JP5758878B2 - Purification method of hyaluronic acid - Google Patents

Description

  The present invention relates to a purification method for hyaluronic acids, a purification agent and activated carbon used in the purification method, and a production method thereof.

  Hyaluronic acid is used as a medicinal product in ophthalmology, orthopedics, dermatology, etc. in addition to a moisturizing agent for cosmetics. Hyaluronic acid can be produced from extracts from animal tissues such as chicken crowns, vitreous bodies of cattle eyes, etc., but chondroitin sulfate etc. are mixed as contaminants, hyaluronidase contained in the tissues, etc. Therefore, it is easy to reduce the molecular weight by culturing microorganisms capable of producing hyaluronic acid, and producing hyaluronic acid from the culture solution (fermentation method) (Non-patent Document 1 and Patent Document 1).

  Since hyaluronic acid produced by an extraction method or a fermentation method contains proteins, pyrogens and the like as impurities, a method for obtaining a high-purity product by separating and removing these has been studied. In particular, the removal of impurities in the initial stage of production is expected to be developed as a method for obtaining a high-purity product that can be used as a pharmaceutical because it can reduce the load of the subsequent purification process. For example, exothermic substances, proteins, and the like are removed from the fermentation broth using, for example, a purification method in which hyaluronic acid is precipitated and separated from impurities by adding an organic solvent such as alcohol to the fermentation broth, or an anion exchange resin. A method for purifying hyaluronic acid is disclosed (Patent Document 2).

Journal of General Microbiology, 85, 372-375, 1976 Japanese Examined Patent Publication No. 4-12960 JP 63-12293 A

  However, in order to obtain high-purity hyaluronic acids that can be used as pharmaceuticals such as injection solutions on an industrial scale, (1) the removal of impurities at the initial stage has not been sufficiently performed, so the load on the subsequent process is large. (2) In addition to the complicated operation, there was a point where further improvement was desired such as a low recovery rate of hyaluronic acid.

  This invention is made | formed in view of the said situation, and it aims at providing the method for refine | purifying hyaluronic acid of high purity simply and with a high yield on an industrial scale. It is another object of the present invention to provide a purification agent, activated carbon, etc. used in the purification method and a method for producing them.

  In order to solve the above-mentioned object, the present inventors have conducted various studies on methods for efficiently separating and removing impurities from a hyaluronic acid-containing liquid and purifying high-purity hyaluronic acid simply and efficiently. It has been found that impurities can be efficiently adsorbed and removed by using activated carbon prepared using a treatment method, and the present invention has been completed.

  That is, according to the present invention, there is provided a method for purifying hyaluronic acid and / or a salt thereof, comprising a step of bringing a hyaluronic acid and / or a salt thereof and a hyaluronic acid solution containing impurities into contact with an activated carbon suspension. . According to this purification method, impurities can be efficiently adsorbed and removed by using activated carbon.

  That is, according to the present invention, the step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, the step of heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more, and the activated carbon suspension after the heating. There is provided a method for purifying hyaluronic acid, comprising a step of stirring and cooling a suspension, and a step of bringing a hyaluronic acid solution containing hyaluronic acid and impurities into contact with an activated carbon suspension after stirring and cooling. According to this purification method, impurities can be efficiently adsorbed and removed by using activated carbon pretreated in a state suitable for purification of hyaluronic acids.

  Further, according to the present invention, the step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, the step of heating the activated carbon suspension at 100 ° C. or more for 20 minutes or more, and the activated carbon suspension after the heating. And a method for producing a purification agent for hyaluronic acid, comprising the step of stirring and cooling the suspension. According to this production method, a purification agent for hyaluronic acid capable of efficiently adsorbing and removing impurities can be produced by pretreating activated carbon into a state suitable for purification of hyaluronic acid.

  Further, according to the present invention, the step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, the step of heating the activated carbon suspension at 100 ° C. or more for 20 minutes or more, and the activated carbon suspension after the heating. There is provided a method for pretreatment of activated carbon, comprising the step of stirring and cooling the suspension. According to this pretreatment method, the activated carbon can be pretreated to a state suitable for purification of hyaluronic acids.

