KR20130056222A - Purification method for hyaluronic acid and/or salts thereof - Google Patents

Abstract

The present invention discloses a method for purifying hyaluronic acid and / or its salt, including a step of dialysis treatment of a hyaluronic acid solution containing a polymer hyaluronic acid, a salt thereof, and an impurity with an ultrafiltration membrane. According to the method of the present invention, high-quality hyaluronic acid and / or its salts from which proteins, nucleic acids, lactic acid, metals, and the like are removed can be produced on an industrial scale in a simple and high yield.

Description

Purification method of hyaluronic acid and / or its salt {PURIFICATION METHOD FOR HYALURONIC ACID AND / OR SALTS THEREOF}

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

In addition to cosmetic moisturizers, hyaluronic acid is used as a medicine in ophthalmology, orthopedics, dermatology and the like. Hyaluronic acid can be produced by extracts from animal tissues, for example chicken broilers, bovine eye vitreous bodies, etc., but low molecular weights such as chondroitin sulfate or the like are mixed as a contaminant or hyaluronidase contained in tissues. Since it is easy to produce, culture | cultivation of the microorganism which has hyaluronic acid production ability, and manufacturing hyaluronic acid from a culture liquid (fermentation method) are also performed (nonpatent literature 1 and patent document 1).

Since hyaluronic acid produced by the extraction method or the fermentation method contains proteins, pyrogenic substances and the like as impurities, a method of separating and removing them to obtain a high-purity product has been studied. In particular, the removal of impurities in the initial stage of manufacture enables the reduction of the load on subsequent purification steps and is expected to be developed as a method of obtaining a high-purity product that can be used as a medicine. A method of purifying hyaluronic acid with high purity, for example, a filter in which a hyaluronic acid solution containing hyaluronic acid, its salts and impurities is positively charged in order to remove by-products such as proteins and nucleic acids and inorganic salts derived from the medium. The method of refine | purifying by adding water-soluble organic solvent, depositing and sedimenting sodium hyaluronate, and extracting a supernatant liquid after passing through is known is known (nonpatent literature 2).

Japanese Patent Publication No. 4-12960 Japanese Patent Publication No. 9-324001

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

However, in order to obtain high-purity hyaluronic acid that can be used as a pharmaceutical on an industrial scale, problems such as low recovery rate of hyaluronic acid remain even in the above-described method.

This invention is made | formed in view of the said situation, and an object of this invention is to provide the method for refine | purifying hyaluronic acid of high purity with an easy and high yield on an industrial scale.

MEANS TO SOLVE THE PROBLEM In order to solve the said objective, the present inventors examined various ways of efficiently separating and removing an impurity from the hyaluronic acid solution containing hyaluronic acid, its salt, and an impurity, and purifying hyaluronic acids of high purity simply and efficiently. As a result, the hyaluronic acid solution was prepared at an acidic pH, followed by dialysis treatment with an ultrafiltration membrane to find that nucleic acids, pyrogenic substances, proteins, lactic acid, inorganic salts, and the like can be effectively removed, thereby completing the present invention. It came to the following.

That is, according to the present invention, a method for purifying hyaluronic acid and / or its salt comprising preparing a hyaluronic acid solution containing hyaluronic acid and salts and impurities thereof at an acidic pH, followed by dialysis treatment with an ultrafiltration membrane Is provided. According to this manufacturing method, impurities can be effectively removed.

[Description of Terms]

In this specification, "hyaluronic acid and / or its salt" and "hyaluronic acid" are synonymous, are used interchangeably, and can be used in the range which does not impair free hyaluronic acid and the objective of this invention. Hyaluronic acid salts (including, but not limited to, metal salts such as sodium salts, potassium salts, calcium salts and lithium salts, and acid adducts such as hydrochlorides, phosphates and citrates), hydrates, and mixtures thereof it means. Here, hyaluronic acid refers to a high molecular weight polysaccharide obtained by repeating a chain of disaccharide units in which N-acetyl-D-glucosamine and D-glucuronic acid are bonded, and various salts are mainly those in which the glucuronic acid moiety is in the form of a salt. Say. Hyaluronic acid is likely to develop in the space by the interaction of the collapsible chain portion and the negative charge of the carboxyl group of the D-glucuronic acid portion, whereby a large amount of water can be combined to form a gel. In addition, even at low concentrations, the intermolecular force is strong, so that it has a relatively high viscosity. From these actions, for example, wetting joints, skin softening action, and the like are playing a role in their physiology.

