TW201139463A - Purification method for hyaluronic acid and/or salts thereof - Google Patents

Abstract

Provided is a purification method for hyaluronic acid and/or salts thereof, which includes a process for performing dialysis on a hyaluronic acid solution comprising a high molecular weight hyaluronic acid and/or salts thereof and impurities, by means of an ultrafiltration membrane. The method is capable of producing a high quality hyaluronic acid and/or salts thereof from which proteins, nucleic acids, lactic acid, metals, etc. have been eliminated, with a high yield in a simple manner on an industrial scale.

Description

201139463 6. INSTRUCTION DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a method for purifying hyaluronic acid and/or a salt thereof. [Prior Art] In addition to being a moisturizer for cosmetics, hyaluronic acid is also used as a medicine in ophthalmology, orthopedics, dermatology, and the like. Although hyaluronic acid can be produced by an extract derived from an animal tissue such as a chicken cockscomb or a vitreous of a bull's eye, it is easy to be low due to the incorporation of chondroitin sulfate as an inclusion or the hyaluronic acid in the tissue. Since the molecular weight is increased, a method of producing hyaluronic acid from a culture solution by culturing a microorganism having hyaluronic acid-producing ability (acid yeast method) is also carried out (Non-Patent Document No. 1 and Patent Document 1). Hyaluronic acid produced by an extraction method or a fermentation method In the case where the protein, the exothermic substance, and the like are present in the form of impurities, a method of separating and removing the product to obtain a high-purity product is being studied. In particular, the removal of impurities in the initial stage of the production can reduce the load of the subsequent purification step. 'It is expected to be developed as a method of obtaining a high-purity product which can be used as a pharmaceutical. As a method of purifying hyaluronic acid with high purity, it is known to remove by-products such as proteins and nucleic acids, and inorganic salts derived from a medium. And the hyaluronic acid solution containing hyaluronic acid and its salts and impurities is passed through a positively charged filter. A method of purifying sodium hyaluronic acid by precipitating, precipitating and extracting a supernatant with a water-soluble organic solvent (Non-Patent Document 2) [Non-Patent Document 1] J〇urnal General Microbiology, μ 372-375, 1976 ' [Patent Document 1] Japanese Patent Publication No. Hei 4_1296 No. 154811.doc 201139463 [Patent Document 2] Japanese Patent Laid-Open Publication No. Hei 9-324〇〇1 [Invention] However, it is used as an industrial grade to be used as a (four) product. In the case of hyaluronic acid of purity, there is also a problem that the recovery rate of hyaluronic acid is low in the above method. The present invention has been made in view of the above circumstances, and an object thereof is to provide a high-purity hyaluronic acid which is purified on an industrial scale in a simple and high-yield manner. In order to achieve the above object, the inventors of the present invention conducted various studies on a method for efficiently and efficiently purifying high-purity hyaluronic acid by efficiently separating and removing impurities from a hyaluronic acid solution containing hyaluronic acid and a salt thereof and impurities. It was found that it was effective by dialysis treatment using an ultrafiltration membrane after adjusting the hyaluronic acid solution to an acidic 2 pH value. The present invention has been completed by removing nucleic acids, exothermic substances, proteins, lactic acid, inorganic salts, and the like. That is, according to the present invention, there is provided a method for purifying hyaluronic acid and/or a salt thereof, which comprises including hyaluronic acid and a salt thereof The hyaluronic acid solution of the impurity is adjusted to a pH value which is acidic, and the dialysis treatment is performed by using an ultrafiltration membrane. According to the production method, impurities can be efficiently removed. [Embodiment] [Description of Terms] "Hyaluronic acid and / or a salt thereof, synonymous with "hyaluronic acid", and used interchangeably, means free hyaluronic acid, and any hyaluronic acid salt which can be used within the scope of the object of the present invention (not limited thereto, such as sodium salt, a metal salt such as a potassium salt, a calcium salt or a lithium salt, or a hydrochloride or a phosphoric acid

