CN114317496A - Hyaluronidase liquid preparation and application thereof - Google Patents

Abstract

The present invention provides a hyaluronidase liquid preparation comprising a hyaluronidase-containing fermentation product and lactate, wherein the liquid preparation uses lactic acid to adjust pH to 5.0-6.0. Compared with the prior art, the present invention provides a minimalist formula of a Bacillus hyaluronidase liquid preparation, which can maintain the stability of the hyaluronidase enzyme activity for a long time, and is an important tool for the production and processing of hyaluronidase. Apps provide convenience.

Description

A kind of hyaluronidase liquid preparation and application thereof

technical field

The invention belongs to the field of biomedicine, and in particular relates to a hyaluronidase liquid preparation and application thereof.

Background technique

Hyaluronidase (HAase), also known as hyaluronidase, is a general term for enzymes that can specifically hydrolyze hyaluronic acid.

Hyaluronidase is a general term for a class of enzymes capable of degrading hyaluronic acid. As early as 1928, Duran-Reynals discovered a "diffusion factor" in the study of mammalian testis and other tissue extracts, which can promote the better diffusion of subcutaneously injected vaccines, dyes, toxins, etc. In 1940 Meyer named this "diffusion factor" Hyaluronidase. Since then, hyaluronidase has been detected in many tissues and organisms, including bacterial viruses, bacteria, fungi, etc., and hyaluronidase is also produced in the venom of invertebrates such as leeches and crustaceans. In vertebrates, hyaluronidase is found in the venom of snakes and lizards, in the testes and other organs such as liver, kidney, lymphatic system and skin.

Hyaluronidase is also an indispensable tool enzyme for hyaluronic acid-related research. Patent CN201811508968.X "A method for determining hyaluronic acid content" provides the use of hyaluronidase to enzymatically hydrolyze hyaluronic acid and related products. Method for the determination of hyaluronic acid content. Patent CN201810999774.8 "A method for measuring the in vitro enzymatic hydrolysis performance of cross-linked hyaluronic acid gel" utilizes the difference in the enzymatic hydrolysis rate of cross-linked hyaluronic acid by hyaluronidase to provide a method for measuring cross-linked hyaluronic acid gelation. Methods for extracolloidal enzymatic performance. Patent CN201911259050.0 "A kind of preparation method of sodium trimetaphosphate cross-linked hyaluronic acid powder and obtained product and application" uses hyaluronidase to confirm that the sodium trimetaphosphate cross-linked hyaluronic acid powder is compared with hyaluronic acid. Anti-enzymatic properties. The patent CN202010243080.9 "Microbial Limit Detection Method for Cross-linked or High Molecular Weight Hyaluronic Acids" uses hyaluronidase to reduce the viscosity of cross-linked or high molecular weight hyaluronic acid solution, and then conducts a method for detecting the microbial limit.

At the same time, hyaluronidase is an indispensable tool enzyme for the production of low-molecular and oligomeric hyaluronic acid and its salts. Therefore, hyaluronidase preparations for production need to be combined with low-molecular and oligomeric hyaluronic acid during the preparation process. In the post-purification process of its salts, additional impurities cannot be introduced, which will affect the quality of the finished products of low-molecular-weight and oligomeric hyaluronic acid and salts.

Hyaluronidase preparations often use complex enzyme activity protectors, stabilizers, and buffers to maintain the stability of their enzyme activities. Patent CN201610081945.X "a liquid preparation of recombinant human hyaluronidase and its preparation method and application" provides a liquid preparation of recombinant human hyaluronidase comprising components: 1400000～500000000IU of recombinant human hyaluronidase, 5～ 10g buffer, 60～180g cryoprotectant, 180～300g excipient, 5～12g enzyme activity protectant and 0.3～5g surfactant. Patent CN201380057659.4 "stabilizer for hyaluronidase and liquid preparation including hyaluronidase" said stabilizer contains: buffer to provide pH 4.0 to 6.0; 0.001 to 0.5v/v% of nonionic a surfactant; and 0.1 to 5 mM of a chelating agent or _MgCl2 . These stabilizers, protective agents, and surfactants will all have a certain impact and interference on the application of hyaluronidase, especially in complex systems containing hyaluronic acid, such as content detection in beverages and cosmetic solutions.

