A method for removing pyrogens

Abstract

The present invention provides a method for removing pyrogens from biocompatible polymers such as hyaluronic acid or its derivatives or their salts using aluminum hydroxychloride, which can not only effectively remove pyrogens but also improve the stability of the aqueous solution of hyaluronic acid or its derivatives or their salts.

Description

Technical Field

[0001] The present invention belongs to the fields of pharmaceutical technology and biological medicine, and particularly relates to a method for removing pyrogens, by which a pyrogen-free hyaluronic acid or its derivative or their salt can be prepared and obtained. Background Art

[0002] Hyaluronic acid, with the English name hyaluronic acid and the English abbreviation HA, is often called hyaluronic acid and is often in the form of sodium salt, namely sodium hyaluronate. Hyaluronic acid is one of the substances with the best moisturizing properties found by humans in nature and is known as the ideal natural moisturizing factor.

[0003] Hyaluronic acid was first isolated from bovine vitreous humor by Meyer et al. in the United States in 1934. It is a linear acidic mucopolysaccharide composed of alternating disaccharide units of N-acetylglucosamine and glucuronic acid. HA is commonly present in the skin, serum, and interstitial fluid of human and animal tissues, and has unique functions such as skin moisturizing, nutrition, anti-aging, stable emulsification, antibacterial and anti-inflammatory, promoting wound healing, and drug carrier. It is a functional biocompatible polymer compound with extremely wide uses and excellent properties in the fields of daily chemicals, pharmaceuticals, medical beauty, biochemistry, and health foods.

[0004] The common application forms of hyaluronic acid in the field of medical beauty are: making sodium hyaluronate gel as a subcutaneous filler to achieve the effects of beauty and filling; being used as the main component of sodium hyaluronate external application patches; being used as the main component of sodium hyaluronate external repair biofilms or external repair patches, etc. The common application forms of hyaluronic acid in the pharmaceutical field include: making eye drops and other drugs as the main drug component, such as sodium hyaluronate eye drops; being used as a pharmaceutical excipient in the prescription composition of various dosage forms; modified or structurally transformed hyaluronic acid can be used as a carrier for a new drug delivery system, etc. Among the various applications of sodium hyaluronate, the most common and well-known at present is injectable sodium hyaluronate gel, and injectable sodium hyaluronate gel is also called hyaluronic acid.

[0005] In China, injectable sodium hyaluronate gel is managed as a medical device. Since its scope of application or intended use is for injecting into the middle to deep layers of facial dermal tissue to correct moderate to severe nasolabial folds, etc., and it is a type of medical device or drug type with potential risks to the human body, therefore, the control or effective removal of various potential risks, various impurity components, or contaminating components in the production process of products such as injectable sodium hyaluronate gel has become a key issue that needs to be focused on in the production process of such products. The complete solution of these problems can thus contribute to avoiding key risk points in the clinical and medical beauty applications of the above-mentioned products, thereby providing products with higher safety for patients and medical beauty users.

[0006] Modified hyaluronic acid can also be called hyaluronic acid derivative. The most common type of modified sodium hyaluronate is cross-linked sodium hyaluronate. Among various cross-linked sodium hyaluronates, there are corresponding cross-linked sodium hyaluronate products due to different cross-linking methods, preparation processes, etc. Modified hyaluronic acids that can still be found in current literature and product listing information include -SH modified hyaluronic acid, PEG modified hyaluronic acid, etc. Oxidized hyaluronic acid is a new category of various modified or structurally modified hyaluronic acids. Oxidized hyaluronic acid can be used not only as the core raw material for constructing cross-linked hydrogels for medical aesthetics, but also as the core raw material for innovative external medical devices and innovative external drugs.

[0007] Oxidized hyaluronic acid, namely aldehyde group hyaluronic acid, is generally abbreviated as AHA. The degree of oxidation can be adjusted accordingly according to the needs of product development and product characteristics. The oxidation sites can be achieved through personalized adjustment of chemical synthesis processes and reach the expected effects. The molecular weight range is related to the initial molecular weight of the hyaluronic acid used as the raw material and can form the characteristics of a gel network through cross-linking reactions.

