Preparation method of butyl-glycine cross-linked sodium hyaluronate gel capable of improving process stability and injection composition of butyl-glycine cross-linked sodium hyaluronate gel

Abstract

The invention discloses a preparation method of a butyl-glycine cross-linked sodium hyaluronate gel for improving process stability and a composition for injection thereof, and aims to solve the technical problems of low product percent of pass and high production cost caused by difficult accurate control of reaction parameters and unstable cross-linking effect in the existing preparation process. Through optimal regulation and control of key parameters such as reaction system temperature, pH value, reaction time, sterilization time and the like, accurate control of cross-linking reaction is realized. According to the improved process, the stability of the production process and the product percent of pass are remarkably improved, the raw material loss and the production cost are effectively reduced, and meanwhile the consistency and reliability of core performance indexes such as viscoelasticity of the product are guaranteed. The process is stable and reliable, is suitable for large-scale production, and has important application value in the field of clinical medical materials for orthopedics and the like.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for preparing a butyric acid-crosslinked sodium hyaluronate gel with improved process stability and an injectable composition thereof. Background Technology

[0002] Hyaluronic acid is a key component of synovial fluid and is essential for maintaining normal joint function. It not only gives synovial fluid good viscoelasticity but also protects articular cartilage, maintaining its ideal cushioning properties. However, in patients with osteoarthritis, the hyaluronic acid in the synovial fluid shows significant abnormalities, including a reduced molecular weight, decreased concentration, and weakened viscoelasticity.

[0003] LG's marketed product, Hyruan ONE (a crosslinked sodium hyaluronate injection), is a medication used to treat osteoarthritis. It provides lubrication to the joints, reduces inflammation, and relieves pain. It is characterized by its long duration of action, maintaining its therapeutic effect in the body for up to 6 months.

[0004] Butylene glycol diglycidyl ether crosslinking sodium hyaluronate is a high-molecular biomaterial. Its molecular structure is modified by crosslinking with butylene glycol diglycidyl ether to form a three-dimensional network macromolecular polymer, which has excellent biocompatibility and viscoelasticity and has important application value in clinical fields such as orthopedics and ophthalmology.

[0005] However, the preparation process of butyric acid crosslinked sodium hyaluronate is significantly complex: the degree and uniformity of the crosslinking reaction directly determine the molecular weight distribution, mechanical properties, and bioactivity of the product, and even slight fluctuations in key parameters such as temperature, pH, reaction time, and sterilization time in the reaction system can significantly affect the crosslinking effect. Existing conventional preparation processes struggle to achieve precise and stable control of these parameters, leading to problems such as insufficient crosslinking, excessive crosslinking, or uneven crosslinking during production. This results in large fluctuations in product quality and a persistently high rate of non-conforming products. The generation of non-conforming products not only significantly increases the costs of raw material loss, rework, and waste disposal but also severely restricts the production line's capacity release and large-scale production efficiency.

[0006] Therefore, optimizing and improving the preparation process of butyric acid crosslinked sodium hyaluronate, breaking through the bottleneck of parameter control in the existing technology, and improving process stability and production qualification rate are of great significance for reducing production costs, ensuring product quality consistency, and meeting clinical application needs. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing butyric acid crosslinked sodium hyaluronate gel and its injectable composition with stable process and high yield.

[0008] The purpose of this invention is to provide a method for preparing cross-linked hyaluronic acid gel with improved process stability, comprising the following steps:

[0009] (1) Mixing: Sodium hyaluronate with a molecular weight of 200-400 million Da is mixed with an alkaline solution, and then 0.5-5% (preferably 0.5-3%, more preferably 0.5-2%) of the mass of sodium hyaluronate is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is (0.1-1):1, preferably (0.2-0.5):1.

[0010] (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 20℃-45℃ for 10-50h to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2mg / g; wherein, the alkaline solution is one of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution or ammonia water;

[0011] (3) Washing: Prepare sodium phosphate chloride physiological buffer solution and add hydrochloric acid solution in equal volume to alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to sodium phosphate chloride physiological buffer solution and stir for 4-12 hours. Then discard the supernatant and wash repeatedly with sodium phosphate chloride physiological buffer solution until the pH is 6-10.