Lactic acid and lactate have no absorption in the ultraviolet region, and have good antiseptic properties at room temperature, and a large amount of lactic acid will be produced during the production of hyaluronic acid fermentation. Therefore, lactic acid and lactate are used as hyaluronidase. The enzyme activity stabilizer and preservative will not introduce additional impurities when applied to the production of low molecular and oligomeric hyaluronic acid and its salts.

SUMMARY OF THE INVENTION

In view of the above-mentioned problems existing in the prior art, the present invention provides a hyaluronidase liquid preparation and application thereof. Specifically, the present invention relates to the following aspects:

1. A hyaluronidase liquid preparation, characterized in that the liquid preparation comprises a hyaluronidase-containing fermentation product and lactate, wherein the liquid preparation uses lactic acid to adjust pH to 5.0-6.0.

2. The liquid preparation according to item 1, wherein, based on the volume of the liquid preparation, the content of the hyaluronidase in the liquid preparation is 10 ⁶ to 10 ⁹ IU/L.

3. The liquid preparation according to item 1, wherein, based on the volume of the liquid preparation, the content of the lactate is 50-300 g/L.

4. The liquid preparation according to item 1, wherein the hyaluronidase specific activity in the hyaluronidase-containing fermentation product is 10 ⁴ IU/mg to 10 ⁶ IU/mg.

5. The liquid preparation according to item 1, wherein the hyaluronidase-containing fermentation product is produced and purified by Bacillus sp. A50 CGMCC NO.5744.

6. The liquid preparation according to item 1, wherein the lactate is selected from one or both of sodium lactate and potassium lactate.

7. The liquid preparation according to item 1, wherein the liquid preparation is composed of a hyaluronidase fermentation product, lactate and purified water, wherein the liquid preparation is adjusted to pH 5.0-6.0 using lactic acid.

8. Application of a hyaluronidase liquid preparation in detection, wherein the hyaluronidase liquid preparation is the liquid preparation described in any one of items 1-7.

9. Application of a hyaluronidase liquid preparation in the production of low molecular or oligomeric hyaluronate, the hyaluronidase liquid preparation being the liquid preparation according to any one of items 1-7.

Compared with the prior art, the present invention provides a minimalist formula of Bacillus hyaluronidase liquid preparation, which can maintain the stability of hyaluronidase enzyme activity for a long time, and has the following advantages:

The hyaluronidase preparation adopts Bacillus hyaluronidase as the main material, uses 50-300 g/L sodium lactate or potassium lactate as stabilizer and preservative, and adopts lactic acid to adjust pH to 5.0-6.0, the composition is single, and is Commonly used inert salts will not interfere with the use of hyaluronidase preparations.

Although the formulation of the hyaluronidase preparation is simple, it can maintain good stability of enzyme activity. Under the freezing condition of -20 °C, the enzyme activity of hyaluronidase can maintain more than 95% after 24 months, and at 25 °C, After 30 days, the enzymatic activity of the hyaluronidase can also maintain more than 95%, which provides convenience for the production, processing and application of the hyaluronidase.

Detailed ways

The present invention will be further described below in conjunction with the examples, and it should be understood that the examples are only used to further illustrate and illustrate the present invention, and are not intended to limit the present invention.

Unless otherwise defined, related technical and scientific terms in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or identical to those described herein can be used in experiments or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions therein, will control, and otherwise, the materials, methods and examples are illustrative and not restrictive. The present invention is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present invention.

