JP5603925B2 - Method for dissolving hyaluronic acid and / or salt thereof - Google Patents

Description

  The present invention relates to a technique for producing an injection solution of hyaluronic acid and / or a salt thereof (hereinafter also collectively referred to as hyaluronic acid) that is compatible with pharmaceuticals from a solution containing hyaluronic acid and / or a salt thereof.

Hyaluronic acid is a high-molecular-weight polysaccharide that is said to have a molecular weight of up to 5 million, which is formed by repeatedly chaining 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)).
Furthermore, it has been known to be effective as a medicine for preventing adhesion after surgery and also in the dermatological and ophthalmological areas, and some of them 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.

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, etc., and filtration or sterilization is difficult. is there. Accordingly, a method for producing a large amount of high molecular weight sodium hyaluronate injection has not been clarified.

  In producing a large amount of hyaluronic acid-containing liquid as an injection solution, the present inventor researched a method for efficiently separating and removing foreign substances from hyaluronic acid-containing liquid to obtain high-purity pharmaceutical grade hyaluronic acid. In the course of the research, when hyaluronic acids were dissolved in an injection solution, using a general stirring tank under normal conditions, the hyaluronic acids were not sufficiently dissolved and aggregates were formed. It has been found that the stirring and dissolving step becomes a bottleneck because the molecular weight of hyaluronic acid is reduced during the dissolution.

This invention was made | formed in view of the said situation, and generally aims at providing the melt | dissolution method of hyaluronic acid which can be implemented suitably when manufacturing the injection solution of hyaluronic acid.
Another object of the present invention is to provide a method for dissolving hyaluronic acids that can sufficiently disperse high molecular weight hyaluronic acids in an injectable solution while suppressing aggregation as much as possible to obtain a clear solution. There is to do.
Furthermore, an object of the present invention is to provide a method for dissolving hyaluronic acids, which can dissolve high molecular weight hyaluronic acids in an injectable solution without reducing the molecular weight as much as possible.

According to the first aspect of the present invention, the following method is provided.
That is,
(1) Select from water for injection, physiological saline, and buffered saline using an agitation tank equipped with an agitating blade selected from turbine type, disper type, disper turbine type, anchor type and saw blade with paddle blade A method for dissolving hyaluronic acid and / or a salt thereof in which hyaluronic acid and / or a salt thereof is dissolved in a kind of injectable solution for injection;
(2) The method for dissolving hyaluronic acid and / or a salt thereof according to (1), wherein a stirring tank in which the axis of the stirring blade is in the center of the container or in an eccentric position is used;
(3) The method for dissolving hyaluronic acid and / or a salt thereof according to (2), wherein the stirring blade is a one-stage or multi-stage stirring tank;
(4) The method for dissolving hyaluronic acid and / or a salt thereof according to (3), wherein a stirring vessel in which the rotation speed of the stirring blade is 100 to 5000 rpm is used;
(5) The method for dissolving hyaluronic acid and / or a salt thereof according to (4), wherein the inner surface material of the stirring tank and the line is Teflon, Teflon lining, or Teflon coating;
(6) The method for dissolving hyaluronic acid and / or salt thereof according to any one of (1) to (5), wherein the hyaluronic acid and / or salt thereof has an average molecular weight of 1,500,000 to 4,000,000;
(7) Hyaluronic acid and / or a salt thereof is produced by a fermentation method using Streptococcus ex FM-100 or Streptococcus ex FM-300, or any one of (1) to (6) Of dissolving hyaluronic acid and / or its salt.

  According to such a method, a high-quality solution of hyaluronic acid that can be used when producing an injection solution can be obtained.

