JP2731545B2 - Method for purifying hyaluronic acid - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for purifying hyaluronic acid from a hyaluronic acid-containing solution. 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 been known for a long time that hyaluronic acid is produced by a group of bacteria belonging to the genus Streptococcus, and there have been many reports (JBWoolcock, Journal of General Microbiology 85 372-).
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, a method of forming an adduct of a quaternary ammonium salt such as cetylpyridinium chloride and hyaluronic acid to separate impurities and further adsorbing the impurities to a column of magnesium silicate such as florisil (Japanese Patent Publication No. 62-50)
No. 1471).

Also disclosed is a method for purifying hyaluronic acid by using a macroreticular type anion exchange resin to remove pyrogens, proteins, and the like from the fermentation broth (JP-A-63-12293).
No.).

The present inventors have also studied a method of purifying hyaluronic acid with high purity, and have proposed a method of bringing a hyaluronic acid-containing solution into contact with alumina (Japanese Patent Application No. 63-144728).

In order to use hyaluronic acid as a medicinal product, pyrogenic substances,
It is necessary to produce high-purity hyaluronic acid with few proteins, nucleic acids, metal impurities and the like.

However, in the above-mentioned methods, pyrogenic substances, proteins, nucleic acids, and the like have an effect of removing to some extent, but the removal of metal impurities is insufficient. That is, metal impurities such as magnesium, calcium, silicon, and iron are detected in incompletely purified hyaluronic acid, and the conventional method has little effect of removing these impurities.

In the method in which the hyaluronic acid-containing liquid is brought into contact with alumina, the effect of removing these metal impurities is large, but aluminum derived from alumina may be mixed.

Therefore, development of a method for obtaining a high-quality product with a small amount of metal impurities that can be used as a pharmaceutical has been awaited.

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

[Means for solving the problem]

The present invention has conducted various studies on a method for purifying high-purity hyaluronic acid containing no metal impurities.As a result of contacting the hyaluronic acid-containing solution with silica gel, metal impurities such as iron and aluminum contained in hyaluronic acid are adsorbed. It has been found that the present invention has been removed, and the present invention has been completed.

In addition, it has been clarified that by contacting a hyaluronic acid-containing solution with alumina beforehand and then with silica gel, high-purity hyaluronic acid containing no exothermic substances, proteins, nucleic acids, metal impurities, etc. can be purified.

The present invention provides: 1. a method for purifying hyaluronic acid, which comprises contacting a hyaluronic acid-containing liquid with silica gel; and 2. the hyaluronic acid according to claim 1, wherein the hyaluronic acid-containing liquid has been brought into contact with alumina in advance. Is a purification method.

The silica gel used in the present invention is produced by decomposing sodium silicate with an inorganic acid, washing the coagulated product with water, removing impurities, and then drying, but is not necessarily limited to this production method. .

Silica gel is used for various purposes such as general dehumidification drying, gas / air drying agent, dehydration of organic solvent, general purification and the like. Depending on the application, grades having different shapes, particle sizes, pore diameters, surface areas, and slurry pHs are commercially available. Silica gel used in the present invention,
It is not limited by such physical properties.

However, the particle size is preferably 30-200μ. Particle size 3
If it is smaller than 0μ, the operability such as the removal operation of silica gel becomes poor, and it becomes difficult to pack the column and perform chromatographic treatment in terms of clogging, etc.
The effect of removing metal impurities becomes worse.

Further, the silica gel to be used is preferably a grade having a small content of metal impurities and a small elution.

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 may be 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 so that the hyaluronic acid has a concentration of 0.1 to 5 g / concentration, known methods, for example, sterilization by centrifugation, sterilization by filtration, sterilization by a flocculant, carbon, celite etc. It is desirable to use a solution that has been disinfected in step 1.

Furthermore, removal of low molecular compounds by dialysis treatment, removal of water-insoluble fine particles by microfiltration treatment or addition of a water-soluble organic solvent such as alcohol, acetone and dioxane to a solution containing hyaluronic acid, precipitation and separation of hyaluronic acid, and then 0.1
A liquid obtained by performing an operation of dissolving hyaluronic acid to a concentration of about 5 g / concentration can be used.

