KR101638662B1 - Purification method for hyaluronic acid andor salts thereof - Google Patents

Abstract

The present invention discloses a method for purifying hyaluronic acid and / or a salt thereof, which comprises a step of dialyzing a hyaluronic acid solution containing a polymer hyaluronic acid and a salt thereof and an impurity with an ultrafiltration membrane. According to the method of the present invention, high-quality hyaluronic acid and / or salt thereof from which protein, nucleic acid, lactic acid, metal and the like have been removed can be produced easily and at a high yield on an industrial scale.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for purifying hyaluronic acid and /

The present invention relates to a method for purifying hyaluronic acid and / or a salt thereof.

Hyaluronic acid is used as a medicine in ophthalmology, orthopedics, dermatology, and the like in addition to moisturizing agents for cosmetics. The hyaluronic acid can be produced by extracting from an animal tissue, for example, a chicken gauge, a vitreous body of a cattle eye, etc. However, it can be produced by incorporating chondroitin sulfate as an impurity or by hyaluronidase contained in a tissue (Fermentation method) of culturing a microorganism capable of producing hyaluronic acid and cultivating hyaluronic acid from a culture medium has also been carried out (Non-Patent Document 1 and Patent Document 1).

Since hyaluronic acid produced by an extraction method or a fermentation method contains a protein or a pyrogenic substance as an impurity, a method of separating and removing the hyaluronic acid to obtain a high-purity product has been studied. Particularly, removal of impurities at the initial stage of production is expected to be developed as a method for reducing the load of the subsequent purification step and obtaining a product of high purity which can be used as a medicine. As a method for purifying hyaluronic acid in high purity, for example, a hyaluronic acid solution containing hyaluronic acid and its salts and impurities to remove inorganic salts derived from a by-product and a medium such as protein and nucleic acid, , A water-soluble organic solvent is added, and sodium hyaluronate is precipitated and settled, and the supernatant is extracted to obtain a purified solution (Non-Patent Document 2).

Japanese Patent Publication No. 4-12960 Japanese Patent Application Laid-Open No. 9-324001

 Journal of General Microbiology, 85, 372-375, 1976

However, in order to obtain high purity hyaluronic acids that can be used as pharmaceuticals on an industrial scale, there is a problem that the recovery rate of hyaluronic acid is low even in the above method.

The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a method for purifying hyaluronic acids of high purity on an industrial scale with a simple and high yield.

In order to solve the above object, the inventors of the present invention have studied various methods for efficiently and efficiently separating and removing impurities from a hyaluronic acid solution containing hyaluronic acid and its salts and impurities and purifying hyaluronic acids of high purity with ease and efficiency As a result, it has been found that a nucleic acid, a pyrogenic substance, a protein, a lactic acid, an inorganic salt and the like can be effectively removed by preparing a solution of hyaluronic acid at a pH of the acidic side and dialyzing it with an ultrafiltration membrane It came to the following.

That is, according to the present invention, there is provided a method for purifying hyaluronic acid and / or a salt thereof, which comprises preparing a hyaluronic acid solution containing hyaluronic acid and its salt and an impurity at a pH of the acidic side, / RTI > According to this manufacturing method, impurities can be effectively removed.

[Description of Terms]

The terms " hyaluronic acid and / or salt thereof " and " hyaluronic acid " in this specification have the same meaning and can be used interchangeably and include free hyaluronic acid and any arbitrary (For example, metal salts such as sodium salts, potassium salts, calcium salts and lithium salts, acid addition salts such as hydrochloride salts, phosphate salts and citric acid salts), hydrates, and mixtures thereof it means. Here, hyaluronic acid refers to a polysaccharide having a high molecular weight composed of repeating two saccharide units bonded with N-acetyl-D-glucosamine and D-glucuronic acid, and various salts are those in which the glucuronic acid moiety is in the form of a salt It says. Hyaluronic acid is liable to develop in the space due to the interaction between the foldable chain portion and the negative charge of the carboxyl group of the D-glucuronic acid moiety, and thus can bond with a large amount of water to form a gel. Further, even at a low concentration, since the intermolecular force is strong, it has a relatively high viscosity. From these actions, for example, they have the wetting action of the joints, the softening action of the skin, and the physiological roles thereof.