154811.doc 4 S 201139463 Salt, citrate and other acid adducts, etc.) or hydrates, mixtures of these. Here, hyaluronic acid refers to a high molecular weight polysaccharide in which N-acetyl-D-glucosamine is repeatedly linked to a D-glucuronic acid-bonded 2-saccharide unit, and various salts mainly refer to a glucuronic acid moiety. Become the form of salt. Hyaluronic acid is easily spread in space due to the interaction of the collapsible chain portion with the negative charge of the carboxyl group of the D-glucuronic acid moiety, whereby it can be combined with a large amount of water to form a gel. In addition, even at a low concentration, the intermolecular force is strong, so that it has a relatively high viscosity. Such an action, for example, has the action of a moist joint, the action of a soft skin, and the like, and also exerts such effects physiologically. It is known that in hyaluronic acid, sodium hyaluronate having a molecular weight of about 2 million Da has an excellent effect on treating deformed knee arthritis, periarthritis of the shoulder, and chronic rheumatoid arthritis as compared with molecular weight. (Pharmacology and Therapy, VoL22, N〇. 9, 289 (1994); Pharmacology and Therapy, Vol. 22, No. 9, 319 (1994)). In addition, it is known that it is generally used clinically as an effect of preventing adhesion after surgery, and also as an effect of pharmaceuticals in the field of dermatology and ophthalmology. In the case of use as a pharmaceutical, it is preferred to use a hyaluronic acid having an average molecular weight of 1,000,000 or more. In view of the ease of obtaining or handling, it is more preferable that the pharmaceutical is a hyaluronic acid having an average molecular weight of 1,000,000 to 5,000,000 Da, and particularly preferably a hyaluronic acid having an average molecular weight of 1.5 to 400,000 Å. Further, when such a high molecular weight hyaluronic acid is used as a cosmetic, it also exerts an excellent effect due to its high moisturizing power. The "average molecular weight" in the present specification means the viscosity average molecular weight 154811.doc 201139463 111 molecular weight can be determined by a method generally used by f unless otherwise specified. / It can be obtained by a measurement method generally used by a Pharmacopoeia of a country, etc., and can be obtained by the method of the Japanese Pharmacopoeia t. 4 For example, in the case where it is desired to break the uric acid sodium, it is similar to the case of the invention of the present application, which is close to the amount of the flat knife (1.5 million to 3.9 million), and the average molecular weight can be obtained by using the following formula, but It is not limited to this. [Formula 1] Average molecular weight

t 22.8 J Dissolved hyaluronic acid As a solution for injection for pharmaceutical use, the injectable solution for use in the Tenth (for example, as confirmed in the Pharmacopoeia of each country) can be suitably used. The solution for injection is based on water for injection, physiological saline, etc. A pH adjuster containing a buffer such as an acid, a base or a phosphate is added thereto. These hyaluronic acids may be produced by an extraction method of extraction by an automatic structure, or may be produced by a fermentation method obtained by producing a microorganism g strain using hyaluronic acid to ferment. However, among the extracts of the animal tissues, other impurities such as mucopolysaccharides are relatively large, and the molecular weight of the hyaluronic acid is also small, so it is preferable to use those obtained by the fermentation method. In one example of the fermentation method suitable for the present invention, for example, a microorganism of the genus Streptococcus can be used and a hyaluronic acid can be obtained by a known method. In the case where 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 is sterilized by a known method such as centrifugation or filtration treatment. The hyaluronic acid may be precipitated and purified by adding a water-soluble organic solvent such as ethanol, as the case may be. In addition, 154811.doc 201139463 can also be used for treatment with alumina or the like. The "Streptococcus" in the present specification includes any strain of the genus Streptococcus which can produce hyaluronic acid. In particular, it is preferable to use a horse chain as described in the patent document 2, Streptococcus equi FM-100 (Microtechnology Research No. 9027), and Japanese Patent Laid-Open No. Hei 2 234 689. The cocci fm-300 (Microtechnology Research No. 2319) is still producing and stably producing a hyaluronic acid mutant. As an example of a bacterium belonging to the genus Streptococcus which is suitable for producing hyaluronic acid, for example, Streptococcus equi, Strept〇c〇ccus zooepidemicus, Strept〇c〇ccus equisimilis And Streptococcus dysgalactiae, Streptococcus py0genes, and the like, but are not limited thereto. The "ultrafiltration membrane" in the present specification refers to a filtration membrane having a pore diameter of 〇 〇〇1 to 〇 μi μηι and/or a filtration membrane having a molecular weight cut off of about 1 〇〇〇 3 〇〇〇〇〇. The material of the ultrafiltration membrane is roughly classified into an inorganic membrane and an organic membrane, and is further classified into a hydrophobicity and a hydrophilicity. Examples of the hydrophobic organic film 'polyhard, polysulfonated sulfone' polyether, polyvinylidene fluoride, ethylene, polypropylene, and the like are not limited thereto. Examples of the hydrophilic organic film include, but are not limited to, polyacrylonitrile, polyacrylamide, and polyamidene cellulose acetate. The shape of its filter media. It includes all modular forms such as flat plate, tubular film, roll film, and hollow fiber (brain-fiber) film. The method of passing data includes endpoint filtering mode and sweeping mode. Endpoint transition mode means the manner in which all water is supplied to the membrane. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The knowing flow method includes a one-pass method, a backlash method, and a reverse method, but is not limited thereto. The so-called single-pass method refers to a method of filtering by using a permeate from an ultrafiltration membrane as shown in Fig. 1A. The so-called back-flushing method means that the permeate from the ultrafiltration membrane is stored as shown in B of the circle 1; in the k-liquid tank, from the permeate tank to the ultra-transition membrane, and the rinsing is attached to the surface of the L membrane. The method of the hyaluronic acid step. The reverse mode refers to a filtration method including a step of rinsing hyaluronic acid attached to the surface of the filtration membrane by backflowing the permeate from the super-membrane by closing the permeate valve as shown in Fig. 1. § “Impurities in the book of the month” refers to substances other than the hyaluronic acid, solvent components other than water, and inorganic salts, especially substances that can cause adverse effects when using the hyaluronic heart as the final product (heat-generating substances, etc.) . The main impurity source is those which are derived from tissues, microorganisms or culture solutions (medium) in the production stage of hyaluronic acid, or in a subsequent purification stage or the like. Examples of the impurities in the present specification include a tissue or a microbial cell: an egg mass, a nucleoside iridium saccharide, a low molecular compound, or an endotoxin, but is not limited thereto. The tissue or the bacterial body as the impurity includes: a tissue piece derived from a tissue used as an extraction raw material used in the extraction method, or the like, or a micro-sheng, Wang Shidong bacterial cell or a bacterial cell sheet used in the fermentation method. However, it is not limited to this. The protein as an impurity and the protein from the above-mentioned tissue, the protein of the fungus, and the protein mixed in the step after the production are not limited to 154811.doc 201139463. The endotoxin as an impurity includes lipopolysaccharides derived from the above-mentioned bacteria, and the like, but is not limited thereto. In the present specification, "low molecular compound U refers to a relatively small compound in a knife compared to hyaluronic acid, for example, a molecular weight of 2 〇〇〇 Da or less, or a 畺 Da 1 Da or a molecular weight of 5 〇〇. The following compounds are not limited thereto. Such low molecular compounds include various amino acids, organic acids (such as lactic acid), sugars (such as glucose), etc. "Removal" in the present specification includes, in addition to complete removal of a target substance. In addition, partial removal (reducing the amount of the substance) is also included. The "purification" of this specification 10 includes the removal of any or specific impurities. Each numerical range in the present specification includes the upper limit and the lower limit by "~". [Embodiment] The present invention relates to, for example, the following embodiments, but is not limited thereto. Embodiment 1. A method for purifying hyaluronic acid and/or a salt thereof, which comprises dialysis treatment by using an ultrafiltration membrane after adjusting a hyaluronic acid solution containing hyaluronic acid and/or a salt thereof and an impurity to an acidic pH value The step of removing impurities. Embodiment 2. The method of Embodiment 1 wherein the pH of the ultrafiltration membrane and the pH of the dialysis treatment using the ultrafiltration membrane satisfy the following formula: dialysis treatment using an ultrafiltration membrane: pH value: -5x1 (Γ5χ (molecular weight cut off) + 4.4978. Embodiments 3. 154811.doc 201139463 The method of embodiment 1 or 2, wherein the ultrafiltration membrane has a molecular weight of 25,000 to 35,000 and the pH of the dialysis treatment is 3 3 The method of Embodiment 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut off of 12,000 to 14000, and the pH value during dialysis treatment is 3 or less. Embodiment 5 _ The method of 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut-off of 9000 to 11 Torr, and the pH of the dialysis treatment is 41 or less. Embodiment 6. The method of the aspect 1 or 2, and the medium μ .+, The molecular weight cutoff of the above-mentioned ultrafiltration cartridge is 6000~8000, and the ρΗ value during dialysis treatment is 4 2 or less. Embodiment 7· As in the method of Embodiment 1 or 2, the above-mentioned ultrafiltration is carried out in the basin. The retention molecule of the membrane is promoted to 4000~5000, and the ρΗ value of dialysis treatment is below 43. The embodiment of the present invention is as follows: wherein the ultrafiltration membrane is a water-based organic membrane in which the above-mentioned treatment is carried out in a manner as described in any one of the first to seventh embodiments. The method of any one of 1 to 8 is a reverse mode. Embodiment 10. The method according to any one of Embodiments 1 to 9 has an overcurrent flow rate of 20 to 50 L/m2.hr. 154811.doc •10· 201139463 As one of the implementation aspects 1 to 10

