A method for storing a cross-linked hyaluronic acid gel

Abstract

The application provides a storage method of cross-linked hyaluronic acid gel, which comprises the following steps: storing the cross-linked hyaluronic acid gel in glycerol. The storage method of the cross-linked hyaluronic acid gel provided by the application stores the cross-linked hyaluronic acid gel in glycerol, thereby meeting the long-term storage requirement of the cross-linked hyaluronic acid gel, and the obtained cross-linked sodium hyaluronate gel not only has higher viscoelasticity, but also has lower epoxy residue, and the use safety is improved.

Description

Technical Field

[0001] This invention relates to the field of medical aesthetic filler materials, specifically to a method for storing cross-linked hyaluronic acid gel. Background Technology

[0002] Sodium hyaluronate, after cross-linking modification, forms a hydrogel called cross-linked hyaluronic acid gel. This is a special dispersion system in which colloidal particles or polymer molecules are interconnected, forming a three-dimensional network structure filled with liquid. Due to the formation of a covalently cross-linked network, the cross-linked sodium hyaluronate gel exhibits swelling rather than dissolving properties. Because this hydrogel contains a large number of hydrophilic groups, it can absorb and store a significant amount of water, resulting in a substantial increase in both weight and volume.

[0003] Cross-linked hyaluronic acid gels possess excellent biocompatibility and biodegradability, overcoming the disadvantage of rapid degradation of uncross-linked hyaluronic acid gels. They also have advantages such as high mechanical strength, good viscoelasticity, and long degradation time, which greatly improves their application range and performance, enabling them to be widely used in cosmetic fillers, anti-adhesion agents, hemostatic agents, ophthalmic viscoelastic agents, and other fields.

[0004] Currently, cross-linked hyaluronic acid gel products on the market are often formulated by combining single cross-linked hyaluronic acid gel with other functional substances such as lidocaine hydrochloride and osmotic pressure regulators, rather than being sold directly as single cross-linked hyaluronic acid gel. This is because cross-linked hyaluronic acid gels often require moist heat sterilization and storage after preparation to meet the requirements for controlling the level of microorganisms and bacterial endotoxins in the gel.

[0005] In existing technologies, each moist heat sterilization of cross-linked hyaluronic acid gel leads to a decrease in the gel's viscoelasticity. Therefore, there is an urgent need in the field to develop a storage method for cross-linked hyaluronic acid gel that can meet the requirements for long-term preservation without moist heat sterilization. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for storing cross-linked hyaluronic acid gel. Before moist heat sterilization, the cross-linked hyaluronic acid gel is soaked in glycerin for preservation, thereby meeting the requirements for long-term preservation of cross-linked hyaluronic acid gel.

[0007] To achieve the above-mentioned objective, the present invention provides a method for storing cross-linked hyaluronic acid gel, comprising the following steps: storing the cross-linked hyaluronic acid gel in glycerin.

[0008] In some embodiments, the weight-to-volume ratio of the cross-linked hyaluronic acid gel to glycerin is 1:(1-12)g / mL, preferably 1:(6-10)g / mL, for example 1:9g / mL.

[0009] In some embodiments, the preparation method of the cross-linked hyaluronic acid gel includes: mixing a cross-linking agent, a hyaluronic acid substance and an alkaline aqueous solution, and then performing a cross-linking reaction to obtain the cross-linked hyaluronic acid gel.

[0010] Wherein, the crosslinking agent is a crosslinking agent containing at least two epoxy groups; the crosslinking agent is selected from crosslinking agents containing two epoxy groups, preferably selected from at least one of 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and poly(dimethylsiloxane) diglycidyl ether.

[0011] The volume-to-mass ratio of the crosslinking agent to the hyaluronic acid is 1.5–35 μL / g, preferably 10–30 μL / g, for example 15 μL / g or 20 μL / g; the mass ratio of the hyaluronic acid to the alkaline aqueous solution is (0.16–0.4):1, preferably (0.25–0.4):1, for example 0.33:1; the mass percentage of alkaline substance in the alkaline aqueous solution is 0.8%–2%.

