JP5690497B2 - Method for producing liquid containing high purity hyaluronic acid and / or salt thereof - Google Patents

Description

  The present invention relates to a method for producing a high-purity hyaluronic acid solution containing “hyaluronic acid and / or a salt thereof” (hereinafter collectively referred to as hyaluronic acid), and particularly relates to a method for producing an injection solution suitable for a pharmaceutical product. It is.

  Hyaluronic acid is a high molecular weight polysaccharide that is said to have a molecular weight of up to 5,000,000, which is formed by repeating and linking disaccharide units in which N-acetyl-D-glucosamine and D-glucuronic acid are linked. Generally, the glucuronic acid is separated and purified as sodium hyaluronate in the form of sodium salt. Sodium hyaluronate with a molecular weight of about 2 million is known to exhibit superior effects in the treatment of knee osteoarthritis, shoulder periarthritis, rheumatoid arthritis, etc., as a pharmaceutical compared with a molecular weight of about 800,000. (Pharmacology and Treatment Vol. 22 No. 9 289 (1994); Pharmacology and Treatment Vol. 22 No. 9 319 (1994)).

  For the purpose of preventing adhesion after surgery, and also in the dermatological and ophthalmological areas, the effects as pharmaceuticals are known and some have been put into practical use. Sodium hyaluronate produced by microbial fermentation is cultured using, for example, a certain genus Streptococcus, the obtained culture solution is diluted, and is obtained in powder form through various purification steps.

Pharmacology and treatment Vol. No. 22 9 289 (1994) Pharmacology and treatment Vol. No. 22 9 319 (1994)

According to the microbial fermentation method, sodium hyaluronate can be purified and obtained with a high molecular weight, but there are various difficult problems when mass-producing sodium hyaluronate injection solutions.
That is, it is difficult to efficiently dissolve high molecular weight sodium hyaluronate in a short time, it is difficult to handle because the viscosity of the solution is very high, and it is unstable to heat and difficult to filter or sterilize. .
Therefore, a method for producing a large amount of a high molecular weight sodium hyaluronate injection solution obtained by such a microbial fermentation method has not been clarified.

  The present inventor has completed the present invention as a result of earnestly examining a method for producing a large amount of a high-purity hyaluronic acid-containing liquid as an injection solution.