According to another aspect of the present invention, the following method is provided.
That is,
(8) Hyaluronic acid in a kind of injection solution selected from water for injection, physiological saline, and buffered physiological saline using a stirring tank equipped with a large pitched turbine blade or a stirring blade of a disper turbine blade And / or a method for dissolving hyaluronic acid and / or a salt thereof for dissolving the salt;
(9) The agitation tank has a substantially vertical cylindrical shape, and the agitation blade is disposed with the shaft positioned at a position eccentrically outward in the radial direction from the center of the tank, and / or the hyaluronic acid according to (8) Salt dissolution method;
(10) The method for dissolving hyaluronic acid and / or a salt thereof according to (9), wherein the eccentric position of the shaft of the stirring blade is a position where the center line is divided into 1: 2;
(11) The stirring tank includes a stirring blade having a stirring blade diameter / tank inner diameter ratio of 0.3 to 0.5, and / or the hyaluronic acid according to any one of (8) to (10). Salt dissolution method;
(12) The melting method according to any one of (8) to (11), wherein the stirring blade is operated at a rotational speed of 1500 to 1800 rpm;
(13) The dissolution method according to any one of (8) to (12), wherein the stirring time is 45 minutes or more;
(14) The melting method according to any one of (8) to (13), wherein at least the inner surface of the stirring tank and the line connected to the stirring tank is made of stainless steel and electropolished.
(15) The dissolution method according to any one of (8) to (14), wherein the hyaluronic acid and / or a salt thereof has an average molecular weight of 1.5 million to 4 million;
(16) The dissolution method according to any one of (8) to (15), wherein the dissolution is performed so that the concentration of hyaluronic acid and / or a salt thereof is 0.75 to 1.25 w / v%;
(17) Any one of (8) to (16), wherein the hyaluronic acid and / or a salt thereof is produced by fermentation using Streptococcus ex FM-100 or Streptococcus ex FM-300. The dissolution method according to 1.

  According to such a method, high molecular weight hyaluronic acids can be sufficiently dispersed in a solution for injection while suppressing aggregation and molecular weight reduction as much as possible, and it is possible to produce injection solutions on a large scale. It becomes.

It is a schematic plan view of the disper turbine type stirring blade installed in the stirring tank for implementing the melt | dissolution method which concerns on one embodiment of this invention. It is a side view of the disper turbine type stirring blade of FIG. It is a figure similar to FIG. 1 which shows the example of another disper turbine type | mold stirring blade. It is a side view of the disper turbine type stirring blade of FIG. It is a figure which shows the state which has arrange | positioned the large pitched turbine blade in the stirring tank, (a) is a schematic sectional side view, (b) is a bb arrow line view in (a). The state which has arrange | positioned the two stage pitched turbine blade to the stirring tank is shown, (a) is a schematic sectional side view, (b) is a bb arrow line view in (a), (c) is (a). FIG. It shows a state where pitched turbine blades and combined blades of paddles are disposed in the stirring tank, (a) is a schematic side sectional view, (b) is a view taken along the line bb in (a), (c) is It is a cc arrow line view in (a). The state which arrange | positioned the disper turbine blade to the stirring tank is shown, (a) is a schematic sectional side view, (b) is a bb arrow line view in (a). The state which has arrange | positioned the Max blend blade to the stirring tank is shown, (a) is a schematic sectional side view, (b) is a bb arrow line view in (a). The state which arrange | positioned the dissolver blade | wing to the stirring tank is shown, (a) is a schematic sectional side view, (b) is a top view of a dissolver blade.

Hereinafter, modes for carrying out the present invention will be described.
The hyaluronic acids used in the present invention include free forms of hyaluronic acid, hyaluronic acid salts, or a mixture of free hyaluronic acid and hyaluronic acid salts. Examples of the salt of hyaluronic acid include sodium salt, potassium salt, calcium salt, lithium salt and the like, and sodium salt is most commonly used. Furthermore, the hyaluronic acid-containing liquid used in the present invention may be extracted from animal tissue or manufactured by a fermentation method, but it is preferable to use a solution manufactured by a fermentation method.

  Hyaluronic acids by fermentation can be obtained by known methods using microorganisms such as bacteria having the ability to produce hyaluronic acid such as Streptococcus. As a strain used in the fermentation method, a microorganism having the ability to produce hyaluronic acid isolated from the natural world, or Streptococcus ex FM-100 described in JP-A No. 63-123392 (No. 9027, Kikoken Bacteria). ), And a mutant strain that stably produces hyaluronic acid at a high yield, such as Streptococcus ex FM-300 (Mikken Kenki No. 2319) described in JP-A-2-23489. Mutant strains are preferably used.

  The hyaluronic acids produced by the above fermentation method and usable in the present invention have a high molecular weight and generally have an average molecular weight of 1,500,000 to 4,000,000. This is because when the average molecular weight is less than 1,500,000, the efficacy as a pharmaceutical agent is lowered, while it is difficult to obtain a compound having an average molecular weight of more than 4,000,000 by the above method. However, this does not mean that the dissolution method according to the present invention cannot be used to dissolve low molecular weight hyaluronic acid.