[0012] In some embodiments, the hyaluronic acid-like substance is selected from hyaluronic acid and hyaluronic acid salts. The hyaluronic acid salt is selected from at least one of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, and zinc hyaluronate.

[0013] In some embodiments, the molecular weight of the hyaluronic acid-like substance is 50WDa to 200WDa, preferably 60WDa to 200WDa, such as 60WDa to 70WDa, 130WDa or 150WDa.

[0014] In some embodiments, the alkaline aqueous solution includes at least one selected from sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, sodium carbonate aqueous solution, and sodium bicarbonate aqueous solution. The water in the alkaline aqueous solution includes at least one selected from deionized water, distilled water, purified water, and water for injection.

[0015] In some embodiments, the mass percentage of alkaline substances in the alkaline aqueous solution is 1% to 2%, preferably 1.5% to 2%.

[0016] In some embodiments, the crosslinking reaction is carried out at a temperature of 20–50°C for 8–24 hours; preferably, the crosslinking reaction is carried out at 40–50°C for 3–5 hours, and then at 20–25°C for 10–14 hours.

[0017] In some embodiments, the cross-linked hyaluronic acid gel is stored in glycerin, including the following steps:

[0018] The cross-linked hyaluronic acid gel can be stored in glycerin in one go, or

[0019] Glycerin was divided into several parts, and cross-linked hyaluronic acid gel was soaked in each part of glycerin in turn, and then stored in the last part of glycerin.

[0020] In some embodiments, glycerol is divided into several portions, and cross-linked hyaluronic acid gel is sequentially immersed in each portion of glycerol and stored in the last portion of glycerol, including:

[0021] The glycerin was divided into three parts, and the cross-linked hyaluronic acid gel was placed into the first part of the glycerin for the first soaking.

[0022] Remove the cross-linked hyaluronic acid gel and soak it a second time in the second part of glycerin.

[0023] Remove the cross-linked hyaluronic acid gel and store it in the third part of glycerin.

[0024] In some embodiments, the volume ratio of the first portion of glycerol, the second portion of glycerol, and the third portion of glycerol is (9-11):(3-6):3, for example, 10:5:3.

[0025] This application also provides the application of cross-linked hyaluronic acid gel obtained by the storage method described above in the preparation of medical or cosmetic products. The medical or cosmetic products include filler / shaping products.

[0026] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0027] Compared with the prior art, the present invention has the following beneficial technical effects:

[0028] 1. The storage method of cross-linked hyaluronic acid gel provided by the present invention involves storing the cross-linked hyaluronic acid gel in glycerin, thereby meeting the requirements for long-term storage of the cross-linked hyaluronic acid gel.

[0029] 2. The cross-linked gel obtained by the storage method of the cross-linked hyaluronic acid gel provided by the present invention not only has high viscoelasticity, but also low epoxy residue, thus improving the safety of use. Detailed Implementation

[0030] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0031] The reagents and raw materials used in the following examples are all commercially available.

[0032] Example 1

[0033] Prepare phosphate buffer solution: Add 26.7g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and 6.9g of sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) to every 1L of water for injection.

[0034] Weigh 5g of sodium hyaluronate (130W) and dissolve it in 25mL of 1% (w / v) sodium hydroxide solution. Add 75µL of 1,4-butanediol diglycidyl ether (BDDE), mix well, and then perform a first crosslinking reaction at 40℃ and under static conditions for 4h. Then, place it at 25℃ and under static conditions for a second reaction for 12h.

[0035] The gel obtained after the secondary cross-linking reaction was placed in a dialysis bag and dialyzed twice with the above-mentioned phosphate buffer solution. The dialysate was replaced with a new one each time, with an interval of 8 hours between dialysis sessions and a gel-to-liquid ratio of 1:100.