[0008] Pyrogen is a pyrogenic substance produced by microorganisms that can cause abnormal elevation of body temperature in warm-blooded animals. Pyrogens include bacterial pyrogens, endogenous high-molecular pyrogens, endogenous low-molecular pyrogens, and chemical pyrogens, etc. The "pyrogen" referred to here mainly refers to bacterial pyrogens, which are metabolites of certain bacteria, bacterial corpses, and endotoxins. The products of Gram-negative bacilli have the strongest pyrogenic ability, followed by Gram-positive bacilli, and Gram-positive cocci are weaker. Molds, yeasts, and even viruses can also produce pyrogens. Pyrogens are usually complexes formed by phospholipid polyols and proteins. Phospholipid polyols are the active centers of the complexes and have the strongest pyrogenic effect. Their chemical compositions vary depending on the bacterial species. The molecular weight is generally 5×10 4 ~5×10 5 Da. The larger the molecular weight, the stronger the pyrogenic effect. When the amount of pyrogen in the injectable drug injected into the human body reaches 1 μg / kg, adverse reactions can occur. The fever reaction usually appears 1 hour after injection into the human body, and it can cause symptoms such as chills, shivering, fever, sweating, nausea, vomiting, etc. Sometimes the body temperature can rise above 40°C, and in severe cases, even coma and collapse may occur. If not rescued in time, it can endanger life. This phenomenon is called "pyrogen reaction". For injectable medical devices, if they carry pyrogens, it will also cause pyrogen reaction in the human body. Bacterial endotoxin is a complex of lipopolysaccharide and trace protein on the cell wall of Gram-negative bacteria. It is a substance with endotoxin biological activity released after the death or disintegration of bacteria.

[0009] The 2000 edition of the Chinese Pharmacopoeia stipulates that the pyrogen test uses the rabbit method, and the bacterial endotoxin test uses the limulus reagent method. In subsequent editions of the pharmacopoeia, the above-mentioned methods have been used for the pyrogen test.

[0010] Since the response of rabbits to pyrogens is basically similar to that of humans, the rabbit method is still the legal method for pyrogen inspection specified in the pharmacopoeias of various countries. The pyrogen inspection method specified in the 2005 edition of the Chinese Pharmacopoeia is to inject a certain dose of the test article intravenously into rabbits and observe the increase in the body temperature of the rabbits within a specified time to determine whether the limit of pyrogens contained in the test article complies with the regulations. The accuracy and consistency of the inspection results depend on the condition of the test animals, the laboratory conditions, and the standardization of the operation. The sensitivity of the rabbit method for detecting endotoxin is 0.001 μg / ml. The test results are close to the actual human situation, but the operation is cumbersome and time-consuming, and it cannot be used for quality control during the production process of injections. Moreover, it is not applicable to cytotoxic drug preparations such as radioactive drugs and tumor inhibitors.

[0011] The bacterial endotoxin inspection method is a method that uses limulus reagents to detect or quantify the bacterial endotoxins produced by Gram-negative bacteria to determine whether the limit of bacterial endotoxins in the test article complies with the regulations. The amount of bacterial endotoxin is expressed in endotoxin units (EU).

[0012] The bacterial endotoxin inspection includes two methods: the gel method and the photometric determination method. The former uses the principle of the agglutination reaction between limulus reagents and bacterial endotoxins to detect or semi-quantify endotoxins. The latter includes the turbidimetry method and the chromogenic substrate method, which respectively use the turbidity change during the reaction between limulus reagents and endotoxins and the amount of chromophore released by a specific substrate by the coagulase produced to determine endotoxins.

[0013] The sensitivity of the limulus reagent method for inspecting endotoxin is 0.0001 μg / ml, which is 10 times more sensitive than the rabbit method. The operation is simple and easy, the test cost is low, the results are rapid and reliable, and it is applicable to the pyrogen control during the production process of injections and some cytotoxic drug preparations that cannot be detected by the rabbit method. However, it is not sensitive to endotoxins other than Gram-negative bacteria and currently cannot completely replace the rabbit method.

[0014] The characteristics of pyrogens are: heat resistance, filterability, water solubility, non-volatility, and they are not resistant to strong acids, strong alkalis, and strong oxidants.

