CN114317497A - Hyaluronidase liquid preparation for detection and application thereof - Google Patents
Hyaluronidase liquid preparation for detection and application thereof Download PDFInfo
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Abstract
The invention provides a hyaluronidase liquid preparation for detection, which comprises a fermentation product containing hyaluronidase, inorganic salt and purified water, wherein the inorganic salt is salt which does not cause absorption in the ultraviolet region of 220-235 nm. Compared with the prior art, the invention has the following advantages: the hyaluronidase preparation adopts bacillus hyaluronidase as a main material, and 30-300 g/L of inorganic salt as a stabilizer and a preservative, has single component and is common inert salt, and does not interfere the use of the hyaluronidase preparation. Meanwhile, the good enzyme activity stability can be kept, the enzyme activity of the hyaluronidase can be maintained by more than 80% within 24 months under the freezing condition of-20 ℃, the enzyme activity can be maintained by more than 80% within 30 days under the condition of 25 ℃, and the enzyme activity can be maintained by more than 80% within 7 days under the condition of 40 ℃, so that the stability under the good storage, use and transportation conditions is ensured.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a hyaluronidase liquid preparation for detection and application thereof.
Background
Hyaluronidase (HAase), also known as hyaluronidase, is a generic term for an enzyme class that specifically hydrolyzes hyaluronic acid.
Duran-Reynals discovered a "diffusion factor" in studies of extracts from mammalian testis and other tissues as early as 1928, which could promote better diffusion of subcutaneously injected vaccines, dyes, toxins, etc. Meyer in 1940 named this "diffusion factor" Hyaluronidase. Thereafter, hyaluronidase was detected in many tissues and organisms, including bacterial viruses, bacteria, fungi, etc., and also produced in the venom of non-vertebrates such as leeches and crustaceans. In vertebrates, hyaluronidase is found in the venom of snakes and lizards, in the testis and in other organs such as the liver, kidney, lymphatic system and skin.
Hyaluronidase is also an indispensable tool enzyme for related research of hyaluronic acid, and patent No. CN201811508968.X discloses a method for measuring hyaluronic acid content, and provides a method for measuring hyaluronic acid content by utilizing hyaluronidase to perform enzymolysis on hyaluronate and related products. Patent CN201810999774.8 is a method for determining in vitro enzymolysis performance of cross-linked hyaluronic acid gel, which utilizes the difference of hyaluronidase on the enzymolysis rate of cross-linked hyaluronic acid, and provides a method for determining in vitro enzymolysis performance of cross-linked hyaluronic acid gel. Patent CN201911259050.0 discloses a preparation method of sodium trimetaphosphate crosslinked hyaluronic acid powder, and the obtained product and application thereof, which utilizes hyaluronidase to confirm the anti-enzymolysis performance of the sodium trimetaphosphate crosslinked hyaluronic acid powder compared with hyaluronic acid. Patent CN202010243080.9 cross-linking or high molecular weight hyaluronic acid substance microorganism limit detection method, which utilizes hyaluronidase to reduce the viscosity of cross-linking or high molecular weight hyaluronic acid solution, and then carries out microorganism limit detection method.
Hyaluronidase preparations often use complex enzyme activity protectors, stabilizers, buffers to maintain their enzyme activity stability. Patent CN201610081945.X A recombinant human hyaluronidase liquid preparation, its preparation method and application thereof provide a recombinant human hyaluronidase liquid preparation, which comprises the following components: 1400000-500000000 IU of recombinant human hyaluronidase, 5-10 g of buffering agent, 60-180 g of cryoprotectant, 180-300 g of excipient, 5-12 g of enzyme activity protectant and 0.3-5 g of surfactant. Patent CN201380057659.4 discloses a stabilizer for hyaluronidase and a liquid formulation comprising hyaluronidase, said stabilizer comprising: a buffer to provide a pH of 4.0 to 6.0; 0.001 to 0.5 v/v% of a nonionicA surfactant; and 0.1 to 5mM of a chelating agent or MgCl2. The stabilizing agent, the protective agent and the surfactant can generate certain influence and interference on the application of the hyaluronidase, especially in a complex system containing the hyaluronic acid, such as content detection in beverages, cosmetic solutions and the like. Meanwhile, if the freeze-dried powder preparation is used for detection, the freeze-dried powder preparation is required to be prepared into a liquid form before use, so that the use is inconvenient, and inaccurate detection is easily caused when the freeze-dried powder preparation is weighed at a low concentration.