The present invention relates to the method described below.
That is,
(1) A method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof, comprising the following 6 steps of A, B, C, D, E and F.
Step A: Step of introducing hyaluronic acid into the stirring tank.
Step B: Step of dissolving hyaluronic acids.
Step C: Step of defoaming the hyaluronic acid-containing liquid.
Step D: A step of heat sterilizing the liquid containing hyaluronic acid.
Step E: Step of removing foreign substances in the hyaluronic acid-containing liquid.
Step F: Step of filling a hyaluronic acid-containing liquid.
(2) A process for producing a high-purity hyaluronic acid-containing liquid, which is processed in the order of the following 6 steps of A, B, C, D, E, and F.
Step A: A step of introducing hyaluronic acid into a stirring tank without turning over the hyaluronic acid filled in an airtight container with a valve and touching the outside air.
Step B: A step of dissolving hyaluronic acids using a stirring tank equipped with a stirring blade selected from a turbine type, a disper type, a disper turbine type, an anchor type, and a saw blade with a paddle plate.
Step C: Step of defoaming the hyaluronic acid-containing liquid by reducing the internal pressure of the stirring tank to atmospheric pressure or lower.
Step D: An inner cylinder through which the liquid to be heated is circulated and an outer cylinder provided so as to surround and surround the outer periphery of the inner cylinder, forming a heating medium passage in the space between the inner and outer cylinders, and in the axial direction An indirect heating device provided with a plurality of fixed stirring mechanisms each having a shaft portion having a length and a plurality of blade pieces projecting radially outward from the shaft portion and having an angle in the axial direction. And heat sterilizing the liquid containing hyaluronic acid.
Step E: A step of removing foreign substances in the hyaluronic acid-containing liquid by filtering the hyaluronic acid-containing liquid through a filtration membrane.
Step F: When filling the liquid containing hyaluronic acid into the injection container and / or vial, the filling pump of the filling machine is a weight valve type, a spring ball valve type, a pinch valve type, a rotary piston pump type, a diaphragm type A step of pressurizing the chemical tank and filling the hyaluronic acid-containing liquid with one selected from the above.
(3) The method for producing a high-purity hyaluronic acid-containing liquid according to (1) or (2), wherein the hyaluronic acid-containing liquid is prepared with water for injection or physiological saline, (4) the valve in the step A (3) The method for producing a high-purity hyaluronic acid-containing liquid according to (3), wherein the connection surface of the attached airtight container is a curved surface, (5) The angle of the charging chute of the airtight container with a valve in the step A is 50 ° or more. (4) A method for producing a high-purity hyaluronic acid-containing liquid according to (4), (6) a method for producing a high-purity hyaluronic acid-containing liquid according to (5), wherein the valve in step A is a butterfly valve, (7) the A (8) The method for producing a high-purity hyaluronic acid-containing liquid according to (6), wherein the material of the inner surface of the airtight container with a valve in the process is stainless steel or Teflon (registered trademark) coating; (7) The method for producing a high-purity hyaluronic acid-containing liquid as described in (7) using a stirring tank in which the axis of the stirring blade is located at the center of the container or at an eccentric position, (9) The stirring blade in the step B is one stage or multistage. A method for producing a high purity hyaluronic acid-containing liquid as described in (8) using a certain stirring tank, (10) a high purity as described in (9) using a stirring tank in which the rotation speed of the stirring blade in the step B is 100 to 5000 rpm (11) The production method of the hyaluronic acid-containing liquid, (11) The internal material of the stirring tank and line in the step B is Teflon (registered trademark), Teflon (registered trademark) lining, Teflon (registered trademark) coating (10) (12) The high-purity hyaluronic acid according to (11), wherein the internal pressure of the stirring layer during the defoaming treatment in step C is reduced to 5 to 20 kPa abs. (13) Containing high-purity hyaluronic acid according to (12), wherein the concentration of the hyaluronic acid-containing liquid during the defoaming treatment in step C is 7.5 to 12.5 g / l (14) The method for producing a high-purity hyaluronic acid-containing liquid according to (13), wherein the indirect heating device in the step D uses an indirect heating device in which a heating unit, a holding unit, and a cooling unit are used. (15) The method for producing a high-purity hyaluronic acid-containing liquid according to (14), wherein the temperature of the holding unit of the indirect heating device in the step D is 115 ° C. to 145 ° C., (16) the indirect heating device in the step D (15) The method for producing a high-purity hyaluronic acid-containing liquid according to (15), wherein the residence time of the hyaluronic acid-containing liquid in the holding section is 2 seconds to 30 minutes, (17) The cooling section outlet of the indirect heating device in the visiting step D of (18) The pressure in the inner cylinder of the indirect heating device in the step D is set higher than the pressure in the outer cylinder. (17) The method for producing a high-purity hyaluronic acid-containing liquid according to (17), (19) The method for producing a high-purity hyaluronic acid-containing liquid according to (18), wherein the material of the indirect heating device in step D is stainless steel or hastelloy. (20) The method for producing a high-purity hyaluronic acid-containing liquid according to (19), wherein the concentration of the hyaluronic acid-containing liquid in Step D is 7.5 to 12.5 g / l, (21) In the Step E (20) The method for producing a high-purity hyaluronic acid-containing liquid according to (20), wherein the pH of the hyaluronic acid-containing liquid during filtration membrane treatment is 2 to 10, (22) of the hyaluronic acid during filtration membrane treatment in the step E (21) The method for producing a high-purity hyaluronic acid-containing liquid according to (21), wherein the temperature of the liquid is 5 to 100 ° C., (23) The concentration of the hyaluronic acid-containing liquid during the filtration membrane treatment in step E is 7.5 The method for producing a high-purity hyaluronic acid-containing liquid according to (22), which is ˜12.5 g / l, (24) the pore size of the filtration membrane in the step E is in the range of 0.2 to 50 μm, according to (23) (25) A method for producing a high purity hyaluronic acid-containing liquid, (25) The high purity hyaluronic acid according to (24), wherein the filter medium shape of the filtration membrane in the step E is one selected from a flat membrane, a filter cartridge, and a disposable filter. (26) The material of the filtration membrane in the E step is polytetrafluoroethylene, polyester, Teflon (registered trademark), polypropylene, polyvinylidene fluoride And a method for producing a high-purity hyaluronic acid-containing liquid according to (25), which is a kind selected from nylon, and (27) when filling the hyaluronic acid-containing liquid in the F step into an injection container and / or vial, (26) The method for producing a high-purity hyaluronic acid-containing liquid according to (26), wherein the filling pump of the filling machine uses a diaphragm mold, (28) When filling the injection liquid and / or vial with the hyaluronic acid-containing liquid in the F step (27) The method for producing a high-purity hyaluronic acid-containing liquid according to (27), wherein the chemical tank is pressurized at 0.01 to 0.50 MPa, (29) The concentration of the hyaluronic acid-containing liquid in step F is 7.5 to (28) The method for producing a high-purity hyaluronic acid-containing liquid according to (28), wherein the molecular weight of the hyaluronic acid is from 1,500,000 to 4,000,000. (31) The method for producing a high-purity hyaluronic acid-containing liquid according to any one of (29), (31) The hyaluronic acid is produced by a fermentation method using Stompococcus equi EFM-100. The method for producing a high-purity hyaluronic acid-containing liquid according to any one of (1) to (29), wherein the hyaluronic acid is Streptococcus equiy FM-300, The method for producing a high-purity hyaluronic acid-containing liquid according to any one of (1) to (29), which is produced by a fermentation method.