As mentioned above, hyaluronidase is an indispensable tool enzyme for hyaluronic acid-related research, and has a wide range of applications in the field of detection, such as the determination of the content of hyaluronic acid or its salts in samples, the determination of hyaluronic acid coagulation The in vitro enzymatic hydrolysis performance of glue, and the anti-enzymatic hydrolysis performance of cross-linked hyaluronidase were determined. When hyaluronidase is currently used for detection, it is usually necessary to prepare a lyophilized preparation of hyaluronic acid as an enzyme solution for use. This method is inconvenient to use, and at the same time, due to the relatively small amount of each weighing and the difference of each operation, it is easy to cause detection errors. However, the preparation of hyaluronidase into a liquid preparation for detection often requires the addition of complex stabilizers or protective agents, resulting in inaccurate detection. In the production of low-molecular and oligomeric hyaluronic acid and its salts, complex stabilizers or protective agents will additionally introduce impurities.

In order to solve the above problems, the present invention provides an easy-to-use liquid formulation of hyaluronidase comprising a hyaluronidase-containing fermentation product and lactate, wherein the liquid formulation uses lactic acid to adjust pH to 5.0 -6.0, eg pH can be adjusted to 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0.

Wherein, when preparing the feed solution, the form of the fermentation product containing hyaluronidase is not limited, for example, it can be fermentation broth supernatant, salting-out reconstituted enzyme solution, ultrafiltration enzyme solution, ion exchange elution enzyme solution, and the like. Among them, the fermentation broth supernatant is the supernatant obtained by centrifugation after hyaluronidase fermentation, and the salting-out reconstituted enzyme solution refers to the enzyme obtained by salting out the fermentation supernatant with ammonium sulfate-centrifugation to obtain a precipitation-precipitation-reconstituted liquid The ultrafiltration enzyme solution refers to the enzyme solution obtained by purifying the fermentation supernatant by ultrafiltration, and the ion exchange enzyme solution is the enzyme solution obtained by purifying the ultrafiltration enzyme solution through an anion exchange resin.

In a specific embodiment, the hyaluronidase-containing fermentation product is a fermentation broth supernatant. In a specific embodiment, the hyaluronidase-containing fermentation product is a salted-out reconstituted enzyme solution.

In a specific embodiment, the hyaluronidase-containing fermentation product is an ultrafiltration enzyme broth. In a specific embodiment, the hyaluronidase-containing fermentation product is an ultrafiltration enzyme broth. In a specific embodiment, the hyaluronidase-containing fermentation product is an ion-exchange eluate solution.

The fermented product containing hyaluronidase described in the present invention can be the fermented product containing hyaluronidase from any source, preferably produced and purified by Bacillus sp. A50 CGMCC NO.5744. Specifically, Bacillus ( For the preparation of hyaluronidase produced by Bacillus sp.A50 CGMCC NO.5744, please refer to CN201210497017.3 (named as a bacillus, a hyaluronidase and its preparation method and use). The purification method can be any known in the art. Known methods, such as centrifugation, filtration, salting out, ultrafiltration, ion exchange, gel chromatography.Therefore, the preparation method of the hyaluronidase freeze-dried preparation has a very wide range of applications, whether it is unpurified The fermentation broth supernatant or the enzyme liquid purified by different steps can be prepared by the method of the present invention.

The specific activity of hyaluronidase in the fermentation product containing hyaluronidase is 10 ⁴ IU/mg～10 ⁶ IU/mg, for example, 10 ⁴ IU/mg, 2×10 ⁴ IU/mg, 5× 10 ⁴ IU/mg, 8 x 10 ⁴ IU/mg, 10 ⁵ IU/mg, 2 x 10 ⁵ IU/mg, 5 x 10 ⁵ IU/mg, 8 x 10 ⁵ IU/mg, 10 ⁶ IU/mg.

Wherein IU is a unit used to express the size of enzyme activity. The enzyme activity determination method can be implemented with reference to the 2020 edition of the Chinese Pharmacopoeia, Part 4 1207 "Hyaluronidase Determination Method".