Among the hyaluronic acids, sodium hyaluronate having a molecular weight of about 2,000,000 Da is known to exert excellent effects in the treatment of deformable knee joint, shoulder joint inflammation, chronic joint rheumatism and the like as a medicine, compared with a molecular weight of about 800,000 Da [ Vol.22, No.9, 289 (1994); Pharmacology and Therapy, Vol.22, No.9, 319 (1994)). In addition to this, the effect as medicine for dermatological and ophthalmic areas is known for preventing adhesion after surgery, and some of them are generally used clinically. When used as a medicine, hyaluronic acid having an average molecular weight of at least 1,000,000 is preferably used. In view of ease of acquisition and handling, hyaluronic acids having an average molecular weight of 1 million to 5 million Da are preferable as pharmaceuticals, and hyaluronic acids having an average molecular weight of 1,500 to 4,000,000 Da are particularly preferable. Such high molecular weight hyaluronic acids also exert excellent effects from their high moisturizing power even when they are used for cosmetic applications.

The term " average molecular weight " in the present specification refers to the viscosity average molecular weight when the average molecular weight of hyaluronic acids is not specified. The viscosity average molecular weight can be determined by a method commonly practiced by those skilled in the art. Preferably, it can be obtained 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, when sodium hyaluronate is expected to have an average molecular weight (1.5 to 3.9 million) similar to that of the present invention, it is not limited to this, but the average molecular weight thereof is determined by using an intrinsic viscosity [?] Can be obtained by the following equation (1).

Examples of the pharmaceutical solution used for dissolving hyaluronic acids include a commonly used solution for injection (for example, water for injection, physiological saline, etc.), a pH adjusting agent containing a buffer such as an acid, an alkali, , Which is recognized in each country's pharmacopeia) can be used appropriately.

These hyaluronic acids may be produced by an extraction method extracted from animal tissues or may be produced by a fermentation method obtained by fermentation using a microorganism strain producing hyaluronic acid. However, in the case of extracting from animal tissues, since impurities such as other mucopolysaccharides are relatively large and the molecular weight of hyaluronic acids is also small, it is preferable to use those obtained by the fermentation method. In one example of a fermentation method suitable for the present invention, hyaluronic acids can be obtained by a known method using microorganisms of the genus Streptococcus, for example.

When the fermentation broth obtained by the fermentation method is used in the method of the present invention or the like, it is preferable to use a solution which has been inactivated by a known method, for example, centrifugation or filtration. In some cases, a water-soluble organic solvent such as alcohol may be added to precipitate and refine hyaluronic acid. Further, it may be treated with alumina or the like.

In the present specification, "Streptococcus" includes any bacterium of Streptococcus or its mutant strains capable of producing hyaluronic acid. In particular, Streptococcus aurei FM-100 (Unphosphorylated Bacteria No. 9027) described in Patent Document 2 and Streptococcus aure FM-300 (Unphosphorylated Bacteria No. 2319) described in Japanese Patent Application Laid- It is preferable to use a mutant strains which stably produce hyaluronic acid at a high yield such as the hyaluronic acid. Examples of the bacterium belonging to the genus Streptococcus which are suitable for the production of hyaluronic acid include, but are not limited to, Streptococcus equi, Streptococcus zooepidemicus, Streptococcus Streptococcus equisimilis, Streptococcus dysgalactiae, Streptococcus pyogenes, and mutant strains thereof.

The term "ultrafiltration membrane" in this specification means a filtration membrane having a pore diameter of 0.001 to 0.01 μm and / or a filtration membrane having a molecular weight cutoff of 1,000 to 300,000. Ultrafiltration membranes are classified into inorganic membranes and organic membranes, and further divided into hydrophobic and hydrophilic. Examples of the hydrophobic organic film include, but are not limited to, polysulfone, polyethersulfone, polyether, polyvinylidene fluoride, polyethylene, and polypropylene. Examples of the hydrophilic organic film include, but are not limited to, polyacrylonitrile, polyamide, polyimide, cellulose acetate and the like. The shape of the filter medium includes all module types such as a flat membrane, a tubular membrane, a spiral membrane, and a hollow fiber membrane.

The filtration system includes a full-volume filtration system and a cross-flow system. The total volume filtration method refers to a method of filtering the entire amount of water supplied to the membrane. On the other hand, the cross-flow system refers to a system in which filtration is performed while suppressing the phenomenon that suspended substances or colloids contained in water supplied to the membrane are deposited on the membrane surface by forming a flow parallel to the membrane surface. The cross flow method includes, but is not limited to, an one-pass method, a back-flush method, and a reverse method. The one-pass method is a method in which filtration is performed without reusing the permeated liquid from the ultrafiltration membrane, as shown in Fig. 1 (A). 1B, the permeate from the ultrafiltration membrane is stored in a permeate tank, transferred from the permeate tank to the ultrafiltration membrane, and the hyaluronic acid attached to the surface of the filtration membrane is cleaned and removed The filtration method including the step of As shown in Fig. 1C, the reverse method is a filtration method including a step of cleaning and removing the hyaluronic acid adhering to the surface of the filtration membrane by causing the permeate liquid from the ultrafiltration membrane to flow backward by locking the permeate valve It says.