  Further, according to the present invention, the step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, the step of heating the activated carbon suspension at 100 ° C. or more for 20 minutes or more, and the activated carbon suspension after the heating. A purification agent for hyaluronic acid is provided, comprising activated carbon produced by a method comprising stirring and cooling a suspension. By including activated carbon pretreated in a state suitable for purification of hyaluronic acids, this purification agent can efficiently adsorb and remove impurities.

BEST MODE FOR CARRYING OUT THE INVENTION

[Explanation of terms]
In the present specification, “hyaluronic acid” includes free hyaluronic acid and any hyaluronic acid salt that can be used within the range not impairing the object of the present invention (for example, but not limited to, sodium salt) Metal salts such as potassium salt, calcium salt and lithium salt, acid adducts such as hydrochloride, phosphate and citrate) and hydrates, and mixtures thereof. Here, hyaluronic acid refers to a high molecular weight polysaccharide in which disaccharide units in which N-acetyl-D-glucosamine and D-glucuronic acid are linked are repeatedly linked, and various salts are mainly composed of glucuronic acid moieties. This is the salt form. Hyaluronic acid is easy to expand into space due to the interaction between the foldable chain portion and the negative charge of the carboxyl group of the D-glucuronic acid portion, and can bind to a large amount of water to form a gel. Even at low concentrations, the intermolecular force is strong, so it has a relatively high viscosity. From such an action, it has, for example, a joint wetting action, a skin softening action and the like, and physiologically plays these roles.

  Among hyaluronic acids, sodium hyaluronate with a molecular weight of about 2 million Da is more effective for treating osteoarthritis of the knee, periarthritis, rheumatoid arthritis, etc. (Pharmacology and treatment, Vol. 22, No. 9, 289 (1994); Pharmacology and treatment, Vol. 22, No. 9, 319 (1994)). In addition, as an anti-adhesion agent after surgery, it is also known to have a medicinal effect in the dermatological and ophthalmological areas, and some are generally used clinically. When used as a pharmaceutical, it is desirable to use hyaluronic acids having an average molecular weight of 1 million or more. Furthermore, considering the availability and handling, hyaluronic acids having an average molecular weight of 1,000,000 to 5,000,000 Da are desirable as pharmaceuticals, and hyaluronic acids having an average molecular weight of 1,500,000 to 4,000,000 Da are particularly desirable. Further, such high molecular weight hyaluronic acids exhibit excellent effects due to their high moisturizing power even when used as cosmetics.

  As an injectable solution for dissolving hyaluronic acids as pharmaceuticals, water for injection, physiological saline, etc. are generally used for injection in which pH adjusting agents including buffers such as acids, alkalis and phosphates are added. A solution (for example, one recognized by the pharmacopoeia of each country) can be used as appropriate.

  These hyaluronic acids may be produced by an extraction method extracted from animal tissue or may be produced by a fermentation method obtained by fermentation using a hyaluronic acid-producing microbial strain. However, those extracted from animal tissues are relatively rich in impurities such as other mucopolysaccharides and have a small molecular weight, so it is desirable to use those obtained by fermentation. In an example of a fermentation method suitable for the present invention, hyaluronic acids can be obtained by a known method using, for example, a Streptococcus microorganism.

  As the solution used in the method of the present invention, it is desirable to use the culture solution as it is. Naturally, a solution sterilized by a known method such as centrifugation or filtration may be used. Depending on the case, operations such as removal of low molecular weight compounds by dialysis treatment, removal of water-insoluble fine particles by microfiltration treatment, etc. may be performed, and a solution obtained by precipitating hyaluronic acid by adding a water-soluble organic solvent such as alcohol is used. May be used. Moreover, you may use what was processed with the alumina etc.