Disclosure of Invention
In view of the above problems in the prior art, the present invention provides a hyaluronidase liquid preparation for detection and applications thereof, and more particularly, the present invention relates to the following aspects:
1. a liquid preparation for detecting hyaluronidase, which comprises a fermentation product containing hyaluronidase, an inorganic salt and purified water, wherein the inorganic salt is a salt that does not cause absorption in the 220-and 235-nm ultraviolet regions.
2. The liquid preparation according to item 1, wherein the amount of hyaluronidase in the liquid preparation is 10 based on the volume of the liquid preparation6～108IU/L。
3. The liquid preparation according to item 1, wherein the inorganic salt is contained in an amount of 10 to 300g/L, preferably 30 to 100g/L, based on the volume of the liquid preparation.
4. The liquid preparation according to item 1, wherein the hyaluronidase-containing fermentation product has a hyaluronidase specific activity of 104IU/mg～106IU/mg。
5. The liquid preparation according to item 1, wherein the fermentation product containing hyaluronidase is produced and purified from Bacillus sp A50 CGMCC NO. 5744.
6. The liquid preparation according to item 1, wherein the inorganic salt is a chlorine-containing inorganic salt.
7. The liquid preparation according to item 6, wherein the inorganic salt is one or both of sodium chloride and potassium chloride.
8. The liquid preparation according to item 1, characterized in that the liquid preparation is composed of a fermentation product containing hyaluronidase, an inorganic salt and purified water.
9. The liquid formulation according to item 1, wherein the liquid formulation is dispensed in a sterile container.
10. Use of a hyaluronidase liquid formulation in an assay, the hyaluronidase liquid formulation of any one of claims 1-9.
Compared with the prior art, the invention provides a simple formula of a bacillus hyaluronidase liquid preparation, which can maintain the stability of the enzyme activity of the hyaluronidase for a long time and has the following advantages:
the hyaluronidase preparation adopts bacillus hyaluronidase as a main material, and 10-300 g/L of inorganic salt as a stabilizer and a preservative, has single component and is common inert salt, and does not interfere the use of the hyaluronidase preparation.
Although the hyaluronidase preparation has a simple formula, the stability of the enzyme activity can be kept well, the enzyme activity of the hyaluronidase can be kept more than 80% within 24 months under the freezing condition of-20 ℃, the enzyme activity can be kept more than 80% within 30 days under the condition of 25 ℃, and the enzyme activity can be kept more than 80% within 7 days under the condition of 40 ℃, so that the stability under the conditions of good storage, use and transportation is ensured.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
As described above, hyaluronidase is an indispensable tool for research on hyaluronic acid and has wide applications in the field of detection, and can be used, for example, for measuring the content of hyaluronic acid or a salt thereof in a sample, measuring the in vitro enzymatic hydrolysis performance of a hyaluronic acid gel, measuring the enzymatic hydrolysis resistance of cross-linked hyaluronidase, and the like. When hyaluronidase is currently used for detection, a lyophilized preparation of hyaluronic acid is usually prepared as an enzyme solution for use. This method is inconvenient to use, and is easy to cause detection errors due to the small amount of weighing each time and the difference of operation each time. The hyaluronidase is prepared into a liquid preparation for detection, and a complex stabilizing agent or a protective agent is often added. In order to solve the above problems, the present invention provides a hyaluronidase liquid preparation which is convenient to use, comprising a fermentation product containing hyaluronidase, an inorganic salt and purified water, wherein the inorganic salt is a salt that does not cause absorption in the 220-and 235-nm ultraviolet region. Further, the inorganic salt may be a chlorine-containing inorganic salt, preferably one or two selected from sodium chloride and potassium chloride. The inorganic salt acts as a stabilizer or protective agent for the hyaluronidase in the hyaluronidase preparation, while not affecting the detection.
