CH718108A2 - Method for preparing a monophasic cross-linked sodium hyaluronate gel. - Google Patents

Abstract

Disclosed is a method for preparing a monophasic cross-linked sodium hyaluronate gel, which belongs to the field of drug preparation technology. Said method includes the following steps: preparing a mixed solution of NaOH containing a crosslinking agent, preparing a solution of sodium hyaluronate, adding the mixed solution of NaOH to the solution of sodium hyaluronate for mixing homogeneous and then manufacture of a sodium hyaluronate gel in a water bath at constant temperature. Sodium hyaluronate gel is cut into small pieces, add PBS solution of hydrochloric acid and place in PBS solution for dialysis to obtain cross-linked phase sodium hyaluronate. The cross-linked phase sodium hyaluronate solution and the non-cross-linked phase sodium hyaluronate solution are mixed, degassed, filled and sterilized. The preparation process that is proposed has the merit not only of effectively reducing human intervention during the production process and reducing the risk of contamination on contact, but also avoids the generation of by-products and effectively reduces the bubbles of air generated during filling, thus providing a high quality sodium hyaluronate gel.

Description

Technical area

The present invention relates to the technical field of the preparation of medicaments, and, more specifically, is a method for preparing a cross-linked sodium hyaluronate gel with a monophasic phase.

State of the prior art

[0002] Hyaluronic acid is an important component of human and animal skin, of the vitreous body, of the lubricating fluid of the joints and of the cartilage tissue. 11 consists of (1-β-4)D-glucuronic acid and (1-β-3)N-acetyl-D-aminoglucose disaccharide units repeatedly linked and is widely used in reconstructive surgery, surgical ocular or as a cosmetic product to fill wrinkles. Hyaluronic acid has good physico-chemical properties and good biocompatibility. However, it is rapidly degraded in the body by the enzymatic action of hyaluronidase and has a short retention time, requiring repeated injections to ensure efficacy. Cross-linked hyaluronic acid is a polymer gel obtained by cross-linking hyaluronic acid with cross-linking agents, which can compensate for the short retention time of hyaluronic acid while having good biocompatibility and efficacy.

[0003] Currently, the current preparation process for sodium hyaluronate generally comprises the following steps: crosslinking reaction - swelling - dialysis - filling - sterilization. This process has a number of drawbacks. The cross-linking phase easily produces by-products and the filling phase allows bubbles to appear, which can easily affect the nature of the gel and its quality, thus seriously damaging the quality of the product.

Disclosure of Invention

Aiming to overcome the shortcomings of the current technique, the present invention aims to provide a method for preparing a monophasic cross-linked sodium hyaluronate gel, which can not only effectively reduce human intervention in the process of production and reduce the risk of contamination, but also avoid the generation of by-products and air bubbles during filling, thus guaranteeing the quality of the sodium hyaluronate gel.

In order to improve the existing technique, the technical solution used in the present invention is as follows:

[0006] S1. The cross-linking agent and NaOH solution are put into the centrifuge tube at a mass ratio of 1:8-1:10, and nitrogen gas is added thereto and mixed well in the centrifuge to obtain a mixed solution of NaOH containing the crosslinking agent and set aside;

[0007] S2. Sodium hyaluronate and NaOH solution of pH > 13 are mixed and dissolved under stirring at a mass ratio of 1:6-1:8 and placed in a three-dimensional mixer for 1~2h to obtain hyaluronate solution sodium.

[0008] S3. Add all the NaOH mixture made in step S1 to the sodium hyaluronate solution, mix well and pass nitrogen gas, then thermostat the water bath at 45-55 for 2-3h to obtain a gel sodium hyaluronate.

[0009] S4. sodium hyaluronate gel was cut into small pieces, added to a PBS solution of hydrochloric acid, mixed in a three-dimensional blender for 24-36 h, stirred well to obtain a crumble paste, packed in bags of dialysis and placed in a solution of pure PBS flowing at 2-8 for 30-36 h to obtain sodium hyaluronate in the cross-linked phase.

[0010] S5. The cross-linked phase sodium hyaluronate solution and non-cross-linked phase sodium hyaluronate solution are mixed well at a mass ratio of 8:1-9:1, degassed, filled and sterilized.

The sodium hyaluronate used in step S2 has a molecular weight of 2.1 to 2.7 MDa, a major specificity of the present invention.

Another major feat of the present invention, the molecular weight of the sodium hyaluronate used in said solution of sodium hyaluronate in non-crosslinked phase is 3.0 MDa.