Embodiment 12. The method of any of the above, wherein the impurities include a low molecular compound or an endotoxin. The above-mentioned method of the method of any one of the first to eleventh, wherein the average molecular weight of the hyaluronic acid and/or its salt is 3.5 million. The concentration of hyaluronic acid and/or its salt in any of the solutions of Examples 1 to 12 is ^58^. Hereinafter, the aspect of the present invention will be described. The aspect of the present invention (for example, the embodiment) is a method for purifying hyaluronic acid and/or a salt thereof, which comprises a hyaluronic acid containing hyaluronic acid and/or a salt thereof and an impurity thereof. After the solution is adjusted to an acidic pH value, the ultrafiltration membrane is used for dialysis treatment to remove impurities. According to this purification method, the loss of hyaluronic acid when filtering by the ultrafiltration membrane can be reduced by adjusting to a pH value which is acidic, and impurities can be efficiently removed. By using the ultrafiltration membrane for dialysis treatment because the molecular weight of the ultrafiltration membrane and the pH value of the ultrafiltration membrane are dialysis treatment, the purification can be carried out without substantially losing hyaluronic acid: pH value $ -5χ10·5χ (molecular weight cut off) +4.4978. The pH value of the ultrafiltration membrane satisfying the above formula and the pH value of the dialysis treatment by the ultrafiltration membrane include, for example, an ultrafiltration membrane having a molecular weight cut off of 35 Å and a pH of 2.7; The molecular weight cut off is 30,000 and the pH is 3.0; the molecular weight cutoff of the ultrafiltration membrane is 25000', the pH value is 3.3; the molecular weight cutoff of the ultrafiltration membrane is 20000', the pH value is 3.5; and the molecular weight of the ultrafiltration membrane is 14 〇〇〇. ? 11 value 154811.doc 201139463 is 3.8; ultrafiltration membrane molecular weight cut-off is 13 〇 () (), pH is 3 9; (d) membrane molecular weight cut-off molecular weight is 12 _, pH value 39; super 遽 membrane molecular weight cutoff _ 〇, PH value is 4.0; the molecular weight cut-off of the super-membrane is _〇, pH is 4.0; the molecular weight cut-off of the super-membrane is _〇, pH is 41; the molecular weight cut-off of the ultrafiltration membrane is 8_, pH 4 The ultrafiltration membrane has a molecular weight cutoff of 7000 'pH of 4.2; the molecular weight of the ultrafiltration membrane is 6 〇〇〇, the pH value is 4.2; the molecular weight cutoff of the ultrafiltration membrane is 5 〇〇〇, and the pH is 4 3 The ultrafiltration membrane has a molecular weight cut-off of 4 Å and a pH of 4.3. Here, "substantially no loss of hyaluronic acid" means that the loss rate of hyaluronic acid is 7% or less (recovery rate is 93% or more), 6% or less (recovery is 94 / 0 or more) '5°/. The following (recovery rate is 95% or more), 4% or less (recovery rate is 96% or more) or 3% or less (recovery rate is 97% or more). When a membrane having a high molecular weight cut off is used, the risk of loss of hyaluronic acid through the ultrafiltration membrane is increased. On the other hand, when a membrane having a relatively low molecular weight cutoff is used, the removal efficiency of impurities such as proteins with respect to a polymer is lowered. According to the method of the above embodiment, the use of a membrane having a higher molecular weight cut-off and the use of a ruthenium having a lower molecular weight cut-off can be carried out by adjusting the pH corresponding to the molecular weight cut off from the membrane without loss of hyaluronic acid. Therefore, the molecular weight cut off of the ultrafiltration membrane used in the method of the above aspect is not particularly limited. For example, in the case of using an ultrafiltration membrane having a molecular weight cut off of 250 〇〇 to 35 Å, 'the pH can be adjusted to 3.3 or less without the loss of hyaluronic acid. purification. When using an ultrafiltration membrane having a molecular weight cutoff of 12,000 to 14,000, the pH can be adjusted to 39 by 154811.doc.