  According to the present invention, it is possible to produce hyaluronic acid injection solution having a molecular weight of 1.5 to 4 million obtained by a microbial fermentation method on a large scale.

The present invention will be further described in detail below.
The hyaluronic acid used in the present invention includes hyaluronic acid, a salt of hyaluronic acid, or a mixture of hyaluronic acid and a salt of hyaluronic acid. Hyaluronic acids may be in a free form or a salt thereof, for example, sodium salt, potassium salt, calcium salt, lithium salt and the like, and sodium salt is preferable. Furthermore, the hyaluronic acid-containing liquid used in the present invention may be extracted from animal tissue or manufactured by fermentation. Preferably, it is produced by a fermentation method.

  Hyaluronic acids by fermentation can be obtained by known methods using, for example, bacteria of the genus Streptococcus.

The strain used in the fermentation method is a microorganism having the ability to produce hyaluronic acid, such as Streptococcus sp. No.) and Streptococcus equii FM-300 described in Japanese Patent Application Laid-Open No. Hei 2-23489 are desirable, such as mutant strains that stably produce hyaluronic acid at a high yield.
Those cultured and purified using the above mutant strains are used.

  The hyaluronic acid used in the present invention has a molecular weight of 1.5 million to 4 million. When the average molecular weight is less than 1,500,000, the efficacy as a pharmaceutical is reduced. It is difficult to obtain more than 4 million by this method.

  As a solution for injection used in the step of dissolving sodium hyaluronate according to the present invention, water for injection, physiological saline, and a Japanese pharmacy added with a pH adjuster containing a buffer such as acid, alkali, and phosphate. The one approved in the section of General Formulations for Injection can be used.

The amount of hyaluronic acid added in the dissolving step is set so that the hyaluronic acid concentration is 0.75 to 1.25 w / v%. When the hyaluronic acid concentration is 0.75 w / v% or less, the viscosity of the hyaluronic acid solution is low, and the production is easy even without the present invention.
Moreover, it is difficult to prepare 1.25 w / v% or more in large quantities from the solubility of hyaluronic acid. A hyaluronic acid concentration of 0.75 to 1.25 w / v% that results in a highly viscous solution corresponds to the production conditions of the present invention.

  The dissolved hyaluronic acid is filled in an airtight container with a valve. The connecting surface for charging is a curved surface, and the angle of the charging chute of the airtight container with a valve is set to be a steep slope of 50 ° or more. The reason is that when the container is turned upside down and hyaluronic acid is charged, there is little loss.