Still specific to the present invention, said PBS hydrochloric acid solution is composed of hydrochloric acid and PBS in which the amount of hydrochloric acid added is calculated by the amount and concentration of sodium hydroxide added to obtain the theoretical quantity required to have neutralization; and PBS is prepared from the hydrates of di-sodium hydrogen phosphate, sodium hydrogen phosphate and sodium chloride.

Also specific to the present invention, said crosslinking agent is 1,4-butanediol diglycidyl ether.

[0015] As a flagship embodiment of the present invention, the specific steps of step S2 are as follows: the mixture of sodium hyaluronate with an NaOH solution of pH > 13 in a mass ratio of 1:6- 1:8 in a polytetrafluoroethylene beaker with a lid is stirred for dissolution, keeping the beaker lid closed, and placed in a three-dimensional mixer for 1~2h to obtain a sodium hyaluronate solution.

[0016] As a key embodiment of the present invention, step S4 replaces the PBS solution 5-6 times during the dialysis process.

[0017] Specificity of the present invention, in step S5: use a stainless steel degassing tank for degassing, connect the stainless steel degassing tank and the filling machine by a silicone pipe, fill the gel in the degassing tank by the filling machine into a glass syringe using vacuum, and put it into a steam-air sterilization cabinet for sterilization using rapid rise and fall of temperature.

Compared to existing technology, the present invention has two major advantages: On the one hand, the preparation method provided by the present invention takes less than 1 hour for the entire manual operation step, which can effectively reduce human intervention in the production process and decrease the risk of contact contamination. On the other hand, the decyclization of the crosslinking BDDE is protected by the passage of nitrogen gas to avoid by-products in the crosslinking process, and the degassing tank and the filling machine are connected by silicone pipes to ensure that no air bubbles are generated during filling. The quality of sodium hyaluronate gel is effectively ensured by using a temperature rise and fall method for terminal sterilization and pressure compensation during sterilization to prevent the displacement of the gel plug and to reduce the effect of the sterilization process on the properties of the gel. In summary, the preparation method of the present invention has few process steps, is easy to operate, has a short cycle time, can effectively improve the quality of the gel product, and is suitable for large-scale industrial production.

Drawings

Figure 1 shows a comparison of the modulus of elasticity of embodiment 1 of the present invention with existing products.

Specific implementation

The present invention is described in greater detail below in connection with specific embodiments.

[0021] The method of preparing a monophasic cross-linked sodium hyaluronate gel comprising the following steps: Adding the BDDE cross-linking agent and the NaOH solution to a centrifuge tube at a mass ratio of 1:8 -1:10, pass nitrogen through it and mix well in a centrifuge to obtain a mixed NaOH solution containing the BDDE crosslinker, and set aside.

[0022] S2, Mixture of sodium hyaluronate with a solution of NaOH of pH > 13 in a mass ratio of 1:6-1:8 in a polytetrafluoroethylene beaker with a lid, and stir to dissolve, keeping the lid on closed beaker and placed in a three-dimensional mixer for 1~2h to obtain a solution of sodium hyaluronate. In said solution, the molecular weight of sodium hyaluronate is 2.1-2.7 MDa.

[0023] S3, Add all the NaOH mixture made in step S1 to the sodium hyaluronate solution, mix well and pass nitrogen through so that the air in the beaker is completely replaced with nitrogen and cover the beaker with a constant temperature water bath at 45-55 for 2-3h to obtain the sodium hyaluronate gel.

[0024] S4, Sodium hyaluronate gel was cut into small pieces, add to a PBS solution of hydrochloric acid, mix in a three-dimensional blender for 24-36h, shake well to obtain a crumble paste, pack into dialysis bags and place in 2-8 fluid pure PBS solution for 30-36h, and the PBS solution should be replaced 5-6 times during dialysis to obtain cross-linked phase sodium hyaluronate in which the solution of PBS of hydrochloric acid is composed of hydrochloric acid and PBS, the amount of hydrochloric acid added being calculated from the amount and concentration of sodium hydroxide added to obtain the theoretical amount necessary for neutralization, i.e. equal amounts of hydrochloric acid and sodium hydroxide substance; PBS is prepared from hydrated disodium hydrogen phosphate, hydrated sodium dihydrogen phosphate and sodium chloride. Preferably, the composition of the PBS is as follows: Na2HPO4•12H2O 0.555 g, NaH2PO4•H2O 0.039 g, NaCl 9 g with water up to 1000 mL.