S •12· 201139463 was purified without loss of hyaluronic acid. In the case of using an ultrafiltration membrane having a molecular weight cut off of 9000 to 11,000, it can be purified without loss of hyaluronic acid by adjusting the pH to 4.1 or less. In the case of using an ultrafiltration membrane having a molecular weight cut off of 6000 to 8000, purification can be carried out without loss of hyaluronic acid by adjusting the pH to 4.2 or less. In the case of using an ultrafiltration membrane having a molecular weight cut off of 4000 to 5,000, purification can be carried out without damaging hyaluronic acid by adjusting the pH to 4.3 or less. Here, the molecular weight cut-off of the ultrafiltration membrane can be determined by filtering using the target materials shown in Table 1 and investigating the respective blocking ratios corresponding to a molecular weight of 90%. [Table 1] Specimen species used to determine the molecular weight cutoff. Molecular weight of molecular weight deterministic sucrose 340 1.1 Raffinose 590 1.3 Vitamin 12 1360 1.7 Subtilin 1410 1.7 Teng Shisu 5700 2.7 Cytochrome C 13000 3.8 Myoglobin 17000 4.0 α - chymotrypsinogen 25000 4.6 pepsin 35000 5.0 ovalbumin 43000 5.6 bovine albumin 66000 6.4 aldolase 142000 8.2 γ-globulin 150000 8.4 The material of the ultrafiltration membrane used in the above method is not particularly limited It is preferable that it is a hydrophobic organic film from the viewpoint of removal of impurities, and more preferably polysulfone, polyethersulfone, polyether, polyvinylidene fluoride, polyethylene, poly 154811.doc -13·201139463 propylene. As the ultrafiltration membrane used in the method of the above aspect, for example, PM-10, PM-50 'PM-100 (manufactured by Koch Co., Ltd.) 'NTU-3050 (manufactured by Jidong Electric Co., Ltd.)' IRIS3065 (Rhone can be used) ·Manufactured by Planck, FS-1〇 (made by Asahi Kasei Corporation), MU-63〇3 (made by Kuraray Co., Ltd.), DUS〇4〇〇 (made by Taijin Chemical Industry Co., Ltd.), SLP-3053 (Asahi Kasei Chemicals) The company manufactures, etc., but is not limited to these. The filtration method in the above method is not particularly limited. However, since the flow rate of the water permeable membrane is easily stabilized, the life of the filtration membrane is prolonged, and the like, it is preferable to use a sweeping method, and particularly preferably a reverse mode. In the above method, the flow rate of the hyaluronic acid solution in the ultrafiltration membrane treatment differs depending on the properties of the hyaluronic acid solution or the type of the ultrafiltration membrane, which cannot be generalized. For example, in the case of purifying hyaluronic acid on an industrial scale, it is preferably 20 L/m2.hr or more, more preferably 25 L/m2.hr or more, and particularly preferably 30 L/m2.hr or more. The flow rate of the hyaluronic acid solution at the time of filtration is preferably 100 L/m2.hr or less, and more preferably 50 L/m2.hr or less, due to the possibility of a decrease in molecular weight due to shearing hyaluronic acid. In the above method, the pressure for transporting the liquid is not limited, and it is usually preferred to pass through the filtration membrane under pressure. In particular, when the pressure is applied by a pump when the liquid is transported, the pressure is not particularly limited as long as the performance is deteriorated without damaging or clogging the filter membrane. The pressure applied to the filtration membrane is preferably 〇1 MPa or more and 0.30 MPa or less, more preferably 0.03 MPa or more and 0.20 MPa or less, and particularly preferably 〇5 Mpa or more and 〇1 〇 MPa or less. 154811.doc -14· 201139463 Furthermore, according to the above method, the molecular weight of hyaluronic acid at the time of purification can be reduced, especially at a high molecular weight (for example, the average amount after purification is 3.5 million to 7 million Da). In the purification of hyaluronic acid, it exerts an excellent effect. Further, even a hyaluronic acid solution having a relatively high concentration (e.g., 〇/~20g/L, 〇.5 to 15g/L, or (7)...) which has been difficult to handle previously can be efficiently treated. Further, according to the method of the above aspect, although the disappearance of hyaluronic acid is suppressed to a low level, as an impurity, not only a low molecular compound (for example, an amino acid, a sugar, an organic acid) but also a polymer which can be removed by an ultrafiltration membrane can be used. Compounds (eg, nucleic acids, endotoxins, proteins) can also be efficiently separated and removed. Further, the above method also exerts an excellent effect in the removal of nucleic acids, endotoxin and or proteins. The hyaluronic acid concentration of the hyaluronic acid solution when using the above method is preferably from 0.^20 g/L, from the viewpoint of the ease of treatment caused by the viscosity of the solution and the solubility of hyaluronic acid. It is hi〇g/L 'but it is not limited to this. Further, in the above method, in order to reduce the viscosity of the hyaluronic acid solution, a salt such as sodium chloride may be coexisted in the hyaluronic acid solution. In this case, it is preferable to avoid the coexistence of the high-concentration salt so that the purification effect is not impaired. As a specific example of the coexistence of such a salt, 0.1 to 5 wt% of sodium chloride is added to the hyaluronic acid solution. The temperature of the hyaluronic acid solution in the case of using the above aspect is preferably 0 to 80 ° C, but is not limited thereto. When the temperature is 8 Torr or less, the decomposition of hyaluronic acid and the decrease in molecular weight during the treatment can be strongly suppressed. 15481I.doc -15- 201139463 In addition, in the ultrafiltration dialysis treatment, it is preferred to use 2% or less of a base (for example, an aqueous sodium hydroxide solution) or a peroxide (for example, sodium hypocarbonate) as a membrane pretreatment. Aqueous solution), surfactant, citric acid, ammonium citrate. A chemical method such as an enzyme cleaner to clean the membrane, or a physical method such as rinsing, sponge ball, or air injection. According to a further aspect of the present invention, in the first aspect, in addition to the above steps, the step of contacting the hyaluronic acid solution with the inorganic adsorbent, the organic adsorbent and/or the activated carbon may be further included. Further, in consideration of the separation, purification step, and the like which are necessary thereafter, it is preferable to avoid the incorporation of components which require additional purification steps. That is, in order to avoid the other aspect of the present invention in which new impurities are mixed, the purification method of the step of contacting the inorganic adsorbent or the organic adsorbent after the ultrafiltration of the hyaluronic acid solution in the first aspect is not included. . In a further aspect of the invention, the step of contacting the hyaluronic acid solution with the inorganic adsorbent or organic adsorbent is not included in the first aspect described above. In the present invention, even if it is ultrafiltration, a sufficient purification effect can be exhibited. Therefore, when it is important to avoid the incorporation of new impurities, it is preferable to carry out treatment by ultrafiltration alone. Of course, in this case, other purification steps may be performed in addition to the super-small. In addition, in a further aspect of the present invention, in the above aspect, the Streptococcus equi subsp. FM-100 (Microtech Research No. 9027) or Streptococcus equi subsp. FM-300 (Microtech Research No. 2319) No.) Production of hyaluronic acid. By using hyaluronic acid produced by such microorganisms as a purification target, a purified hyaluronic acid having less impurities and a high molecular weight can be obtained, and in particular, it is excellent in use as a medicine. -16- 154811.doc