[0015] Pyrogens include bacterial endotoxins, which are inevitable harmful components that can cause adverse reactions in pharmaceutical injections and injectable medical devices.

[0016] In the production process of pharmaceutical injections and injectable medical devices, typical pyrogen contamination routes include: 1. Introduction from solvents, which is the main cause of pyrogen contamination in pharmaceutical injections and injectable medical devices. The following factors: unreasonable distiller structure, improper operation, and excessive storage time of injection water, can all bring about pyrogen contamination. Therefore, in the production process of pharmaceuticals and medical devices, fresh injection water should be used, and it is best to use it immediately after distillation. 2. Introduction from raw materials. Drugs that are prone to growing microorganisms, such as glucose, etc., often cause pyrogen contamination due to long storage time and damaged packaging; drugs manufactured by biological methods, such as dextran, hydrolyzed protein, antibiotics, etc., often bring pyrogens into subsequent injections or medical device production due to incomplete removal of pyrogen components. 3. Introduction from containers, utensils, pipelines, and devices. 4. Introduction during the preparation process. During the preparation process, due to inadequate environmental control in the workshop, long operation time, and insufficiently sealed devices, etc., the opportunity for bacterial contamination is increased, thus leading to the generation of pyrogens. 5. Introduction from infusion sets. This type of pyrogen contamination generally occurs during the use of pharmaceutical injections and injectable medical devices, but for pre-filled injectable medical devices, pyrogens may also be introduced from infusion sets during the pre-filling production process. 6. Other situations that may introduce pyrogen contamination into the product.

[0017] For the pharmaceutical or medical device products prepared from hyaluronic acid or its derivatives or their salts involved in the present invention, during their production process, the above-listed 5 pyrogen contamination routes may all be involved. However, for the 1st, 3rd, and 4th cases, they can all be eliminated in the production environment control and production quality control links; for the 5th case, it can be eliminated in the selection and quality control links of infusion sets; only the 2nd case is due to pyrogens brought in by the raw materials used, and pyrogens should be removed in the raw material preparation link, and thus the pyrogen contamination should be solved before the production of medical device products begins.

[0018] The definition of removing pyrogens is: removing all substances that can raise the patient's body temperature. Currently, in the pharmaceutical production process, there are practical methods for removing pyrogen components, mainly physical removal methods and passivation methods. Physical removal methods include: distillation method, ultrafiltration method, activated carbon adsorption, hydrophobic adsorption method, oxidation method, etc. A typical passivation method is chemical passivation with acids and bases to remove surface pyrogens from containers, utensils, devices, etc.

[0019] The hyaluronic acid, its derivatives or their salts involved in the present invention, and the pharmaceutical and medical device products produced from them. Due to the nature of these products themselves, it is difficult to remove the pyrogens contained in the products in the prior art. In particular, the hyaluronic acid, its derivatives or their salts for injection, although belonging to the management category of medical devices, will be injected into the human body during their actual application, and the general requirements for injectable drugs also need to be referred to. Moreover, with the rapid development of the international and Chinese medical beauty markets in recent years, developing a new and effective method for removing pyrogens from hyaluronic acid, its derivatives or their salts has become an inevitable requirement in the industry. Summary of the Invention

[0020] The object of the present invention is to provide a method for removing pyrogens from biocompatible polymers such as hyaluronic acid, its derivatives or their salts, which can solve the inevitable pyrogen contamination problem in the production process of the above biocompatible polymers, thereby solving the safety risks of products such as pharmaceuticals and medical devices prepared from the above biocompatible polymers due to the inevitable presence or contamination of pyrogens, and avoiding the occurrence of adverse events.

[0021] The present invention discovers that aluminum hydroxychloride can effectively remove pyrogens from hyaluronic acid, its derivatives or their salts. In particular, it is unexpectedly found that the stability of the solution of hyaluronic acid, its derivatives or their salts after removing pyrogens with aluminum hydroxychloride is improved, and the technical effect of improving stability is most obvious when [[Al2(OH)5Cl]] m , m is a positive integer and m ≤ 10.