[0025] S5, The cross-linked phase sodium hyaluronate solution and the non-cross-linked phase sodium hyaluronate solution with a molecular weight of 3.0 MDa were mixed in a mass ratio of 8:1- 9:1, degassed using a stainless steel degassing tank, connected to the stainless steel degassing tank and the filling machine by a silicone pipe, and the gel in the degassing tank was filled into a glass syringe by the vacuum filling machine, and placed in a steam-air sterilization cabinet using a rapid elevator. Sterilization is carried out in a steam-air sterilization cabinet with a rapid rise and fall in temperature.

Embodiment 2 of the invention:

[0026] The method of preparing a single-phase cross-linked sodium hyaluronate gel comprising the following steps:

[0027] S1. 700 μL of BDDE crosslinking agent and 5 g of NaOH solution were added to a centrifuge tube, passed through nitrogen and mixed well in a centrifuge to obtain a mixed solution of NaOH containing the agent of BDDE crosslinking, and set aside.

[0028] S2. Dissolve 10 g of powdered sodium hyaluronate with a molecular weight of 2.1 MDa in a PTFE beaker in 90 g of 0.3 mol/L sodium hydroxide solution, then stir and dissolve on a three-dimensional mixer at 70 rpm .

[0029] S3. Dissolve with sufficient stirring then add the NaOH mixture, stir well and squirt nitrogen into the beaker and immediately cover the beaker and transfer to a constant temperature water bath at 45 for 3 hours to obtain a sodium hyaluronate gel .

[0030] S4. The gel was cut into 0.5×0.5cm pieces and added to 9g of 3mol/L hydrochloric acid and 160g of PBS solution and placed on a 3D mixer at 70rpm for 6 hours, after which the gel was finely mixed with 80 mesh sieve and put into 8000Da-12000Da dialysis bags and put into fluid PBS solution at 20°C for dialysis. And the dialysate is changed and weighed every 6 hours until the gel mass reaches about 500g and the dialysis is completed.

[0031] S5. Add 50g of 20mg/ml PBS sodium hyaluronate solution and mix well; mix and fill in 60ml disposable plastic syringes with 2/3 of syringe volume and place in degassing device for 5 minutes, then fill in 1ml syringes and place in autoclave for 3min at 129°C to obtain sample A.

Embodiment 1 of the invention:

[0032] The method of preparation of a single-phase cross-linked sodium hyaluronate gel comprising the following steps:

[0033] S1. Add 70µL of BDDE crosslinking agent and 5g of NaOH solution into the centrifuge tube, add nitrogen to it and mix well in a centrifuge to obtain a mixed NaOH solution containing the BDDE crosslinking agent, and we put aside.

[0034] S2. Dissolve 10g of sodium hyaluronate powder with a molecular weight of 2.5 MDa in a PTFE beaker in 90g of 0.3 mol/L sodium hydroxide solution, then stir and dissolve on a three-dimensional mixer at 70 rpm.

[0035] S3. Dissolve with sufficient stirring then add the NaOH mixed solution, stir well and pass nitrogen through the beaker and immediately cover the beaker and transfer to a constant temperature water bath at 50°C for 2 hours to obtain a gel of sodium hyaluronate.

[0036] S4. Cut the gel into 0.5×0.5cm pieces and add to 9g of 3mol/L hydrochloric acid and 160g of PBS solution and place on a 3D mixer at 70rpm for 6 hours, after which the gel is finely mixed with a 80 mesh sieve and filled into 8000Da-12000Da dialysis bags and placed in common PBS solution at 20°C for dialysis, and the dialysate is changed and weighed every 6 hours until the gel mass reaches approximately 500 g and the dialysis is complete.

[0037] S5. Add 50g of 20mg/ml PBS sodium hyaluronate solution and stir well; mix and fill in 60ml disposable plastic syringes with 2/3 of syringe volume and place in degassing device for 5 minutes, then fill in 1ml syringes and place in autoclave for 3min at 129 to obtain l sample B.

Embodiment 3 of the invention:

The method of preparing a monophasic phase cross-linked sodium hyaluronate gel comprising the following steps:

[0039] S1. Mix 750µL of BDDE crosslinking agent with 5g of NaOH solution in a centrifuge tube and pass nitrogen gas through it, then place it in a centrifuge to obtain a mixture of NaOH containing BDDE crosslinking agent and put aside.

[0040] S2. Dissolve 10g of sodium hyaluronate powder with a molecular weight of 2.5 MDa in a PTFE beaker in 90g of 0.3 mol/L sodium hydroxide solution, then stir and dissolve on a three-dimensional mixer at 70 rpm.