Among hyaluronic acids, sodium hyaluronate having a molecular weight of about 2 million Da is known to exert an excellent effect in the treatment of deformed knee joint, periarthritis of the shoulder, chronic joint rheumatism, etc. as a medicine compared to a molecular weight of about 800,000 Da (document [ Pharmacology and Treatment, Vol. 22, No. 9, 289 (1994); Pharmacology and Treatment, Vol. 22, No. 9, 319 (1994)]. In addition, as an anti-adhesion after a surgical operation, the effect as a medicine is known also in a dermatological area and an ophthalmic area, and some are used clinically generally. When using as a pharmaceutical, it is preferable to use hyaluronic acid whose average molecular weight is 1 million or more. In addition, in view of ease of availability and handling, hyaluronic acids having an average molecular weight of 1 million to 5 million Da are preferable as pharmaceutical products, and hyaluronic acids having an average molecular weight of 1.5 million to 4 million Da are particularly preferable. In addition, such high molecular weight hyaluronic acid exhibits an excellent effect from its high moisturizing power even when used as a cosmetic use.

About the "average molecular weight" in this specification, when showing the average molecular weight of hyaluronic acid unless it mentions specially, a viscosity average molecular weight is said. A viscosity average molecular weight can be calculated | required by the method normally performed by a person skilled in the art. Preferably, it can obtain | require by the measuring method generally used by the pharmacopoeia etc. of each country, More preferably, it can obtain | require by the measuring method used by Japanese Pharmacopoeia. As an example, for example, when sodium hyaluronate is expected to have an average molecular weight (1.5 million to 3.9 million) close to the present invention, the average molecular weight is not limited to this, and the following average molecular weight is obtained using the intrinsic viscosity [η]. It can obtain | require by Formula (1).

As a medicament, as an injectable solution for dissolving hyaluronic acid, a commonly used injectable solution (for example, a pH adjuster containing a buffer such as an acid, an alkali, or a phosphate, etc.) is added to water for injection, physiological saline (for example, 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 hyaluronic acid producing microorganism strain. However, it is preferable to use what is obtained by the fermentation method because the thing extracted from animal tissues has comparatively many impurities, such as other mucopolysaccharides, and the molecular weight of hyaluronic acid is small. In an example of the fermentation method suitable for the present invention, hyaluronic acid can be obtained by a known method using, for example, a microorganism of the genus Streptococcus.

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 that has been sterilized by a known method such as centrifugation or filtration. In some cases, a precipitated and purified hyaluronic acid may be used by adding a water-soluble organic solvent such as alcohol. Moreover, the thing processed with alumina etc. can also be used.

The "streptococcus" in the present specification includes any bacteria of the genus Streptococcus capable of producing hyaluronic acid and its mutant strains. In particular, Streptococcus aquim FM-100 (U.S. bacterium 9027) described in patent document 2, Streptococcus aqui FM-300 (U.S. microparticles No. 2319) described in Unexamined-Japanese-Patent No. 2-234689. It is preferable to use a mutant strain that stably produces hyaluronic acid at a high yield such as). Examples of bacteria of the genus Streptococcus suitable for the production of hyaluronic acid include, but are not limited to, for example, Streptococcus equi, Streptococcus zooepidemicus, Streptococcus e. Streptococcus equisimilis, Streptococcus dysgalactiae, Streptococcus pyogenes, and mutants thereof.

The term "ultrafiltration membrane" in the present specification means a filtration membrane having a pore size of 0.001 to 0.01 µm and / or a filtration membrane having a fractional molecular weight of about 1000 to 300000. The material of the ultrafiltration membrane is largely divided into inorganic membrane and organic membrane, and further divided into hydrophobicity and hydrophilicity. Examples of the hydrophobic organic film include, but are not limited to, polysulfone, polyether sulfone, polyether, polyvinylidene fluoride, polyethylene, polypropylene, and the like. 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 (hollow fiber) membrane.

