JPH02103204A - Purification of hyaluronic acid - Google Patents

Abstract

PURPOSE:To simply obtain high-quality hyaluronic acid useful for cosmetic humectants and medicines by bringing a hyaluronic acid-contg. solution into contact with silica gel. CONSTITUTION:A hyaluronic acid-contg. solution is pref., in advance, brought into contact with alumina, followed by contact with silica gel to accomplish the objective purification into high-purity hyaluronic acid free from pyrogens, proteins, nucleic acid, metallic impurities, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for purifying hyaluronic acid from a hyaluronic acid-containing liquid. In addition to cosmetic moisturizing agents, hyaluronic acid is also being used as a pharmaceutical in ophthalmology, orthopedics, dermatology, and other fields.

[Conventional technology and problems to be solved]

Conventionally, hyaluronic acid has been produced by extraction from animal tissue, for example, chicken crest on an industrial scale. Polymers that are easily molecularized and purified to a high degree of purity are expensive.

In order to solve these problems, in recent years, hyaluronic acid has been produced by fermentation.

It has been known for a long time that hyaluronic acid is produced by a group of bacteria of the genus Strezotococcus, and there are many reports (J. Woolcock, J. B., W.
oolccck), Journal of General Microbiology 85 372-375 197
6).

Compared to the extraction method, hyaluronic acid produced by the fermentation method is produced using fixed raw materials and using a fixed method, so the quality of the product is kept constant, so it has great industrial utility value.

However, polymer compounds exist as impurities in the fermentation liquid, and methods of separating and removing them to obtain a highly pure product have been studied.

For example, an adduct of a quaternary ammonium salt such as cetylpyridinium chloride and hyaluronic acid is formed to separate impurities, and then the impurities are adsorbed on a column of magnesium silicate such as 7EI Lysyl (Published Patent Publication No. 1986-
501471).

In addition, a method for purifying hyaluronic acid using a macroreticular anion exchange resin to remove pyrogens, proteins, etc. from a fermentation liquid has been disclosed (Japanese Patent Application Laid-Open No. 63-122).
Publication No. 93).

The present inventors also studied methods for purifying hyaluronic acid to a high purity, and proposed a method for bringing a hyaluronic acid-containing liquid into contact with alumina (Japanese Patent Application No. 144,728/1983).
issue).

In order to use hyaluronic acid as a pharmaceutical product, it is necessary to produce highly pure hyaluronic acid containing few pyrogens, proteins, nucleic acids, metal impurities, etc.

However, although the above-mentioned methods are effective in removing pyrogens, proteins, nucleic acids, etc. to some extent, they are insufficient in removing metal impurities. In other words, metal impurities such as magnesium, calcium, silicon, and iron are detected in insufficiently purified hyaluronic acid, and conventional methods are less effective at removing these metal impurities. Although it is large, there is a possibility that aluminum derived from alumina may be mixed in.

Therefore, it has less metal impurities and can be used as a medicine.
The development of a method to obtain high-quality products was awaited.

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

[Means to solve the problem]

As a result of various studies on methods for purifying high-purity hyaluronic acid that does not contain metal impurities, the present invention was developed by bringing a hyaluronic acid-containing liquid into contact with silica gel to adsorb metal impurities such as iron and aluminum contained in hyaluronic acid. It was discovered that this can be removed, and the present invention was completed.

It was also revealed that by contacting a hyaluronic acid-containing solution with alumina in advance and then contacting it with silica gel, highly pure hyaluronic acid containing no pyrogens, proteins, nucleic acids, metal impurities, etc. could be purified. .

The present invention provides the following steps: 1. A method for purifying hyaluronic acid, which comprises bringing a hyaluronic acid-containing liquid into contact with silica gel; This is a method for purifying acids.

The silica gel used in the present invention is produced by decomposing sodium silicate with an inorganic acid, washing the solidified product with water, and removing impurities.
Although it is manufactured by drying, this manufacturing method is not necessarily used.

Silica gel is used for a variety of purposes, including numerical dehumidification and drying, a desiccant agent for gas and air, dehydration of organic solvents, and general purification. Depending on the application, shape, particle size, pore size,
Different grades of surface area and slurry are commercially available. In the present invention, the silica gel used is not limited by such physical properties.

However, a particle size of 30-200μ is preferred. If the particle size is smaller than 60μ, the operability of removing silica gel will be poor, and it will be difficult to perform chromatography by filling a column due to clogging, etc.If it exceeds 200μ, the removal effect of metal impurities will be poor. I feel sick.

Furthermore, the silica gel used is preferably of a grade that has a low content of metal impurities and also has a low elution rate.

The hyaluronic acid-containing liquid used in the present invention can be extracted from animal tissues or manufactured by fermentation, but in order to stably manufacture high-quality products at industrially low cost, It is preferable to use fermentation methods.

Hyaluronic acid produced by fermentation can be obtained by known methods using bacteria such as Strezotococcoa.