  “Streptococcus” in the present specification includes any bacterium belonging to the genus Streptococcus capable of producing hyaluronic acid and its mutant strain. In particular, Streptococcus ex FM-100 described in Japanese Patent Application Laid-Open No. Hei 10 (No. 9027), and Streptococcus ex FM-300 described in Japanese Patent Application Laid-Open No. 2-23489 (No. 90). It is desirable to use a mutant that stably produces hyaluronic acid with a high yield such as No. 2319). Examples of bacteria belonging to the genus Streptococcus suitable for the production of hyaluronic acid include, but are not limited to, for example, Streptococcus equi, Streptococcus zooepidemicus, Streptococcus zooepidemicus, (Streptococcus equisimilis), Streptococcus dysgalactiee, Streptococcus pyogenes, and mutants thereof.

  Unless otherwise specified, “average molecular weight” in the present specification refers to the viscosity average molecular weight when indicating the average molecular weight of hyaluronic acids. The viscosity average molecular weight can be determined by a method usually performed by those skilled in the art. Preferably, it can be determined by a measurement method generally used in the pharmacopoeia of each country, and more preferably by a measurement method used in the Japanese pharmacopoeia. For example, the present invention is not limited to this. For example, the average molecular weight can be obtained by the following equation using the intrinsic viscosity [η].

Formula 1

  In this specification, “activated carbon” refers to porous carbon that is commercially available and generally used for separation, removal, and purification. It is derived from raw materials such as coal or wood, activated chemicals, or water vapor. It is not limited to manufacturing methods, such as activation. Furthermore, it is not limited to shapes such as powdered activated carbon and granular activated carbon, and properties such as dry and wet. Although it is not limited to this in the method of this invention, etc., it is desirable to use activated carbon derived from wood. Similarly, it is desirable to use one produced by steam activation. Similarly, it is desirable to use powdered activated carbon.

  Specific examples of the activated carbon include, but are not limited to, for example, Tsurumi Coal GL-30S, Tsurumi Coal HC-30S [more granular, manufactured by Tsurumi Co., Ltd.], white birch WH5C8 / 32, white birch LH2C20 / 48, white birch WH2C8 / 32SS [above, granular, manufactured by Takeda Pharmaceutical Co., Ltd.], white birch A, white birch P, purified white birch, characteristic white birch, carborafine [over, powdered, manufactured by Takeda Pharmaceutical Co., Ltd.], Hokuetsu SD, Hokuetsu GSA [above, powdered, manufactured by Hokuetsu Carbon Industry Co., Ltd.], silver A, flower F, snow A [above, powdered, manufactured by Daisan Industry Co., Ltd.], and the like.

  The term “impurities” as used herein refers to substances other than hyaluronic acids, water and other solvent components, inorganic salts, particularly substances that can give disadvantages when using hyaluronic acids as final products (such as pyrogenic substances). That means. Examples of main impurity sources include those derived from tissues, microorganisms, or culture solutions (medium) in the production stage of hyaluronic acids, or those mixed in subsequent purification stages. Examples of impurities in the present specification include, but are not limited to, tissues or cells, proteins, nucleic acids, polysaccharides, low molecular compounds, endotoxins, and the like. The tissue or fungus body as an impurity is not limited to this, but a tissue piece derived from a tissue as an extraction raw material used in the extraction method or the fungus body or fungus body of a microorganism used in the fermentation method, respectively. Includes pieces. Proteins as impurities include, but are not limited to, proteins derived from the tissues and fungi, and proteins mixed in the post-production process. Endotoxins as impurities include, but are not limited to, lipopolysaccharides derived from the above bacteria.

  The “low molecular weight compound” in the present specification refers to a compound having a relatively small molecular weight as compared to hyaluronic acids. For example, the molecular weight is not limited to 2000 Da or less, or a molecular weight of 1000 Da, Alternatively, it refers to a compound having a molecular weight of 500 Da or less. Such low molecular weight compounds include various amino acids, organic acids (for example, lactic acid), sugars (for example, glucose) and the like.

