and metals are efficiently separated and removed with good reproducibility when performing a purification treatment, and high purity and low molecular weight do not easily occur in the purification process. As a result of diligent examination, when purifying hyaluronic acid, by performing some or all of the steps using an organic solvent that is miscible with water, impurities such as exothermic substances are stably removed, The inventors have found that the object has been achieved, and have completed the present invention.

That is, the present invention is a method for purifying hyaluronic acid, comprising treating an aqueous solution of an organic solvent containing hyaluronic acid with alumina, activated carbon, or florisil.

The organic solvent used in the present invention is not particularly limited as long as it is an organic solvent miscible with methanol, ethanol, alcohol such as propanol, and water such as acetone. Is preferred. The present invention is characterized in that an organic solvent is added to a hyaluronic acid-containing aqueous solution, or that hyaluronic acid is dissolved in a predetermined concentration of a water-containing organic solvent for purification treatment. The concentration of the organic solvent added at this time can be used in a wide concentration range up to a concentration at which hyaluronic acid does not precipitate.

However, for the most stable effect, 20-50%
The range of the degree is preferable.

The hyaluronic acid-containing liquid used in the present invention can be used either as extracted from animal tissues or as produced by a fermentation method.However, in order to stably produce high-quality products at low cost industrially. Those manufactured by fermentation are desirable.

Hyaluronic acid by fermentation can be obtained by a known method using bacteria such as Streptococcus. The strain used in the fermentation method is a microorganism having hyaluronic acid-producing ability, such as Streptococcus sp. Isolated from the natural world, or Streptococcus ex FM-100 (microscopic) described in JP-A-63-123392. A mutant that stably produces hyaluronic acid at a high yield, such as Koken Bacteria No. 9027) is preferred.

A microorganism having such a hyaluronic acid-producing ability is obtained by aerobically culturing in a medium using glucose, a carbon source such as sucrose, a nitrogen source such as peptone, polypeptone, and yeast extract, vitamins, and inorganic salts. After diluting the culture solution to have a hyaluronic acid concentration of 0.1 to 5 g / concentration, known methods, such as sterilization by centrifugation, sterilization by filtration, sterilization by a flocculant, carbon, sterilization by carbonite, etc. It is desirable to use a solution that has been sterilized by the method.

More desirably, removal of low molecular compounds by dialysis treatment, removal of water-insoluble fine particles by microfiltration treatment, and addition of a water-soluble organic solvent such as alcohol, acetone, dioxane to a hyaluronic acid-containing solution to precipitate and separate hyaluronic acid Is to dissolve hyaluronic acid to a concentration of 0.1 to 5 g / concentration again.

As a purification treatment method of hyaluronic acid in the present invention, an organic solvent aqueous solution containing hyaluronic acid, alumina,
The feature is that the treatment is performed using activated carbon or florisil. According to this method, a particularly stable effect is obtained.

The alumina, activated carbon, and florisil used here are:
It is used in a conventional purification treatment method, and its type, shape, and the like are not particularly limited. As a treatment method, a batch type in which these are added to a hyaluronic acid-containing liquid in the form of powder or granules and agitated, and a granulated or molded one in a packed tower or the like are filled, and then a hyaluronic acid-containing liquid is passed through the liquid. Combination and repetition are possible, but the packed tower system is more effective than the batch system. The pH at this time is preferably 6 to 8, and the SV is most effectively 0.1 to 0.2.

〔Example〕

 Next, examples of the present invention will be described.

Reference example (blank) Streptococcus equii FM-100
No. 027) was diluted to 50 with pure water (hyaluronic acid concentration: 1.10 g /), centrifuged, and hollow fiber type ultrafiltration was performed to remove bacterial cells and medium components.

Dissolve 15 g of sodium chloride in 500 ml of this hyaluronic acid-containing solution, pH 7
Then, the precipitate was precipitated with 750 ml of ethanol, washed with 100 ml of ethanol, and dried at 40 ° C. under vacuum to obtain 0.49 g of sodium hyaluronate. The analysis results are shown in Table 1.

Example 1 1.0 g of the partially purified hyaluronic acid obtained in Reference Example was
After dissolving in 700 ml, ethanol (300 ml) was added to obtain a 0.1% hyaluronic acid ethanol mixed solution.

On the other hand, a column having an inner diameter of 11.3 cm and a height of 15 cm was packed with 300 g of alumina (A-13-S) manufactured by Showa Denko KK, and thoroughly washed with 30% ethanol. 60 ml / h)
500 ml was added with 15 g of common salt, and precipitated and dried with 750 ml of ethanol to obtain 0.4 g of sodium hyaluronate. The analysis results are shown in Table 1. The same operation was performed ten times, and the results were all the values shown in Table 1.

Comparative Example 1 The same operation was performed as in Example 1 except that ethanol water was used (however, ethanol for precipitation was used).
This operation was performed 10 times, and the average value of the analysis results was shown.

In Comparative Example 1, hyaluronic acid having a quality equivalent to that of Example 1 was obtained in some cases, but there was no reproducibility and the results shown in Table 1 were averaged.

Example 2 The same procedure was performed as in Example 1 except that ethanol was replaced by acetone (however, ethanol for precipitation was used).

Example 3 0.1 g of partially purified hyaluronic acid in Reference Example was dissolved in 70 ml of water, and 30 ml of ethanol was added to obtain a 0.1% hyaluronic acid-ethanol mixed solution. After adding 3 g of salt thereto, 0.1 g of activated carbon (manufactured by Wako, special grade) was added, and the mixture was stirred at 30 ° C. for 1 hour.
Then, after performing a 0.2 μ filter treatment, 1.5 times the amount of ethanol was added to precipitate. The results of analysis of the precipitated hyaluronic acid are shown in Table 3. The same operation was performed 10 times, and the results were all the values shown in Table 3.

Comparative Example 2 The same operation was performed 10 times except that ethanol was replaced with water in Example 3 (but ethanol for precipitation was used).
The average of the results was taken as Comparative Example 2.

Example 4 0.2 g of partially purified hyaluronic acid of the reference example was dissolved in 140 ml of water, and then 60 ml of ethanol was added, and 12 g of sodium chloride was dissolved therein. The solution was passed through a column (1.5 cm in diameter) packed with 15 g of 60-100 mesh Florisil at SV = 1.

This solution was precipitated by adding 1.5 volumes of ethanol, and the analysis results of the precipitated hyaluronic acid are shown in Table 4. The same operation was performed 10 times, and the results were all the values shown in Table 4.

Comparative Example 3 The same operation was performed 10 times except that ethanol was changed to water in Example 4 (however, ethanol for precipitation was used). The average value of the results was used as Comparative Example 3.

Measuring method 1) Protein content: Purified hyaluronic acid was dissolved in 0.1N sodium hydroxide, and subjected to a Lowry method.

2) Nucleic acid: 26% of 0.1% sodium hyaluronate solution
The absorbance at 0 nm was measured.

3) Exothermic substance: Performed by colorimetric analysis using a Toxicolor system manufactured by Seikagaku Corporation.

〔The invention's effect〕

According to the present invention, high-quality hyaluronic acid can be stably obtained. The hyaluronic acid obtained by this method is expected to be used in medicine.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-60-133894 (JP, A) (58) Field surveyed (Int. Cl. ⁶ , DB name) C08B 37/08

Claims (1)

(57) [Claims] 1. A method for purifying hyaluronic acid, comprising treating an aqueous solution of an organic solvent containing hyaluronic acid with alumina, activated carbon, or florisil.