In a specific embodiment, the lactate is selected from one or both of sodium lactate and potassium lactate.

In a specific embodiment, based on the volume of the liquid preparation, the content of the hyaluronidase in the liquid preparation is 10 ⁶ -10 ⁹ IU/L, for example, 10 ⁶ IU/L, 2×10 ⁶ IU/L, 5×10 ⁶ IU/L, 8×10 ⁶ IU/L, 10 ⁸ IU/L, 2×10 ⁸ IU/L, 5×10 ⁸ IU/L, 8×10 ⁸ IU/L, 10 ⁹ IU/L, etc.

In a specific embodiment, based on the volume of the liquid preparation, the content of the lactate is 50-300g/L, such as 50g/L, 60g/L, 70g/L, 80g/L , 90g/L, 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, 150g/L, 160g/L, 170g/L, 180g/L, 190g/L, 200g/L, 210g /L, 220g/L, 230g/L, 240g/L, 250g/L, 260g/L, 270g/L, 280g/L, 290g/L, 300g/L.

The hyaluronidase liquid preparation can be used directly or packaged in suitable packaging equipment.

Further, the present invention also provides a hyaluronidase liquid preparation composed of a hyaluronic acid fermentation product, lactate and purified water, wherein the liquid preparation uses lactic acid to adjust pH to 5.0-6.0. Wherein, the selection and definition of the hyaluronidase product and lactate are as described above.

In a specific embodiment, the hyaluronidase liquid formulation consists of a hyaluronidase liquid formulation consisting of a hyaluronic acid fermentation product, lactate and purified water, wherein the liquid formulation uses lactic acid to adjust the pH to 6.0, wherein the content of hyaluronidase in the liquid preparation is 5×10 ⁷ IU/L, and the fermentation product containing hyaluronidase is a fermentation broth supernatant, wherein the specific activity of hyaluronidase is 10 ⁴ IU/mg , the lactate is sodium lactate, the content is 100g/L.

In a specific embodiment, the hyaluronidase liquid formulation consists of a hyaluronidase liquid formulation consisting of a hyaluronic acid fermentation product, lactate and purified water, wherein the liquid formulation uses lactic acid to adjust the pH to 6.0, wherein the content of hyaluronidase in the liquid preparation is 5×10 ⁷ IU/L, and the fermentation product containing hyaluronidase is a salted-out reconstituted enzyme solution, wherein the specific activity of hyaluronidase is 2×10 ⁵ IU/mg, the lactate is sodium lactate, the content is 100g/L.

In a specific embodiment, the hyaluronidase liquid formulation consists of a hyaluronidase liquid formulation consisting of a hyaluronic acid fermentation product, lactate and purified water, wherein the liquid formulation uses lactic acid to adjust the pH to 6.0, wherein the content of hyaluronidase in the liquid preparation is 5×10 ⁷ IU/L, and the fermentation product containing hyaluronidase is an ultrafiltration enzyme liquid, wherein the specific activity of hyaluronidase is 6×10 ⁵ IU /mg, lactate is potassium lactate, the content is 100g/L.

In a specific embodiment, the hyaluronidase liquid formulation consists of a hyaluronidase liquid formulation consisting of a hyaluronic acid fermentation product, lactate and purified water, wherein the liquid formulation uses lactic acid to adjust the pH to 5.0, wherein the content of hyaluronidase in the liquid preparation is 5×10 ⁷ IU/L, and the fermentation product containing hyaluronidase is an ion-exchange elution enzyme solution, wherein the specific activity of hyaluronidase is 2×10 ⁶ IU/mg, the lactate is sodium lactate, the content is 300g/L.

The present invention also provides the application of the above-mentioned hyaluronidase liquid preparation in detection.

The detection is selected from quantitative or qualitative detection of hyaluronic acid and its derivatives, quantitative or qualitative detection of cross-linked hyaluronic acid, quantitative or qualitative detection of chondroitin sulfate, quantitative or qualitative analysis and detection of impurities, And one or two or more of the anti-enzymatic performance tests.