  Water for injection, physiological saline and buffered physiological saline can be used as an injectable solution used in the step of dissolving hyaluronic acid, and pH adjustment including a buffer such as acid, alkali, and phosphate is particularly possible. Those approved by the Japanese Pharmacopoeia General Preparations for Injection, including drugs, etc. 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 hyaluronic acid solution has a low viscosity and is easy to produce. On the other hand, if it is 1.25 w / v% or more, it is difficult to prepare a large amount from the solubility of hyaluronic acids. Therefore, the hyaluronic acid concentration of 0.75 to 1.25 w / v%, which becomes a highly viscous solution, corresponds to the production conditions targeted by the dissolution method of the present invention.

  It is preferable that the hyaluronic acid to be dissolved is filled in an airtight container equipped with a valve and charged into a stirring tank by a charging chute via the valve. The angle of the charging chute is preferably a steep slope of 50 ° or more. When the container is inverted and hyaluronic acid is charged, the loss is reduced. As the valve, a butterfly valve is preferably used. By switching the valve, hyaluronic acid can be aseptically charged into the agitation tank without touching the outside air. The material of the airtight container with a valve is preferably stainless steel, the inner surface thereof coated with Teflon, or the inner surface thereof subjected to electrolytic polishing finish, from the viewpoint of cleanability, ease of handling, and the like.

  The agitation tank used for the dissolution is a general substantially vertical cylindrical tank body with a vertical axis agitation device, and the agitation device is arranged inside the tank body with the axis line in the vertical direction. A shaft disposed on the shaft, a stirring blade attached substantially horizontally to the lower end of the shaft (and in some cases a middle part), and a drive device attached to the upper end of the shaft and installed on the canopy portion of the tank body It comprises.

  In the stirring / dissolving step in the stirring tank, the solubility of hyaluronic acids in an injectable solution is poor, the solution is highly viscous, and further, the molecular weight is often lowered during dissolution. Therefore, there was a problem to be solved as described above. In view of this, the present inventor conducted various comparative studies using various types of stirrers under various conditions in the above-described stirring tank. As a result, it has been found preferable to use a particular type of stirrer under certain conditions.

That is, as the stirring device, a stirrer 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 blade can be used. Then, it was found that those having turbine type, disper type, and disper turbine type agitating blades were preferable, and in particular, large pitched turbine blades and disper turbine blades were preferable.
In addition, the position where the stirring blade is disposed (the position of the shaft of the stirring blade) can be approximately the center position of the tank body, as usual, but if the shaft of the stirring blade is installed eccentrically outward in the radial direction of the tank body It was found preferable because the dissolution rate of hyaluronic acids is increased. For example, the eccentric position can be a position where the tank diameter on the center line of the tank body is divided by 1: 2, a position divided by 1: 3, a position divided by 1: 4, a position divided by 1: 5, etc. : It turned out that the position divided into 2 is preferable.

  Further, the rotation speed of the stirring blade is generally 100 to 5000 rpm, for example, 800 to 2000 rpm, and particularly preferable for the large pitched turbine blade and the disper turbine blade among the stirring blades, the rotation speed is 1500 to 1800 rpm. Was found to be preferable. If the rotational speed is too small, the dispersibility of hyaluronic acids in a solution for injection becomes poor. On the contrary, even if the rotational speed is increased, the stirring blade does not rotate because the molecular weight of the hyaluronic acid is too high. In addition, it is often effective to warm simultaneously with stirring during dissolution, but in the case of hyaluronic acids, troublesome physical property changes such as molecular weight reduction can occur due to heating. Furthermore, when the stirring is not sufficient, it is conceivable to increase the stirring time. However, when the stirring time is extended, physical property changes such as a decrease in molecular weight may occur. However, in addition to adjusting the stirring blade shape, position, other operating conditions, etc., by setting the rotation speed within the above range, it can dissolve in a short time under mild conditions without heating. it can.