[0012] (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 6-20 hours to obtain a gel mixture;

[0013] (5) Crushing: The gel mixture obtained from swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles;

[0014] (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121℃ for 10-30 min; thus, cross-linked sodium hyaluronate gel is obtained.

[0015] Another object of the present invention is to provide a method for preparing a cross-linked hyaluronic acid gel injection composition with improved process stability, comprising the following steps:

[0016] (1) Mixing: Sodium hyaluronate with a molecular weight of 2,000,000 to 4,000,000 Da is mixed with an alkaline solution, and then 0.5-5% (preferably 0.5-3%, more preferably 0.5-2%) of a crosslinking agent by mass of sodium hyaluronate is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is (0.1-1):1, preferably (0.2-0.5):1;

[0017] (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 20℃-45℃ for 10-50h to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2mg / g; wherein, the alkaline solution is one of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution or ammonia water;

[0018] (3) Washing: Prepare sodium phosphate chloride physiological buffer solution and add hydrochloric acid solution in equal volume to alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to sodium phosphate chloride physiological buffer solution and stir for 4-12 hours. Then discard the supernatant and wash repeatedly with sodium phosphate chloride physiological buffer solution until the pH is 6-10.

[0019] (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 6-20 hours to obtain a gel mixture;

[0020] (5) Crushing: The gel mixture obtained from swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles;

[0021] (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121℃ for 10-30 minutes to obtain cross-linked sodium hyaluronate gel.

[0022] (7) Filling: The cross-linked sodium hyaluronate gel particles sterilized in step (6) are filled into a pre-filled syringe and then subjected to moist heat sterilization at 121°C for 10-60 min to obtain a cross-linked sodium hyaluronate gel injection composition.

[0023] Further, in step (2), the crosslinking agent is any one of 1,4-butanediol diglycidyl ether, glycerol trioxypropyl ether, resorcinol diglycidyl ether, allyl glycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether or neopentyl glycol diglycidyl ether.

[0024] Furthermore, the crosslinking agent is preferably 1,4-butanediol diglycidyl ether.

[0025] Further, in step (3), the sodium chloride phosphate physiological buffer solution includes 6-12 g / L sodium chloride, 0.01-0.1 g / L potassium dihydrogen phosphate or sodium dihydrogen phosphate, 0.1-0.5 g / L disodium hydrogen phosphate or potassium dihydrogen phosphate, and the remainder is water for injection.

[0026] Furthermore, in step (5), the D50 particle size distribution range of the cross-linked sodium hyaluronate gel particles is 440-1000 μm, specifically including 440-650 μm, 600-850 μm, and 800-1000 μm.

[0027] The cross-linked hyaluronic acid gel prepared by this invention has the following characteristics: 1) The hyaluronic acid gel should be a colorless, clear, viscous liquid; 2) Its hyaluronic acid content is 1.5%–2.5% (w / w); 3) Its pH range is 6.5–8.5; 4) It has a storage modulus of 200–800 Pa and a loss modulus of 40–160 Pa; 5) It has a complex viscosity of 10,000–100,000 cp; 6) Its residual cross-linking agent should be less than 0.0002%; 7) Its heavy metal content should be less than 20 ppm; 8) Its osmolar concentration is 250–350 mOsmol / kg; 9) Its bacterial endotoxin content should be less than 0.5E. 10) It has a viscosity of 5000–30000 mPa·s; 11) Its Tanδ value is 0.05–0.5; 12) Its free sodium hyaluronate content should be less than 1.0% (w / w); 13) Its adhesion is 0.1–5 N; 14) Its critical strain is 0.1%–20%; 15) Its cohesive force (upward pull) is 0.1–3 N; 16) Its pulling capacity is 5000 Pa·gf–600000 Pa·gf; 17) Its cohesive force (compressive force) is greater than 10 gf; 18) Its degree of crosslinking is 0.1%–10%; 19) Its crosslinking ratio is 0.05–0.5; 20) Its degree of modification is 0.5%–5%.