S 201139463 The purification method of the above-mentioned aspect can be alleviated in the initial stage of the industrial process of manufacturing by using the purification method of the above aspect to reduce the negative # of the separation and purification steps of the uric acid. Further, the purification method described by the above embodiment and aspects is not limited to the inventors, and (5) is intended to be exemplified and disclosed. The technique of the present invention is stipulated by the special (four) riding regulations, and the manufacturer can make various design changes within the technical scope of the invention disclosed in the patent application scope. & For example, the above purification method may be a method of producing hyaluronic acid or the like by further including other steps or further performing other steps after the above purification method. As such a step and method, for example, a step of cultivating a hyaluronic acid producing microorganism strain; a step of producing a culture filtrate from a hyaluronic acid-producing microbial strain culture solution; a step of centrifuging the purification target liquid; a step of neutralizing the target liquid; and a micro-purification a step of the target liquid; a step of permeating, treating the target liquid; a step of adding an aromatic adsorption resin to the purification target liquid, and mixing and ultrafiltration; a step of purifying the target liquid by chromatography; and an activated carbon from the target liquid The step of separating; the step of removing activated carbon from the target liquid; the step of adding an organic solvent to make the hyaluronic acid swell; the step of crystallizing the hyaluronic acid; and the step of drying the hyaluronic acid. [Embodiment] The present invention will be described more specifically by way of example, but the invention is not limited thereto. Example 1 45 L of the culture medium cultured using spherinella ball FM-100 (Microtechnical Research No. 9〇27) was diluted with pure water to 80 L (sodium hyaluronate concentration 2〇g/L) and 154811 .doc •17- 201139463 Centrifugal separation removes bacteria. After the obtained crude hyaluronic acid was adjusted to pH 2.9, an ultrafiltration membrane (PM-100 manufactured by K〇ch Co., Ltd.) of 2 m 2 having a molecular weight cutoff of 30,000 and a polysulfone was used, and the flow rate was 30 L/m 2 , hr. A concentration-equal dilution operation of 2 times the volume ratio was performed, and the treatment was performed in the same manner as the number of times of dialysis i. 2.4 kg of salt was dissolved in a solution obtained by 80 L. After adjusting to pH 7, it was precipitated with 240 L of ethanol and washed with 8 L of ethanol, and vacuum dried at 40 ° C to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2. Example 2 After the crude hyaluronic acid used in Example 1 was adjusted to pH 2.9, an ultrafiltration membrane (manufactured by Asahi Kasei Co., Ltd., FS-10) having a molecular weight cutoff of 30,000 and a polyethersulfone was used, 5 m2 '30 L/ The permeation flow rate of m2.hr was repeatedly diluted with a volume ratio of 2 times, diluted with pure water, and treated in the reverse order of the number of times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and hyaluronic acid recovery are shown in Table 2. Example 3 After the crude hyaluronic acid used in Example 1 was adjusted to pH 3.3, an ultrafiltration membrane (NTU-3050 manufactured by Nippon Electric Co., Ltd.) 3 m2 using a polysulfone having a molecular weight cut off of 20,000 was used, and 30 L/m2 was used. The permeation flow rate of .hr was repeated with a volume ratio of 2 times concentrated, diluted with pure water, and treated in the reverse direction with 8 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the recovery of sodium hyaluronate are shown in Table 2. 154811.doc