As the valve, a butterfly valve is used, and hyaluronic acids can be added aseptically to a stirring tank for dissolution without touching the outside air by switching. The material of the inner surface of the airtight container with a valve is preferably stainless steel or its Teflon (registered trademark) coating.
Therefore, the container is easy to clean and easy to handle.

  For dissolution, a stirring tank equipped with a stirring device is used. Because of the poor solubility of hyaluronic acids in injection solutions and the high viscosity of the solution, the stirrer is of the turbine type, anchor type, saw blade with paddle plate, disper type or disper turbine type. What has a stirring blade is preferable.

  The shaft of the stirring blade is attached at the center of the container or at an eccentric position. The stirring blades may be one stage or multistage. The rotation speed of the stirring blade is 100 to 5000 rpm, preferably 800 to 2000 rpm. When the rotational speed is smaller than the above range, the penetration of hyaluronic acid into the solution for injection is poor, and it takes a long time to completely dissolve. When the rotational speed is larger than the above range, the hyaluronic acid is poorly dispersed in the injectable solution, the scattering to the upper part of the interface in the stirring tank is increased, and the dissolution does not proceed smoothly. In order to increase the dissolution rate, it is not necessary to heat from the outside of the stirring tank, and dissolution can be performed in a short time. Thus, since it can melt | dissolve on mild conditions in a short time, there are few physical-property changes like the molecular weight fall of hyaluronic acid.

In the dissolving operation, the inside of the stirring vessel is preferably depressurized to atmospheric pressure or lower as appropriate. It removes hyaluronic acids and bubbles in the liquid, and is effective in increasing the dissolution rate.
The hyaluronic acid solution has a high viscosity, but it is preferable to reduce the pressure to about 5 to 20 kPa abs by using a normal pressure-reducing means such as a vacuum pump for defoaming. When the temperature is increased or the solution is stirred together, the effect is further improved.

  The material of the inner surface of the stirring tank for dissolution includes stainless steel, glass, Teflon (registered trademark), etc. due to the corrosion resistance to saline solution and the cleanability of the inner surface after dissolution, etc., but the hyaluronic acid solution adheres to the material surface. From the viewpoint, Teflon (registered trademark), Teflon (registered trademark) lining or Teflon (registered trademark) coating is preferable. Since Teflon (registered trademark) has less adherence of hyaluronic acid solutions than other materials, it is suitable for discharging the solution from the stirring tank and washing the stirring tank.

  In the sterilization process of the present invention, a heating medium passage is formed in the space between the inner and outer cylinders, comprising an inner cylinder through which the liquid to be heated is circulated and an outer cylinder provided so as to surround and surround the outer periphery of the inner cylinder. Indirectly provided with a plurality of fixed agitating mechanisms, each of which has a shaft portion having a length in the axial direction and a plurality of blade pieces projecting radially outward from the shaft portion and having an angle in the axial direction. Use a heating device.

In sterilization of hyaluronic acid solutions, a reduction in the molecular weight of hyaluronic acid must be minimized. For this purpose, the temperature and residence time of the hyaluronic acid solution in the sterilizer are important requirements.
Therefore, in the sterilization process, it is necessary to rapidly heat and rapidly cool the hyaluronic acid solution. As the sterilizer to be used, for example, an indirect heating apparatus as disclosed in Japanese Patent Publication No. 6-15953 can be used.

  That is, it consists of a double tube, and has a structure in which a hyaluronic acid solution can be continuously passed through the inside and a heating medium or a cooling medium can be passed through the outside, and a portion for heating the hyaluronic acid solution (hereinafter referred to as a heating unit). ) It is composed of a part for maintaining the temperature for a certain period of time (hereinafter referred to as a hold part) and a part for rapid cooling (hereinafter referred to as a cooling part).

  The residence time can be selected in the range of 2 seconds to 30 minutes in combination with the temperature of the hold unit in the sterilizer. The temperature of the hold part in the sterilizer is 115 ° C to 145 ° C, preferably 130 ° C to 140 ° C. When the temperature at the holding part of the hyaluronic acid solution is lower than 115 ° C., a sufficient sterilization effect cannot be expected. When the temperature is higher than 145 ° C., the hyaluronic acid is decomposed by heat, and the molecular weight is greatly reduced.