The term " impurities " in this specification refers to substances other than hyaluronic acids, water and other solvent components and inorganic salts, particularly substances (pyrogenic substances) which can cause disadvantages when using hyaluronic acids as final products It says. Examples of the main impurity source include those derived from tissues, microorganisms or culture medium (medium) in the stage of production of hyaluronic acids, or those which have been introduced in the subsequent purification step or the like. Examples of the impurities in the present specification include, but are not limited to, tissues or cells, proteins, nucleic acids, polysaccharides, low molecular compounds or endotoxins. The tissue or cells as the impurities include, but are not limited to, tissue pieces derived from the tissue as an extraction raw material used in the extraction method, microbial cells or microbial cells used in the fermentation method, and the like. Proteins as impurities include, but are not limited to, proteins derived from the above tissues and bacteria, proteins incorporated in a post-production process, and the like. Endotoxin as an impurity includes, but is not limited to, lipopolysaccharide derived from the above-mentioned bacteria.

Refers to a compound having a relatively small molecular weight as compared with a hyaluronic acid and includes, for example, a compound having a molecular weight of 2000 Da or less, a molecular weight of 1,000 Da, or a molecular weight of 500 Da or less Compound. Such low-molecular compounds include various amino acids, organic acids (for example, lactic acid), sugars (for example, glucose) and the like.

As used herein, the term " removing " includes not only removing the target material completely, but also partially removing (reducing the amount of the material). The term " tablet " in this specification includes removing any or specific impurities.

In the present specification, each numerical range includes an upper limit value and a lower limit value indicated by "to".

[Embodiment]

The present invention is not limited to this but, for example, relates to the following embodiments.

Embodiments 1.

A process for purifying hyaluronic acid and / or its salt, which comprises the step of preparing a solution of hyaluronic acid containing hyaluronic acid and / or salt thereof and impurities at a pH of the acidic side and then removing the impurities by dialysis with an ultrafiltration membrane.

Embodiment 2.

The fractional molecular weight of the ultrafiltration membrane and the pH at the dialysis treatment by the ultrafiltration membrane are given by Equation

pH? ^-5? 10 ^-5 (fraction molecular weight) +4.4978

Wherein the dialysis treatment with the ultrafiltration membrane is carried out under the condition satisfying the following conditions:

Embodiment 3.

The method according to claim 1 or 2, wherein the fractionated molecular weight of the ultrafiltration membrane is 25000 to 35000 and the pH at the dialysis treatment is 3.3 or less.

The method according to Claim 1 or 2, wherein the ultrafiltration membrane has a cut molecular weight of 12000 to 14000 and a dialysis treatment pH of 3.9 or less.

Embodiment 4.

The method according to Claim 1 or 2, wherein the ultrafiltration membrane has a fraction molecular weight of 9000 to 11000 and a pH at the dialysis treatment of 4.1 or less.

Embodiment 5

The method according to Claim 1 or 2, wherein the ultrafiltration membrane has a fraction molecular weight of 6000 to 8000 and a pH at the dialysis treatment of 4.2 or less.

Embodiment 6.

The method according to Claim 1 or 2, wherein the ultrafiltration membrane has a cut molecular weight of 4000-5000 and a pH during dialysis treatment of 4.3 or lower.

Embodiment 7.

The method according to any one of claims 1 to 6, wherein the ultrafiltration membrane is a hydrophobic organic membrane.

Embodiment 8

The method according to any one of embodiments 1 to 7, wherein the filtration method of the treatment is a reverse method.

Embodiment 9

The process according to any one of the embodiments 1 to 8, wherein the permeate flux during the treatment is 20 to 50 L / m ² 시간 hour.

Embodiment 10.

11. The method according to any one of claims 1 to 9, wherein the impurity comprises cells, proteins, nucleic acids, low molecular weight compounds or endotoxins.

Embodiment 11.

The method according to any one of embodiments 1 to 10, wherein the average molecular weight of the hyaluronic acid and / or its salt after purification is 3.5 to 7 million Da.

Embodiment 12.

The method according to any one of the embodiments 1 to 11, wherein the concentration of hyaluronic acid and / or its salt in the hyaluronic acid solution is 1 to 5 g / L.

Hereinafter, aspects of the present invention will be described.

In an embodiment of the present invention (for example, embodiment 1), a hyaluronic acid solution containing hyaluronic acid and / or a salt thereof and an impurity is prepared at the pH of the acidic side and then dialyzed with an ultrafiltration membrane to remove impurities And / or a salt thereof. According to this purification method, the production at the pH of the acidic side reduces the loss of hyaluronic acid in the filtration by the ultrafiltration membrane, and the impurities can be efficiently removed.