For example, CN202010547909.4 describes a method for rapidly determining the content of hyaluronic acid in fermentation broth, that is, a method for quantitatively detecting hyaluronan and its derivatives. The method uses hyaluronidase to completely enzymatically hydrolyze hyaluronic acid in the fermentation broth into 4,5-unsaturated disaccharides, and then detects the UV absorption value of the fermentation broth before and after enzymatic hydrolysis, and then calculates the hyaluronic acid content in the fermentation broth . CN201811508968.X also relates to a detection method for quantifying hyaluronic acid, and describes a method for enzymatic hydrolysis of a sample to be tested containing hyaluronic acid by using hyaluronidase, and detecting the hyaluronic acid after enzymatic hydrolysis by hyaluronidase, And further calculate the content of hyaluronic acid contained in the sample to be tested based on the detection result. CN202010243080.9 describes a microbial limit detection method for cross-linked or high-molecular-weight hyaluronic acid substances. The method performs enzymatic hydrolysis on the cross-linked or high-molecular-weight hyaluronic acid substances to be tested under low-temperature weak acid conditions to reduce cross-linking. Or the viscosity of the high molecular weight hyaluronic acid solution, and then the enzymatic hydrolysis solution with reduced viscosity is used to prepare a test solution to realize the microbial limit detection of the sample to be tested. CN201811508981.5 provides a method for determining the content of hyaluronic acid in a solution containing citric acid, comprising: using hyaluronidase to enzymatically hydrolyze a sample to be tested containing hyaluronic acid and citric acid; The acid ion exchange column uses a mobile phase without salt to detect the hyaluronic acid enzymatically hydrolyzed by the hyaluronidase; and calculates the content of the hyaluronic acid based on the detection result. CN201810999774.8 describes a method for measuring the in vitro enzymatic hydrolysis performance of cross-linked hyaluronic acid gel, the method utilizes hyaluronidase to enzymatically hydrolyze the cross-linked hyaluronic acid gel, and then adopts UV-visible spectrophotometry to measure the enzymatic hydrolysis The enzymatic hydrolysis solution produced in the process is used to test the anti-enzymatic hydrolysis performance. In vitro enzymatic hydrolysis rate of cross-linked hyaluronic acid gel was calculated by absorbance.

The present invention also provides the application of the above-mentioned hyaluronidase liquid preparation in the production of low molecular or oligomeric hyaluronate.

The hyaluronidase liquid preparation of the present invention uses 50-300 g/L sodium lactate or potassium lactate as a stabilizer and a preservative, has a single component, and is a commonly used inert salt, which will not interfere with the use of the hyaluronidase preparation. The hyaluronidase liquid preparation has excellent stability. Under the freezing condition of -20 ℃, the hyaluronidase enzyme activity can maintain more than 95% after 24 months, and at 25 ℃, the hyaluronidase enzyme activity is the same after 30 days More than 95% can be maintained.

Example

(1) Preparation of fermentation broth supernatant containing hyaluronidase

Hyaluronidase fermentation was carried out with reference to the method described in CN201210497017.3 (a Bacillus, a hyaluronidase and its preparation method and use), and the fermentation broth supernatant was obtained after centrifugation. The hyaluronidase specific activity of the fermentation broth supernatant containing hyaluronidase was measured to be 10 ⁴ IU/mg.

Among them, the enzymatic activity determination method of the fermentation products containing hyaluronidase shall be carried out with reference to the 2020 edition of the Chinese Pharmacopoeia, Part 4, 1207 "Hyaluronidase Determination Method", and the protein determination method shall be the Coomassie brilliant blue method.

Wherein, the specific activity of hyaluronidase (IU/mg) = the enzyme activity of hyaluronidase/protein content.