As a filtration system, a whole quantity filtration system and a crossflow system are included. The whole amount filtration method is a method of filtering the entire amount of water supplied to the membrane. In contrast, the cross-flow method refers to a method of filtration while forming a parallel flow with respect to the membrane surface while suppressing a phenomenon in which suspended substances or colloids contained in water supplied to the membrane deposit on the membrane surface. The cross flow method includes, but is not limited to, one-pass method, back flush method and reverse method. As shown by A of FIG. 1, the one-pass system means the system which filters without perusing the permeate liquid from an ultrafiltration membrane. As shown in FIG. 1B, the back flush system stores permeate from the ultrafiltration membrane in a permeate tank, transfers the permeate from the permeate tank to the ultrafiltration membrane, and washes and removes hyaluronic acid adhered to the surface of the filter membrane. The filtration method including the process of doing. The reverse method is a filtration method including a step of washing and removing hyaluronic acid adhered to the surface of the filtration membrane by backflowing the permeate from the ultrafiltration membrane by closing the permeate valve as shown in FIG. 1C. Say.

As used herein, the term "impurity" refers to a substance (pyrogenic substance, etc.) which may be disadvantageous when using hyaluronic acid, solvents other than water, solvents other than inorganic salts, and especially hyaluronic acid as a final product. Say. Examples of the main impurity source include those derived from tissues at the production stage of hyaluronic acid, microorganisms or culture medium (medium), or subsequent purification steps. Examples of the impurities in the present specification include, but are not limited to, tissues or cells, proteins, nucleic acids, polysaccharides, low molecular weight compounds or endotoxins. The tissue or cell as an impurity includes, but is not limited to, a tissue fragment derived from a tissue as an extract raw material used in the extraction method, or a cell or cell fragment of a microorganism used in the fermentation method. The protein as an impurity includes, but is not limited to, a protein derived from the tissue, a bacterium, a protein mixed in a post-production step, and the like. Endotoxins as impurities include, but are not limited to, lipopolysaccharides derived from the above bacteria.

As used herein, the term "low molecular weight compound" refers to a compound having a relatively low molecular weight compared to hyaluronic acids, and is not limited thereto, for example, but has a molecular weight of 2000 Da or less, or a molecular weight of 1000 Da or a molecular weight of 500 Da or less. Say a compound. Such low molecular weight compounds include various amino acids, organic acids (eg, lactic acid), sugars (eg, glucose), and the like.

"Removal" in this specification includes not only removing a target substance completely, but also removing it partially (reducing the quantity of the substance). "Refining" in this specification includes removing arbitrary or specific impurity.

About each numerical range in this specification, the upper limit and lower limit shown by "-" shall be included, respectively.

[Embodiment]

Although this invention is not limited to this, For example, it is related with the following embodiment.

Embodiment 1.

A method for purifying hyaluronic acid and / or salts thereof, comprising the step of preparing a hyaluronic acid solution containing hyaluronic acid and / or its salt and impurities to pH on the acidic side and then removing the impurities by dialysis treatment with an ultrafiltration membrane.

Embodiment 2.

Fractional molecular weight of ultrafiltration membrane and pH at dialysis treatment by ultrafiltration membrane

pH≤-5 × 10 ^-5 × (fractional molecular weight) +4.4978

The method as described in Embodiment 1 in which the dialysis process by an ultrafiltration membrane is performed on condition to satisfy | fill.

Embodiment 3.

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

The fractional molecular weight of the said ultrafiltration membrane is 12000-14000, and the method of embodiment 1 or 2 whose pH at the time of dialysis process is 3.9 or less.

Embodiment 4.

The fractional molecular weight of the said ultrafiltration membrane is 9000-11000, and the method of embodiment 1 or 2 whose pH at the time of a dialysis process is 4.1 or less.

Embodiment 5.

The fractional molecular weight of the said ultrafiltration membrane is 6000-8000, and the method of embodiment 1 or 2 whose pH at the time of a dialysis process is 4.2 or less.

Embodiment 6.

The fractional molecular weight of the said ultrafiltration membrane is 4000-5000, and the method of embodiment 1 or 2 whose pH at the time of a dialysis process is 4.3 or less.

Embodiment 7.

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

Embodiment 8.

The method in any one of embodiment 1-7 whose filtration system of the said process is a reverse system.

Embodiment 9.

The method according to any one of embodiments 1 to 8, wherein the permeation flow rate during the treatment is 20 to 50 L / m ² · hour.