The fractional molecular weight of the ultrafiltration membrane and the pH at the dialysis treatment by the ultrafiltration membrane are given by Equation

pH? ^-5? 10 ^-5 (fraction molecular weight) +4.4978

The dialysis treatment with the ultrafiltration membrane can be carried out without substantially losing hyaluronic acid. For example, in the ultrafiltration membrane having a cut-off molecular weight of 35000 and a pH of 2.7 and a cutoff molecular weight of 30000, the cut-off molecular weight of the ultrafiltration membrane satisfying the above-mentioned formula and the pH of the dialysis treatment are pH 3.0 and 25000 In the ultrafiltration membrane having a pH of 3.3 and a molecular weight cutoff of 20,000, the pH of the ultrafiltration membrane was pH 3.8, the pH of the ultrafiltration membrane was 13.0, the pH of the ultrafiltration membrane was 3.9, and the cutoff molecular weight of the ultrafiltration membrane was pH 3.9. In the ultrafiltration membrane of pH 4.0 and molecular weight of 10000, the pH of the ultrafiltration membrane of pH 4.0 and the molecular weight of cutoff of 9000 was 4.1, the pH of the ultrafiltration membrane was 4.1, the pH of the ultrafiltration membrane was 8.4, the pH of the ultrafiltration membrane was 7, In the ultrafiltration membrane having pH 4.2 and the cutoff molecular weight 5000, pH 4.3 and cutoff molecular weight 4000 The pH of the ultrafiltration membrane is 4.3.

Here, "substantially without causing loss of hyaluronic acid" means that the loss rate of hyaluronic acid is 3% or less (recovery rate is 97% or more).

When a membrane having a high fractionation molecular weight is used, the risk of loss of hyaluronic acids through the ultrafiltration membrane is increased, while the removal efficiency of impurities of relatively high polymers such as proteins is lowered when a membrane having a low molecular weight fraction is used. According to the method of the above embodiment, even when a film having a high cut-off molecular weight is used, even if a film having a low cut-off molecular weight is used, impurities can be removed without loss of hyaluronic acid by adjusting the pH to a fractional molecular weight of the film. The fraction molecular weight of the ultrafiltration membrane used in the above-described method is not particularly limited. For example, in the case of using an ultrafiltration membrane having a cut-off molecular weight of 25000 to 35000, the pH can be adjusted to 3.3 or less to purify without losing hyaluronic acid species. When an ultrafiltration membrane having a cut-off molecular weight of 12000 to 14000 is used, purification can be performed without loss of hyaluronic acid by adjusting the pH to 3.9 or less. When an ultrafiltration membrane having a cut-off molecular weight of 9000 to 11000 is used, purification can be performed without loss of hyaluronic acid by adjusting the pH to 4.1 or lower. When an ultrafiltration membrane having a cut-off molecular weight of 6000 to 8000 is used, purification can be performed without loss of hyaluronic acid by adjusting the pH to 4.2 or less. When an ultrafiltration membrane having a cut-off molecular weight of 4000 to 5000 is used, purification can be performed without loss of hyaluronic acid by adjusting the pH to 4.3 or less.

Here, the fraction molecular weight of the ultrafiltration membrane can be determined by performing filtration using an indicator material as shown in Table 1 and irradiating a molecular weight corresponding to 90% of each of the blocking rates.

The material of the ultrafiltration membrane used in the method of the above embodiment is not particularly limited, but a hydrophobic organic film is preferable from the viewpoint of removing impurities, and polysulfone, polyethersulfone, polyether, polyvinylidene fluoride, polyethylene, More preferable.

Examples of the ultrafiltration membrane used in the method of the above embodiment include PM-10, PM-50, PM-100 (manufactured by Koch), NTU-3050 (manufactured by Nitto Denko Corporation), IRIS3065 (Manufactured by Asahi Kasei Chemicals Co., Ltd.), FS-10 (manufactured by Asahi Kasei Corporation), MU-6303 (manufactured by Kuraray Co., Ltd.), DUSO400 .

The filtration system in the method of the above embodiment is not particularly limited, but a cross flow system is preferable from the standpoint that the membrane permeation flux of water is easily stabilized and the lifetime of the filtration membrane is extended. Among them, the reverse system is particularly preferable Do.

In the method of this embodiment, the permeation flux at the time of the ultrafiltration membrane treatment of the hyaluronic acid solution differs depending on the properties of the hyaluronic acid solution and the kind of the ultrafiltration membrane and can not be uniformly defined. For example, Preferably 20 L / m ² 시간 hour or more, more preferably 25 L / m ² 시간 hour or more, and particularly preferably 30 L / m ² 시간 hour or more in the purification of the hyaluronic acid of the present invention. The flow rate of the hyaluronic acid solution in the filtration is preferably 100 L / m ² 시간 hour or less, more preferably 50 L / m ² 시간 hour or less, since the hyaluronic acid is sheared and the molecular weight is lowered Do.