  The “aqueous liquid” in the present specification refers to a liquid mainly composed of water (solvent), and includes liquids containing various salts, buffers, small amounts of organic solvents, and the like. As the aqueous liquid, any liquid can be used as long as the object of the present invention is not impaired. However, water is preferable from the viewpoint of preventing unnecessary substances from being mixed when using the hyaluronic acid as a final product.

  In the present specification, “stirring cooling” refers to cooling a target liquid by continuing stirring. Here, the temperature achieved after stirring and cooling is defined as a cooling completion temperature.

“Clean environment” in this specification refers to the production of products that are extremely unlikely to contaminate products with impurities such as bacteria and endotoxins derived from the environment, especially products that are equivalent to pharmaceuticals (bacteria and endotoxins) by air conditioning management. It refers to the environment.
As an example of the clean environment, for example, an environment in which the target substance is regulated as follows can be given.

  As used herein, “removal” includes not only removing a target substance completely, but also partially removing (reducing the amount of the substance). As used herein, “purification” includes removing any or specific impurities.

  In this specification, “purifying agent” refers to an agent used for purification, such as an adsorbent, and the expression “purifier for substance A” refers to an agent for purifying substance A. Shall mean. The purification agent may be in the form of a purification composition. Similarly, the removal agent, the separation agent, and the like refer to a drug used for removal and a drug used for separation, respectively, for example, a term including an adsorbent, a carrier, etc. The composition may be in the form of a composition for separation or a composition for separation.

  Each numerical range in the present specification includes an upper limit value and a lower limit value indicated by “to”. For example, the description “A to B” means A or more and B or less. “Contains” includes “consisting essentially of” and “consisting of”.

Embodiment
Although this invention is not restricted to this, For example, it is related with the following embodiments.

Embodiment 1 FIG.
Suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension;
Heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more;
Stirring and cooling the activated carbon suspension after the heating;
A method for purifying hyaluronic acid and / or a salt thereof, comprising a step of contacting a hyaluronic acid solution containing hyaluronic acid and / or a salt thereof and impurities with an activated carbon suspension after stirring and cooling.

Embodiment 2 FIG.
Suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension;
Heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more;
A method for producing a purification agent for hyaluronic acid and / or a salt thereof, comprising a step of stirring and cooling the activated carbon suspension after the heating.

Embodiment 3 FIG.
Suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension;
Heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more;
A pretreatment method for activated carbon, comprising a step of stirring and cooling the activated carbon suspension after the heating.

Embodiment 4 FIG.
The method according to any one of Embodiments 1 to 3, wherein in the stirring and cooling step, the cooling completion temperature is 60 ° C. or lower.

Embodiment 5 FIG.
The method according to any one of Embodiments 1 to 4, wherein in the step of stirring and cooling, stirring and cooling are performed at a temperature of 10 to 60 ° C for 1 hour or longer.

Embodiment 6 FIG.
Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein at least one of the above steps is performed in a clean environment.

Embodiment 7 FIG.
The method according to any one of Embodiments 1 to 6, wherein hyaluronic acid and / or a salt thereof is produced by a fermentation method.

Embodiment 8 FIG.
The method according to any one of Embodiments 1 to 7, wherein the hyaluronic acid and / or salt thereof after purification has an average molecular weight of 3.5 to 7 million Da.

Embodiment 9 FIG.
Suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension;
Heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more;
A purification agent for hyaluronic acid and / or a salt thereof, comprising activated carbon produced by a method comprising a step of stirring and cooling the activated carbon suspension after the heating.

  Hereinafter, embodiments of the present invention will be described.

  The first aspect of the present invention (for example, Embodiment 1) includes a step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, and heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or longer. A method of purifying hyaluronic acid, comprising: a step of stirring and cooling the activated carbon suspension after heating; and a step of bringing the hyaluronic acid solution containing hyaluronic acid and impurities into contact with the activated carbon suspension after stirring and cooling. It is. According to this purification method, impurities can be efficiently adsorbed and removed by using activated carbon pretreated in a state suitable for purification of hyaluronic acids.