The form of the fermentation product containing hyaluronidase is not particularly limited when preparing the feed solution, and examples thereof include supernatant of fermentation broth, salting-out redissolution, ultrafiltration solution, ion-exchange elution solution, and the like. Wherein the fermentation liquid supernatant is supernatant obtained by centrifuging after hyaluronidase fermentation, the salting-out redissolution is enzyme liquid obtained by salting-out the fermentation supernatant by ammonium sulfate and centrifuging to obtain precipitation-precipitation redissolution, the ultrafiltration enzyme liquid is enzyme liquid obtained by purifying the fermentation supernatant by ultrafiltration, and the ion exchange enzyme liquid is enzyme liquid obtained by purifying the ultrafiltration enzyme liquid by anion exchange resin.
The fermentation product containing the hyaluronidase can be a fermentation product containing the hyaluronidase from any source, and is preferably produced and purified by Bacillus (Bacillus sp.) A50 CGMCC NO. 5744. Specifically, the preparation of hyaluronidase produced by Bacillus (Bacillus sp.) a50 CGMCC No.5744 can refer to CN201210497017.3 (named as a Bacillus, a hyaluronidase, its preparation method and application). The purification method may be any method known in the art, and may be, for example, centrifugation, filtration, salting out, ultrafiltration, ion exchange, gel chromatography. Therefore, the preparation method of the hyaluronidase freeze-dried preparation has a wide application range, and the purified fermentation broth supernatant or the enzyme solution purified by different steps can be prepared by the method.
In a specific embodiment, the hyaluronidase-containing fermentation product has a hyaluronidase specific activity of 104IU/mg～106IU/mg, for example, may be 104IU/mg、2×104IU/mg、5×104IU/mg、8×104IU/mg、105IU/mg、2×105IU/mg、5×105IU/mg、8×105IU/mg、106IU/mg。
Wherein IU is a unit for expressing the magnitude of enzyme activity. The method for measuring the enzyme activity can be performed with reference to the 2020 edition "Chinese pharmacopoeia" fourth part 1207 "hyaluronidase assay".
In a specific embodiment, the inorganic salt is a chlorine-containing inorganic salt.
In a preferred embodiment, the inorganic salt is selected from one or both of sodium chloride and potassium chloride, and may be, for example, sodium chloride, or potassium chloride, or a mixture of sodium chloride and potassium chloride.
In a specific embodiment, the hyaluronidase is present in the liquid formulation in an amount of 10 based on the volume of the liquid formulation6～108IU/L may be, for example, 106IU/L、2×106IU/L、5×106IU/L、8×106IU/L、107IU/L、2×107IU/L、5×107IU/L、8×107IU/L、108IU/L. The content of the inorganic salt is 10-300 g/L, the preferable content is 30-100g/L, for example10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, 100g/L, 150g/L, 200g/L, 250g/L, 300 g/L.
The hyaluronidase liquid preparation can be directly used, and can also be subpackaged in proper aseptic packaging equipment, preferably the liquid filling amount is 1 ml/piece, and the hyaluronidase liquid preparation can be directly used for one-time detection.
Further, the present invention provides a hyaluronidase liquid preparation comprising a fermentation product containing hyaluronidase, an inorganic salt and purified water. Wherein the hyaluronidase product and inorganic salt are selected and defined as described above.
The invention also provides application of the hyaluronidase liquid preparation in detection.