S -18-201139463 Example 4 After the crude hyaluronic acid used in Example 1 was adjusted to pH 2 9 , a molecular weight of 3G0 () () was used, and the material was a super-deposited film of polyethylene. Planck made .IRIS3065) 5 m2, diluted with 3 〇 [/^ according to the flow rate repeatedly into a volume ratio of 2 times concentrated, equal times of pure water, and 9 times of dialysis, reversed deal with. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the recovery rate of hyaluronic acid are shown in Table 2. Example 5 After adjusting the crude hyaluronic acid used in Example 1 to pH Η 2·7, an ultrafiltration membrane (DUSO40G manufactured by Taijin Chemical Industry Co., Ltd.) 5 m2 having a molecular weight cut off of 40,000 and a polyether sulfone was used. The permeation flow rate of 30 L/m2.hr was reversed into a concentration of 2 times concentrated, equal-purified pure water, and treated in the reverse direction with 11 times of dialysis. The resulting liquid was treated in the same manner as in the working example to obtain 15 g of sodium hyaluronate. The analysis results and the recovery rate of sodium hyaluronate are shown in Table 2. Example 6 The crude hyaluronic acid used in Example 1 was adjusted to pH 3 7 and then subjected to an ultrafiltration membrane (manufactured by Kuraray Co., Ltd., MU_63〇3) 5 m2 having a molecular weight of 13 GGG and a polyfluorene. The permeation flow rate of 〇L/m2.hr was repeatedly diluted with a volume ratio of 2 times, diluted with pure water, and treated in the reverse order of dialysis times. The resulting solution was treated in the same manner as in the examples to obtain sodium hyaluronate. The analysis results and the recovery rate of sodium hyaluronate are shown in Table 2. 154811.doc 201139463 Example 7 After the crude hyaluronic acid used in Example 1 was adjusted to pH 3.7, an ultrafiltration membrane having a molecular weight cut off of 10,000 and made of polysulfone (manufactured by Asahi Kasei Chemical Co., Ltd., 8Ι^Ρ-3053) was used. M2, at a flow rate of 30 L/m2.hr, was subjected to a concentration ratio of 2 times concentration, diluted with pure water, and treated in a reversed manner with 11 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the recovery rate of sodium hyaluronate are shown in Table 2. Comparative Example 1 After the crude hyaluronic acid used in Example 1 was adjusted to pH 5.5, a hydrophobic ultrafiltration membrane having a molecular weight cut off of 10,000 and made of polysulfone was used (〇11; manufactured by 么-1〇) 2 M2' was repeatedly subjected to a concentration and equal-fold dilution operation at a permeation flow rate of 30 L/m2.hr, and was treated in the reversed manner by the number of dialysis times. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and hyaluronic acid recovery are shown in Table 2. Comparative Example 2 After the crude hyaluronic acid used in Example 1 was adjusted to pH 3 6 , a hydrophilic ultrafiltration membrane (manufactured by DDS Co., Ltd., CA6〇〇pp) having a molecular weight cut off of 20,000 and a cellulose acetate content of 45 m 2 was used. The volumetric ratio was doubled to a concentration of 2 〇L/m2hr, and the mixture was diluted with twice the pure water, and treated in the reverse direction of the number of dialysis persons n. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2. 154811.doc 201139463 Comparative Example 3 After the crude hyaluronic acid obtained in Example 1 was adjusted to pH 3.3, a hydrophilic ultra-ruthenium film having a molecular weight of 20,000 and a polyethylenimine acetate was used (manufactured by Jidong Electric Co., Ltd.). NTU-4220) 5 m2, with a volumetric ratio of 30 L/m2.hr, a concentration ratio of 2 times concentrated, equal-fold dilution of pure water, and treatment with 9 times of dialysis and reversed. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2. Comparative Example 4 After the crude hyaluronic acid obtained in Example 1 was adjusted to ρΗ 3·7, a hydrophobic ultrafiltration membrane (manufactured by Kuraray Co., Ltd. MU-6303) having a molecular weight cutoff of 13,000 and a material of polyacetic acid was used, 5 m 2 , The volume ratio was doubled to a concentration of 2 L/m2.hr, and the mixture was diluted with twice the volume of pure water. The treatment was carried out in a one-way manner with 11-person dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2. Comparative Example 5 The crude hyaluronic acid used in Example 1 was adjusted to ? After 11 3 7 , using a hydrophobic ultrafiltration membrane (manufactured by Kuraray Co., Ltd., MU-6303) with a molecular weight cutoff of 13,000 and a molecular weight of 13,000, the volume ratio was reversed at a flow rate of 15 L/m2.hr. Diluted with 2 times concentrated, equal times pure water, and treated with 11 times of dialysis and backflushing. The obtained solution was treated in the same manner as in Example 1 to obtain hyaluronic acid. The analysis results and the hyaluronic acid recovery rate are shown in Table 2. 154811.doc -21 - 201139463 [(Νΐ 154811.doc ζ.·ε