In the case of producing hyaluronic acid that can be used as a pharmaceutical, it is desirable to carry out the above treatment and then carry out an alumina treatment.

The alumina used in the present invention is so-called aluminum oxide, which is generally produced by dehydrating and firing aluminum hydroxide at a high temperature, but is not necessarily limited to this production method.

Alumina is classified into fine particles (10μ or less), normal particles (40-60μ), and coarse particles (70μ or more) according to the particle size. Depending on the alumina content or the content of impurities, commercially available alumina has a purity of 99.99% or more compared to a general alumina pure content of about 90% to 99%. There is a so-called low soda alumina having a sodium oxide content of 0.1% or less, which is usually 0.3-0.4%. Also, the surface area per 1g
Although there is activated alumina having a large adsorption capacity of 50 to 400 m ² , the alumina used in the present invention is not limited to the above.

Regarding the mineral forms of α, β, and γ and the shapes of grains such as honeycomb bodies, granules, and spheres, any of them can be used in the present invention.

In performing the alumina treatment of the hyaluronic acid-containing liquid, the pH of the aqueous solution is preferably 3 to 10, particularly 6 to 9, the temperature is 0 to 40 ° C, and the concentration of hyaluronic acid is 0.1 to 5 g / particularly 0.1 to 2 g /.

As the alumina treatment method, powdery or granular alumina is added to the hyaluronic acid-containing liquid, and the mixture is stirred in a batch type. Alternatively, the granular or shaped alumina is filled in a packed tower or the like, and then the hyaluronic acid-containing liquid is passed through. Although a liquid treatment method, and a combination or repetition thereof are also possible, one treatment is usually sufficient depending on the selection of treatment conditions.

Compared with the batch type, the packed tower method is more effective, but when treating the hyaluronic acid-containing liquid by filling alumina into a packed tower or the like, according to the particle size of the alumina used,
The bed height of the packed tower and the linear velocity of the liquid containing hyaluronic acid must be considered. Therefore, the processing speed is preferably SV = 0.1 to 2 in consideration of column clogging and processing efficiency.

In treating the hyaluronic acid-containing solution with silica gel, the pH of the hyaluronic acid solution is preferably 3-12, the temperature is 0-40 ° C, and the concentration of hyaluronic acid is preferably 0.1-5 g / hour and 0.5-2 g / hour.

If the pH of the hyaluronic acid-containing solution is less than 3, hyaluronic acid may be decomposed or the viscosity may increase, resulting in poor operability. Become.

If the temperature also exceeds 40 ° C., hyaluronic acid is decomposed and the molecular weight decreases.

When the concentration of hyaluronic acid is less than 0.1 g /, the treatment efficiency is inferior. When it exceeds 5 g /, the viscosity becomes high and the operation becomes difficult.

As the treatment method of the present invention, a hyaluronic acid-containing liquid
A method of adding silica gel and stirring with a batch type and a method of passing a hyaluronic acid-containing liquid after filling the packed tower,
Although a combination or repetition thereof is also possible, a method using a packed tower is preferable, and the number of treatments is sufficient by selecting one condition, and one treatment is sufficient.

When treating a hyaluronic acid-containing liquid by packing silica gel in a packed tower, the bed height of the packed tower and the linear velocity of the hyaluronic acid-containing liquid must be taken into account according to the particle size of the silica gel and the concentration of hyaluronic acid. No, but the processing speed is SV =
0.1 to 3 is preferred.

The amount of silica gel to be used relative to the amount of hyaluronic acid needs to be determined according to the content of metal impurities in hyaluronic acid. However, when 1 g of hyaluronic acid is purified, silica gel requires 10 g to 300 g.

When treating a hyaluronic acid-containing liquid with alumina or silica gel, dissolving an organic solvent mixed with water in the hyaluronic acid-containing liquid may improve the treatment effect in some cases.

Examples of the organic solvent to be mixed with water include alcohols such as methanol and ethanol and acetone, and these are dissolved to a concentration of 5 to 50%.

If it is less than 5%, the effect is small, and if it exceeds 50%, the viscosity of the solution becomes high and hyaluronic acid may be precipitated.