The bacterial strain used in the fermentation method is a microorganism with hyaluronic acid-producing ability such as the genus Strezotococcus isolated from nature, or Streptococcus equi FM-100 (microorganism) described in JP-A-63-123392. Kenbokuyori No. 9
Mutant strains that stably produce hyaluronic acid with high yield, such as No. 027), are preferred.

Microorganisms capable of producing hyaluronic acid are aerobically cultured in a medium containing carbon sources such as glucose and sucrose, nitrogen sources such as hefton, polypeptone, and yeast extract, vitamins, and inorganic salts. After diluting the culture solution to a concentration of hyaluronic acid of 1 to 5 g/l of skin, it is sterilized using known methods such as sterilization by centrifugation, sterilization by filtration,
It is desirable to use a solution that has been sterilized by a method such as sterilization using a flocculant, carbon, celite, or the like.

Further, low-molecular compounds are removed by dialysis treatment, water-insoluble fine particles are removed by precision filtration treatment, and hyaluronic acid is precipitated and separated by adding a water-soluble organic solvent such as alcohol, acetone, or dioxane to the hyaluronic acid-containing solution, and then 0. 1~
It is possible to use a solution that has been subjected to an operation such as dissolving hyaluronic acid to a concentration of 5 g/l.

When producing hyaluronic acid that can be used as a pharmaceutical product, it is desirable to perform alumina treatment after the above treatment.

The alumina used in the present invention is so-called aluminum oxide, and generally aluminum hydroxide is dehydrated at high temperature.
Although it is manufactured by firing, this manufacturing method is not necessarily used.

Alumina is classified into fine grains (10μ or less), regular grains (40-60μ), and coarse grains (70μ or more) depending on the particle size. Commercially available alumina varies depending on its alumina content or the amount of impurities it contains, compared to general alumina grades with a purity of about 90 to 99 degrees, as well as those with a purity of 99.99% or higher, which are called high-purity alumina, and alumina. The sodium oxide content in K is 0.1% compared to the usual 0.3-0.4% K.
There is something called low soda alumina that has been refined to less than %. There is also activated alumina which has a large surface area of 5050-400 per gram and has a high adsorption capacity, but the alumina used in the present invention is not limited to the above.

Regarding the mineral morphology of α, β, and γ, and the shape of particles such as honeycomb bodies, granules, and spherical shapes, any of them can be used in the present invention.

When performing alumina treatment on a hyaluronic acid-containing solution, the - of the aqueous solution is 3 to 10, especially 6 to 9, the temperature is 0 to 40°C, and the concentration of hyaluronic acid is 0.1 to 5! l/1 especially 0.5~29
/l is good.

There are two methods for alumina treatment: adding powdered or granular alumina to a hyaluronic acid-containing liquid and stirring it batchwise; or filling a packed tower with granular or molded alumina and then passing the hyaluronic acid-containing liquid through it. Although liquid treatment methods, combinations, and repetitions thereof are possible, one treatment is usually sufficient depending on the selection of treatment conditions.

The packed column method is more effective than the batch method, but
When processing a hyaluronic acid-containing liquid by filling a packed tower with alumina, the bed height of the packed tower and the linear velocity of the hyaluronic acid-containing liquid must be considered depending on the particle size of the alumina used. Therefore, the processing speed is preferably s v = 0.1 to 2 in consideration of column clogging and processing efficiency.

When treating a hyaluronic acid-containing liquid with silica gel, the temperature of the hyaluronic acid solution is 3-12, the temperature is 0-40℃, and the concentration of hyaluronic acid is 0.1-5 g/l. It will rise and the operability will deteriorate, -
When the number exceeds 12, decomposition of hyaluronic acid becomes a problem.

When the temperature exceeds 40°C, hyaluronic acid decomposes and its molecular weight decreases.

If the concentration of hyaluronic acid is less than 0.1 g/II, the treatment efficiency will be poor, and if it exceeds 59/l, the viscosity will become high and operation will become difficult.

The treatment method of the present invention includes a method of adding silica gel to a hyaluronic acid-containing liquid and stirring it batchwise, a method of filling a packed tower and passing the hyaluronic acid-containing liquid through it, and combinations and repetitions thereof. Although it is possible, a method using a packed column is preferable, and the number of times of treatment depends on the selection of conditions, and one treatment is sufficient.

When processing a hyaluronic acid-containing liquid by filling a packed tower with silica gel, the bed height of the packed tower and the linear velocity of the hyaluronic acid-containing liquid must be considered, depending on the particle size of the silica gel, the concentration of hyaluronic acid, etc. However, the processing speed is 8 V.
=0.1-3 is preferable.

The amount of silica gel to be used relative to the amount of hyaluronic acid needs to be determined depending on the content of metal impurities in hyaluronic acid, but normally, when refining 1 g of hyaluronic acid, 10 g - 300.9 g of silica gel is required.

When treating a hyaluronic acid-containing liquid with alumina or silica gel, the treatment effect may be improved if an organic solvent that mixes with water is dissolved in the hyaluronic acid-containing liquid.

Examples of organic solvents that can be mixed with water include alcohols such as methanol and ethanol, and acetone, which are dissolved in a concentration of 5 to 50%.