The detection is one or more than two of quantitative or qualitative detection of hyaluronic acid and derivatives thereof, quantitative or qualitative detection of cross-linked hyaluronic acid, quantitative or qualitative detection of chondroitin sulfate, quantitative or qualitative analysis detection of impurities, and test of enzymolysis resistance. For example, CN202010547909.4 describes a method for rapidly determining the content of hyaluronic acid in a fermentation broth, i.e., a method for quantitatively detecting hyaluronate and its derivatives. The method adopts hyaluronidase to carry out complete enzymolysis on hyaluronic acid in fermentation liquor into 4, 5-unsaturated disaccharide, then detects the ultraviolet absorption value of the fermentation liquor before and after enzymolysis, and further calculates the content of hyaluronic acid in the fermentation liquor. Cn201811508968.x also relates to a method for quantitatively detecting hyaluronic acid, and describes a method for performing enzymolysis on a sample to be detected containing hyaluronic acid by using hyaluronidase, detecting hyaluronic acid after the enzymolysis by the hyaluronidase, and further calculating the content of hyaluronic acid contained in the sample to be detected based on the detection result. CN202010243080.9 describes a method for detecting microbial limit of crosslinked or high molecular weight hyaluronic acid substance, which comprises performing enzymolysis treatment on the crosslinked or high molecular weight hyaluronic acid substance to be detected under low temperature and weak acid condition to reduce viscosity of the crosslinked or high molecular weight hyaluronic acid substance solution, and then preparing a detection solution from the enzymolysis solution with reduced viscosity to realize microbial limit detection of the sample to be detected. CN201811508981.5 provides a method for determining the content of hyaluronic acid in a solution containing citric acid, which comprises: carrying out enzymolysis on a sample to be detected containing hyaluronic acid and citric acid by using hyaluronidase; detecting hyaluronic acid after enzymatic hydrolysis by hyaluronidase using an ion exchange column for analyzing organic acids using a mobile phase not containing salt; and calculating the content of hyaluronic acid based on the detection result. CN201810999774.8 describes a method for determining the in vitro enzymolysis performance of a cross-linked hyaluronic acid gel, which utilizes hyaluronidase to carry out enzymolysis on the cross-linked hyaluronic acid gel, and then adopts an ultraviolet-visible spectrophotometry method to determine enzymolysis liquid generated in the enzymolysis process, thereby testing the enzymolysis resistance. And calculating the in-vitro enzymolysis rate of the crosslinked hyaluronic acid gel through absorbance.
The hyaluronidase liquid preparation disclosed by the invention adopts bacillus hyaluronidase as a main material, and 10-300 g/L of inorganic salt as a stabilizer and a preservative, so that the hyaluronidase liquid preparation is single in component and cannot interfere with the use of the hyaluronidase preparation. The hyaluronidase has good enzyme activity stability, the enzyme activity of the hyaluronidase can be maintained by more than 80% within 24 months under the freezing condition of-20 ℃, the enzyme activity can be maintained by more than 80% within 30 days under the condition of 25 ℃, and the enzyme activity can be maintained by more than 80% within 7 days under the condition of 40 ℃, so that the hyaluronidase is more convenient to store, detect and use.
Examples
(1) Preparation of fermentation supernatant containing hyaluronidase
Hyaluronidase fermentation was performed according to the method described in CN201210497017.3 (a bacillus, a hyaluronidase, methods of preparation and uses thereof), and the supernatant of the fermentation broth was obtained after centrifugation. Wherein the enzyme specific activity of the supernatant of the fermentation broth containing hyaluronidase was measured to be 1.4X 104IU/mg～3.0×104IU/mg。
The method for measuring the enzyme activity of the fermentation product containing hyaluronidase is executed by referring to 2020 version of Chinese pharmacopoeia, fourth part 1207, hyaluronidase measuring method, and the method for measuring protein adopts Coomassie brilliant blue method.
Wherein, the specific activity (IU/mg) of the hyaluronidase is the enzyme activity/protein content of the hyaluronidase.