[0022] The present invention provides a method for removing pyrogens from hyaluronic acid, its derivatives or their salts, which includes the following steps:

[0023] (1) Dissolve aluminum hydroxychloride in water to obtain an aqueous solution of aluminum hydroxychloride;

[0024] (2) Dissolve hyaluronic acid, its derivatives or their salts in water to obtain an aqueous solution of hyaluronic acid, its derivatives or their salts;

[0025] (3) While stirring, add the solution obtained in step (1) dropwise to the solution obtained in step (2), let it stand, take the supernatant, and filter successively with a 0.45 μm microporous filter membrane and a 0.22 μm microporous filter membrane.

[0026] Preferably, it further includes step (4) to remove water.

[0027] According to the present invention, the hyaluronic acid derivative is a modified hyaluronic acid, such as cross-linked hyaluronic acid, oxidized hyaluronic acid (i.e., aldehyde-modified hyaluronic acid), mercapto-modified hyaluronic acid, PEGylated hyaluronic acid, etc.

[0028] According to the present invention, the salt of hyaluronic acid or its derivative is preferably an alkali metal salt or an alkaline earth metal salt, more preferably a sodium salt or a potassium salt.

[0029] According to the present invention, the hyaluronic acid or its derivatives or their salts are preferably sodium hyaluronate.

[0030] The hyaluronic acid or its derivatives or salts thereof of the present invention may be of synthetic or semi-synthetic origin or of natural origin, and may be commercially available or homemade.

[0031] According to the present invention, the weight average molecular weight of hyaluronic acid or its derivatives or their salts is preferably 50,000-2,000,000 Daltons, preferably 100,000-500,000 Daltons.

[0032] Aluminum hydroxychloride, also known as basic aluminum chloride, is a multi-base multivalent electrolyte with a molecular formula of [Al2(OH) n Cl 6-n ] m , m, n are positive integers, n = 1-5, m ≤ 10. In the present invention, if aluminum hydroxychloride containing crystal water is used, when preparing its solution, it should be converted into aluminum hydroxychloride without crystal water. For example, a 2.5% aluminum hydroxychloride solution should be prepared by adding 2.5g of aluminum hydroxychloride without crystal water to 100mL.

[0033] According to the present invention, aluminum hydroxychloride is preferably [Al2(OH) n Cl 6-n ] m , where n = 5, m is a positive integer and m ≤ 10, i.e. [Al2(OH)5Cl] m , m is a positive integer and m≤10.

[0034] According to the present invention, the water is preferably depyrogenated water, such as water for injection.

[0035] According to the present invention, in step (1), the concentration of aluminum hydroxychloride is preferably 1.5%-6.0% by weight / volume, more preferably 2.0%-5.5%, most preferably 2.5%-5.0%.

[0036] According to the present invention, in step (2), the concentration of hyaluronic acid or its derivatives or their salts is preferably 0.01%-2.5%, more preferably 0.2%-2.2% by weight / volume.

[0037] According to the present invention, in step (3), the aluminum hydroxychloride is preferably 0.005%-0.12% by weight / volume (total volume of the product formed in step (3)), further preferably 0.01%-0.10%, more preferably 0.02%-0.08%, and most preferably 0.03%-0.07%.

[0038] According to the present invention, in step (2), the pH of the aqueous solution of hyaluronic acid or its derivative or their salt is preferably between 5.0 and 8.5, more preferably between 6.1 and 7.5. If necessary, a pH regulator can be used. Examples of the pH regulator include aqueous sodium carbonate solution, aqueous sodium bicarbonate solution, aqueous hydrochloric acid solution, etc.

[0039] According to the present invention, in step (3), it is preferably allowed to stand for 0.5 hour to 8 hours.

[0040] According to another aspect of the present invention, the present invention provides hyaluronic acid or its derivative or their salt, or an aqueous solution thereof, prepared by the above method.

[0041] The beneficial effect of the present invention is that the hyaluronic acid, modified hyaluronic acid, or their salt obtained by the preparation method of the present invention has the property of being pyrogen-free and more suitable for the preparation of injectable products. The injectable products can be medical devices or drugs.