In the method of the above embodiment, there is no limitation on the pressure for sending, but it is generally preferable to pass the filtration membrane through pressurization. Particularly, it is preferable that the pressure is transmitted by a pump or the like. So long as it does not deteriorate performance by breaking or clogging of the filtration membrane when pressure is applied to the filtrate at the time of pumping. The pressure applied to the filtration membrane is preferably from 0.01 MPa to 0.30 MPa, more preferably from 0.03 MPa to 0.20 MPa, and particularly preferably from 0.05 MPa to 0.10 MPa.

Further, according to the method of the above-described embodiment, it is possible to reduce the molecular weight of the hyaluronic acid at the time of purification and, in particular, to reduce the molecular weight of the hyaluronic acid (for example, And exhibits excellent effects in purification. In addition, it is possible to efficiently treat hyaluronic acid solutions having a relatively high concentration (for example, from 0.1 to 20 g / L, from 0.5 to 15 g / L, and from 1 to 10 g / L) conventionally difficult to treat.

Further, according to the above-described method, not only a low molecular compound (for example, an amino acid, a sugar, or an organic acid) that is generally capable of being removed by an ultrafiltration membrane as an impurity while suppressing the disappearance of hyaluronic acid, For example, nucleic acid, endotoxin, protein) can be efficiently separated and removed. In addition, the method of the above embodiment exerts an excellent effect on elimination of nucleic acid, endotoxin and / or protein.

The concentration of the hyaluronic acid in the solution of the hyaluronic acid solution when using the method of the above embodiment is not particularly limited in view of the difficulty in handling due to the height of the solution viscosity and the solubility of the hyaluronic acid, L is preferable, and 1 to 10 g / L is most preferable.

In addition, in the method of the above embodiment, for the purpose of lowering the viscosity of the hyaluronic acid solution, a salt such as sodium chloride may coexist in the hyaluronic acid solution. In this case, it is preferable to avoid coexistence of salt at a high concentration so that the refining effect is not impaired. As a specific example of the coexistence of such a salt, 0.1 to 5% by weight of sodium chloride is added to the hyaluronic acid solution.

The temperature of the hyaluronic acid solution when the above-described method is used is not limited to this, but is preferably 0 to 80 ° C. When the temperature is 80 占 폚 or lower, decomposition of the hyaluronic acids and degradation of the molecular weight during the treatment can be strongly suppressed.

When the ultrafiltration is carried out, the membrane is treated with an alkali (for example, sodium hydroxide solution) of 2% or less, a peroxide (for example, sodium hypochlorite solution), a surfactant, citric acid, ammonium citrate Or a physical method such as flushing, sponge ball, air injection method, or the like.

In another aspect of the present invention, in the first aspect, the method may further include a step of bringing the hyaluronic acid solution into contact with the inorganic adsorbent, the organic adsorbent, and / or the activated carbon separately from the above-described process.

Further, in consideration of the separation and purification steps required thereafter, it is desirable to avoid the incorporation of components requiring further purification steps. That is, another aspect of the present invention for avoiding incorporation of a new impurity is a purification method that does not include a step of contacting the inorganic adsorbent or the organic adsorbent after ultrafiltration of the hyaluronic acid solution in the first embodiment. In another aspect of the present invention, in the first aspect, the step of contacting the hyaluronic acid solution with an inorganic adsorbent or an organic adsorbent is not included. In the present invention, a sufficient purification effect can be exhibited even by ultrafiltration. Therefore, when it is important to avoid the incorporation of new impurities, it is preferable to perform ultrafiltration alone. Of course, in this case as well, it is possible to carry out another purification treatment or the like separately from the ultrafiltration.

In another embodiment of the present invention, in the above embodiment, the hyaluronic acid is selected from the group consisting of Streptococcus equi FM-100 (Unphosphorylated Bacteria No. 9027) or Streptococcus aure FM-300 (Unphosphorylated Bacteria No. 2319) . By using the hyaluronic acids produced by these microorganisms as purification targets, it is possible to obtain refined products of hyaluronic acids with less impurities and high molecular weight, and in particular, exhibit excellent effects when used as medicines.

By using the purification method of the above-described embodiment, the burden of the separation and purification process of hyaluronic acids can be reduced. Therefore, the purification method according to the above embodiment is particularly effective when used in a relatively early stage of the industrial process of production.