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[%] «1-^0VH #1 201139463 Determination method (1) Nucleic acid content: The absorbance of 0.1% sodium hyaluronate at 260 nm was measured. (2) Protein content: The sodium hyaluronate was dissolved in 0.1 N sodium hydroxide and measured by the Loley method. (3) Lactic acid: The sodium hyaluronate was dissolved at a concentration of 0.1%, and the WL-LDH (L-Lactate Dehydrogenase, L-lactate dehydrogenase) method was used for measurement. (4) Metal: Sodium hyaluronate was dissolved in 8 N nitric acid at a concentration of 0.05, and subjected to ICP (Inductively Coupled Plasma) emission spectrum analysis. (5) Ultimate viscosity: sodium hyaluronate is 0.02 ° /. The concentration was dissolved in 0.2 Torr of sodium chloride and the ultimate viscosity at 30 ° C was measured. [Table 3] Evaluation item small medium polynucleic acid [260 nm / E] ^ 0.02 0.02 < Nucleic acid < 10 10 $ Nucleic acid < 100 Endotoxin [EU / mL] Core 0.001 0.001 < ET +1 < 0.01 0.01 ^ET*1 <50000 Protein [pg/mL] ^0.04 0.04 < protein < 0.20 0.20 ^protein <0.5 Lactic acid [W%] ^0.01 0.01<Lactic acid <10 10$ Lactic acid <100 Amino group Acid [pg/mL] 5 < amino acid < 1000 1000^ amino acid < 150000 Metal * 2 [ppm] ^ 10 10 < Metal < 500 500 S Metal < 1000 ^ET : Endotoxin * 2Ca, Fe In total, the composition of Mg, Cr, Si, Al, As, Cu, and Pb was as follows. The crude hyaluronic acid used in Example 1 was purified under the following conditions using a hydrophobic ultrafiltration membrane having various molecular weight cut-offs. The relationship between the pH value of ultrafiltration 154811.doc -23·201139463 and the recovery rate of hyaluronic acid was investigated. HA solution conditions

HA concentration: 2 g/L Molecular weight: 4.4 million (limit viscosity: 55 dL/g) Filtration conditions Line speed: 1 m/s Transmission flow rate: 30 L/(m2.hr)

