JP2893451B2 - Method for producing high molecular weight hyaluronic acid - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a high-molecular-weight hyaluronic acid from a hyaluronic acid-containing raw material such as a chicken crown in high purity and high yield.

(Prior Art) Hyaluronic acid is a kind of complex polysaccharide composed of amino sugar and uronic acid. Widely distributed in animal tissues, especially mesenchymal tissues, vitreous, humorous body, umbilical cord, synovial fluid, pleural fluid,
It is abundantly contained in skin, chicken cockscomb (so-called crow), and in animal tissues, it is a binding substance that gels as a free acid and salt to fill cells and tissues. Hyaluronic acid, which has properties such as viscosity, water retention, and lubricity, is in great demand as a pharmaceutical or cosmetic base material, has high viscosity,
High purity is required.

Conventionally, to obtain hyaluronic acid, a method is known as a basic method in which a raw material is extracted with water, and the resulting solution is precipitated with cetylpyridinium chloride, dissolved in a sodium chloride solution, and then reprecipitated with ethanol. I have.

In addition, in order to produce high-quality mucopolysaccharides in good yield and in large quantities, it is necessary to carry out pretreatment of appropriate raw materials. Therefore, many methods have been proposed. This is described in detail in the specification of Sho 62-273194. For example, a method of subjecting a connective tissue to heat treatment at about 70 to 130 ° C. in its original form, followed by proteolytic enzyme treatment, and usual treatment (Japanese Patent Publication No. 609042), a method in which a cockscomb is kept in its original form or thinly After heating at 35-65 ° C and performing protease treatment,
Hereinafter, a method of treating with a conventional method (Japanese Patent Publication No. 61-8808) and a method of pasting a hyaluronic acid-containing raw material that has been previously heat-treated into a paste and treating with a protease (JP-A-60-24)
No. 194).

Each of these methods is a simple and efficient method for mass production, but the hyaluronidase mixed in the raw material and the production process acts to reduce the molecular weight of hyaluronic acid, Therefore, there is a problem that the quality is deteriorated.

(Problems to be Solved by the Invention) On the other hand, there is an ultrafiltration membrane treatment as one of the means for purifying polysaccharides. This is to remove low molecular impurities such as inorganic salts coexisting in the solution prior to treating the polysaccharide with a precipitant such as ethanol, thereby obtaining a high-purity polysaccharide.

However, the ultrafiltration membrane treatment of the polysaccharide solution requires high pressure because the polysaccharide solution is generally very viscous. There are drawbacks such as a significant decrease in the membrane permeation rate with an increase in the concentration rate, and in order to prevent this, it is necessary to dilute the treatment solution with a large amount of water in advance and frequently wash the membrane. Therefore, it is not always an efficient method. In addition, ultrafiltration membranes (pore diameter is usually 0.001
As a filtration membrane having a larger pore diameter than that of a microfiltration membrane, there is a so-called microfiltration membrane. This is usually 0.05-10 μm
Although it has a small pore size, it also allows the passage of a considerable amount of high-molecular substances, so that it is often used for the germ reduction of culture solutions, etc., and it has never been used in the purification of polysaccharides. I didn't.

(Means for Solving the Problems) In view of the above-mentioned drawbacks in the ultrafiltration membrane treatment, the present inventors conducted various studies and found that a filtration material having a pore size of 0.05 to 1 μm (hereinafter referred to simply as MF membrane) Has been found to be able to solve the above problem.

That is, the present invention is a method for producing high molecular weight hyaluronic acid, which comprises contacting a solution containing hyaluronic acid with a filter medium having a pore size of 0.05 to 1 μm and treating a non-permeate by a conventional method.

 Hereinafter, the present invention will be described in detail.