  In order to increase the efficiency of heat exchange between the hyaluronic acid solution, the heating medium, and the cooling medium, a fixed stirring mechanism is incorporated inside the heating unit and the cooling unit through which the hyaluronic acid solution passes. Ordinary steam or hot water is used as the medium of the heating part of the sterilizer, and water is appropriate as the cooling medium. The heating unit is heated so that the temperature of the hyaluronic acid solution reaches the temperature of the hold unit, and the cooling unit is cooled so that the hyaluronic acid solution reaches 60 ° C. or less from the temperature of the hold unit. The holding portion does not need to be heated theoretically, but it is appropriate to heat it slightly in order to prevent the temperature effect due to heat radiation to the surroundings.

  In order to obtain a uniform sterilization effect with the sterilizer of the present invention, it is important to supply the hyaluronic acid solution continuously at a constant flow rate. For this purpose, a metering pump or a flow controller is used in accordance with a conventional method. Under the sterilization operation conditions of the present invention, it is essential that the internal pressure of the hyaluronic acid solution in the sterilizer is always kept higher than the pressure of the heating medium or the cooling medium by the pressure control valve attached to the outlet of the sterilizer Is the condition. This is because it is possible to prevent the heating medium and the cooling medium from being mixed into the hyaluronic acid solution even if there is an obstacle on the equipment.

  The material of the sterilizer is selected from materials that exhibit corrosion resistance to high-temperature saline, such as stainless steel and hastelloy.

  The filtration membrane used in the foreign matter filtration step of the present invention preferably has a pore size of 0.2 to 50 μm. If the pore size is smaller than 0.2 μm, it is difficult to pass the membrane because the sterilizing solution obtained in the previous step is a very high viscosity solution, and if the pore size is larger than 50 μm, foreign matter filtration is not possible. This is not preferable because it becomes complete and insoluble foreign matter that can be visually discerned is mixed in the injection solution.

  The material of the filtration membrane can be selected from polytetrafluoroethyne, polyester, Teflon (registered trademark), polypropylene, polyvinylidene fluoride, and nylon.

  The filter medium shape of the filtration membrane can be selected from a flat membrane, a filter cartridge, and a disposable filter. The filtrate can be diluted with a solution for injection to adjust the concentration.

Prior to filling, the inside of the chemical tank is pressurized after the large bubbles are removed by standing, thereby stabilizing the chemical feeding liquid at the time of filling and improving the filling accuracy.
As a pressurizing method, any method may be used as long as it does not cause a change in quality by contact with a chemical solution such as pumping of clean air or pumping of inert gas such as nitrogen gas.
The pressure condition varies depending on the concentration and viscosity of the liquid containing hyaluronic acid and / or its salt, but good results can be obtained by controlling the pressure to 0.01 to 0.50 MPa.

  As the filling machine used in the filling process of the present invention, a filling machine comprising a portion for filling a container with a hyaluronic acid solution and a sealed portion for plugging a rubber stopper into the container after filling or sealing the container is used. .

  As a filling pump, a well-known weight valve type, spring ball valve type, pinch valve type, rotary piston pump type, diaphragm type, etc. can be used. Hyaluronic acid solutions are, for example, high viscosity liquids of 300 to 600 poise. Therefore, it is preferable to use a spring ball valve type having a suck back mechanism that has good filling accuracy and is effective in preventing stringing, which is characteristic of hyaluronic acid solutions.

As the container for injection solution used in the present invention, a general ampoule, a vial or, for example, a Duphage ecto type or a prefilled syringe can be used.
These injection solution containers are preferably coated with polydimethylsiloxane or the like in order to minimize the amount of hyaluronic acid injection solution remaining in the container when used in a medical institution.

  As the material of the rubber stopper for sealing the vial or syringe, butyl rubber, butyl chloride rubber, and butadiene rubber widely used for pharmaceuticals can be used. However, those coated with silicone are preferable from the viewpoint of less elution of impurities.