Concentration: 2 times concentration Temperature: 25 ° C A graph showing the relationship between pH value and hyaluronic acid recovery rate in ultrafiltration is shown in Fig. 2 . Further, with respect to the film having the molecular weight cut off, Formulas 1 to 5 indicating the relationship between the pH value at the time of ultrafiltration and the hyaluronic acid loss rate were obtained. The pH value of the hyaluronic acid in the ultra-transition membrane which does not substantially cause each molecular weight cutoff is calculated by the following formula, and is purified by using an ultrafiltration membrane within the range of the pH value (optimum pH value). Purification was carried out without loss of hyaluronic acid. When using an ultrafiltration membrane with a molecular weight cut off of 30,000 (Formula 1), the loss rate (%) of hyaluronic acid = 44.86 χ (pH at ultrafiltration) - 131.79 (optimal pH ^ 2.9) In the case of an ultrafiltration membrane of I3,000 (Formula 2), the loss rate of hyaluronic acid (%) = 40.84x (the value of ultrafiltration is ΡΗ) 86.92 (optimal pH value S3.7) using a molecular weight cutoff of 10,000 When the filter is used

154811.doc • 24· S 201139463 (Formula 3) Loss rate of hyaluronic acid (%)=24.36χί#ϊ, 士士(pH at ultrafiltration)_ 97.19 (Optimum pH S 4.0) In the case of 7,000 ultrafiltration membranes (Formula 4) Loss rate of hyaluronic acid (%) = 7. G9x (pH at ultrafiltration) _M (10) (Optimum pH S4.1) 'With a molecular weight cutoff of 5,000 In the case of a filter (Equation 5), the loss rate of hyaluronic acid (%) = 0.79x (when ultrafiltration is used (optimal pH value: 4.2). The loss rate of hyaluronic acid is 3% or less of pHi and super-membrane. The relationship between the molecular weight cut off and the molecular weight cut off is shown in Fig. 3. In addition, it is clear that the relationship between the pH value of the hyaluronic acid loss rate of 3% or less and the molecular weight cut off of the ultrafiltration membrane satisfies the following formula 6. (Formula 6) The loss rate of hyaluronic acid becomes pH value below 3% = _5 χ丨〇 · 5 χ (molecular weight cut off) + 4.4978 By using Equation 6, the upper limit of the optimum molecular value relative to the molecular weight cut off of the ultrafiltration membrane can be determined by using the optimum pH value The membrane is subjected to dialysis, and the hyaluronic acid can be purified at a loss rate of 3% or less. According to the above experiment, it can be confirmed that the purification method of the present invention is used. The high molecular weight hyaluronic acid can be purified by efficiently removing impurities from the hyaluronic acid solution. The present invention has been carried out based on the examples. This embodiment is always an example. The practitioner understands that various modifications can be implemented, and such modifications are also It is included in the scope of the present invention. I54811.doc • 25· 201139463 [Simple description of the diagram], the backlash method (B) The relationship between the pH value of the graph and the ultrafiltration diagram 1 indicates the one-way included in the sweep mode. Figure (2) and the reverse mode (C) filter mode; Figure 2 shows the pH value and hyaluronic acid loss table during ultrafiltration; and Figure 3 shows that the loss rate of hyaluronic acid is 3% or less. A graph of the relationship between molecular weights. 1548ll.doc 26-

Claims (1)

201139463 VII. Patent application scope: 1. A method for purifying hyaluronic acid and/or its salt, which comprises using an ultrafiltration membrane by adjusting a hyaluronic acid solution containing hyaluronic acid and/or its salt and impurities to an acidic pH value. The step of performing dialysis treatment to remove impurities. 2. The method of claim 1, wherein the dialysis treatment is performed by using an ultrafiltration membrane under the condition that the molecular weight of the ultrafiltration membrane and the pH of the dialysis treatment using the ultrafiltration membrane satisfy the following formula: pH value _5xl〇- 5x (molecular weight cut off) +4.4978. 3. The method of claim 1 or 2, wherein the ultrafiltration membrane has a molecular weight cutoff of 25,000 to 35,000 and a pH of 33 or less during dialysis treatment. The method of claim 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut off of 12,000 to 14,000 and a dialysis treatment value of 3 or less. For example, the method of "monthly term 1 or 2, wherein the molecular weight cut off of the above super-ruthenium film is 9000~11000, and the pH value during dialysis treatment is below. Such as. The method of claim 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut off of 6000 to 8000 Å and a pH of 4.2 or less during dialysis treatment. The method of claim 1 or 2, wherein the ultrafiltration membrane has a molecular weight cutoff of 4000 to 5000, and the pH of the dialysis treatment is 4.3 or less. ' wherein the ultrafiltration membrane is hydrophobic, wherein the treatment is excessive The method of the present invention, wherein the method of any one of claims 1 to 8 is a reverse mode. The method of any one of the above-mentioned methods, wherein the above-mentioned impurities include the bacterial protein, the method of any one of the above-mentioned impurities, wherein the above-mentioned impurities include the bacterial protein. The nucleic acid, the low molecular compound or the endotoxin. The method of the above-mentioned hyaluronic acid and/or its salt after purification has an average molecular weight of 3,500,000 to 7 million Da. The method of any one of items 1 to 12, wherein the concentration of hyaluronic acid and/or a salt thereof in the hyaluronic acid solution is 丨~5 g/L. 154811.doc