(2) Preparation of ultrafiltration enzyme solution containing hyaluronidase

The supernatant of the fermentation broth is ultrafiltered with a 30KDa-50KDa ultrafiltration membrane to obtain an ultrafiltration enzyme solution. The specific enzyme activity of the ultrafiltration enzyme solution containing hyaluronidase was determined to be 1×10 ⁵ IU/mg to 4×10 ⁵ IU/mg.

(3) Preparation of salting-out reconstituted enzyme solution containing hyaluronidase

The supernatant of the fermentation broth is subjected to 25%-35% ammonium sulfate fractional precipitation and centrifugation, and then redissolved with an equal volume of purified water to obtain a salted-out reconstituted enzyme solution. Wherein, the enzyme specific activity of the salted-out reconstituted enzyme solution containing hyaluronidase was determined to be 2×10 ⁵ IU/mg to 6×10 ⁵ IU/mg.

(4) Preparation of ion-exchange enzyme solution containing hyaluronidase

The ultrafiltration enzyme solution or the salting-out reconstituted enzyme solution is adsorbed by DEAE-FAST FLOW ion resin, 0-1M NaCl gradient elution to obtain the ion exchange eluate, which is obtained by dialysis and desalting. The specific activity of the ion-exchange enzyme solution containing hyaluronidase was measured to be 2×10 ⁵ IU/mg to 1×10 ⁶ IU/mg.

Example 1

Add 100 g of sodium lactate to the fermentation supernatant containing 5×10 ⁶ IU of hyaluronidase in the step, mix well, add a certain amount of purified water, set the volume to 1L, and adjust the pH to 6 with lactic acid to obtain hyaluronidase Liquid preparations.

Example 2-4

The difference between Examples 2-4 and Example 1 lies in the specific activity of hyaluronidase in the fermented product containing hyaluronidase, and other reaction conditions are the same as those in Example 1. Specifically, in Example 2, the hyaluronidase was salted out and reconstituted enzyme solution, and the specific activity of the enzyme was 2.7×10 ⁵ IU/mg.

In Example 3, the hyaluronidase was an ultrafiltration enzyme solution, and its specific enzyme activity was 1.6×10 ⁵ IU/mg. Wherein, the supernatant of the fermentation broth in Example 1 was ultrafiltered with an ultrafiltration membrane of 30KDa to 50KDa to obtain an ultrafiltration enzyme liquid.

In Example 4, the hyaluronidase was an ion-exchange enzyme solution, and its specific enzyme activity was 9.2×10 ⁵ IU/mg. Wherein, the ultrafiltration enzyme solution in Example 3 was adsorbed by DEAE-FAST FLOW ion resin, 0-1M NaCl gradient elution was performed to obtain ion exchange eluent, and the ion exchange eluate was obtained by dialysis and desalting.

Embodiment 5-6

Examples 5-6 differ from Example 4 in that the content of hyaluronidase in the liquid formulations is different. Other reaction conditions were the same as in Example 4. Specifically, the content of hyaluronidase in Example 5 was 5×10 ⁸ IU/L, and the content of hyaluronidase in Example 6 was 1×10 ⁹ IU/L.

Example 7

The difference between Example 7 and Example 1 is that the same content of potassium lactate was used instead of sodium lactate in the hyaluronidase liquid preparation. Other reaction conditions are the same as in Example 1.

Examples 8-11

The difference between Examples 8-11 and Example 1 is that the content of sodium lactate in the hyaluronidase liquid preparation is different. Other reaction conditions are the same as in Example 1. Specifically, the content of lactate in Example 8 is 50g/L, the content of hyaluronate in Example 9 is 150g/L, and the content of hyaluronate in Example 10 is 200g/L, The content of the hyaluronate in Example 11 was 300 g/L.

Example 12

The difference between Example 12 and Example 1 is that the pH of the hyaluronidase liquid preparation is different, the pH is 5, and other reaction conditions are the same as those of Example 1.