[0028] The cross-linked hyaluronic acid gel injection composition prepared by this invention has the following characteristics: 1) the injection composition containing cross-linked hyaluronic acid gel should be a colorless, clear, viscous liquid; 2) its hyaluronic acid content is 75.0%–125.0%; 3) its pH range is 6.0–8.0; 4) it has a storage modulus of 80–400 Pa and a loss modulus of 10–80 Pa; 5) it has a complex viscosity of 5000–70000 cp; 6) its residual cross-linking agent should be less than 0.0002%; 7) its heavy metal content should be less than 20 ppm; 8) its osmolar concentration is 250–350 mOsmol / kg; 9) its bacterial endotoxin content should be less than 0.5 EU. / ml; 10) It has a viscosity of 5000-30000 mPa·s; 11) Its Tanδ value is 0.05-1.0; 12) Its free sodium hyaluronate content should be less than 50%; 13) Its adhesion is 0.1-5 N; 14) Its critical strain is 0.1%-20%; 15) Its cohesive force (uplift) is 0.1-3 N; 16) Its lifting capacity is 5000 Pa·gf-600000 Pa·gf; 17) Its cohesive force (compression force) is greater than 10 gf; 18) Its degree of crosslinking is 0.1%-10%; 19) Its crosslinking ratio is 0.05-0.5; 20) Its degree of modification is 0.5%-5%; 21) It has an injection force of 1-60 N.

[0029] The butyric acid crosslinked sodium hyaluronate gel and its injectable composition of the present invention are used to relieve osteoarthritis. Further, the injectable composition or the crosslinked sodium hyaluronate gel injection is administered via intra-articular injection. More specifically, the osteoarthritis refers to the knee joint.

[0030] Compared with existing technologies, the beneficial effects of this application are as follows: By optimizing the preparation process parameters of butyric acid-crosslinked sodium hyaluronate, this invention achieves precise control over the crosslinking reaction process, effectively solving the technical pain point of large product quality fluctuations in conventional processes. The improved process significantly enhances the stability of the production process, greatly reduces the product defect rate, reduces raw material loss and rework costs, and improves the efficiency of large-scale production. Simultaneously, it ensures the consistency and reliability of key performance indicators such as molecular weight distribution and viscoelasticity, meeting the stringent quality requirements of clinical applications, and possesses significant economic value and application prospects. Detailed Implementation

[0031] The present application is further illustrated below with specific embodiments. It should be understood that the embodiments of the present application are merely illustrative and not intended to limit the scope of the application. Experimental methods in the following embodiments without specified conditions are generally performed under conventional conditions or as recommended by the manufacturer. Raw materials and excipients in the following embodiments without specified sources are generally commercially available conventional products. Any simple improvements to the present application or equivalent substitutions of conventional methods or components based on the technical solutions of the present application are within the scope of protection of this application.

[0032] Example 1

[0033] A method for preparing a cross-linked hyaluronic acid gel injection composition with improved process stability includes the following steps:

[0034] (1) Mixing: Sodium hyaluronate with a molecular weight of 2 million Da is mixed with an alkaline solution, and then 0.5% of the mass of sodium hyaluronate crosslinking agent is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is 0.2:1;

[0035] (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 20°C for 50 h to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2 mg / g; wherein the alkaline solution is an aqueous solution of sodium hydroxide; wherein the crosslinking agent is 1,4-butanediol diglycidyl ether.

[0036] (3) Washing: Prepare sodium chloride phosphate physiological buffer and add hydrochloric acid solution in equal volume to alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to sodium chloride phosphate physiological buffer and stir for 6 hours. Then discard the supernatant and wash repeatedly with sodium chloride phosphate physiological buffer until the pH is 6. The sodium chloride phosphate physiological buffer includes 6 g / L sodium chloride, 0.01 g / L potassium dihydrogen phosphate or sodium dihydrogen phosphate, 0.1 g / L disodium hydrogen phosphate or potassium dihydrogen phosphate, and the remainder is water for injection.

[0037] (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 9 hours to obtain a gel mixture;

[0038] (5) Crushing: The gel mixture obtained by swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles; wherein the D50 particle size distribution range of the cross-linked sodium hyaluronate gel particles is 440-650μm.