  The following examples are provided to further illustrate the present invention but are not intended to limit the invention in any way.

Example 1
1580 g of sodium hyaluronate having a molecular weight of 2.37 million was charged into an airtight container equipped with a 20 liter butterfly valve. A disperser turbine type stirring blade is attached to a 200-liter stainless steel dissolution tank whose inner surface is coated with Teflon (registered trademark) at a 1: 2 position, and a 2 mM sodium phosphate buffer solution of pH 7.3 is added. 149 liters of the physiological saline solution (solution for injection) contained was charged into the dissolution tank. The container filled with sodium hyaluronate was attached upside down to the raw powder inlet of the dissolution tank, the butterfly valve was opened, and sodium hyaluronate was charged into the dissolution tank. Stirring was performed at 1800 rpm for 50 minutes to completely dissolve sodium hyaluronate. In order to remove bubbles in the liquid, the pressure in the dissolution tank was maintained at a vacuum level of 15 kPa abs or lower for 20 minutes to remove bubbles, and then returned to normal pressure. The sodium hyaluronate was completely dissolved in 1 hour after the start of stirring. When the sodium hyaluronate concentration was measured by the carbazole sulfate method, it was 1.00%. It was 33.8 dl / g when the intrinsic viscosity of this liquid was measured according to the 13th revision Japanese Pharmacopoeia, and it was 2.37 million when converted into molecular weight.

This solution was continuously sterilized with a kids cooker continuous sterilizer manufactured by Kikkoman. This device consists of a double tube, the inner tube has an inner diameter of 23 mm, a fixed stirrer is built in, the heating unit volume is 3.4 liters, the hold unit volume is 0.6 liters, and the cooling unit volume is 2.6 liters. there were. The hot water in the heating unit outer tube was adjusted so that the temperature of the holding unit was 135 ° C., and the metering pump at the heating unit inlet was controlled so that the residence time in the holding unit was 34 seconds.
The cooling unit adjusted the water in the cooling unit outer tube so that the outlet temperature was 40 ° C. or lower. The cooling pressure at the outlet of the cooling unit was controlled by a pressure control valve so that the pressure was 0.33 MPa, and the cooled hyaluronic acid solution was filtered with a Millidisk 40 manufactured by Nihon Millipore, which was made of a nylon filter membrane having a pore size of 5 μm.

  The filtrate was maintained under pressure with purified air in a chemical tank 0.02 MPa. Next, 2.85 ml each of silicone-coated 3 ml vials were filled with a vial filling and sealing machine having a diaphragm-type filling pump, a rubber stopper, and a tightening mechanism. The rubber stopper was plugged with silicone-coated butyl rubber (Daikyo Seiko Co., Ltd.).

  When the quality test of the product was conducted according to the 13th revised Japanese Pharmacopoeia, the difference from the set amount of the maximum filling capacity in the actual capacity deviation test was 0.01 ml, and the difference from the minimum filling capacity was 0.0 lml. Further, the pass rate of the in-process total insoluble foreign matter test was 99.5%, the sterility test was negative, the sodium hyaluronate molecular weight was 2,260,000, and the sodium hyaluronate concentration was 1.0%.

Comparative Example 1
In Example 1, the sterilization temperature for continuous sterilization was set to 110 ° C., and the residence time of the holding part was changed to 20 minutes and 160 minutes, and the same test was performed. The sterility test was positive at 20 minutes and negative at 160 minutes. Further, the molecular weight before sterilization of 2.37 million decreased to 1.6 million at a residence time of 20 minutes and to 1 million at 160 minutes.

Comparative Example 2
In Example 1, without passing through the foreign matter filtration membrane, the insoluble foreign matter test pass rate of 6000 filled and sealed vials was 65.3%.

Comparative Example 3
In Example 1, using a weight spring ball valve instead of a diaphragm type filling pump as the filling pump, the same procedure was performed with 720 filling speeds per hour. As a result, the liquid dripped outside.

Comparative Example 4
In Example 1, as a result of filling without pressurizing the inside of the chemical liquid tank, air bubbles accumulated in the tube connected to the filling needle due to air bubbles in the liquid, resulting in poor liquid feeding and frequent abnormal stops. did.