[0042] In addition, the present invention unexpectedly finds that after removing pyrogens with aluminum hydroxychloride, the aqueous solution of hyaluronic acid or its derivative or their salt remains homogeneous and stable after being stored at room temperature for more than 7 days. In contrast, for the aqueous solution of hyaluronic acid or its derivative or their salt without removing pyrogens with aluminum hydroxychloride, or after removing pyrogens with activated carbon, sedimentation will occur to a certain extent after being stored at room temperature, manifested as: the concentration in the lower part of the solution increases, that is, it becomes thicker, while the concentration in the upper part of the solution decreases, that is, it becomes thinner. Therefore, using aluminum hydroxychloride to remove pyrogens can improve the stability of the aqueous solution of hyaluronic acid or its derivative or their salt, where [Al2(OH)5Cl] m , and the technical effect of improving stability is most obvious when m≤10. Specific Embodiments

[0043] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the present invention.

[0044] Preparation Example 1 Preparation of Aluminum Hydroxychloride Aqueous Solution 1

[0045] Weigh an appropriate amount of aluminum hydroxychloride and dissolve it in water to prepare an aluminum hydroxychloride aqueous solution with a w / v concentration of 5.0%, denoted as aluminum hydroxychloride aqueous solution 1. The aluminum hydroxychloride used is [Al2(OH)5Cl] m , where m is a positive integer and m≤10.

[0046] Preparation Example 2 Preparation of Aluminum Hydroxychloride Aqueous Solution 2

[0047] Weigh an appropriate amount of aluminum hydroxychloride and dissolve it in water to prepare an aluminum hydroxychloride aqueous solution with a w / v concentration of 4.0%, denoted as aluminum hydroxychloride aqueous solution 2. The aluminum hydroxychloride used is [Al2(OH)5Cl] m , where m is a positive integer and m ≤ 10.

[0048] Preparation Example 3: Prepare aluminum hydroxychloride aqueous solution 3

[0049] Weigh an appropriate amount of aluminum hydroxychloride and dissolve it in water to prepare an aluminum hydroxychloride aqueous solution with a w / v concentration of 2.5%, denoted as aluminum hydroxychloride aqueous solution 3. The aluminum hydroxychloride used is [Al2(OH)5Cl] m , where m is a positive integer and m ≤ 10.

[0050] Preparation Example 4: Prepare aluminum hydroxychloride aqueous solution 4

[0051] Weigh an appropriate amount of aluminum hydroxychloride and dissolve it in water to prepare an aluminum hydroxychloride aqueous solution with a w / v concentration of 4.0%, denoted as aluminum hydroxychloride aqueous solution 4. The aluminum hydroxychloride used is [Al2(OH)4Cl2] m , where m is a positive integer and m ≤ 10.

[0052] Preparation Example 5: Prepare aluminum hydroxychloride aqueous solution 5

[0053] Weigh an appropriate amount of aluminum hydroxychloride and dissolve it in water to prepare an aluminum hydroxychloride aqueous solution with a w / v concentration of 5.0%, denoted as aluminum hydroxychloride aqueous solution 5. The aluminum hydroxychloride used is [Al2(OH)2Cl4] m , where m is a positive integer and m ≤ 10.

[0054] Example 1

[0055] Sodium hyaluronate is an externally purchased commercial product, of cosmetic grade, with a weight-average molecular weight of 370,000 Daltons.

[0056] Weigh an appropriate amount of sodium hyaluronate and dissolve it in pyrogen-free injection water to prepare a sodium hyaluronate aqueous solution with a w / v concentration of 2.0%, and its pH is 6.51;

[0057] Under stirring, slowly add 20 mL of the aluminum hydroxychloride aqueous solution prepared in Preparation Example 1 to 980 mL of the above sodium hyaluronate aqueous solution, let it stand for 2 hours, take the supernatant, and filter it successively with a 0.45 μm microporous membrane and a 0.22 μm microporous membrane to obtain 900 mL of filtrate;

[0058] Perform pyrogen inspection according to the method of Example 11, and the result is negative; perform bacterial endotoxin inspection according to the method of Example 12, and the result shows that it is qualified. Remove water to obtain sodium hyaluronate in the form of a white powdery substance.

[0059] Examples 2 - 6

[0060] Examples 2 - 6 all refer to Example 1, as shown in Table 1 below:

[0061] Table 1

[0062]

[0063] Example 7

[0064] The oxidized sodium hyaluronate was self - made, at the cosmetic grade, with a weight - average molecular weight of 300,000 Daltons.