[0041] S3. Dissolve with sufficient stirring then add the NaOH mixed solution, stir well and pass nitrogen through the beaker and immediately cover the beaker and transfer to a constant temperature water bath at 50°C for 2 hours to obtain a gel of sodium hyaluronate.

[0042] S4. Cut the gel into 0.5×0.5cm pieces and add to 9g of 3mol/L hydrochloric acid and 160g of PBS solution and place on a 3D mixer at 70rpm for 6 hours, after which the gel was finely mixed with a 80 mesh sieve and filled into 8000Da-12000Da dialysis bags and placed in a fluid PBS solution at 20°C for dialysis, and the dialysate is changed and weighed every 6 hours until the mass of gel reaches approximately 500 g and the dialysis is complete.

[0043] S5. Add 50g of 20mg/ml PBS sodium hyaluronate solution and stir well; mix and fill in 60ml disposable plastic syringes with 2/3 of syringe volume and place in degassing device for 5 minutes, then fill in 1ml syringes and place in autoclave for 3min at 129 to obtain l sample C.

Performance Comparison Experience:

Comparison of degree of swelling and modulus of elasticity

[0044] The swelling and the modulus of elasticity were determined for the samples obtained in the embodiments 1 to 3, respectively, and the results are presented in Table 1.

Table 1: Results of dissolution and modulus of elasticity tests for examples 1 to 3

[0045] Realization 1 31 295Pa Realization 2 26 353Pa Realization 3 21 412Pa

[0046] The lower the degree of swelling laterally, the higher the degree of cross-linking of the product, the more the modulus of elasticity reflects the ability of the product to resist the influence of external forces, and the better the supporting effect. in the body, while the elastic modulus should not be too high, too high elastic modulus will affect the naturalness of the face after injection. Therefore, from the results in Table 1, it can be seen that Design 2 has a better modulus of elasticity and a better degree of swelling.

Experience of in vitro degradation

[0047] The in vitro enzymatic degradation performance of the samples of embodiments 1 to 3 was compared by carrying out an in vitro enzymatic degradation test at 37 5" wi="3" file="CH00718108-P00009.tif" orientation=" portrait" alt="custom character" img-content="character" img-format="tif" inline="yes"/> using 5U/mL of hyaluronidase, and detecting and recording the degree of enzymatic degradation up to to complete degradation at different time points to compare the in vitro degradation performance of samples from embodiments 1 to 3. The degree of enzymatic degradation was observed by detecting the concentration of glucuronide in the enzyme solution at different time points.

[0048] The glucuronide content detected in the enzymatic digestion was uniformly converted to sodium hyaluronate content, and the G0 sample was used as a blank, and the sample with 24 h of enzymatic digestion was used as the digestion point complete enzymatic digestion, and the degree of enzymatic digestion at each time point was obtained by calculating the sodium hyaluronate content of the enzymatic digestion obtained at each time point against the content of the enzymatic digestion for 24 h and in the sample tube G0 by the following equation, and the results are shown in Table 2.

In the above equation: p is the value of the mass concentration of sodium hyaluronate in the sample to be tested, in mg/ml; C1 is the glucuronide content in the sample tube, in µg/ml; m is the mass of the cross-linked sodium hyaluronate gel, in g; m1 is the amount of enzyme solution added; ρ1 is the density of the cross-linked sodium hyaluronate gel, in 1.01 g/ml.

[0050] In said gong equation, p is the mass concentration value of the sodium hyaluronate in the article to be tested, mg/ml; ρ0 is the mass concentration value of sodium hyaluronate in the sample tube G0, mg/ml; ρ24 is the mass concentration value of sodium hyaluronate in the sample tube after 24 h of enzymatic digestion, mg/ml. 0 0 0 0 1 18.3 11.7 5.6 3 30.6 29.8 26.8 6 58.9 53.1 52.1 12 100 100 90.3 24 100 100 100

The results in Table 2 show that embodiment 3 has the best resistance to enzymatic digestion, reflecting its better retention in vivo, and that embodiment 1 has the least resistance to enzymatic digestion, reflecting its poor retention in vivo. Run 2 has better resistance to enzymatic digestion and has better modulus of elasticity and swelling, so the sample produced by Run 2 is of the highest quality.

Comparison experiences with existing products

Using the monophasic product Biohyalux, a product of Bloomage Biotechnology, as reference 1 and the monophasic product Voluma, a product of Allergan, as reference 2, a comparative elastic modulus experiment was carried out with the realization 2 of the present invention: by using the scanning mode under the oscillation test of the rotational rheometer, the modulus of elasticity G' was scanned from 0.08 Hz to 5 Hz and the corresponding modulus of elasticity was recorded, and the results are shown in Figure 1.