  The second aspect of the present invention (for example, Embodiment 2) includes a step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, and heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or longer. It is a manufacturing method of the refinement | purification agent of hyaluronic acid including the process and the process of stirring and cooling the activated carbon suspension after the heating. According to this production method, a purification agent for hyaluronic acid capable of efficiently adsorbing and removing impurities can be produced by pretreating activated carbon into a state suitable for purification of hyaluronic acid.

  The third aspect of the present invention (for example, Embodiment 3) is a step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, and heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or longer. It is a pretreatment method of activated carbon including a step and a step of stirring and cooling the activated carbon suspension after the heating. According to this pretreatment method, the activated carbon can be pretreated to a state suitable for purification of hyaluronic acids.

  The fourth aspect of the present invention (for example, Embodiment 9) includes a step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, and heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or longer. A purification agent for hyaluronic acids, comprising activated carbon produced by a method comprising a step and a step of stirring and cooling the activated carbon suspension after the heating. By including activated carbon pretreated in a state suitable for purification of hyaluronic acids, this purification agent can efficiently adsorb and remove impurities.

  Purification using the purification method of the first aspect, the purification agent produced by the production method of the second aspect, the activated carbon pretreated by the pretreatment method of the third aspect, or the purification agent of the fourth aspect Since the method (hereinafter referred to as “the purification method of the above embodiment”) has a higher impurity adsorption power than the purification method using untreated activated carbon, more effective impurity removal can be performed. .

  That is, when the purification method of the above embodiment is used, only high molecular compounds (for example, bacterial cells, proteins, nucleic acids, endotoxins) that can generally be removed by activated carbon as impurities while suppressing the disappearance of hyaluronic acids to a low level. In addition, even low molecular weight compounds (for example, amino acids, sugars, organic acids) could be efficiently separated and removed. Moreover, the purification method of the said aspect has a remarkable removal effect especially with respect to a living microbe, protein, and endotoxin compared with the purification method using untreated activated carbon.

  Moreover, the purification method of the said aspect can also reduce the fall of the molecular weight of hyaluronic acid compared with the case where untreated activated carbon is used. It is possible to reduce the decrease in the molecular weight of hyaluronic acid during purification, in particular, the hyaluronic acid having a high molecular weight (for example, an average molecular weight after purification (for example, after filtration following adsorption treatment) of 3.5 to 7 million Da). Excellent effect in purification. Moreover, it can process efficiently also about the hyaluronic acid solution of comparatively high concentration (for example, 1g / L-10g / L) conventionally difficult to process.

  Furthermore, when the purification method of the above aspect is used, the culture solution itself can be purified (impurities / cells etc. are removed) without performing sterilization treatment, dialysis treatment, purification treatment, etc., and the culture solution itself It also has an excellent effect when separating and purifying hyaluronic acids from sucrose. By using the culture solution as it is without performing complicated operations such as centrifugation and filtration, it is possible to obtain further efficiency and cost reduction effects.

  The hyaluronic acid concentration of the hyaluronic acid solution when performing the purification method of the above embodiment is not limited to this from the viewpoint of difficulty in handling due to the high solution viscosity and the solubility of hyaluronic acid, 0.1 to 20 g / L is desirable, 0.5 to 15 g / L is more desirable, and 1 to 10 g / L is most desirable.

  The pH of the hyaluronic acid solution that is the target of the purification method of the above embodiment is not limited to this from the viewpoint of preventing the degradation of hyaluronic acid and preventing the molecular weight from decreasing, but it is preferably 3-12. ~ 9 is most desirable. Furthermore, from the viewpoint of further enhancing the purification (adsorption) effect, the pH is preferably 3.5 or more and / or 7 or less, although not limited thereto.

  The temperature of the hyaluronic acid solution when using the purification method of the above embodiment is not limited to this, but is preferably 0 to 80 ° C. If temperature is 80 degrees C or less, decomposition | disassembly and the fall of molecular weight of hyaluronic acid during processing can be suppressed strongly.