A connective tissue containing a large amount of hyaluronic acid is used as a raw material. Examples of such raw materials include a cockscomb, skin, ligament ligament, synovial fluid, umbilical cord, vascular wall, cartilage, vitreous body, and the like. Raw materials are
As the number of days after collection increases, hyaluronidase (hyaluronic acid degrading enzyme) mixed in the raw material acts to lower the molecular weight of hyaluronic acid.
After collection, it is desirable to use immediately or to freeze and store.

These raw materials are, for example, in the form of about 70 to 130 ° C.
A water dispersion is prepared by a known method such as a method of mincing after heat treatment (Japanese Patent Publication No. 609042). In addition, as an aqueous dispersion of the raw material, the above-mentioned rapidly frozen raw material is cut into pieces using a meat chopper or the like without being thawed, and further converted into a paste using a homogenizer or an ultrafine crusher (such as a supermass colloider). And this is 70
If a sterilized product is used at ~ 140 ° C for 1 to 60 seconds, the action of hyaluronidase in the extraction step is prevented, and complete inactivation is achieved, so that a high molecular weight hyaluronic acid which is not available in the conventional method can be obtained. This is particularly advantageous.

In the present invention, a solution containing hyaluronic acid obtained from the above-mentioned raw materials by a conventional protease treatment or the like is subjected to MF membrane treatment after removing solids by centrifugation or filtration or the like. Prior to membrane treatment, if protease is added to the filtrate again to decompose the mixed proteinaceous impurities, the subsequent purification efficiency can be further improved.
As the conditions for the MF film treatment, a hyaluronic acid concentration in the treatment solution of 0.005 to 0.2% and a temperature of 4 to 50 ° C. are generally employed.

Examples of the filter material applicable to the present invention include polysulfone-based, polypropylene-based, cellulose acetate-based, polyvinyl alcohol-based, and polyethylene-based MF membranes, as well as known membrane modules such as metal, glass, and ceramic-based sintered filter media. Can be used, but the pore size is 0.05-1μ
m. That is, a filtration material having a filtration rate lower than the lower limit of this range has a low permeation speed and severe clogging, so that the treatment liquid is diluted with a large amount of water, an extremely high pressure is required for the treatment, and the filtration material is frequently washed. is necessary. Conversely, in the case of a filter medium exceeding the above range, hyaluronic acid permeates through the filter medium, so that the yield of the product is remarkably reduced. As a filter material satisfying such conditions, specifically, NSF-M manufactured by Kuraray Co., Ltd.
8202 (pore diameter 0.3 μm) and the like.

The hyaluronic acid fraction obtained as a non-permeated liquid by the treatment with such a filtering material can be dried as it is or after further undergoing a precipitation treatment by a conventional method to obtain a powder product. As the precipitation treatment, for example, a treatment was carried out such that a precipitate formed by adding a quaternary ammonium salt (for example, cetylpyridinium chloride) to the permeate as it is or as necessary was redissolved in a saline solution. Thereafter, ethanol is added so that the final concentration becomes 50 to 85% (V / V).

(Example) Example 1 A cockscomb collected immediately after slaughter was rapidly frozen with liquid nitrogen using a spray-type freezing apparatus. 1 kg of the frozen cockscomb was chopped by a meat chopper. 2 kg of flake ice was added to this, and the mixture was pasted with a supermass colloider (manufactured by Masuko Sangyo Co., Ltd.). The obtained paste was sterilized at 130 ° C. for 3 seconds and immediately cooled immediately. Add 2 l of water and adjust the pH to 7.0
Then, 30 mg of actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) was added as a protease, the mixture was incubated at 50 ° C. for 3 hours, and the obtained solution was filtered. The filtrate obtained by repeating the above operation three times was collected to obtain 9.5 l.