The results in Figure 1 show that the elastic modulus of the sodium hyaluronate gel prepared by Embodiment 2 of the present invention is greater than both Substance 1 and Substance 2, which shows that the sodium hyaluronate gel of Embodiment 2 has more excellent physical properties, and also reflects that the gel prepared by the present invention has better effect in shaping, maintenance and resistance to external forces.

[0054] In summary, the sodium hyaluronate gel produced by the method of preparation provided by the present invention has a better modulus of elasticity and degree of swelling, and good anti-enzymatic properties. The preparation method of the present invention has few steps in its process, is easy to operate, has a short cycle time, can effectively improve the quality of the produced gel, and is suitable for large-scale industrial production.

[0055] Said embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of applications of the present invention, and all non-substantial modifications and substitutions made by a person skilled in the art on the basis of the present invention are within the scope of the properties claimed by the present invention.

Claims (8)

1. The mode of preparation of a monophasic cross-linked sodium hyaluronate gel is specific in that it includes the following steps: S1, The cross-linking agent and NaOH solution are put into a centrifuge tube at a mass ratio of 1:8-1:10 and nitrogen is added thereto, then placed in a centrifuge and mixed well to obtain a mixed solution of NaOH containing the crosslinking agent and set aside. S2, Sodium hyaluronate and NaOH solution of pH > 13 are mixed and dissolved under stirring at a mass ratio of 1:6-1:8 and placed in a three-dimensional mixer for 1~2h to obtain a solution of sodium hyaluronate. S3, Add all the NaOH mixture made in step S1 to the sodium hyaluronate solution, mix well and pass nitrogen gas, then thermostat the water bath at 45-55 for 2-3h to obtain a sodium hyaluronate gel. S4, Sodium hyaluronate gel is cut into small pieces, added to a PBS solution of hydrochloric acid, mixed in a three-dimensional blender for 24-36h, stirred well to obtain a crumble paste, packed in bags of dialyzed and placed in 2-8 running pure PBS solution for 30-36h to obtain cross-linked phase sodium hyaluronate. S5, The cross-linked phase sodium hyaluronate solution and the non-cross-linked phase sodium hyaluronate solution are mixed well at a mass ratio of 8:1-9:1, degassed, filled and sterilized. 2. The method of preparation of a monophasic cross-linked sodium hyaluronate gel according to claim is characterized by: the molecular weight of the sodium hyaluronate used in step S2 is 2.1 ~ 2.7 MDa. 3. The mode of preparation of a monophasic phase cross-linked sodium hyaluronate gel according to claim 1 is characterized by the molecular weight of the sodium hyaluronate used in said non-cross-linked phase sodium hyaluronate solution which is 3.0 Mda. 4. The mode of preparation of a monophasic cross-linked sodium hyaluronate gel according to claim 1; the peculiarity lies in the fact that the PBS solution of hydrochloric acid is composed of hydrochloric acid and PBS in which the PBS is prepared from the hydrates of di-sodium hydrogen phosphate, sodium hydrogen phosphate and sodium chloride . 5. The mode of preparation of a monophasic cross-linked sodium hyaluronate gel according to claim 1; the specificity lies in the fact that: said crosslinking agent is 1,4-Butanediol diglycidyl ether. 6. The method of preparation of a monophasic cross-linked sodium hyaluronate gel according to claim 1 the particularity is justified in that the specific phases of step S2 are: mixture of sodium hyaluronate with an NaOH solution of pH > 13 in a mass ratio of 1:6-1:8 in a polytetrafluoroethylene beaker with a lid, and shaking for dissolution with the lid closed, then placing in a three-dimensional mixer for 1~2h to obtain the sodium hyaluronate solution. 7. The mode of preparation of a monophasic cross-linked sodium hyaluronate gel according to Claims 1, is characterized in that at step S4 the PBS solution is replaced 5 to 6 times during the dialysis process. 8. The mode of preparation of a monophasic cross-linked sodium hyaluronate gel according to Claims 1, characterized in that step S5 is carried out by degassing using a stainless steel degassing tank, by connecting the degassing tank stainless steel tube and the filling machine through a silicone hose, filling the gel in the degassing tank through the filling machine into a glass syringe using vacuum, and placing it into a steam-air sterilization cabinet for sterilization using rapid temperature rise and fall.