  In the purification method of the above aspect, when the activated carbon suspension is brought into contact with the hyaluronic acid solution to be purified, it may be suspended once and kept as it is, but it is desirable to continuously suspend and stir. . When stirring, although not limited to this, stirring for 10 minutes-1 hour is desirable, and stirring for 20-40 minutes is further more desirable.

  In the heat treatment of the activated carbon suspension in each method of the above aspect, the ability to remove impurities is improved by heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or longer. However, in order to enhance the effect of the heat treatment, the temperature is not limited to this, but is more preferably 120 ° C. or higher, and most preferably 121 ° C. or higher. In order to enhance the effect of the heat treatment, it is desirable that the atmospheric pressure during heating is high. Such high atmospheric pressure is not limited to this, but is preferably 0.1 MPa or more, and more preferably 0.2 MPa or more. In order to enhance the effect of the heat treatment, the heating time is not limited to this, but is more preferably 30 minutes or more, and most preferably 1 hour or more. Further, at the time of heating, it is desirable to heat while continuing stirring, and in that case, although not limited to this, it is desirable to stir at 50 to 1000 rpm, and further stir at 100 to 600 rpm. desirable.

  In each method of the said aspect, when stirring and cooling the activated carbon suspension after a heating, the heated activated carbon suspension is cooled by continuing stirring. When cooling with stirring in the pretreatment, a decrease in molecular weight during purification of hyaluronic acids can be greatly reduced as compared to cooling without stirring. The ambient temperature at the time of stirring and cooling is not limited to this, but is preferably 0 ° C to 80 ° C, more preferably 10 ° C to 60 ° C, and most preferably 15 to 40 ° C. Moreover, you may operate at room temperature which is the temperature which does not perform operation especially. The stirring time is not limited to this, but is preferably 1 hour or more, more preferably 2 hours or more, still more preferably 3 hours or more, and 4 hours or more. Is most desirable. Even if the cooling completion temperature is the same temperature, the effect of reducing the molecular weight of hyaluronic acids when used for purification is greater when stirring for an appropriate stirring time. Further, the cooling completion temperature is desirably lower because of the influence on the molecular weight reduction of hyaluronic acid. Although not limited to this, the cooling completion temperature is desirably 80 ° C. or less, and the cooling completion temperature is more desirably 60 ° C. or less (0 ° C. or more).

  The amount of the activated carbon used in the purification method of the above embodiment is not limited to this, but is preferably 0.5 to 20.0% by weight with respect to 100% by weight of the hyaluronic acid solution. More desirably, it is ˜20.0% by weight. Within these ranges, higher purification (adsorption) action can be expected.

  Furthermore, the raw material for activated carbon is preferably derived from wood from the viewpoint of impurities that can be a problem particularly when applied to a living body, and the shape is preferably powder from the viewpoint of dispersibility and adsorption efficiency.

  Further, in a further aspect of the present invention, in the above aspect, the hyaluronic acid is Streptococcus ex FM-100 (Mikken Kenjo No. 9027) or Streptococcus ex FM-300 (Maiko Kenjo No. 2319). Produced by. By using hyaluronic acid produced by these microorganisms as a purification target, a purified product with high molecular weight hyaluronic acid can be obtained with less impurities, and exhibits an excellent effect particularly when used as a medicine.

  By using the purification method of the above aspect, it is possible to reduce the load of the separation / purification process of hyaluronic acids. Therefore, the purification method according to the above aspect is particularly used at a relatively early stage of the industrial process of production. It is effective.

  Furthermore, by performing the purification method of the above aspect in a clean environment, it is possible to efficiently purify high-quality hyaluronic acids required for pharmaceuticals on an industrial scale, which has been difficult in the past. Such a purification method has extremely high industrial utility in the production of hyaluronic acids for pharmaceuticals.

  In addition, the purification method etc. which are demonstrated by the said embodiment and aspect do not limit this invention, and are indicated with the intention of illustrating. The technical scope of the present invention is defined by the description of the claims, and those skilled in the art can make various design changes within the technical scope of the invention described in the claims.