The filtrate is subjected to a precision filtration device MR manufactured by ULVAC SERVICE CO., LTD.
61-0015 (MF membrane NSF-M8202 manufactured by Kuraray Co., Ltd. (pore size 0.3μ)
m) use), temperature 30 ℃, liquid supply pressure 0.5 kg / cm ²
Then, the circulation treatment was performed until the treatment liquid became 1/3 of the original. Inner liquid
2.1 was collected, and the inside of the module was further washed with 2 l of pure water.
This was combined with the above internal solution. 1.8 l of a 5 (w / v)% cetylpyridinium chloride solution was added thereto.

The resulting precipitate was collected by filtration and redissolved in a 0.5 M sodium chloride solution. Ethanol was added to this solution to a concentration of 60 (v / v)%, and the resulting precipitate was collected by filtration and dried.

Example 2 10 μl of Neutrase (manufactured by Novo Industries Japan) was further added to 9 l of the filtrate after actinase E treatment in Example 1, and the mixture was kept at 50 ° C. for 1 hour. After cooling the reaction solution, twice the volume of pure water was added, and the same MF as in Example 1 was used.
It was subjected to membrane treatment. Concentration was performed until the internal solution was reduced to 3 liters, followed by freeze-drying to obtain a dried product.

Comparative Example 1 5 liters of the filtrate after actinase E treatment in Example 1
0.6 l of a (w / v)% cetylpyridinium chloride solution was added, and the resulting precipitate was collected by filtration and redissolved in a 0.5 M sodium chloride solution. Ethanol was added to this solution to a concentration of 60 (v / v)%, and the resulting precipitate was collected by filtration and dried.

Comparative Example 2 Ultrafiltration membrane MOLSEP fiber manufactured by Daicel Chemical Industries, Ltd., FC-01 FUS1541 (molecular weight cut off) as a membrane module
150,000) except that a filtration device HSD-05A equipped with the same treatment as in Example 2 was used, and the obtained non-permeate was dried.
A powdery hyaluronic acid product was obtained.

Yield of hyaluronic acid obtained in Examples and Comparative Examples,
The purity and molecular weight are shown in Table 1.

Note 1) Yield per kg of raw material (g) 1) Calculated by multiplying the uronic acid amount measured by the carbazole sulfate method by 2.06.

3) Calculated from the intrinsic viscosity measured using an Ubbelohde viscometer.

(Effect of the Invention) The filtering material having a pore size of 0.05 to 1 μm used in the present invention is expected to pass the same molecule based on the molecular weight of hyaluronic acid (1 to 2 million daltons). As shown in FIG. 1, there is almost no decrease in the permeation rate due to clogging during processing, which is remarkable in the conventional ultrafiltration membrane, as shown in FIG. 1, and low molecular impurities such as inorganic salts. And proteins were efficiently removed, and the purity was found to be significantly improved.

Moreover, more surprisingly, it is thought that the above treatment promptly removes metal ions and the like derived from tissues and suppresses the degradation of hyaluronic acid mediated by these treatments. It was found that a very high molecular product was obtained. This is clear from the comparison of the molecular weights of the examples and comparative examples shown in Table 1.

[Brief description of the drawings]

FIG. 1 shows the concentration ratio when the extract of hyaluronic acid of Example 1 obtained by treating a cockscomb with a protease (actinase E) was treated with the MF membrane used in the present invention and a conventional ultrafiltration membrane. It is a graph which shows the change of the transmission speed (vertical axis) accompanying the change of (horizontal axis). The permeation rate is the unit area of the filtration membrane,
It is the amount of transmission per hour. The solid line (indicated by a circle) in the figure is an MF membrane [NSF-M820 manufactured by Kuraray Co., Ltd.] used in the examples of the present invention.
2), the dotted line (x mark) shows the change in the control ultrafiltration membrane (MOLSEP fiber, FC-01 FUS1541 manufactured by Daicel Chemical Industries, Ltd.), respectively.

Claims (1)

(57) [Claims] 1. A solution containing hyaluronic acid having a pore size of 0.05 to
A method for producing high-molecular-weight hyaluronic acid, comprising contacting a non-permeate with a 1 μm filter medium by a conventional method.