[0039] (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121°C for 10 min; thus, cross-linked sodium hyaluronate gel is obtained.

[0040] (7) Filling: The cross-linked sodium hyaluronate gel particles sterilized in step (6) are filled into a pre-filled syringe and then subjected to moist heat sterilization at 121°C for 60 min to obtain a cross-linked sodium hyaluronate gel injection composition.

[0041] Example 2

[0042] A method for preparing a cross-linked hyaluronic acid gel injection composition with improved process stability includes the following steps:

[0043] (1) Mixing: Sodium hyaluronate with a molecular weight of 3 million Da is mixed with an alkaline solution, and then 1% of the mass of sodium hyaluronate crosslinking agent is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is 0.35:1;

[0044] (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 35°C for 30 h to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2 mg / g; wherein the alkaline solution is an aqueous solution of sodium hydroxide; wherein the crosslinking agent is 1,4-butanediol diglycidyl ether.

[0045] (3) Washing: Prepare a sodium chloride phosphate physiological buffer solution and add an equal volume of hydrochloric acid solution to the alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to the sodium chloride phosphate physiological buffer solution and stir for 8 hours. Then discard the supernatant and wash repeatedly with sodium chloride phosphate physiological buffer solution until the pH is 7.5. The sodium chloride phosphate physiological buffer solution includes 9 g / L sodium chloride, 0.05 g / L potassium dihydrogen phosphate or sodium dihydrogen phosphate, 0.3 g / L disodium hydrogen phosphate or potassium dihydrogen phosphate, and the remainder is water for injection.

[0046] (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 12 hours to obtain a gel mixture;

[0047] (5) Crushing: The gel mixture obtained by swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles; wherein the D50 particle size distribution range of the cross-linked sodium hyaluronate gel particles is 600-850μm.

[0048] (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121°C for 20 min; thus, cross-linked sodium hyaluronate gel is obtained.

[0049] (7) Filling: The cross-linked sodium hyaluronate gel particles sterilized in step (6) are filled into a pre-filled syringe and then subjected to moist heat sterilization at 121°C for 35 min to obtain a cross-linked sodium hyaluronate gel injection composition.

[0050] Example 3

[0051] A method for preparing a cross-linked hyaluronic acid gel injection composition with improved process stability includes the following steps:

[0052] (1) Mixing: Sodium hyaluronate with a molecular weight of 4 million Da is mixed with an alkaline solution, and then 5% of the mass of sodium hyaluronate crosslinking agent is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is 1:1;

[0053] (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 45°C for 10 h to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2 mg / g; wherein the alkaline solution is an aqueous solution of sodium hydroxide; wherein the crosslinking agent is 1,4-butanediol diglycidyl ether.

[0054] (3) Washing: Prepare a sodium chloride phosphate physiological buffer solution and add an equal volume of hydrochloric acid solution to the alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to the sodium chloride phosphate physiological buffer solution and stir for 12 hours. Then discard the supernatant and wash repeatedly with sodium chloride phosphate physiological buffer solution until the pH is 10. The sodium chloride phosphate physiological buffer solution includes 12 g / L sodium chloride, 0.1 g / L potassium dihydrogen phosphate or sodium dihydrogen phosphate, 0.5 g / L disodium hydrogen phosphate or potassium dihydrogen phosphate, and the remainder is water for injection.

[0055] (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 20 hours to obtain a gel mixture;

[0056] (5) Crushing: The gel mixture obtained by swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles; wherein the D50 particle size distribution range of the cross-linked sodium hyaluronate gel particles is 800-1000μm.

[0057] (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121°C for 30 min; thus, cross-linked sodium hyaluronate gel is obtained.

[0058] (7) Filling: The cross-linked sodium hyaluronate gel particles sterilized in step (6) are filled into a pre-filled syringe and then subjected to moist heat sterilization at 121°C for 10 min to obtain a cross-linked sodium hyaluronate gel injection composition.