Embodiment 10.

The method according to any one of embodiments 1 to 9, wherein the impurities include cells, proteins, nucleic acids, low molecular weight compounds, or endotoxins.

Embodiment 11.

The method as described in any one of embodiments 1-10 whose average molecular weights of the said hyaluronic acid after refine | purification and / or its salt are 3.5 million-7 million Da.

Embodiment 12.

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

EMBODIMENT OF THE INVENTION Hereinafter, the aspect of this invention is demonstrated.

According to an aspect of the present invention (for example, Embodiment 1), after preparing a hyaluronic acid solution containing hyaluronic acid and / or its salt and impurities at pH on the acidic side, impurities are removed by dialysis treatment with an ultrafiltration membrane. It is a purification method of hyaluronic acid and / or its salt containing the process of making it. According to this purification method, the loss of hyaluronic acid in the filtration by the ultrafiltration membrane can be reduced by making the pH at the acidic side, and impurities can be efficiently removed.

Fractional molecular weight of ultrafiltration membrane and pH at dialysis treatment by ultrafiltration membrane

pH≤-5 × 10 ^-5 × (fractional molecular weight) +4.4978

By carrying out the dialysis treatment with the ultrafiltration membrane under the conditions satisfying the above condition, it is possible to purify the hyaluronic acids without losing substantially. As the fractional molecular weight of the ultrafiltration membrane satisfying the above formula and the pH at the time of dialysis treatment with the ultrafiltration membrane, for example, the ultrafiltration membrane having a fractional molecular weight of 35000 has a pH of 2.7 and the fractional molecular weight of 30000 has a pH of 3.0 and a fractional molecular weight of 25000. Ultrafiltration membranes with a pH of 3.3 and fractional molecular weight of 20000 have a pH of 3.5, fraction molecular weight of 14000 with an ultrafiltration membrane of pH 3.8, fractional molecular weight of 13000 with an ultrafiltration membrane of pH 3.9 and fractional molecular weight of 12000 with an pH of 3.9 and fractional molecular weight of 11000. Ultrafiltration membranes with pH 4.0, fraction molecular weight 10000, ultrafiltration membranes with pH 4.0, fraction molecular weight 9000, ultrafiltration membranes with pH 4.1, fraction molecular weight 8000 with ultrafiltration membranes with pH 4.1, fraction molecular weight 7000 with ultrafiltration membranes with pH 4.2 and fraction molecular weight 6000 with For ultrafiltration membranes, pH 4.2, fractional molecular weight 5000. For ultrafiltration membranes, pH 4.3, fractional molecular weight 4000. In the ultrafiltration membrane, pH 4.3 can be mentioned.

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

The use of a membrane having a high fractional molecular weight increases the risk of loss of hyaluronic acids through the ultrafiltration membrane, while the use of a membrane having a low fractional molecular weight lowers the removal efficiency of relatively high molecular impurities such as proteins. According to the method of the above embodiment, even when a membrane having a high fractional molecular weight or a membrane having a low molecular weight is used, impurities can be removed without losing hyaluronic acids by adjusting to a pH suitable for the molecular weight of the membrane. The fractional molecular weight of the ultrafiltration membrane used in the method of the said aspect is not specifically limited. For example, when using the ultrafiltration membrane which has a fractional molecular weight of 25000-35000, it can refine | purify without losing hyaluronic acid by adjusting pH to 3.3 or less. When using an ultrafiltration membrane having a fractional molecular weight of 12000 to 14000, it is possible to purify without losing hyaluronic acids by adjusting the pH to 3.9 or less. In the case of using an ultrafiltration membrane having a fractional molecular weight of 9000 to 11000, it is possible to purify without losing hyaluronic acids by adjusting the pH to 4.1 or less. In the case of using an ultrafiltration membrane having a fractional molecular weight of 6000 to 8000, the pH can be purified to 4.2 or lower without losing hyaluronic acids. When using an ultrafiltration membrane having a fractional molecular weight of 4000 to 5000, it is possible to purify without losing hyaluronic acid by adjusting the pH to 4.3 or less.

Here, the fractional molecular weight of an ultrafiltration membrane can be determined by performing filtration using the indicator substance shown in Table 1, and irradiating the molecular weight which each blocking rate corresponds to 90%.