Comparison 5
In Example 1, when the chemical tank was pressurized at 1.0 MPa, the chemical dripping occurred from the filling needle, resulting in poor filling accuracy as well as contamination of the filling container and the filling machine.

Example 2
In Example 1, instead of the vial filling and sealing machine, a syringe filling and sealing machine was used to fill 2.75 ml each of the silicone-coated Arte Duphage Ecto syringe, and a silicone-coated butyl rubber made by Daikyo Seiko Co., Ltd. The rubber plug was plugged.
When an actual volume deviation test was performed according to the 13th revised Japanese Pharmacopoeia, the difference between the minimum filling capacity and the set quantity was 0.01 ml, and the difference between the maximum filling capacity was 0.01 ml. The pass rate of the insoluble foreign matter test was 99.6%.

Claims (31)

A method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof, which is processed in the order of the following 6 steps of A, B, C, D, E and F.
Process A: A process in which hyaluronic acid and / or a salt thereof filled in an airtight container equipped with a valve is put into a stirring tank without inverting the container and touching the outside air.
Step B: a step of dissolving hyaluronic acid and / or a salt thereof using a stirring tank equipped with a stirring blade selected from a turbine type, a disper type, a disper turbine type, an anchor type, and a saw blade with a paddle plate.
Step C: Step of defoaming the hyaluronic acid and / or its salt-containing liquid by reducing the internal pressure of the stirring tank to atmospheric pressure or lower.
Step D: An inner cylinder through which the liquid to be heated is circulated and an outer cylinder provided so as to surround and surround the outer periphery of the inner cylinder, forming a heating medium passage in the space between the inner and outer cylinders, and in the axial direction An indirect heating device provided with a plurality of fixed stirring mechanisms each having a shaft portion having a length and a plurality of blade pieces projecting radially outward from the shaft portion and having an angle in the axial direction. And heat sterilizing the liquid containing hyaluronic acid and / or a salt thereof.
Step E: A step of removing foreign substances in the hyaluronic acid and / or salt-containing liquid by filtering the hyaluronic acid and / or salt-containing liquid with a filtration membrane.
Step F: When filling the injection container and / or vial with the liquid containing hyaluronic acid and / or its salt, the filling pump of the filling machine is a weight valve type, a spring ball valve type, a pinch valve type, and a rotary piston. A step of filling a liquid containing hyaluronic acid and / or a salt thereof by pressurizing a chemical tank using one selected from a pump type and a diaphragm type. Hyaluronic acid and / or containing liquid water for injection of a salt thereof, saline, and high purity hyaluronic acid according to claim 1 Symbol placement are those prepared by one selected from phosphate-buffered saline, and / or A method for producing the salt-containing liquid. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 2 , wherein the connection surface of the airtight container with a valve in the step A is a curved surface. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 3 , wherein the angle of the charging chute of the airtight container with a valve in the step A is 50 ° or more. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 4 , wherein the valve in the step A is a butterfly valve. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 5 , wherein the material of the inner surface of the airtight container with a valve in the step A is stainless steel or Teflon (registered trademark) coating. The method for producing a high-purity hyaluronic acid and / or salt-containing liquid according to claim 6 , wherein a stirring tank in which the shaft of the stirring blade in the step B is in the center of the container or in an eccentric position is used. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 7 , wherein the stirring blade in the step B uses a stirring tank having one or more stages. The manufacturing method of the high purity hyaluronic acid and / or its salt containing liquid of Claim 8 using the stirring tank whose rotation speed of the stirring blade in the said B process is 100-5000 rpm. The high purity hyaluronic acid and / or salt thereof according to claim 9, wherein the internal material of the stirring tank and line in the step B is Teflon (registered trademark), Teflon (registered trademark) lining, or Teflon (registered trademark) coating. Liquid manufacturing method. The manufacturing method of the high purity hyaluronic acid and / or its salt containing liquid of Claim 10 which reduces the internal pressure of the stirring layer at the time of the defoaming process in the said C process to 5-20 kPa abs of vacuum degree. The high purity hyaluronic acid and / or its salt-containing liquid according to claim 11 , wherein the concentration of the hyaluronic acid and / or its salt-containing liquid during the defoaming treatment in the step C is 7.5 to 12.5 g / l. Manufacturing method. The method for producing a high-purity hyaluronic acid and / or salt-containing liquid according to claim 12, wherein the indirect heating apparatus in the step D uses an indirect heating apparatus in which a heating section, a holding section, and a cooling section are used. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 13 , wherein the temperature of the holding part of the indirect heating device in the step D is 115 ° C to 145 ° C. Containing solution having a high purity hyaluronic acid and / or its salt of the D step claim 14, wherein the residence time of the solution containing hyaluronic acid and / or salts thereof of the hold portion of the indirect heating device is 2 seconds to 30 minutes in the Manufacturing method. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 15 , wherein the temperature at the outlet of the cooling part of the indirect heating device in step D is 60 ° C or lower. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 16 , wherein the pressure in the inner cylinder of the indirect heating device in step D is higher than the pressure in the outer cylinder. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 17 , wherein a material of the indirect heating device in the step D is stainless steel or hastelloy. The method for producing a high-purity hyaluronic acid and / or salt-containing liquid according to claim 18 , wherein the concentration of the hyaluronic acid and / or salt-containing liquid in the step D is 7.5 to 12.5 g / l. The method for producing a high-purity hyaluronic acid and / or salt-containing liquid according to claim 19 , wherein the pH of the hyaluronic acid and / or salt-containing liquid during filtration membrane treatment in the step E is 2 to 10. 21. The method for producing a high-purity hyaluronic acid and / or salt-containing liquid according to claim 20 , wherein the temperature of the liquid containing hyaluronic acid and / or the salt thereof during filtration membrane treatment in the step E is 5 to 100 ° C. The high purity hyaluronic acid and / or its salt-containing liquid according to claim 21 , wherein the concentration of the hyaluronic acid and / or its salt-containing liquid during filtration membrane treatment in the E step is 7.5 to 12.5 g / l. Manufacturing method. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 22, wherein the pore size of the filtration membrane in the step E is in the range of 0.2 to 50 µm. The method for producing a liquid containing high purity hyaluronic acid and / or a salt thereof according to claim 23, wherein the shape of the filter medium in the step E is one selected from a flat membrane, a filter cartridge, and a disposable filter. Wherein E Material polytetrafluoroethylene filter membrane in step, polyester, teflon, polypropylene, high purity hyaluronic acid according to claim 24, wherein the one selected from polyvinylidene fluoride and nylon, and / or A method for producing a salt-containing liquid. When filling the hyaluronic acid and / or liquid containing a salt thereof in injection containers and / or vials in the F step, filling pump of the filling machine, highly pure hyaluronic acid and / or according to claim 25, wherein using a diaphragm-type A method for producing the salt-containing liquid. 27. The high-purity hyaluronic acid and the high-purity hyaluronic acid according to claim 26 , wherein the chemical liquid tank is pressurized at 0.01 to 0.50 MPa when filling the injection container and / or vial with the hyaluronic acid and / or salt-containing liquid in the step F. / Or production method of the salt-containing liquid. 28. The method for producing a high-purity hyaluronic acid and / or salt-containing liquid according to claim 27 , wherein the concentration of the hyaluronic acid and / or salt-containing liquid in step F is 7.5 to 12.5 g / l. The method for producing a high-purity hyaluronic acid and / or salt-containing liquid according to any one of claims 1 to 28 , wherein the molecular weight of hyaluronic acid and / or a salt thereof is 1.5 to 4 million. The high-purity hyaluronic acid and / or the hyaluronic acid and / or salt thereof according to any one of claims 1 to 28 , wherein the hyaluronic acid and / or a salt thereof is produced by fermentation using Streptococcus equi FM-100. Or the manufacturing method of the liquid containing the salt. The high-purity hyaluronic acid and / or the hyaluronic acid and / or salt thereof according to any one of claims 1 to 28 , wherein the hyaluronic acid and / or a salt thereof is produced by a fermentation method using Streptococcus equi FM-300. Or the manufacturing method of the liquid containing the salt.