Furthermore, it has been found that there is an optimum range for the size of the stirring blade. That is, the stirring blade preferably has a stirring blade diameter (d) / tank inner diameter (D) ratio (d / D) of 0.3 to 0.5. This is because when the ratio is less than 0.3, the stirring effect is not sufficient and the solubility and dispersibility are deteriorated. On the other hand, when the ratio exceeds 0.5, problems such as a decrease in molecular weight occur.
Similarly, if the stirring time is short, the stirring effect is not sufficient, and the solubility and dispersibility deteriorate. On the other hand, if the stirring time is too long, problems such as a decrease in molecular weight occur. Illustratively, it is preferably 45 minutes or longer, up to about 100 minutes, particularly up to about 60 minutes.
Further, as described above, if the type, size, installation position, number of rotations, and the like of the stirring blade are appropriately designed as described above, one step is sufficient, but this does not exclude the fact that the stirring blade is multi-staged.

In the dissolving operation, it is preferable to depressurize the inside of the stirring vessel as appropriate. This is to remove hyaluronic acids and bubbles in the liquid, but is also effective to increase the dissolution rate.
Although the hyaluronic acid solution has a high viscosity, it is preferable to reduce the pressure to 5 to 20 kPa abs 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 may be stainless steel, glass, Teflon (registered trademark), etc. due to the corrosion resistance to saline solution, 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. This is because Teflon (registered trademark) has less adherence of a hyaluronic acid solution compared to other materials, and is suitable for discharging the solution from the stirring tank or washing the stirring tank.
Alternatively, it is also preferable to use stainless steel, particularly SUS316L, in place of Teflon (registered trademark). In that case, when the inner surface is electropolished, performance equivalent to or higher than that of Teflon (registered trademark) can be obtained.

After stirring and dissolving, steps of sterilization, foreign matter filtration, defoaming, and filling are performed.
Briefly describing these steps, the hyaluronic acid solution is sterilized before removing foreign substances or after filling a container such as a vial.
Foreign matter filtration is performed by filtration. The filtration membrane used for filtration preferably has a pore size of 0.2 to 50 μm. If the pore size is smaller than that range, it is difficult to pass the membrane because the sterilizing liquid obtained in the previous step is very viscous, and if the pore size is larger than that range, 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 polytetrafluoroethylene, polyester, Teflon (registered trademark), polypropylene, polyvinylidene fluoride, nylon, and the like, but polyvinylidene fluoride, polypropylene, or nylon is preferable. As the shape of the filtration membrane, any of a flat membrane, a filter cartridge, and a disposable filter can be used. However, when processing in a large amount, a filter cartridge or a disposable filter is preferable. Specific examples of the filtration membrane that can be used in the present invention include Millipak and Durapore Millidisk manufactured by Nihon Millipore.

  Arbitrary conditions are selected in the pH of a hyaluronic acid containing liquid being 2-10, and temperature being 5-100 degreeC. The flow rate and pressure at the time of liquid flow are set in consideration of pressure resistance according to the type of filter, but care must be taken because foreign matter may flow out of the filter when pressure is applied. For the millidisc 40, a flow rate of 50 to 300 L / hr and a processing pressure of 0.01 to 0.50 MPa are preferable. The filtrate can be diluted with a solution for injection to adjust the concentration.

  In the filling step, a filling machine comprising a portion for filling the container with the hyaluronic acid solution and a sealed portion for plugging the rubber stopper into the container after filling or sealing the container is used. As the container for injection solution to be filled, a general ampoule, vial, Duffer Jocto type or prefilled syringe is used.

  EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not limited to the content of description of this Example.