[0065] Weigh an appropriate amount of oxidized sodium hyaluronate and dissolve it in pyrogen - free injection water to prepare an oxidized sodium hyaluronate aqueous solution with a w / v concentration of 2.0%, and its pH is 6.35;

[0066] Under stirring, slowly add 20 mL of the aluminum hydroxy chloride aqueous solution prepared in Preparation Example 1 to 980 mL of the above - mentioned oxidized sodium hyaluronate aqueous solution, let it stand for 3 hours, take the supernatant, and filter it successively with a 0.45 - μm microporous membrane and a 0.22 - μm microporous membrane to obtain 900 mL of filtrate;

[0067] Conduct a pyrogen test according to the method of Example 11, and the result is negative; conduct a bacterial endotoxin test according to the method of Example 12, and the result shows it is qualified. Remove water to obtain white to off - white powdery oxidized sodium hyaluronate.

[0068] Example 8 Removing pyrogens from the hyaluronic acid solution by activated carbon adsorption method (comparative example)

[0069] Take the same batch number of sodium hyaluronate used in Example 1, at the cosmetic grade, with a weight - average molecular weight of 370,000 Daltons.

[0070] Weigh an appropriate amount of sodium hyaluronate and dissolve it in pyrogen - free injection water to prepare a sodium hyaluronate aqueous solution with a w / v concentration of 2.0%, and its pH is 6.51;

[0071] Under stirring, add 5.0 g of activated carbon for injection to 1000 mL of the above - mentioned sodium hyaluronate aqueous solution heated to 50 °C, keep it warm and continuously stir for 30 minutes, and then filter it successively with a 0.45 - μm microporous membrane and a 0.22 - μm microporous membrane to obtain the filtrate.

[0072] Example 9

[0073] Take the same batch number of sodium hyaluronate used in Example 1, at the cosmetic grade, with a weight - average molecular weight of 370,000 Daltons.

[0074] Weigh an appropriate amount of sodium hyaluronate and dissolve it in pyrogen-free water for injection to prepare an aqueous sodium hyaluronate solution with a w / v concentration of 2.0%, and its pH is 6.51;

[0075] Under stirring, slowly add 20 mL of the aqueous aluminum hydroxychloride solution prepared in Preparation Example 4 to 980 mL of the above-mentioned aqueous sodium hyaluronate solution, let it stand for 2 hours, take the supernatant, and filter it successively through a 0.45 μm microporous membrane and a 0.22 μm microporous membrane to obtain 900 mL of filtrate;

[0076] Perform pyrogen test according to the method of Example 11, and the result is negative; perform bacterial endotoxin test according to the method of Example 12, and the result shows compliance. Remove water to obtain sodium hyaluronate in the form of a pale white powder.

[0077] Example 10

[0078] Take the sodium hyaluronate of the same batch number used in Example 1, cosmetic grade, with a weight average molecular weight of 370,000 Daltons.

[0079] Weigh an appropriate amount of sodium hyaluronate and dissolve it in pyrogen-free water for injection to prepare an aqueous sodium hyaluronate solution with a w / v concentration of 2.0%, and its pH is 6.51;

[0080] Under stirring, slowly add 20 mL of the aqueous aluminum hydroxychloride solution prepared in Preparation Example 5 to 980 mL of the above-mentioned aqueous sodium hyaluronate solution, let it stand for 2 hours, take the supernatant, and filter it successively through a 0.45 μm microporous membrane and a 0.22 μm microporous membrane to obtain 900 mL of filtrate;

[0081] Perform pyrogen test according to the method of Example 11, and the result is negative; perform bacterial endotoxin test according to the method of Example 12, and the result shows compliance. Remove water to obtain sodium hyaluronate in the form of a pale white powder.

[0082] Pyrogen test of Example 11

[0083] Respectively take the sample solutions before and after pyrogen removal in Examples 1 - 10, and refer to the pyrogen test method in the Chinese Pharmacopoeia (Appendix 1142, 2015 Edition) to perform pyrogen test.