In addition, the purification method and the like described in the above embodiments and modes are not intended to limit the present invention, but are intended to be exemplified. The technical scope of the present invention is defined by the description of the claims, and a person skilled in the art can make various design changes in the technical scope of the invention described in the claims.

For example, the purification method may include another step, or may be a method of producing hyaluronic acids or the like by performing another process or method subsequent to the purification method. Such processes and methods include, for example, a step of culturing a hyaluronic acid-producing microorganism strain, a step of producing a culture filtrate from a hyaluronic acid-producing microorganism culture medium, a step of centrifuging the liquid to be purified, A step of refining the liquid to be purified, a step of dialyzing the object liquid, a step of adding an aromatic adsorption resin to the liquid to be purified and stirring and filtering, a step of purifying the object liquid by chromatography, A step of removing the activated carbon from the liquid, a step of precipitating the hyaluronic acid by adding an organic solvent, a step of crystallizing the hyaluronic acid, a step of drying the hyaluronic acid, and the like.

[Example]

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

Example 1

45 L of the cultured medium was diluted with 80 L of pure water (sodium hyaluronate concentration: 2.0 g / l) using Streptococcus equi FM-100 (Untreated bacterial strain No. 9027) and the cells were removed by centrifugation. After adjusting the pH of the resulting hyaluronic acid to 2.9, the filtrate was subjected to filtration with a flow rate of 30 L / m ² 시간 hour, a volume ratio of 2 times, 2 m ² of an ultrafiltration membrane (PM-100, manufactured by Kotetsu Co., Ltd.) The dilution operation was repeated, and the dialysis was performed 10 times and the reverse mode. 2.4 kg of sodium chloride was dissolved in 80 L of the obtained solution, and the pH of the solution was adjusted to 7, followed by precipitation with 240 L of ethanol, 8 L of ethanol and vacuum drying at 40 캜 to obtain sodium hyaluronate. The analytical results and hyaluronic acid recovery rates are shown in Table 2.

Example 2

Examples used in the first preparation was adjusted to hyaluronic acid to pH 2.9, cut-off molecular weight: 30,000 Material polyethersulfone of the ultrafiltration membrane (manufactured by Asahi Kasei manufacturers and FS-10) 5 m ² with flux 30 L / m ² and hour, The volume ratio was doubled, and the pure dilution was repeated, and the dialysis was performed 11 times and the reverse mode. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and hyaluronic acid recovery rates are shown in Table 2.

Example 3

After adjusting the pH of the prepared hyaluronic acid used in Example 1 to 3 m ² using an ultrafiltration membrane (NTU-3050, manufactured by Nitto Denko Corporation) having a molecular weight cut-off of 20,000 and a permeation flow rate of 30 L / m ² 시간 hour, 2 times concentration, and pure dilution was repeatedly performed, and the dialysis was performed 8 times and reverse method. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and the recovery rate of sodium hyaluronate are shown in Table 2.

Example 4

The pH of the prepared hyaluronic acid used in Example 1 was adjusted to 2.9, and then the filtrate was permeated at a flow rate of 30 L / m ² 시간 hour with 5 m ² of an ultrafiltration membrane of polyvinylidene fluoride (manufactured by Ron Poulenc Company, IRIS 3065) Volume ratio of 2 times, and dilution with pure water was repeated, and the dialysis was repeated 9 times and the reverse method. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and the recovery rate of sodium hyaluronate are shown in Table 2.

Example 5

Example 1 After adjusting the preparation of hyaluronic acid used in a pH 2.7, cut-off molecular weight: 40,000 Material polyethersulfone of the ultrafiltration membrane (Daicel Kagaku Kogyo manufacture and DUSO400) 5 m transmitted to the ^second flow rate of 30 L / m ² and hour , The volume ratio was doubled, and the pure dilution was repeated, and the dialysis was performed 11 times and the reverse mode. The obtained solution was treated in the same manner as in Example 1 to obtain 150 g of sodium hyaluronate. The analytical results and the recovery rate of sodium hyaluronate are shown in Table 2.

Example 6

Example 1 After adjusting the preparation of hyaluronic acid used in a pH 3.7, cut-off molecular weight: 13,000 Material polysulfone of the ultrafiltration membrane (manufactured by Kuraray Co., Ltd. and MU-6303) 5 m ² with flux 30 L / m ² and hour, the ratio 2 Fold dilution, and so on. The dilution was repeated 11 times and the reverse method was used. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and the recovery rate of sodium hyaluronate are shown in Table 2.