Comparative Example 1

The difference between Comparative Example 1 and Example 1 is that 100 g/L of mannitol is used instead of sodium lactate in the hyaluronic acid liquid formulation. The other reaction conditions were the same as in Example 1.

Comparative Example 2

The difference between Comparative Example 2 and Example 1 is that each 100 g/L of trehalose was used instead of sodium lactate in the hyaluronic acid liquid preparation. The other reaction conditions were the same as in Example 1.

Comparative Example 3

The difference between Comparative Example 3 and Example 1 is that the pH of the hyaluronidase liquid preparation is different, the pH is 4, and other reaction conditions are the same as those of Example 1.

Comparative Example 4

The difference between Comparative Example 4 and Example 1 is that the pH of the hyaluronidase liquid preparation is different, and the pH is 8.2, that is, the pH of the liquid preparation is not adjusted with lactic acid. Other reaction conditions are the same as in Example 1.

The specific main reaction conditions of each embodiment and comparative example are shown in Table 1.

The reaction conditions of each embodiment and comparative example of table 1

Experimental example

The enzyme activity stability of the subpackaged hyaluronidase liquid preparation under different conditions was determined, wherein the enzyme activity percentage of the liquid preparation was used to characterize the storage stability of the hyaluronidase liquid preparation at different temperatures, which refers to the The ratio of hyaluronidase activity to the initial hyaluronidase activity in the liquid formulation after storage of the hyaluronidase liquid formulation under different temperature conditions for a certain period of time.

Specifically: the hyaluronidase liquid preparation was placed in a freezing condition of -20°C, and the enzyme activity after 24 months was measured; the hyaluronidase liquid was placed at 25°C, and the enzyme activity after 30 days was measured.

Among them, the specific enzyme activity determination method is implemented with reference to the 2020 edition of the "Chinese Pharmacopoeia", Part 4, 1207 "Hyaluronidase Determination Method".

Table 2 shows the enzymatic activity measurement results of the liquid preparations of hyaluronidase obtained in the above examples and comparative examples.

Table 2 Enzyme activity stability of hyaluronidase liquid preparation

Among them, the enzyme activity percentage of the liquid preparation is used to characterize the storage stability of the hyaluronidase liquid preparation at different temperatures, which means that when the hyaluronidase liquid preparation is stored at different temperatures for a certain period of time, the Ratio to initial hyaluronidase activity in liquid formulation.

Claims (9)

1. A hyaluronidase liquid formulation that includes a fermentation product that includes hyaluronidase and a lactate salt, wherein the liquid formulation uses lactic acid to adjust the pH to 5.0-6.0.

2. The liquid preparation according to claim 1, wherein the hyaluronidase is present in the liquid preparation in an amount of 10 based on the volume of the liquid preparation⁶～10⁹IU/L。

3. The liquid preparation according to claim 1, wherein the lactate is contained in an amount of 50 to 300g/L based on the volume of the liquid preparation.

4. The liquid formulation of claim 1, wherein the hyaluronidase-containing fermentation product has a hyaluronidase specific activity of 10⁴IU/mg～10⁶IU/mg。

5. The liquid formulation of claim 1, wherein the fermentation product comprising hyaluronidase is produced and purified from Bacillus sp A50 CGMCC NO. 5744.

6. The liquid preparation according to claim 1, wherein the lactate is selected from one or both of sodium lactate and potassium lactate.

7. The liquid formulation of claim 1, wherein the liquid formulation is comprised of a hyaluronidase fermentation product, lactate, and purified water, wherein the liquid formulation uses lactic acid to adjust the pH to 5.0-6.0.

8. Use of a hyaluronidase liquid formulation in an assay, the hyaluronidase liquid formulation of any one of claims 1-7.

9. Use of a liquid formulation of hyaluronidase in the manufacture of a low molecular or oligomeric hyaluronate salt, the liquid formulation of hyaluronidase being a liquid formulation according to any one of claims 1-7.