[0084] Take 3 suitable rabbits. Within 15 minutes after measuring their normal body temperatures, slowly inject the sample solutions of the specified doses warmed to about 38 °C into the ear veins. Then measure their body temperatures once every 30 minutes for a total of 6 times. Subtract the normal body temperature from the highest one of the 6 body temperatures, which is the increase in body temperature (°C) of the rabbit. If the body temperature of 1 out of 3 rabbits rises by 0.6 °C or higher, or the total increase in body temperature of the 3 rabbits reaches 1.3 °C or higher, 5 more rabbits should be taken for retesting, and the test method is the same as above.

[0085] Result judgment: Among the 3 rabbits in the initial test, the increase in body temperature was less than 0.6°C for each rabbit, and the total increase in body temperature of the 3 rabbits was less than 1.3°C; or among the 5 rabbits in the retest, no more than 1 rabbit had an increase in body temperature of 0.6°C or higher, and the total increase in body temperature of the 8 rabbits in the initial test and retest combined was 3.5°C or lower. In both cases, the pyrogen test of the test article is judged to meet the requirements.

[0086] If, among the 3 rabbits in the initial test, more than 1 rabbit had an increase in body temperature of 0.6°C or higher; or if, among the 5 rabbits in the retest, more than 1 rabbit had an increase in body temperature of 0.6°C or higher; or if the total increase in body temperature of the 8 rabbits in the initial test and retest combined exceeded 3.5°C, the pyrogen test of the test article is judged not to meet the requirements. When the increase in the body temperature of the rabbit is negative, it is counted as 0°C.

[0087] The results of the pyrogen test are shown in Table 2 below.

[0088] Example 12 Bacterial endotoxin test

[0089] Separate samples of the solutions before and after pyrogen removal in Examples 1-10 were taken and subjected to the bacterial endotoxin test in accordance with the 2015 Edition of the Chinese Pharmacopoeia (Appendix 1143). The amount of endotoxin contained in each 1 mg of hyaluronic acid or its hyaluronic acid derivatives or their salts was less than 0.5 EU to be qualified, otherwise it was unqualified.

[0090] The results of the bacterial endotoxin test are shown in Table 2 below.

[0091] Table 2 Results of pyrogen and bacterial endotoxin tests

[0092]

[0093] The above Table 2 shows that for the samples in Examples 1-10, before pyrogen removal, they could all cause pyrogenic reactions in rabbits and contained excessive bacterial endotoxins, while after pyrogen removal, they did not cause pyrogenic reactions in rabbits and the bacterial endotoxins all met the requirements.

[0094] Example 13 Homogeneity and stability

[0095] Method for determining the homogeneity and stability of samples: Respectively take the solutions before removing pyrogens (i.e., raw materials) in Example 1 (i.e., 8, 9, 10) and the samples solutions after removing pyrogens in Example 1, 2, 7, 8, 9, 10. Take 3 parallel samples for each solution, and the sampling volume for each is 25 mL. Place them in 25-mL Nessler tubes respectively. After sealing and standing at room temperature for 7 days, without shaking the Nessler tubes, it can be observed that in the Nessler tubes of Example 1, 2, 7, 9, 10 after removing pyrogens, the upper and lower parts of the solution show a basically homogeneous state. In contrast, in the Nessler tube of Example 8 after removing pyrogens, the upper part of the solution shows a thinner state and the lower part shows a thicker state. In the Nessler tubes of each example before removing pyrogens, the upper part of the solution shows a thinner state and the lower part shows a thicker state. Compare with the light transmittance in Example 1, 2, 7, 9, 10. Among them, the homogeneity and stability of Example 1, 2, 7 are better than those of Example 9, 10. The light transmittance test is specifically as follows: Without shaking the Nessler tube, gradually suck the aqueous solution of hyaluronic acid or hyaluronic acid derivative in the Nessler tube from top to bottom. The suction amounts are 8 mL for the upper part, 8 mL for the middle part, and 8 mL for the lower part respectively. For the solutions obtained by suction, measure the light transmittance at 550 nm according to the method in Appendix General Rules 0401 of the Chinese Pharmacopoeia 2015 Edition, and record the light transmittance values. When the measured values of the light transmittance differ by 0.1%, there is a significant difference.