<Example 1>
1580 g of sodium hyaluronate having a molecular weight of 2,370,000 obtained by fermentation using Streptococcus ex FM-100 (Mikkenken Bunkyo No. 9027) was filled into an airtight container equipped with a 20 L butterfly valve. A disperser turbine type stirring blade is attached to a 200 L stirring tank made of stainless steel whose inner surface is coated with Teflon, and 149 L of physiological saline (solution for injection) containing 2 mM sodium phosphate buffer at pH 7.3 is stirred. The tank was charged.
The above-mentioned airtight container filled with sodium hyaluronate was attached upside down to the raw powder inlet of the stirring tank, the butterfly valve was opened, and sodium hyaluronate was put into the stirring tank. 1 and 2 are plan views of a disperser turbine type stirring blade (dissolver blade diameter 275 mm, consisting of 12 blades and 6 pitched paddle blades).
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 stirring tank was maintained at a vacuum degree of 15 kPa abs for 20 minutes, and after the bubbles were removed, the pressure was returned to normal pressure. When the sodium hyaluronate concentration of this solution 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 15th 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 apparatus consisted of a double tube, the inner tube had an inner diameter of 23 mm, a fixed stirrer was incorporated, and the heating unit volume was 3.4 L, the holding unit volume was 0.6 L, and the cooling unit volume was 2.6 L. 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 pressure of the cooling section outlet pressure is controlled by a pressure control valve so that the pressure is 0.33 MPa, and the cooled sodium hyaluronate solution is flowed using a Millidisk 40 manufactured by Nihon Millipore, which is made of a polyvinylidene chloride filter membrane having a pore size of 5 μm Filtration was performed at 60 L / hr.
The filtrate was stirred and mixed at 144 rpm for 30 minutes. Next, 2.85 L of the liquid was filled into each vial by a vial filling and sealing machine having a filling part having a diaphragm type filling pump, a stopper for a rubber stopper, and a tightening mechanism. The rubber stopper was stoppered with butyl rubber (Daikyo Seiko Co., Ltd.).
The product quality test was conducted according to the 15th revised Japanese Pharmacopoeia, General rules for preparations / injections, and when insoluble foreign matter inspection was performed, the pass rate was 99.9% or more.

  Further, instead of the disperser turbine type agitating blade shown in FIGS. 1 and 2, a disper turbine type agitating blade as shown in FIGS. 3 to 4 (having a blade diameter of 243 mm, 12 blades, a blade diameter of 170 mm, and 4 blades) ) Was performed under the same conditions, and it was confirmed to be effective as well.

<Example 2>
A dissolution test was conducted in order to obtain various optimum stirring blades and stirring conditions by variously changing the configuration of the stirrer in the stirring tank. In the dissolution test, 1580 g of sodium hyaluronate (intrinsic viscosity 35.0 dL / g) was charged into a stirring tank having an inner diameter of 550 mm charged with 149 L of physiological saline, and this was dissolved by operating the stirrer. As a result of the dissolution test, dispersibility of sodium hyaluronate, dissolution possibility, and molecular weight reduction are evaluated according to the criteria described below.

[Dispersibility]
The behavior of hyaluronic acid is visually confirmed and evaluated according to the following criteria.
○: Hyaluronic acid is dispersed throughout the tank △: Hyaluronic acid is dispersed near the bottom of the tank and not dispersed throughout the tank ×: Most of the hyaluronic acid is dispersed at the bottom of the tank Is almost unacceptable

[Dissolvability]
The following determination criteria a to c are comprehensively determined. If all the criteria are satisfied, ◯, if not one, Δ, and if all are not satisfied, ×.
a: No agglomerates are observed at visual level during dissolution of hyaluronic acids b: The hyaluronic acid solution is colorless and transparent c: Undissolved hyaluronic acids are visually observed on the wall surface of the stirring tank Not allowed

[Degree of molecular weight reduction (Δ molecular weight)]
The Δ molecular weight (= (the intrinsic viscosity before dissolution−the intrinsic viscosity after dissolution) / the intrinsic viscosity before dissolution) is determined according to the following criteria.
○: Δmolecular weight ≦ 0.03
Δ: 0.03 <Δmolecular weight ≦ 0.15
×: 0.15 <Δ molecular weight

[Test on shape of stirring blade]
A large pitched turbine blade, two-stage pitched turbine blade, combined use of pitched turbine blade and paddle, disper turbine blade, max blend blade, and dissolver blade are prepared as agitating blades, attached to a stirring tank, and a stirring speed of 1800 rpm. The dissolution test was performed. The results are shown in Table 1 below.