  For example, the purification method or the like may include another step, or may be a method for producing hyaluronic acid or the like by performing another step or method subsequent to the purification method. Examples of such processes and methods include, for example, a step of culturing a hyaluronic acid-producing microbial strain, performed simultaneously or separately, a step of producing a culture filtrate from a hyaluronic acid-producing microbial strain culture solution, and centrifuging a solution to be purified. Process, neutralizing the target liquid, adding salt to the target liquid, microfiltration of the target liquid to be purified, dialysis of the target liquid, adding aromatic adsorption resin to the target liquid to be purified, target The process of ultrafiltration of the liquid, the process of purifying the target liquid by chromatography, the process of adding the activated carbon adsorbent to the target liquid, the process of separating the activated carbon from the target liquid, the process of removing the activated carbon from the target liquid, the organic solvent In addition, a step of precipitating hyaluronic acid, a step of crystallizing hyaluronic acid, a step of drying hyaluronic acid and the like can be mentioned.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

  150 ml of a fermented liquid cultured with Streptococcus ex FM-100 (Microtechnological Bacteria No. 9027) (sodium hyaluronate concentration 3.5 g / l, pH 4.0) was collected in a 300 ml glass beaker, and Table 2 Alternatively, treatment with an adsorbent (activated carbon or the like) prepared under the conditions shown in 3 was performed, and suspension stirring was performed at 400 rpm for 30 minutes using a magnetic stirrer. The treated liquid was filtered using filter paper, and after adding 4.5 g (3.0 wt%) of sodium chloride to 150 ml of the filtrate, sodium hyaluronate was precipitated with 600 ml of ethanol. This precipitated sodium hyaluronate was washed with 300 ml of ethanol and then vacuum dried at 40 ° C. to obtain 0.5 g of sodium hyaluronate.

  The sodium hyaluronate thus obtained was evaluated using the evaluation items and evaluation criteria shown in Table 4. The purification conditions and evaluation results are shown in Tables 2 and 3 (Experimental Examples 1 to 17).

[Table 2]

[Table 3]

*ET：エンドトキシン

* ET: Endotoxin

  From the results of the above experiments, it was confirmed that impurities can be effectively adsorbed and removed from the hyaluronic acid solution by purification using activated carbon pretreated by a certain pretreatment method.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (9)

Suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension;
Heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more;
A step of stirring and cooling the activated carbon suspension after the heating, and a pretreatment step of the activated carbon,
A method for purifying hyaluronic acid and / or a salt thereof, comprising a step of contacting a hyaluronic acid solution containing hyaluronic acid and / or a salt thereof and impurities with an activated carbon suspension after stirring and cooling. Suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension;
Heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more;
Comprising the pre-process and process of including activated carbon to stir cooled activated carbon suspension after the heating, the production method of the hyaluronic acid and / or purification agent salt thereof containing activated carbon. Suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension;
Heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more;
A pretreatment method of activated carbon for use in purification of hyaluronic acid and / or a salt thereof, comprising a step of stirring and cooling the activated carbon suspension after the heating.   The method according to any one of claims 1 to 3, wherein in the stirring and cooling step, a cooling completion temperature is 60 ° C or lower.   The method according to any one of claims 1 to 4, wherein in the step of stirring and cooling, stirring and cooling are performed at a temperature of 10 to 60 ° C for 1 hour or longer.   The method according to claim 1, wherein at least one of the steps is performed in a clean environment. The method according to any one of claims 1 to 6, wherein the hyaluronic acid and / or a salt thereof is produced by a fermentation method. The method according to any one of claims 1 to 7, wherein the hyaluronic acid and / or a salt thereof has a molecular weight of 3.5 to 7 million Da. Suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension;
Heating the activated carbon suspension at 100 ° C. or higher for 20 minutes or more;
A purification agent for hyaluronic acid and / or a salt thereof, comprising activated carbon produced by a method comprising a step of stirring and cooling the activated carbon suspension after the heating.