[0059] Comparative Example 1

[0060] A method for preparing a cross-linked hyaluronic acid gel injection composition with improved process stability includes the following steps:

[0061] (1) Mixing: Sodium hyaluronate with a molecular weight of 3 million Da is mixed with an alkaline solution, and then 0.3% of the mass of sodium hyaluronate crosslinking agent is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is 0.08:1;

[0062] (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 10°C for 60 h to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2 mg / g; wherein the alkaline solution is an aqueous solution of sodium hydroxide; wherein the crosslinking agent is 1,4-butanediol diglycidyl ether.

[0063] (3) Washing: Prepare a sodium chloride phosphate physiological buffer solution and add an equal volume of hydrochloric acid solution to the alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to the sodium chloride phosphate physiological buffer solution and stir for 2 hours. Then discard the supernatant and wash repeatedly with sodium chloride phosphate physiological buffer solution until the pH is 5. The sodium chloride phosphate physiological buffer solution includes 10 g / L sodium chloride, 0.05 g / L potassium dihydrogen phosphate or sodium dihydrogen phosphate, 0.3 g / L disodium hydrogen phosphate or potassium dihydrogen phosphate, and the remainder is water for injection.

[0064] (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 5 hours to obtain a gel mixture;

[0065] (5) Crushing: The gel mixture obtained by swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles; wherein the D50 particle size distribution range of the cross-linked sodium hyaluronate gel particles is 300-400μm.

[0066] (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121°C for 5 min; thus, cross-linked sodium hyaluronate gel is obtained.

[0067] (7) Filling: The cross-linked sodium hyaluronate gel particles sterilized in step (6) are filled into a pre-filled syringe and then subjected to moist heat sterilization at 121°C for 70 min to obtain a cross-linked sodium hyaluronate gel injection composition.

[0068] Comparative Example 2

[0069] A method for preparing a cross-linked hyaluronic acid gel injection composition with improved process stability includes the following steps:

[0070] (1) Mixing: Sodium hyaluronate with a molecular weight of 3 million Da is mixed with an alkaline solution, and then 6% of the mass of sodium hyaluronate crosslinking agent is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is 1.5:1;

[0071] (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 50°C for 8 hours to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2mg / g; wherein the alkaline solution is an aqueous solution of sodium hydroxide; wherein the crosslinking agent is 1,4-butanediol diglycidyl ether.

[0072] (3) Washing: Prepare a sodium chloride phosphate physiological buffer solution and add an equal volume of hydrochloric acid solution to the alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to the sodium chloride phosphate physiological buffer solution and stir for 15 hours. Then discard the supernatant and wash repeatedly with sodium chloride phosphate physiological buffer solution until the pH is 10.5. The sodium chloride phosphate physiological buffer solution includes 10 g / L sodium chloride, 0.05 g / L potassium dihydrogen phosphate or sodium dihydrogen phosphate, 0.3 g / L disodium hydrogen phosphate or potassium dihydrogen phosphate, and the remainder is water for injection.

[0073] (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 25 hours to obtain a gel mixture;

[0074] (5) Crushing: The gel mixture obtained by swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles; wherein the D50 particle size distribution range of the cross-linked sodium hyaluronate gel particles is 1050-1500μm.

[0075] (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121℃ for 40 min; thus, cross-linked sodium hyaluronate gel is obtained.

[0076] (7) Filling: The cross-linked sodium hyaluronate gel particles sterilized in step (6) are filled into a pre-filled syringe and then subjected to moist heat sterilization at 121°C for 8 minutes to obtain a cross-linked sodium hyaluronate gel injection composition.

[0077] Comparative Example 3

[0078] A hyaluronic acid gel filler was prepared according to Example 1 of patent CN113164652B.

[0079] Comparative Example 4

[0080] Injectable crosslinked hyaluronic acid gel was prepared according to Example 5 of patent CN103834053B, except that the molecular weight of hyaluronic acid in Comparative Example 4 was 3 million Da.

[0081] Experimental Example

[0082] 1. Sample Sampling: Samples were taken from the raw materials (hyaluronic acid gel) and formulations (hyaluronic acid gel injection composition or filler) prepared by the methods of Examples 1-3 and Comparative Examples 1-4, respectively, as experimental samples. Three production batches were set up for each group, and 50 samples were randomly selected from each batch. All samples were tested for all indicators according to the detection methods specified in Table 2-3. The specific sampling method is shown in Table 1.