Moreover, here, the shape of each said stirring blade used by the present Example, and the attachment position in the stirring tank are shown to FIGS.
FIG. 5 shows a state in which the large pitched turbine blades 5 are arranged in the stirring tank 4, wherein (a) is a schematic side sectional view, and (b) is a view taken along the line bb in (a). Medium, H = 754 mm, h = 522 mm, D = 550 mm, d = 180 mm (d / D = 0.33). In the test concerning the shape of the stirring blade, the large pitched turbine blade 5 was attached to the eccentric position (e = 90 mm) of the stirring tank 4. This eccentric position corresponds to the eccentricity (1: 2) described later.
FIG. 6 shows a state in which the two-stage pitched turbine blades 6 are arranged in the stirring tank 4. FIG. 6A is a schematic side sectional view, FIG. 6B is a view taken along the line bb in FIG. ) Is a cc arrow view in (a), in which H = 754 mm, h1 = 290 mm, h2 = 232 mm, D = 550 mm, d ₁ (top) = 155 mm, d ₂ (bottom) = 180 mm (D ₁ /D=0.28 (top); d ₂ /D=0.33 (bottom)). Also in this test, the two-stage pitched turbine blade 6 was attached to an eccentric position (e = 90 mm).
FIG. 7 shows a state in which a pitched turbine blade and a paddle combined blade 7 are disposed in the stirring tank 4. FIG. 7A is a schematic sectional side view, and FIG. 7B is a view taken along the line bb in FIG. (C) is a cc arrow line view in (a), in which H = 754 mm, h ₁ = 283 mm, h ₂ = 85 mm (paddle height), h ₃ = 154 mm, D = 550 mm, d ₁ (Paddle diameter) = 155 mm, d ₂ (turbine blade diameter) = 180 mm (d ₁ /D=0.28 (paddle); d ₂ /D=0.33 (turbine blade)). Also in this test, the combined blade 7 was attached to an eccentric position (e = 90 mm).
FIG. 8 shows a state in which the disper turbine blades 8 are arranged in the stirring tank 4, (a) is a schematic side sectional view, (b) is a view taken along the line bb in (a), H = 754 mm, h = 522 mm, D = 550 mm, d = 180 mm (d / D = 0.33). Also in this test, the disperser turbine blade 8 was attached to an eccentric position (e = 90 mm).
FIG. 9 shows a state in which the Max Blend blade 9 is disposed in the stirring tank 4, wherein (a) is a schematic side sectional view and (b) is a view taken along the line bb in (a). H = 677 mm, h (blade height) = 450 mm, D = 550 mm, d = 290 mm (d / D = 0.53).
FIGS. 10A and 10B show a state in which the dissolver blade 10 is disposed in the stirring tank 4. FIG. 10A is a schematic side sectional view, and FIG. 10B is a plan view of the dissolver blade 10 in which H = 677 mm, e = 137.5 mm, D = 550 mm, d = 250 mm (d / D = 0.53).

  As can be seen from the results in Table 1, the large pitched turbine blade and the disper turbine blade were excellent in all items of dispersibility of sodium hyaluronate, possibility of dissolution, and molecular weight reduction. On the other hand, the two-stage pitched turbine blade is inferior in dispersibility of sodium hyaluronate and also has a decrease in molecular weight, and the combined use of the remaining pitched turbine blade and paddle is either dispersible or dissolvable. The molecular weight drop was also observed, and the degree of molecular weight drop was particularly large in the Max Blend blade.

表２の結果から分かるように、大ピッチドタービン翼及びディスパータービン翼の何れについても偏心１：２の位置が撹拌翼の最適取り付け位置であった。 [Stirring blade position]
For each of the large pitched turbine blade and the disperser turbine blade that had excellent results of the dissolution test on the shape of the stirring blade, the stirring blade was attached at a position of eccentricity 1: 2, eccentricity 1: 3, and eccentricity 1: 5. The influence of the position of the stirring blade on the dissolution was investigated. The results are shown in Table 2 below.

As can be seen from the results in Table 2, the position of eccentricity 1: 2 was the optimum mounting position of the stirring blade for both the large pitched turbine blade and the disperser turbine blade.

表３の結果から分かるように、撹拌回転数としては１５００ｒｐｍと１８００ｒｐｍが同等の性能を示したが、溶解時間が短い点を考慮すると、１８００ｒｐｍが最も優れていた。 [Agitating speed]
Dispersion turbine blades that were excellent in the tests so far were installed at a position of eccentricity 1: 2 that was excellent in performance, and the dissolution test was performed by varying the number of rotations of stirring in the range of 1000 rpm to 2500 rpm. The results are shown in Table 3 below.

As can be seen from the results in Table 3, 1500 rpm and 1800 rpm showed the same performance as the number of stirring rotations, but 1800 rpm was the most excellent considering the short dissolution time.