Although the material of the ultrafiltration membrane used in the method of the said aspect is not specifically limited, A hydrophobic organic membrane is preferable from a viewpoint of the removal of an impurity, and polysulfone, polyether sulfone, polyether, polyvinylidene fluoride, polyethylene, polypropylene More preferred.

As an ultrafiltration membrane used in the method of the said aspect, For example, PM-10, PM-50, PM-100 (made by Kotsu), NTU-3050 (made by Nito Denko Co., Ltd.), IRIS3065 (Lon-Pulrangsa) Manufactured), FS-10 (manufactured by Asahi Kasei Co., Ltd.), MU-6303 (manufactured by Kuraray Corporation), DUSO400 (manufactured by Daicel Chemical Industries, Ltd.), SLP-3053 (manufactured by Asahi Kasei Chemicals Co., Ltd.), etc. It is not limited to.

Although the filtration system in the method of the said aspect is not specifically limited, The crossflow system is preferable from the point which the membrane permeation flow rate of water is easy to stabilize, the lifespan of a filtration membrane, etc. are especially preferable, and the reverse system is especially preferable. Do.

In the method of the above aspect, the permeation flow rate during the ultrafiltration membrane treatment of the hyaluronic acid solution differs depending on the properties of the hyaluronic acid solution and the type of the ultrafiltration membrane, but cannot be uniformly defined, for example, on an industrial scale. In the purification of hyaluronic acid, it is preferable that it is 20 L / m <^{2> *} hour or more, It is more preferable that it is 25 L / m <^{2> *} hour or more, It is especially preferable that it is 30 L / m <^{2> *} hour or more. Since the hyaluronic acid shears and the molecular weight decreases, the permeation flow rate in the filtration of the hyaluronic acid solution is preferably 100 L / m ² · hour or less, more preferably 50 L / m ² · hour or less. Do.

Although there is no limitation regarding the pressure for liquid feeding in the method of the said aspect, In general, it is preferable to pass a filtration membrane by pressurization. Particularly, the method of transmitting pressure to a pump or the like is preferable. The pressure is not particularly limited as long as the filtration membrane is damaged or clogged when depressurizing the pump to deteriorate its performance. The pressure applied to the filtration membrane is preferably 0.01 MPa or more and 0.30 MPa, more preferably 0.03 MPa or more and 0.20 MPa, particularly preferably 0.05 MPa or more and 0.10 MPa.

Moreover, according to the method of the said aspect, the fall of the molecular weight of hyaluronic acid at the time of refinement | purification can be reduced, and especially the hyaluronic acid of high molecular weight (for example, the average molecular weight after refinement is 3.5 million-7 million Da) It has an excellent effect in purification. Moreover, the hyaluronic acid solution of the comparatively high density | concentration (for example, 0.1-20 g / L, 0.5-15 g / L, 1-10 g / L) which was conventionally difficult to process can be processed efficiently.

In addition, according to the method of the above aspect, not only the low molecular compound (e.g., amino acid, sugar, organic acid) that can be removed by the ultrafiltration membrane as an impurity, while the loss of hyaluronic acid is suppressed low, as well as a high molecular compound (e.g., For example, even nucleic acids, endotoxins, and proteins can be efficiently separated and removed. Moreover, the method of the said aspect shows the outstanding effect also in the removal of a nucleic acid, an endotoxin, and / or a protein.

The hyaluronic acid concentration of the hyaluronic acid solution at the time of using the method of the said aspect is not limited to this from the viewpoint of the difficulty of handling and the solubility of hyaluronic acid resulting from the height of solution viscosity, but it is 0.1-20 g / L is preferred, with 1-10 g / L being most preferred.

Moreover, in the method of the said aspect, salts, such as sodium chloride, can also coexist in a hyaluronic acid solution for the purpose of reducing the viscosity of a hyaluronic acid solution. In this case, it is preferable to avoid coexistence of a high concentration of salt so that the purification effect is not impaired. As a specific example of coexistence of such salt, adding 0.1-5 weight% of sodium chloride to a hyaluronic acid solution is mentioned.

Although the temperature of the hyaluronic acid solution at the time of using the method of the said aspect is not limited to this, It is preferable that it is 0-80 degreeC. If temperature is 80 degrees C or less, decomposition | disassembly of hyaluronic acid in a process and the fall of molecular weight can be suppressed strongly.