If it is less than 5%, the effect will be small, and if it exceeds 50%, the viscosity of the liquid will increase and hyaluronic acid may precipitate.

The hyaluronic acid-containing liquid treated with silica gel can be adjusted to have a pore size of 0.2 to 1 μm or dried, if necessary.

〔Example〕

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

Reference Example Culture medium 151 cultured using Streptococcus equi FM-100 (Feikoken 1, No. 9027) was diluted to 501 with pure water (hyaluronic acid concentration 1.10 g/l),
Cells and medium components were removed by centrifugation and hollow fiber ultrafiltration.

15 g of common salt was dissolved in this hyaluronic acid-containing liquid 500 Ilt, and after adjusting the pH to 7, it was precipitated with 750 M ethanol, washed with 100 M ethanol, and vacuum-dried at 40° C. to obtain sodium hyaluronate of 0.499. The analysis results are shown in Table 1.

Example 1 500 wLt of the hyaluronic acid-containing liquid obtained in Reference Example 1, excluding the bacterial cells and culture medium components, was placed in a glass column with an inner diameter of 5 mm and a height of 30 mm and an activated alumina for chromatography manufactured by Wako Tsumugi Yakuhin Co., Ltd. (3 mm).
00 mesh) was filled, and after thorough washing with pure water, the solution was passed through at 5v=0.3 (45m/hour). The column-passing liquid was packed in the same column with 1501 parts of Wako Tsumugi Pharmaceutical Co., Ltd.'s Silica Ru Wakodel Q-50, and the voltage was set at 8V = 0.6 (9
0+z/h).

500 WLt of this column-passing liquid was collected, 15 g of common salt was added, the p# was adjusted to 7, precipitated with 750 v of ethanol, washed with 1001 ethanol, and vacuum-dried at 40°C.
0.40.9 of sodium hyaluronate was obtained. The analysis results are shown in Table 1.

Comparative Example 1 In Example 1, 50011Lt of alumina column passing liquid was
A similar operation was performed without silica gel treatment to obtain sodium hyaluronate 0.47, P.

The analysis results are shown in Table 1.

Example 2 25rnt of cetylpyridinium chloride 10% solution in 0.15M sodium chloride was added to 500ml of the hyaluronic acid-containing liquid from Example 1, from which the bacterial cells and medium components had been removed, and the precipitate was collected by centrifugation. The same operation as in Example 2 was performed except that the silica gel column treatment was not performed. The resulting 0.69 g of hyaluronic acid (IJ) was dissolved in 50 DU of 6% saline.

This solution was passed through a 20 El column (diameter 1.5 cm) packed with 70 lysyl of 60-100 mesh at SV:1.

This solution was desalted using a hollow fiber with a molecular weight cut off of 100,000, and then placed in a column vertically packed with 150 silica del Wacodel Q-50 manufactured by Wako Tsumugi Co., Ltd. at SV = 0.
6 (90 mt 7 o'clock) and stop the liquid flow.

Collect this column-passing liquid and add 15 g of salt.
After adjusting the pH to 7, precipitation was performed with 75 CIIILt of ethanol, washed with 100 ethanol meters, and vacuum dried at 40°C to obtain 0.38 g of sodium hyaluronate. The analysis results are shown in Table 2.

Comparative Example 2 Example 6 100mz ethanol was added to 400ml of the hyaluronic acid-containing liquid from Example 1 except for the bacterial cells and medium components to make it sooty.
After mz, alumina treatment and silica gel treatment were performed in the same manner. After adding IFM salt to this treatment solution, 0
．． Precise filtration was performed using a membrane filter with a pore size of 2 microns, and precipitation was performed with 75ON ethanol, washed with 100ml of ethanol, and vacuum-dried at 40°C. The amount of sodium hyaluronate obtained was 0.37 g. The analysis results of this sodium hyaluronate were as follows.

Protein content Nucleic acid Pyrogenic substances Metal impurities Aluminum Calcium Magnesium Iron Silicon 1 ppm or less ppm Not detected Not detected Not detected Not detected Not detected Not detected 5.1 P9 Measurement method 1) Protein content: Purified hyaluronic acid was added to 0.1 ppm. It was dissolved in IN sodium hydroxide and carried out by the Lauseau method.

2) Cedar Acid The absorbance of a 0.1% sodium hyaluronate solution at 260 nm was measured.

6) Pyrogen: This was carried out by comparative one-color analysis using a Toxicolor system manufactured by Seikagaku Corporation.

4) Metal impurities: Based on emission analysis.

〔Effect of the invention〕

According to the present invention, high quality hyaluronic acid can be produced by a simple method. In addition, the hyaluronic acid obtained by this method is expected to have pharmaceutical applications.Patent applicant: Denki Kagaku Kogyo Co., Ltd.

Claims (1)

[Scope of Claims] 1. A method for purifying hyaluronic acid, which comprises bringing a hyaluronic acid-containing liquid into contact with silica gel. 2. The method for purifying hyaluronic acid according to claim 1, wherein the hyaluronic acid-containing liquid is brought into contact with alumina in advance.