表４から分かるように、撹拌翼径／槽内径の比が０．３のものと０．５のものが同じ優れた性能を示したが、該比が０．１のものは溶解性が悪く、該比が１のものは分子量低下が大であった。 [Agitator blade diameter / tank inner diameter]
The size of the disperser turbine blade, which was excellent in the previous tests, was changed in the range of 0.1 to 1 in the ratio of stirring blade diameter / tank inner diameter, and the eccentricity 1: 2 position, which was excellent in performance, stirring rotation The dissolution test was performed at several 1800 rpm. The results are shown in Table 4 below.

As can be seen from Table 4, those having a stirring blade diameter / tank inner diameter ratio of 0.3 and 0.5 showed the same excellent performance, but those having a ratio of 0.1 had poor solubility. When the ratio was 1, the molecular weight was greatly reduced.

表５から分かるように、撹拌時間が３０分では溶解性が十分ではないが、逆に撹拌時間が１２０分になると、分子量の低下が顕著になった。これに対して、撹拌時間４５分及び６０分では、全ての基準を満たした。
[Stirring time]
Dispersion turbine blades having a stirring blade diameter / tank inner diameter ratio of 0.33 were attached at a position of eccentricity 1: 2, and the stirring time was changed in the range of 30 minutes to 120 minutes, and a dissolution test was performed. The results are shown in Table 5 below.

As can be seen from Table 5, the solubility is not sufficient when the stirring time is 30 minutes, but conversely, when the stirring time is 120 minutes, the decrease in molecular weight becomes significant. On the other hand, all the criteria were satisfied at the stirring time of 45 minutes and 60 minutes.

DESCRIPTION OF SYMBOLS 1 Dissolver blade 2 Shaft hole 3 Pitched paddle blade 4 Stirrer tank 5 Large pitched turbine blade 6 Two stage pitched turbine blade 7 Pitched turbine blade and paddle combined blade 8 Disper turbine blade 9 Max blend blade 10 Dissolver blade

Claims (11)

Using an agitation tank equipped with a large pitched turbine blade or a disperse turbine blade stirring blade, a kind of injection solution selected from water for injection, physiological saline, and buffered physiological saline has an average molecular weight of 1.5 million to A method for dissolving hyaluronic acid and / or a salt thereof, wherein 4 million hyaluronic acid and / or a salt thereof are dissolved so as to have a concentration of 0.75 to 1.25 w / v% . The method for dissolving hyaluronic acid and / or a salt thereof according to claim 1, wherein the stirring blade is a single-stage or multistage stirring tank. The method for dissolving hyaluronic acid and / or a salt thereof according to claim 1 or 2 , wherein a stirring tank in which the rotation speed of the stirring blade is 800 to 2000 rpm is used. The method for dissolving hyaluronic acid and / or a salt thereof according to any one of claims 1 to 3 , wherein the inner surface material of the stirring tank and the line is Teflon, Teflon lining, or Teflon coating. The hyaluron according to any one of claims 1 to 4, wherein the agitation tank has a substantially vertical cylindrical shape, and the agitation blade is disposed with a shaft located at a position eccentrically radially outward from the center of the tank. Method for dissolving acid and / or salt thereof. The method for dissolving hyaluronic acid and / or a salt thereof according to claim 5 , wherein the eccentric position of the shaft of the stirring blade is a position dividing the center line into 1: 2. The method for dissolving hyaluronic acid and / or a salt thereof according to any one of claims 1 to 6 , wherein the stirring tank comprises a stirring blade having a stirring blade diameter / tank inner diameter ratio of 0.3 to 0.5. The method for dissolving hyaluronic acid and / or a salt thereof according to any one of claims 1 to 7 , wherein the stirring blade is operated at a rotational speed of 1500 to 1800 rpm. The method for dissolving hyaluronic acid and / or a salt thereof according to any one of claims 1 to 8 , wherein the stirring time is 45 minutes or more. The dissolution of hyaluronic acid and / or a salt thereof according to any one of claims 1 to 3 and 5 to 9 , wherein at least an inner surface of the stirring tank and a line connected to the stirring tank is made of stainless steel and electropolished. Method. The hyaluronic acid and / or a salt thereof is produced by a fermentation method using Streptococcus ex FM-100 or Streptococcus ex FM-100, The hyaluronic acid according to any one of claims 1 to 10 And / or a method for dissolving the salt thereof.

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