In addition, when performing ultrafiltration dialysis treatment, 2% or less of an alkali (for example, sodium hydroxide aqueous solution), a peroxide (for example, sodium hypochlorite aqueous solution), surfactant, a citric acid, ammonium citrate, an enzyme detergent is carried out as a membrane pretreatment. It is preferable to perform by the chemical method of washing | cleaning a film | membrane with chemical | medical agents, such as these, and physical methods, such as a flushing, a sponge ball, and an air injection method.

Another aspect of the present invention may further include the step of contacting the hyaluronic acid solution with an inorganic adsorbent, an organic adsorbent and / or activated carbon in the first aspect, in addition to the above-described steps.

In addition, in consideration of the separation / purification step required after that, it is preferable to avoid mixing of components that require an additional purification step. That is, the other aspect of this invention for avoiding mixing of a new impurity is the refinement | purification method which does not include the process of contacting with an inorganic adsorption agent or an organic adsorption agent after the ultrafiltration of the hyaluronic acid solution in the said 1st aspect. In another aspect of the present invention, the first aspect does not include a step of contacting the hyaluronic acid solution with an inorganic adsorbent or an organic adsorbent. In the present invention, since it is possible to exert a sufficient purification effect even by ultrafiltration, it is preferable to treat with ultrafiltration alone when focusing on avoiding the incorporation of new impurities. Of course, also in this case, processes, such as a purification process other than ultrafiltration, can be performed.

In another aspect of the present invention, in the above aspect, hyaluronic acid is added to Streptococcus aqui FM-100 (Micro-No. 9027) or Streptococcus aqui FM-300 (Micro-No. 2319). Are produced by By using hyaluronic acid produced by these microorganisms as a refining target, it is possible to obtain less high impurity and high molecular weight hyaluronic acid purified product, and particularly exhibits excellent effects when used as a medicine.

Since the load of the separation and purification process of hyaluronic acid can be reduced by using the purification method of the said aspect, it is especially effective to use the purification method concerning the said aspect at the comparatively early stage of the industrial process of manufacture.

In addition, the refinement | purification method etc. which were demonstrated by the said embodiment, aspect do not limit this invention, It intends to illustrate, and is disclosed. 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 in the technical scope of the invention described in the claims.

For example, the said purification method may include another process, or another process and method may be performed following the said purification method, and the method of manufacturing hyaluronic acid etc. may be sufficient. As such a process and method, for example, a step of culturing a hyaluronic acid producing microorganism strain, a step of preparing a culture filtrate from a hyaluronic acid producing microorganism strain culture solution, a step of centrifuging the purification target liquid, a step of neutralizing the target liquid, A process of finely filtering the target liquid, a process of dialysis treatment of the target liquid, a process of stirring and filtering by adding an aromatic adsorption resin to the target liquid, a step of purifying the target liquid by chromatography, and separating activated carbon from the target liquid. The process, the process of removing activated carbon from a target liquid, the process of precipitating hyaluronic acid by adding an organic solvent, the process of crystallizing hyaluronic acid, the process of drying hyaluronic acid, etc. are mentioned.

[Example]

Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to this.

Example 1

45 L of the culture medium incubated with Streptococcus aqui FM-100 (Micro-No. 9027) was diluted to 80 L with pure water (sodium hyaluronate concentration 2.0 g / 1), and the cells were removed by centrifugation. After adjusting the obtained prepared hyaluronic acid to pH 2.9, the permeation flow rate 30 L / m <^2> time, volume ratio double concentration, and equal distribution with 2 m <^2> of ultrafiltration membranes (PM-100 by Kotsu Corp.) of polysulfone of fractional molecular weight 30000 material. The dilution operation was repeated and treated with 10 times of dialysis and reverse method. 2.4 kg of salts were dissolved in 80 L of the obtained solution, adjusted to pH 7, and precipitated with 240 L of ethanol, washed with 8 L of ethanol, and dried under vacuum at 40 ° C. to obtain sodium hyaluronate. Table 2 shows the analysis results and hyaluronic acid recovery.