(2) Preparation of an Ultrafiltration enzyme solution containing Hyaluronidase
And ultrafiltering the supernatant of the fermentation liquor by adopting an ultrafiltration membrane of 30-50 KDa to obtain ultrafiltration enzyme liquid. Wherein the enzyme specific activity of the ultrafiltrate containing hyaluronidase is measured to be 1X 105IU/mg～4×105IU/mg。
(3) Preparation of salting-out redissolution containing hyaluronidase
And (3) after supernatant of the fermentation liquor and fractional precipitation and centrifugation of 25-35% ammonium sulfate, redissolving by using purified water with the same volume to obtain salting-out redissolution. Wherein the enzyme specific activity of the salting-out reconstituted enzyme solution containing hyaluronidase was measured to be 2X 105IU/mg～6×105IU/mg。
(4) Preparation of ion-exchange enzyme solution containing hyaluronidase
The ultrafiltration enzyme solution or salting-out redissolution enzyme solution is absorbed by DEAE-FAST FLOW ion resin, and is subjected to gradient elution with 0-1M NaCl to obtain ion exchange eluent, and is subjected to dialysis and desalting to obtain the final product. Wherein the enzyme specific activity of the ion exchange enzyme solution containing hyaluronidase was measured to be 2X 105IU/mg～1×106IU/mg。
Example 1
The content of 6.7X 10 in the step (4)630g of sodium chloride is added into IU hyaluronidase ion exchange enzyme liquid, and after the sodium chloride is fully dissolved, the mixture is filtered and sterilized by a sterile filter membrane of 0.2 micron. Adding purified water sterilized and cooled at 121 ℃ for 20min into the solution, and metering the volume to 1L to obtain the hyaluronidase liquid preparation. And further subpackaging the prepared hyaluronidase liquid preparation into pre-sterilized 3ml penicillin bottles, wherein the liquid filling amount is 1 ml/piece.
Examples 2 to 4
Examples 2 to 4 differ from example 1 in the amount of hyaluronidase in the liquid formulation. Other reaction conditions were the same as in example 1. Specifically, the hyaluronidase content in example 2 was 5.8 × 107IU/L, hyaluronidase content in example 3 was 2.4X 108IU/L, hyaluronidase content in example 4 was 5X 106IU/L。
Examples 5 to 7
Examples 5 to 7 differ from example 1 in that hyaluronidase specific activity is present in the fermentation product containing hyaluronidase. Other reaction conditions were the same as in example 1.
Specifically, the hyaluronidase in example 5 is a salting-out redissolution, and the specific activity thereof is 5.3X 105IU/mg。
In example 6, hyaluronidase was ultrafiltrated enzyme solution, and its specific activity was 4.4X 105IU/mg。
In example 7, hyaluronidase was a fermentation supernatant having an enzymatic specific activity of 2.6X 104IU/mg。
Example 8
Example 8 differs from example 7 in that the same amount of potassium chloride was used in place of sodium chloride in the hyaluronidase liquid formulation. Other reaction conditions were the same as in example 1.
Examples 9 to 12
Examples 9 to 12 differ from example 7 in the content of sodium chloride in the hyaluronidase liquid formulation. Other reaction conditions were the same as in example 7. Specifically, the content of sodium chloride in example 9 was 100g/L, the content of sodium chloride in example 10 was 50g/L, the content of sodium chloride in example 11 was 250g/L, and the content of sodium chloride in example 12 was 10 g/L.
Comparative example 1
Comparative example 1 differs from example 7 in that mannitol, trehalose each 15g/L was used as a protective agent in place of sodium chloride in the hyaluronic acid solution formulation. Other reaction conditions were the same as in example 1.
Comparative example 2
Comparative example 2 differs from example 7 in that sodium citrate is used instead of sodium chloride in the hyaluronic acid liquid formulation, the total amount being 30 g/L. Other reaction conditions were the same as in example 1.
The main reaction conditions of the specific examples and comparative examples are shown in table 1.
TABLE 1 reaction conditions for each example and comparative example
Experimental example 1
And measuring the enzyme activity stability of the subpackaged hyaluronidase liquid preparation under different conditions, wherein the enzyme activity percentage of the liquid preparation is used for representing the storage stability of the hyaluronidase liquid preparation at different temperatures, and the ratio of the hyaluronidase activity to the initial hyaluronidase activity in the liquid preparation after the hyaluronidase liquid preparation is stored for a certain time at different temperatures is referred to.