Example 7

After adjusting the pH of the prepared hyaluronic acid used in Example 1 to 4.5, a permeation flow rate of 30 L / m ² 시간 hour, 4.5 m ² of an ultrafiltration membrane (SLP-3053, manufactured by Asahi Kasei Chemicals Co., Ltd.) The volume ratio was doubled, and the pure dilution was repeated, and the dialysis was performed 11 times and the reverse mode. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and the recovery rate of sodium hyaluronate are shown in Table 2.

Comparative Example 1

The pH of the prepared hyaluronic acid used in Example 1 was adjusted to pH 5.5, and then the filtrate was permeated at a flow rate of 30 L / m ² 시간 hour, 2 m ² of a hydrophobic ultrafiltration membrane (PM-10, manufactured by Kotetsu Co., Ltd.) 2-fold concentration, and 1-fold dilution were repeated, and the dialysis was repeated 15 times and the reverse method. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and hyaluronic acid recovery rates are shown in Table 2.

Comparative Example 2

The prepared hyaluronic acid used in Example 1 was adjusted to pH 3.6, and then 4.5 m ² of a hydrophilic ultrafiltration membrane (CA600PP, manufactured by DDS Corporation) having a molecular weight cut-off of 20000 and a permeation flow rate of 30 L / m ² 시간 hour, Concentration, and so on. The dilution was repeated ten times and the dialysis was repeated 10 times. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and hyaluronic acid recovery rates are shown in Table 2.

Comparative Example 3

Example 1 After adjusting the preparation of hyaluronic acid used in the pH 3.3, cut-off molecular weight: 20,000 Material acetate polyimide hydrophilic ultrafiltration membranes of the imide (Nitto Den Tommy Seiko manufacture and NTU-4220) 5 m transmitted to the ^second flow rate of 30 L / m ² and hour , The volume ratio was doubled, and the dilution with pure water was repeated, and the dialysis was repeated 9 times and the reverse mode. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and hyaluronic acid recovery rates are shown in Table 2.

Comparative Example 4

Carried out after adjusting the preparation of hyaluronic acid used in Example 1 in pH 3.7, cut-off molecular weight: 13,000 Material acetate poly hydrophobic ultrafiltration membrane of polysulfone (manufactured by Kuraray Co., Ltd. and MU-6303) 5 m ² flux 5 L / m ² and hour, Volume ratio was doubled, and pure dilution was repeated, and the dialysis was performed 11 times and one pass. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and hyaluronic acid recovery rates are shown in Table 2.

Comparative Example 5

Carried out after adjusting the preparation of hyaluronic acid used in Example 1 in pH 3.7, cut-off molecular weight: 13,000 Material acetate poly hydrophobic ultrafiltration membrane of polysulfone (manufactured by Kuraray Co., Ltd. and MU-6303) 5 m ² permeation flow rate 15 L / m ² and hour, The volume ratio was doubled, and pure dilution was repeated, and the dialysis was performed 11 times and the back flush method. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analytical results and hyaluronic acid recovery rates are shown in Table 2.

How to measure

(1) Nucleic acid content: The absorbance of sodium 0.1% hyaluronate at 260 nm was measured.

(2) Protein content: Sodium hyaluronate was dissolved in 0.1 N sodium hydroxide and subjected to the Lori method.

(3) Lactic acid: Sodium hyaluronate was dissolved at a concentration of 0.1%, and L-LDH method was used.

(4) Metal: Sodium hyaluronate was dissolved in 8 N nitric acid at a concentration of 0.05%, and ICP emission spectrochemical analysis was performed.

(5) Intrinsic viscosity: Sodium hyaluronate was dissolved in 0.2 M sodium chloride at a concentration of 0.02%, and the intrinsic viscosity at 30 캜 was measured.

Example 8

The prepared hyaluronic acid used in Example 1 was purified by using a hydrophobic ultrafiltration membrane of polysulfone having various cut molecular weights under the following conditions to investigate the relationship between the pH at the ultrafiltration and the recovery rate of hyaluronic acid.

HA solution condition

HA concentration: 2 g / L

Molecular weight: 4.40 million (intrinsic viscosity: 55 dL / g)

Filtration conditions

Speed: 1 m / s

Permeate flow rate: 30 L / (m ² ㆍ hour)

Concentration: 2x concentration

Temperature: 25 ℃

A graph showing the relationship between the pH at the time of ultrafiltration and the hyaluronic acid recovery rate is shown in Fig. Further, for the membranes having the respective cut-off molecular weights, the formulas 1 to 5 showing the relationship between the pH during ultrafiltration and the hyaluronic acid loss rate were obtained. The pH at which the loss of hyaluronic acids in the ultrafiltration membrane of each cut-off molecular weight is substantially not generated is calculated using the following formulas 1 to 5, and purification by ultrafiltration is carried out within the range of the pH (optimum pH) , It is possible to purify it without losing hyaluronic acids.