Hyaluronic acid-containing liquid treated with silica gel
If necessary, after performing pH adjustment and microfiltration using a membrane filter having a pore size of 0.2 to 1 μm, the product can be separated by an organic solvent precipitation method or the like, and then dried by a vacuum drying method or the like.

〔Example〕

 Next, reference examples and examples of the present invention will be described.

Reference example 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 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. Table 1 shows the analysis results.

Example 1 500 ml of a hyaluronic acid-containing liquid obtained by removing the cells and medium components obtained in Reference Example 1 was placed in a glass column having an inner diameter of 5 cm and a height of 30 cm, and activated alumina for chromatography (300 mesh) manufactured by Wako Pure Chemical Industries, Ltd.
After filling with 150 ml of water and washing thoroughly with pure water, SV = 0.3 (45 ml /
At the time). The same column was filled with 150 ml of Wako Pure Chemical Industries silica gel Wakogel Q-50 and passed through the column at SV = 0.6 (90 ml / hr). Liquid passing through this column
After collecting 500 ml and adding 15 g of sodium chloride to adjust the pH to 7, it was precipitated with 750 ml of ethanol, washed with 100 ml of ethanol, and dried in vacuo at 40 ° C. to obtain 0.40 g of sodium hyaluronate.
Table 1 shows the analysis results.

Comparative Example 1 The same operation as in Example 1 was carried out on 500 ml of the liquid passed through the alumina column without silica gel treatment, to obtain 0.47 g of sodium hyaluronate.

 Table 1 shows the analysis results.

Example 2 25 ml of a 10% solution of cetylpyridinium chloride in 0.15 M sodium chloride was added to 500 ml of the hyaluronic acid-containing solution from which the cells and the medium components of Example 1 had been removed, and the precipitate was collected by centrifugation. , And dissolved in 500 ml of 6% saline. 750 ml of ethanol was added for precipitation, and the precipitate was dissolved in 500 ml of 6% saline. 20 g of this solution,
Column filled with 60-100 mesh florisil (diameter
1.5 cm) at SV = 1.

This solution was desalted using a hollow fiber having a molecular weight cut off of 100,000, and then applied to a column packed with 150 ml of silica gel Wakogel Q-50 manufactured by Wako Pure Chemical Industries, Ltd., to give SV = 0.6 (90 ml / 90 ml).
At the time). Collect 500 ml of the liquid passed through the column, and add
5 g was added to adjust the pH to 7, followed by precipitation with 750 ml of ethanol, washing with 100 ml of ethanol, and vacuum drying at 40 ° C. to obtain 0.38 g of sodium hyaluronate. Table 2 shows the analysis results.

Comparative Example 2 The same operation was performed as in Example 2, except that the silica gel column treatment was not performed. Table 2 shows the result of analysis of the obtained 0.39 g of sodium hyaluronate.

Example 3 100 ml of ethanol was added to 400 ml of the hyaluronic acid-containing liquid excluding the cells and the medium components of Example 1 to make 500 ml, and then similarly treated with alumina and silica gel.
After adding 15 g of sodium chloride to this treatment solution, the solution was subjected to microfiltration with a membrane filter having a pore size of 0.2 μm, precipitated with 750 ml of ethanol, washed with 100 ml of ethanol, and washed with 40 ml of ethanol.
Vacuum dried at ℃. The resulting sodium hyaluronate is
0.37g. The analysis results of this sodium hyaluronate were as follows.

Protein content Not detected Nucleic acid Not detected Pyrogenic substance 5.1 Pg / mg Metal impurities Aluminum 1 ppm or less Calcium 1 ppm Magnesium Not detected Iron Not detected Silicon Not detected Measurement method 1) Protein content: purified hyaluronic acid, 0.1N water It was dissolved in sodium oxide, and was carried out by the Lowry method.

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

3) Exothermic substance: This was carried out by colorimetric analysis using a Toxicolor system manufactured by Seikagaku Corporation.

4) Metal impurities: Emission analysis.

〔The invention's effect〕

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

Claims (2)

(57) [Claims] 1. A method for purifying hyaluronic acid, comprising contacting a hyaluronic acid-containing liquid with silica gel. 2. The method for purifying hyaluronic acid according to claim 1, wherein the hyaluronic acid-containing liquid has been brought into contact with alumina in advance.