Example 2

After adjusting the prepared hyaluronic acid used in Example 1 to pH 2.9, an ultrafiltration membrane (FS-10, manufactured by Asahi Kasei Co., Ltd., FS-10) of fractional molecular weight 30000 material 5 m ² permeation flow rate 30 L / m ² ㆍ hour, The volumetric ratio was concentrated twice, concentrated equal dilution was repeated 11 times the number of dialysis, the reverse treatment. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and hyaluronic acid recovery.

Example 3

Example 1 After adjusting the preparation of hyaluronic acid used in the pH 3.3, cut-off molecular weight: 20,000 Material polysulfone of the ultrafiltration membrane (Nitto Den Tommy Seiko manufacture and NTU-3050) 3 m ² permeation flow rate 30 L / m ² in and time, the ratio Two-fold concentration, equal distillation of pure dilution was repeated and treated with reverse dialysis 8 times. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and the recovery of sodium hyaluronate.

Example 4

Example 1 After adjusting the preparation of hyaluronic acid used in a pH 2.9, cut-off molecular weight: 30,000 Material poly ultrafiltration membrane of vinylidene fluoride (Ron and pool rangsa manufacture and IRIS3065) permeation flow rate 30 L / m ² and hour to 5 m ^2, The volumetric ratio was concentrated twice, concentrated equal dilution was repeated 9 times the dialysis number, it was treated in reverse. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and the recovery of sodium hyaluronate.

Example 5

After adjusting the prepared hyaluronic acid used in Example 1 to pH 2.7, an ultrafiltration membrane (DUSO400, manufactured by Daicel Chemical Industries, Ltd., DUSO400) of fractional molecular weight 40000 material polyethersulfone was passed through at a flow rate of 30 L / m ^{2 at} 5 m ² . The volume ratio was twice concentrated, the same dilution pure dilutions were repeated 11 times of dialysis and treated in reverse. The obtained liquid was processed like Example 1 and 150 g of sodium hyaluronate was obtained. Table 2 shows the analysis results and the recovery of sodium hyaluronate.

Example 6

The hyaluronic acid prepared in Example 1 was adjusted to pH 3.7, and the permeation flow rate was 30 L / m ^{2 at} 5 m ² of ultrafiltration membrane (MU-6303, manufactured by Kuraresa Co., Ltd.) of fractional molecular weight 13000 material. The concentration of pears was repeated, diluting purely evenly, and then treated with reverse dialysis 11 times. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and the recovery of sodium hyaluronate.

Example 7

After adjusting the prepared hyaluronic acid used in Example 1 to pH 3.7, an ultrafiltration membrane of fractional molecular weight 10000 polysulfone (SLP-3053, manufactured by Asahi Kasei Chemicals Co., Ltd., SLP-3053) was passed through 4.5 m ² at a flow rate of 30 L / m ² . The volumetric ratio was concentrated twice, concentrated equal dilution was repeated 11 times the number of dialysis, the reverse treatment. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and the recovery of sodium hyaluronate.

Comparative Example 1

Example 1 After adjusting the preparation of hyaluronic acid used in the pH 5.5, cut-off molecular weight: 10,000 Material hydrophobic ultrafiltration membrane of polysulfone (co cheusa manufacture and PM-10) ² m 2 permeation flow rate 30 L / m ² and hour, the ratio Two-fold concentration and equal dilution operations were repeated to treat 15 times of dialysis and reverse treatment. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and hyaluronic acid recovery.

Comparative Example 2

After adjusting the prepared hyaluronic acid used in Example 1 to pH 3.6, the hydrophilic ultrafiltration membrane of the fractional molecular weight 20000 material cellulose acetate (CA600PP manufactured by DDS, CA600PP) was 4.5 m ² and the permeation flow rate was 30 L / m ^2. Concentration, equal dilution pure dilution was repeated 10 times of dialysis, the reverse treatment. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and hyaluronic acid recovery.

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 volumetric ratio was concentrated twice, concentrated equal dilution was repeated 9 times the dialysis number, the reverse treatment. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and hyaluronic acid recovery.

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, The volumetric ratio was concentrated twice, concentrated equal dilution was repeated, and treated with 11 times of dialysis and the one-pass method. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and hyaluronic acid recovery.

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 volumetric ratio was concentrated twice, concentrated equal dilution was repeated 11 times the dialysis number, and treated by a back flush method. The obtained liquid was processed similarly to Example 1, and obtained sodium hyaluronate. Table 2 shows the analysis results and hyaluronic acid recovery.