[0096] The results are shown in Table 3 below.

[0097] Table 3 Light transmittance measurement results before and after removing pyrogens

[0098]

[0099] In summary, for hyaluronic acid or hyaluronic acid derivatives with pyrogens removed by aluminum hydroxy chloride, their aqueous solutions are more homogeneous and stable than before removing pyrogens and those with pyrogens removed by activated carbon. That is, removing pyrogens with aluminum hydroxy chloride not only removes pyrogens and bacterial endotoxins, but also improves the homogeneity and stability of the solution of hyaluronic acid or its derivatives.

[0100] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for removing pyrogens from hyaluronic acid or its derivatives or their salts, characterized in that, It includes the following steps: (1) Dissolve aluminum hydroxychloride in water to obtain an aqueous solution of aluminum hydroxychloride; (2) Dissolve hyaluronic acid or its derivatives or their salts in water to obtain an aqueous solution of hyaluronic acid or its derivatives or their salts; (3) While stirring, add dropwise the solution obtained in step (1) to the solution obtained in step (2), let it stand, take the supernatant, and filter successively with a 0.45 μm microporous membrane and a 0.22 μm microporous membrane.

2. The method according to claim 1, characterized in that It further includes step (4) of removing water.

3. The method according to claim 1 or 2, characterized in that The hyaluronic acid derivatives are cross-linked hyaluronic acid, oxidized hyaluronic acid, mercaptohyaluronic acid, and PEGylated hyaluronic acid.

4. The method according to claim 1 or 2, characterized in that, The salts of hyaluronic acid or its derivatives are alkali metal salts or alkaline earth metal salts.

5. The method according to claim 1 or 2, characterized in that, The salts of hyaluronic acid or its derivatives are sodium salts or potassium salts.

6. The method according to claim 1 or 2, characterized in that The hyaluronic acid or its derivatives or their salts are sodium hyaluronate.

7. The method according to claim 1 or 2, characterized in that, The weight-average molecular weight of hyaluronic acid or its derivatives or their salts is 50,000 - 2,000,000 Daltons.

8. The method according to claim 7, characterized in that, The weight-average molecular weight of hyaluronic acid or its derivatives or their salts is 100,000 - 500,000 Daltons.

9. The method according to claim 1 or 2, characterized in that, Hydroxyaluminum chloride is [Al2(OH)5Cl] m , where m is a positive integer and m ≤ 10.

10. The method according to claim 1 or 2, characterized in that, In step (1), the concentration of aluminum hydroxychloride is 1.5% - 6.0% by weight / volume.

11. The method according to claim 10, characterized in that, In step (1), the concentration of aluminum hydroxychloride is 2.0% - 5.5% by weight / volume.

12. The method according to claim 11, wherein In step (1), the concentration of aluminum hydroxychloride is 2.5% - 5.0% by weight / volume.

13. The method according to claim 1 or 2, characterized in that, In step (2), the concentration of hyaluronic acid or its derivatives or their salts is 0.01% - 2.5% by weight / volume.

14. The method according to claim 13, wherein In step (2), the concentration of hyaluronic acid or its derivatives or their salts is 0.2% - 2.2% by weight / volume.

15. The method according to claim 1 or 2, characterized in that, In step (2), the pH of the aqueous solution of hyaluronic acid or its derivatives or their salts is 5.0 - 8.

5.

16. The method according to claim 15, wherein In step (2), the pH of the aqueous solution of hyaluronic acid or its derivatives or their salts is 6.1 - 7.

5.

17. The method according to claim 1 or 2, characterized in that, In step (3), aluminum hydroxychloride is 0.005% - 0.12% by weight / volume.

18. The method according to claim 17, wherein In step (3), aluminum hydroxychloride is 0.01% - 0.10% by weight / volume.

19. The method according to claim 18, wherein In step (3), aluminum hydroxychloride is 0.02 - 0.08% by weight / volume.

20. The method according to claim 19, wherein In step (3), aluminum hydroxychloride is 0.03% - 0.07% by weight / volume.

21. Hyaluronic acid or its derivatives or their salts, or their aqueous solutions, prepared by the method according to any one of claims 1 - 20.

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