[0036] After dialysis, the gel was removed, weighed, and placed in the first glycerol bath at a weight-to-volume ratio of 1:5 g / mL. The gel was mechanically stirred at 100 rpm for 5 hours for the first soaking. The gel was then removed and placed in the second glycerol bath at a weight-to-volume ratio of 1:2.5 g / mL. This second soaking was performed by mechanically stirring at 100 rpm for 5 hours. The gel was then removed and placed in the third glycerol bath at a weight-to-volume ratio of 1:1.5 g / mL for the third soaking. Finally, the gel was stored in a 40°C stability test chamber for one month. The weight of the gel is calculated based on its initial weight before the first glycerol bath, and the glycerol used is injection-grade gel.

[0037] After the expiration date, remove the gel and continue dialysis for 6 more times under the above conditions. Adjust the total hyaluronic acid content in the gel to 2% using the above phosphate buffer (calculated based on the amount of hyaluronic acid added). After sieving and granulating the gel, fill it with a pre-filled syringe and then sterilize it by moist heat (121℃, 15min).

[0038] Example 2

[0039] Compared to Example 1, the only difference lies in the way the glycerin is stored, as follows:

[0040] After dialysis, the gel was removed, weighed, and then immersed in glycerol at a weight-to-volume ratio of 1:9. It was then stored in a 40°C stability test chamber for one month. The amount of glycerol used here is equivalent to the total amount of glycerol used in the three parts of Example 1.

[0041] Comparative Example 1

[0042] Compared with Example 1, the difference is that the gel was not stored in glycerol, nor was it stored in a 40°C stability test chamber for 1 month. Instead, it was directly dialyzed 8 times, weighed, sieved and granulated, and then subjected to moist heat sterilization.

[0043] Comparative Example 2

[0044] Compared to Example 1, the difference is that the gel was not stored in glycerol, but instead was dialyzed eight times, then treated with moist heat sterilization (121°C, 15 min), and then stored in a 40°C stability test chamber for one month. After the expiration date, the gel was weighed, sieved, granulated, and then treated with moist heat sterilization again.

[0045] Performance testing

[0046] The performance of the cross-linked hyaluronic acid gels prepared in Examples 1-2 and Comparative Examples 1-2 was measured, specifically including the viscoelasticity and total modification degree of the cross-linked hyaluronic acid gel, the free HA content, total HA content, epoxy residue, glycerol residue and bacterial endotoxin content in the cross-linked hyaluronic acid gel.

[0047] Viscoelasticity: refers to the deformation and flow properties of the cross-linked gel under external force. Specifically, it is tested using a rheometer in oscillation mode at a test temperature of 25℃, a rotor spacing of 0.3mm, a starting frequency of 10Hz, an ending frequency of 0.1Hz, a shear strain of 0.5%, and 10 sampling points for each order of magnitude. The elastic modulus G' (Pa), viscous modulus G'' (Pa), and complex viscosity η* (Pa.s) at 1Hz are observed.

[0048] Overall level of refinement:

[0049] Total modification degree describes the ratio of the total amount of cross-linking agent bound to HA to the total amount of repeating HA disaccharide units, that is, the ratio of all cross-linking agents bound to at least one end of the cross-linked hyaluronic acid gel to all repeating HA disaccharide units in the cross-linked hyaluronic acid gel.

[0050] This method uses NMR spectroscopy to determine the total degree of modification by detecting the ratio of HA disaccharide groups to BDDE groups on the cross-linked hyaluronic acid gel product catalyzed by hyaluronidase degradation. Soluble HA, residual (unbound) cross-linking agents, and their derivatives are removed by filtration before the degradation of the cross-linked hyaluronic acid gel. The cross-linked hyaluronic acid gel is then degraded by hyaluronidase treatment at 40°C. After degradation, the sample is lyophilized and then analyzed by 1H-NMR spectroscopy. 1H-NMR spectroscopy is performed by detecting the δ-axis... H The signal at 1.6 ppm (from four protons in the BDDE molecule) and at δ HThe degree of modification was calculated by integrating the signal at 2.0 ppm (from the three protons in the CH3 group on the N-acetylglucosamine residue of the HA disaccharide). After correcting for the number of protons responsible for each signal, the total degree of modification was calculated as follows:

[0051]

[0052] Epoxy residue: Epoxy residue was measured using high performance liquid chromatography-mass spectrometry (SEC-MS).