[0083] Table 1

[0084]

[0085] 2. Testing Indicators: The raw materials (hyaluronic acid gel) and formulations (hyaluronic acid gel injection compositions or fillers) prepared by the methods in Examples 1-3 and Comparative Examples 1-4 were tested according to the testing methods in Table 2-3, and the relevant test results were recorded. A single sample was deemed qualified if all test indicators met the acceptable standards in Table 2-3; if any indicator failed to meet the standards, it was deemed unqualified.

[0086] Table 2. Determination methods and standards for raw materials

[0087]

[0088]

[0089] Table 3. Determination methods and standards for the formulation

[0090]

[0091]

[0092] 3. Experimental Results: The raw material test results are shown in Table 4, and the formulation test results are shown in Table 5. As can be seen from Tables 4-5, the pass rates of both the raw materials and formulations of the cross-linked hyaluronic acid gel prepared using the methods described in Examples 1-3 of this invention are ≥90%, which is significantly higher than the pass rates of Comparative Examples 1-4.

[0093] Table 4 Raw material testing results

[0094]

[0095] Table 5. Results of Formulation Testing

[0096]

Claims

1. A method for preparing cross-linked hyaluronic acid gel with improved process stability, characterized in that, Includes the following steps: (1) Mixing: Sodium hyaluronate with a molecular weight of 200-400 million Da is mixed with an alkaline solution, and then 0.5-5% of the mass of sodium hyaluronate crosslinking agent is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is (0.1-1):1; (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 20℃-45℃ for 10-50h to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2mg / g; wherein the alkaline solution is one of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution or ammonia water; (3) Washing: Prepare sodium phosphate chloride physiological buffer solution and add hydrochloric acid solution in equal volume to alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to sodium phosphate chloride physiological buffer solution and stir for 4-12 hours. Then discard the supernatant and wash repeatedly with sodium phosphate chloride physiological buffer solution until the pH is 6-10. (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 6-20 hours to obtain a gel mixture; (5) Crushing: The gel mixture obtained by swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles; (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121℃ for 10-30 min; thus, cross-linked sodium hyaluronate gel is obtained.

2. A method for preparing a cross-linked hyaluronic acid gel injection composition with improved process stability, characterized in that, Includes the following steps: (1) Mixing: Sodium hyaluronate with a molecular weight of 200-400 million Da is mixed with an alkaline solution, and then 0.5-5% of the mass of sodium hyaluronate crosslinking agent is added and mixed evenly with the sodium hyaluronate solution; wherein, the mass ratio of sodium hyaluronate to alkaline solution is (0.1-1):1; (2) Crosslinking treatment: The mixture obtained in step (1) is subjected to crosslinking treatment at 20℃-45℃ for 10-50h to prepare crosslinked sodium hyaluronate gel; the remaining amount of crosslinking agent is <0.2mg / g; wherein the alkaline solution is one of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution or ammonia water; (3) Washing: Prepare sodium phosphate chloride physiological buffer solution and add hydrochloric acid solution in equal volume to alkaline solution. Add the cross-linked sodium hyaluronate gel obtained in step (2) to sodium phosphate chloride physiological buffer solution and stir for 4-12 hours. Then discard the supernatant and wash repeatedly with sodium phosphate chloride physiological buffer solution until the pH is 6-10. (4) Swelling: The cross-linked sodium hyaluronate gel washed in step (3) was allowed to swell for 6-20 hours to obtain a gel mixture; (5) Crushing: The gel mixture obtained by swelling in step (4) is crushed and sieved to obtain cross-linked sodium hyaluronate gel particles; (6) Sterilization: After the cross-linked sodium hyaluronate gel particles pulverized in step (5) are stirred and homogenized, they are subjected to moist heat sterilization at 121℃ for 10-30 minutes to obtain cross-linked sodium hyaluronate gel. (7) Filling: The cross-linked sodium hyaluronate gel particles sterilized in step (6) are filled into a pre-filled syringe and then subjected to moist heat sterilization at 121°C for 10-60 min to obtain a cross-linked sodium hyaluronate gel injection composition.