Fraction molecular weight of 30,000 Han Filter membrane  When using

(Formula 1) Loss rate (%) of hyaluronic acids = 44.86 占 (pH at ultrafiltration) -131.79

(Optimum pH? 2.9)

Fractional molecular weight of 13,000 Han Filter membrane  When using

(Equation 2) Loss rate (%) of hyaluronic acids = 40.84 x (pH at ultrafiltration) -86.92

(Optimum pH? 3.7)

Fraction molecular weight of 10,000 Han Filter membrane  When using

(Formula 3) Loss rate (%) of hyaluronic acids = 24.36 占 (pH at ultrafiltration) -97.19

(Optimum pH? 4.0)

Fractional molecular weight of 7,000 Han Filter membrane  When using

(Formula 4) Loss rate (%) of hyaluronic acids = 7.09 占 (pH at ultrafiltration) -29.02

(Optimum pH? 4.1)

Fractional molecular weight of 5,000 Han Filter membrane  When using

(Equation 5) Loss rate (%) of hyaluronic acids = 0.79 x (pH at ultrafiltration) -2.01

(Optimum pH? 4.2)

3 shows the relationship between the pH at which the loss rate of hyaluronic acids is 3% or less and the fraction molecular weight of the ultrafiltration membrane. It has also become clear that the relationship between the pH at which the loss rate of the hyaluronic acids is 3% or less and the fraction molecular weight of the ultrafiltration membrane satisfies the following expression (6).

(Equation 6)

PH at which the loss rate of hyaluronic acids is 3% or less = -5 × 10 ^-5 × (fraction molecular weight) +4.4978

By using Equation 6, the upper limit of the optimal pH for the cut-off molecular weight of the ultrafiltration membrane can be obtained, and the hyaluronic acid can be purified at a loss rate of 3% or less by performing dialysis with an ultrafiltration membrane at an optimum pH.

From the above-mentioned experiments, it was confirmed that by using the purification method according to the present invention, impurities can be effectively removed from the hyaluronic acid solution to purify hyaluronic acid having a high molecular weight.

The present invention has been described based on the embodiments. It is to be understood by those skilled in the art that this embodiment is merely illustrative and that various modifications are possible, and that such modifications are also within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS Fig.

Fig. 1 is a view showing filtering methods of the one-pass method (A), the backflush method (B), and the reverse method (C) included in the cross flow method.

2 is a graph showing the relationship between pH and hyaluronic acid loss rate during ultrafiltration.

3 is a graph showing the relationship between the pH at which the loss factor of hyaluronic acids is 3% or less and the fraction molecular weight of the ultrafiltration membrane.

Claims (13)

A step of preparing a hyaluronic acid solution containing hyaluronic acid and / or a salt thereof and an impurity at a pH of the acidic side, and then removing the impurities by dialysis with an ultrafiltration membrane,
The cut molecular weight of the ultrafiltration membrane and the pH at the dialysis treatment with the ultrafiltration membrane satisfy the following equations
pH? ^-5? 10 ^-5 (fraction molecular weight) +4.4978
Wherein the dialysis treatment with the ultrafiltration membrane is carried out under the condition that the hyaluronic acid and / or the salt thereof is satisfactory. The method according to claim 1, wherein the ultrafiltration membrane has a fraction molecular weight of 25000 to 35000 and a pH at the dialysis treatment of 3.3 or less. The method according to claim 1, wherein the ultrafiltration membrane has a cut molecular weight of 12000 to 14000 and a dialysis treatment pH of 3.9 or less. The method according to claim 1, wherein the ultrafiltration membrane has a cut-off molecular weight of 9000 to 11000 and a dialysis treatment pH of 4.1 or less. The method according to claim 1, wherein the ultrafiltration membrane has a fraction molecular weight of 6000 to 8000 and a pH at the dialysis treatment of 4.2 or less. The method according to claim 1, wherein the ultrafiltration membrane has a cutoff molecular weight of 4000-5000 and a dialysis treatment pH of 4.3 or lower. The method of claim 1, wherein the ultrafiltration membrane is a hydrophobic organic membrane. The method of claim 1, wherein the filtering of the process is a reverse method. The process according to claim 1, wherein the permeate flux during the treatment is 20 to 50 L / m ² 시간 hour. 2. The method of claim 1, wherein the impurity comprises cells, proteins, nucleic acids, low molecular weight compounds or endotoxins. The method according to claim 1, wherein the average molecular weight of the hyaluronic acid and / or its salt after purification is 3.5 to 7 million Da. The method according to claim 1, wherein the concentration of hyaluronic acid and / or its salt in the hyaluronic acid solution is 1 to 5 g / L. delete

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