[0053] Bacterial endotoxins: determined according to the dynamic turbidity method in the bacterial endotoxin test method of 2020 edition 1143.

[0054] The performance test results of the cross-linked hyaluronic acid gels prepared in Examples 1-2 and Comparative Examples 1-2 are shown in Table 1.

[0055] Table 1

[0056]

[0057]

[0058] According to the measurement results in Table 1, both Examples 1 and 2 are cross-linked hyaluronic acid gels preserved by soaking in glycerol, and their viscoelasticity is higher than that of the gel in Comparative Example 2. Comparative Example 2 is a cross-linked hyaluronic acid gel that has undergone two sterilization processes. Compared with Comparative Example 1, after the gel was stored for one month and then sterilized by moist heat, its viscoelasticity decreased significantly, and the proportion of free HA in the gel increased from 27% to 51%.

[0059] In addition, compared with Comparative Example 1, the epoxy residue in the gels of Examples 1 and 2 was reduced, and the reduction was greater than that in the gel of Comparative Example 2. This indicates that although moist heat sterilization can reduce epoxy residue, glycerol soaking can reduce epoxy residue even more effectively.

[0060] Example 1 is a cross-linked gel that has been soaked in glycerol three times, and Example 2 is a cross-linked gel that has been directly soaked in glycerol. The epoxy residue in the gel of Example 1 is lower than that in the gel of Example 2, and the cross-linked gel is safer.

[0061] Glycerin can absorb moisture from the gel. When using a glycerin gradient soak as in Example 1, the third glycerin-gel system contains less moisture, which may be more conducive to reducing epoxy residue.

[0062] Glycerin is often used as an osmotic pressure regulator in injection solutions. It is an endogenous substance in the human body. Low concentrations of glycerin are harmless to the human body. 2.6% glycerin is an isotonic solution, and 10% glycerin is an isotonic solution. Therefore, in Examples 1 and 2, the residue of glycerin will not have any impact on the use of the gel. In addition, glycerin in the gel can be further removed by increasing the number of dialysis sessions.

[0063] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0064] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A method for storing cross-linked hyaluronic acid gel, characterized in that, Includes the following steps: Store the cross-linked hyaluronic acid gel in glycerin.

2. The storage method as described in claim 1, characterized in that, The weight-to-volume ratio of the cross-linked hyaluronic acid gel to glycerin is 1:(1-12)g / mL.

3. The storage method as described in claim 1, characterized in that, The preparation method of the cross-linked hyaluronic acid gel includes: mixing a cross-linking agent, a hyaluronic acid substance and an alkaline aqueous solution, and then performing a cross-linking reaction to obtain the cross-linked hyaluronic acid gel.

4. The storage method as described in claim 3, characterized in that, The crosslinking agent is a crosslinking agent containing at least two epoxy groups; the volume-to-mass ratio of the crosslinking agent to the hyaluronic acid is 1.5–35 μL / g; the mass ratio of the hyaluronic acid to the alkaline aqueous solution is (0.16–0.4):1; and the mass percentage of alkaline substances in the alkaline aqueous solution is 0.8%–2%.

5. The storage method as described in claim 1, characterized in that, Includes the following steps: The cross-linked hyaluronic acid gel can be stored in glycerin in one go, or Glycerin was divided into several parts, and cross-linked hyaluronic acid gel was soaked in each part of glycerin in turn, and then stored in the last part of glycerin.

6. The storage method as described in claim 5, characterized in that, Includes the following steps: The glycerin was divided into three parts, and the cross-linked hyaluronic acid gel was placed into the first part of the glycerin for the first soaking. Remove the cross-linked hyaluronic acid gel and soak it a second time in the second part of glycerin. Remove the cross-linked hyaluronic acid gel and store it in the third part of glycerin.

7. The storage method as described in claim 6, characterized in that, The volume ratio of the first part of glycerol, the second part of glycerol and the third part of glycerol is (9-11):(3-6):

3.

8. The use of a cross-linked hyaluronic acid gel obtained by the storage method according to any one of claims 1 to 7 in the preparation of medical or cosmetic products.

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