How to measure

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

(2) Protein content: Sodium hyaluronate was dissolved in 0.1 N sodium hydroxide and carried out by the Laurie method.

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

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

(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 ° C was measured.

Example 8

The prepared hyaluronic acid used in Example 1 was purified using hydrophobic ultrafiltration membranes of polysulfone having various fractional molecular weights under the following conditions, and the relationship between pH during ultrafiltration and recovery of hyaluronic acid was investigated.

HA solution conditions

HA concentration: 2 g / L

Molecular weight: 4.4 million (intrinsic viscosity: 55 dL / g)

Filtration conditions

Speed: 1 m / s

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

Thickening: 2x Thickening

Temperature: 25 ℃

The graph which shows the relationship between pH at the time of ultrafiltration, and hyaluronic acid recovery is shown in FIG. Moreover, about the membrane | membrane of each fractional molecular weight, Formulas 1-5 which show the relationship between the pH at the time of ultrafiltration, and the loss rate of hyaluronic acid were obtained. Using the following formulas 1 to 5, a pH at which no loss of hyaluronic acid in the ultrafiltration membrane of each fractional molecular weight is substantially generated is calculated, and purification by ultrafiltration is performed within the pH range (optimum pH). By carrying out, it can refine | purify without losing hyaluronic acid.

Fractional molecular weight of 30,000 Han Filtration membrane  If used

Loss rate (%) of hyaluronic acid = 44.86 x (pH at the time of ultrafiltration) -131.79

(Optimum pH≤2.9)

Fractional molecular weight of 13,000 Han Filtration membrane  If used

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

(Optimum pH≤3.7)

Fractional molecular weight of 10,000 Han Filtration membrane  If used

Loss rate (%) of hyaluronic acid = 24.36 x (the pH at the time of ultrafiltration) -97.19

(Optimum pH≤4.0)

Fractional molecular weight of 7,000 Han Filtration membrane  If used

Loss rate (%) of hyaluronic acids = 7.09 x (pH at the time of ultrafiltration) -29.02

(Optimum pH≤4.1)

Fraction molecular weight of 5,000 Han Filtration membrane  If used

Loss rate (%) of hyaluronic acid (formula 5) = 0.79 × (pH at the time of ultrafiltration) -2.01

(Optimum pH≤4.2)

FIG. 3 shows the relationship between the pH at which the loss rate of hyaluronic acids is 3% or less and the fractional molecular weight of the ultrafiltration membrane. In addition, it became clear that the relationship between the pH at which the loss rate of hyaluronic acids became 3% or less and the molecular weight of the fraction of the ultrafiltration membrane satisfied the following formula (6).

(Equation 6)

PH = -5x10 <^-5> (fractional molecular weight) +4.4978 which loss rate of hyaluronic acids becomes 3% or less

By using Formula 6, the upper limit of the optimal pH with respect to the fractional molecular weight of an ultrafiltration membrane can be calculated | required, and hyaluronic acid can be refine | purified with a loss rate of 3% or less by performing dialysis by an ultrafiltration membrane at an optimal pH.

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

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

BRIEF DESCRIPTION OF THE DRAWINGS Fig.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the filtration system of the one-pass system (A), the back flush system (B), and the reverse system (C) contained in a crossflow system.

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 rate of hyaluronic acids becomes 3% or less and the fractional molecular weight of the ultrafiltration membrane.

Claims (5)

A method for purifying hyaluronic acid and its salt, comprising the step of removing impurities by dialysis treatment of a hyaluronic acid solution containing high molecular weight hyaluronic acid and / or its salt and impurities with an ultrafiltration membrane. The method of claim 1 wherein the impurities comprise cells, proteins, nucleic acids, small molecule compounds or endotoxins. The method according to claim 1 or 2, wherein the hyaluronic acid and / or salt thereof has a molecular weight of 1.5 million to 4 million Da. The method according to any one of claims 1 to 3, wherein the concentration of hyaluronic acid and / or salt thereof in the hyaluronic acid solution is 1 to 10 g / L. The method of any one of Claims 1-4 whose temperature of the hyaluronic acid solution at the time of treatment is 0-80 degreeC.

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