Specifically, the method comprises the following steps: placing the hyaluronidase liquid preparation under-20 deg.C freezing condition, and measuring enzyme activity after 24 months; placing the hyaluronidase liquid at 25 deg.C, and measuring enzyme activity after 30 days; the hyaluronidase liquid was placed at 40 ℃ and the enzyme activity was measured after 7 days.
Wherein, the specific enzyme activity measuring method is executed according to 2020 edition Chinese pharmacopoeia 1207 hyaluronidase measuring method in the fourth part.
The results of measuring the enzyme activity of the liquid preparations of hyaluronidase obtained in the above examples and comparative examples are shown in table 2.
TABLE 2 enzyme activity stability of hyaluronidase liquid formulations
Experimental example 2
The hyaluronidase preparations of example 7 and comparative example 2 were used to measure the hyaluronic acid content of the fermentation broth, and the specific measurement method was performed according to the method described in example 7 of CN202010547909.4 (a method for rapidly measuring the hyaluronic acid content of the fermentation broth). And (4) measuring the content of the hyaluronic acid in the hyaluronic acid fermentation liquor of the streptococcus equi subsp zooepidemicus of one batch, and parallelly measuring for three times. The determination result shows that the content of hyaluronic acid is 6.75g/L respectively; 6.88 g/L; 6.84 g/L; the hyaluronidase formulation of comparative example 2, however, resulted in a higher amount of absorbance at 232nm due to the high sodium citrate content, which resulted in the inability to measure hyaluronic acid.
Claims (10)
1. A liquid preparation for detecting hyaluronidase, which comprises a fermentation product containing hyaluronidase, an inorganic salt and purified water, wherein the inorganic salt is a salt that does not cause absorption in the 220-and 235-nm ultraviolet regions.
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 preparation6～108IU/L。
3. The liquid preparation according to claim 1, wherein the inorganic salt is contained in an amount of 10 to 300g/L, preferably 30 to 100g/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 formulation of claim 1, wherein the inorganic salt is a chlorine-containing inorganic salt.
7. The liquid formulation of claim 6, wherein the inorganic salt is selected from one or both of sodium chloride and potassium chloride.
8. The liquid formulation according to claim 1, wherein the liquid formulation is composed of a fermentation product containing hyaluronidase, inorganic salts and purified water.
9. The liquid formulation of claim 1, wherein the liquid formulation is dispensed in a sterile container.
10. Use of a hyaluronidase liquid formulation in an assay, the hyaluronidase liquid formulation of any one of claims 1-9.
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| GB730929A (en) * | 1952-12-20 | 1955-06-01 | American Home Prod | Stabilised aqueous hyaluronidase solutions and method of preparing them |
| WO2006067608A1 (en) * | 2004-12-22 | 2006-06-29 | Laboratoire Medidom S.A. | Aqueous formulations based on sodium hyaluronate for parenteral use |
| CN103255076A (en) * | 2012-02-21 | 2013-08-21 | 华熙福瑞达生物医药有限公司 | Bacillus spp and hyaluronidase and preparation method and purpose thereof |
| CN103468662A (en) * | 2013-09-29 | 2013-12-25 | 惠觅宙 | Recombined human hyaluronidase, production and purification method and preparations thereof, use method and application |
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2021
- 2021-12-14 CN CN202111526893.XA patent/CN114317497A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB730929A (en) * | 1952-12-20 | 1955-06-01 | American Home Prod | Stabilised aqueous hyaluronidase solutions and method of preparing them |
| WO2006067608A1 (en) * | 2004-12-22 | 2006-06-29 | Laboratoire Medidom S.A. | Aqueous formulations based on sodium hyaluronate for parenteral use |
| CN103255076A (en) * | 2012-02-21 | 2013-08-21 | 华熙福瑞达生物医药有限公司 | Bacillus spp and hyaluronidase and preparation method and purpose thereof |
| CN103468662A (en) * | 2013-09-29 | 2013-12-25 | 惠觅宙 | Recombined human hyaluronidase, production and purification method and preparations thereof, use method and application |
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