3. The method according to claim 1 or 2, characterized in that, The crosslinking agent mentioned in step (2) is any one of 1,4-butanediol diglycidyl ether, glycerol trioxypropyl ether, resorcinol diglycidyl ether, allyl glycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether or neopentyl glycol diglycidyl ether.

4. The preparation method according to claim 3, characterized in that, The crosslinking agent is preferably 1,4-butanediol diglycidyl ether.

5. The method according to claim 1 or 2, characterized in that, The sodium chloride phosphate physiological buffer solution mentioned in step (3) includes 6-12 g / L sodium chloride, 0.01-0.1 g / L potassium dihydrogen phosphate or sodium dihydrogen phosphate, 0.1-0.5 g / L disodium hydrogen phosphate or potassium dihydrogen phosphate, and the remainder is water for injection.

6. The method according to claim 1 or 2, characterized in that, The D50 particle size distribution range of the cross-linked sodium hyaluronate gel particles in step (5) is 440-1000 μm.

7. The cross-linked hyaluronic acid gel prepared by the method according to any one of claims 1-6, having the following characteristics: 1) the hyaluronic acid gel should be a colorless, clear, viscous liquid; 2) its hyaluronic acid content is 1.5% to 2.5% (w / w); 3) its pH range is 6.5 to 8.5; 4) it has a storage modulus of 200 to 800 Pa and a loss modulus of 40 to 160 Pa; 5) it has a complex viscosity of 10,000 to 100,000 cp; 6) its residual cross-linking agent should be less than 0.0002%; 7) its heavy metal content should be less than 20 ppm; 8) it has an osmolar concentration of 250 to 350 mOsmol / kg; 9) its bacterial endotoxin content is... 10) Its viscosity is 5000–30000 mPa·s; 11) Its Tanδ value is 0.05–0.5; 12) Its free sodium hyaluronate content is less than 1.0% (w / w); 13) Its adhesion is 0.1–5 N; 14) Its critical strain is 0.1%–20%; 15) Its cohesive force (upward pull) is 0.1–3 N; 16) Its pulling capacity is 5000 Pa·gf–600000 Pa·gf; 17) Its cohesive force (compressive force) is greater than 10 gf; 18) Its degree of crosslinking is 0.1%–10%; 19) Its crosslinking ratio is 0.05–0.5; 20) Its degree of modification is 0.5%–5%.

8. The cross-linked hyaluronic acid gel injection composition prepared by the method according to any one of claims 1-6, having the following characteristics: 1) the injection composition containing cross-linked hyaluronic acid gel shall be a colorless, clear, viscous liquid; 2) its hyaluronic acid content is 75.0% to 125.0%; 3) its pH value range is 6.0 to 8.0; 4) it has a storage modulus of 80 to 400 Pa and a loss modulus of 10 to 80 Pa; 5) it has a complex viscosity of 5000 to 70000 cp; 6) its residual cross-linking agent content shall be less than 0.0002%; 7) its heavy metal content shall be less than 20 ppm; 8) it has an osmolar concentration of 250 to 350 mOsmol / kg; 9) its bacterial endotoxin content is... It should be less than 0.5 EU / ml; 10) It has a viscosity of 5000-30000 mPa·s; 11) Its Tanδ value is 0.05-1.0; 12) Its free sodium hyaluronate content should be less than 50%; 13) Its adhesion is 0.1-5 N; 14) Its critical strain is 0.1%-20%; 15) Its cohesive force (uplift) is 0.1-3 N; 16) Its lifting capacity is 5000 Pa·gf-600000 Pa·gf; 17) Its cohesive force (compression force) is greater than 10 gf; 18) Its degree of crosslinking is 0.1%-10%; 19) Its crosslinking ratio is 0.05-0.5; 20) Its degree of modification is 0.5%-5%; 21) It has an injection force of 1-60 N.

9. The use of a cross-linked hyaluronic acid gel or injectable composition as described in any one of claims 1-8 in relieving osteoarthritis.

10. The application as described in claim 9, characterized in that, The cross-linked hyaluronic acid gel injection composition is administered via intra-articular injection.

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