Purification method for hyaluronic acid sodium salt carried out in organic solvent

Abstract

To provide a method for purifying pharmaceutical grade hyaluronic acid or its salts by a simple process in an organic medium without decomposition or the like. [Solution] A method for purifying pharmaceutical grade hyaluronic acid or a salt thereof, comprising the steps of: a) dissolving hyaluronic acid or a salt thereof in an organic solvent at a temperature of 50 to 100°C and stirring at that temperature for 1 to 48 hours; b) lowering the temperature to room temperature and keeping the solution under stirring for 1 hour to 48 hours; c) adding a C1-C4 alkyl alcohol or the like to precipitate hyaluronic acid or a salt thereof; and d) subsequent washing in a mixture of C1-C4 alkyl alcohol and water to remove impurities, etc.; A purification method comprising:

Description

[Technical Field]

[0001] The present invention relates to a method for preparing pharmaceutical, injectable or ophthalmic grade hyaluronic acid or its salts for use in the dermocosmetic or pharmaceutical fields or in medical devices, comprising dissolving hyaluronic acid or its salts in an organic solvent, thermal cycling and precipitation in an organic solvent and subsequent recovery of the product by washing. [Background technology]

[0002] The present invention surprisingly discloses a method for purifying hyaluronic acid (HA) or its salts by heat treatment in an organic medium, which removes both bacteria and endotoxins, i.e., lipopolysaccharides present in the outer membrane of Gram-negative bacteria, which are responsible for in vivo inflammatory responses, and protects native hyaluronic acid from hydrolysis-induced degradation of the polysaccharide chains. The hyaluronic acid prepared by this method has a microbiological profile compatible with that of pharmaceutical, injectable, or ophthalmic grade hyaluronic acid. Because this method is carried out in a non-aqueous organic solvent, it offers advantages from both a chemical and biological perspective by preventing hydrolysis. This method also has a small environmental and economic impact, since it does not require the large volumes typical of existing techniques and allows for complete recovery of the solvent for reuse. Finally, the extreme simplicity of the process, which does not involve expensive filtration, diafiltration, or ion exchange techniques, has a significant beneficial effect on its cost, thus expanding the possible economically sustainable applications of high-purity sodium hyaluronate and thus increasing the safety of products containing this polysaccharide, generally intended for human use.

[0003] Hyaluronan is a glycosaminoglycan consisting of repeating units of glucuronic acid and N-acetylglucosamine, linked via glycosidic bonds β1→4 and β1→3. It is an essential component of connective tissue and is also present in synovial fluid, vitreous humor, and umbilical cord.

[0004] Hyaluronic acid was isolated by Karl Meyer and John Palmer in 1934 and has many topical uses, injectable uses, implants (subcutaneous or intra-articular), and uses in the ophthalmological field. The acceptable bioburden depends on the end use of hyaluronic acid: for topical application or application to external mucous membranes, the number of bacterial colonies and yeasts (10 per gram) should be within the range of 1000 to 10000. 3 colony forming units, i.e., 10 3 "Cosmetic grade" is used with limits defined as total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) for bacterial counts (up to CFU / g), while no endotoxin unit limit is defined; for more invasive applications, "pharmaceutical," "ophthalmic," or "injectable" grade is used, with stricter bioburden limits for bacterial count and an endotoxin content of less than 0.5 EU / mg for parenteral applications and 0.05 EU / mg for intra-articular or intraocular applications. Considering the many uses of hyaluronic acid in the ophthalmology field and as a subcutaneous or intra-articular injectable, the importance of researching simple, reliable, and inexpensive methods for purifying hyaluronic acid is clear.

[0005] WO2013 / 132506 discloses a method for purifying endotoxins by treatment with bentonite and filtration with activated carbon; WO00 / 44925 reports tangential filtration followed by sterile filtration, while US2018 / 014041 uses diafiltration techniques with highly dilute solutions; CA02225866 discloses the use of arrays of negatively charged resins. All these methods are very complex and require very expensive instrumentation and equipment (ultrafiltration units, ion exchange columns, and freeze dryers), making them difficult to apply to anything other than very expensive industrial processes. In the case of dilute solutions (US2018 / 014041), the required quantities also have a significant environmental impact.

[0006] EP 2865395 discloses a heating cycle in water followed by sterile filtration. The process times described range from 1 to 6 days, which limits the process's potential given the susceptibility of hyaluronic acid to temperature-dependent hydrolysis.

[0007] EP 2039777 uses an extraction process in organic solvents, which is a heterogeneous process with a high risk of ineffectiveness, since it is difficult to completely eliminate microbial impurities trapped in the amorphous solid of hyaluronic acid, even though it is swollen.

[0008] Finally, US 5,079,236 discloses the preparation of injectable preparations, starting from hyaluronic acid that has already been microbiologically pure by dissolving it in water to a suitable concentration in the presence of paraben preservatives.The method described in this case does not involve actual purification, but is a preservation method that uses ingredients that may have safety contraindications, especially in the case of injectable medical devices.

[0009] Generally, all known methods involve the use of machinery that is difficult to implement in industrial processes due to both their technical complexity and their high costs. Furthermore, said methods use large amounts of aqueous and organic solvents, which entail disposal and recovery costs and environmental burdens.

[0010] Furthermore, all of the above methods are carried out in water or aqueous and organic mixtures and require long processing times and temperatures above room temperature, which adversely affect the molecular weight of the hyaluronic acid by hydrolysis.

[0011] Finally, no methods have been described that involve using cosmetic grade hyaluronic acid or its salts and increasing its microbiological purity to a level compatible with pharmaceutical, injectable or ophthalmic grade hyaluronic acid. Summary of the Invention

[0012] A method for preparing hyaluronic acid or its salts has now been found that results in a product with a microbial profile compatible with pharmaceutical, injectable or ophthalmic grade hyaluronic acid, starting from a product with a lower (cosmetic) grade microbial purity.

[0013] The method according to the present invention eliminates the complexity of existing processes because it is carried out in a homogeneous phase in an organic solvent. The efficiency of this method limits the volume of solvent used, resulting in favorable economic and environmental benefits. The use of a non-aqueous system prevents the typical hydrolysis that adversely affects standard processes. The possibility of obtaining a higher grade of microbial purity starting from hyaluronic acid or its salts with a lower (cosmetic) grade of microbial purity without the need for a fermentation process exponentially increases the versatility of the process, increases the value of the starting substrate by means of a cost-effective process with low environmental impact, and allows for different levels of microbial purity to be obtained from the same starting material, thus enabling its use in fields of application with different levels of invasiveness, with clear implications in terms of efficiency, safety, and cost.

[0014] An object of the present invention is a process for the preparation of pharmaceutical, injectable or ophthalmic grade hyaluronic acid or its salts, comprising the following steps: a) dissolving hyaluronic acid or a salt thereof in a solvent selected from dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and formamide at a temperature of 50 to 100°C, and maintaining the solution at that temperature for 1 to 48 hours with stirring; b) lowering the temperature to room temperature and maintaining the solution under stirring for 1 hour to 48 hours; c) precipitating the hyaluronic acid or its salt by adding a solvent selected from (C1-C4) alkyl alcohols, such as methanol or isopropanol, or acetone; and d) Removal of impurities and by-products by subsequent washing in a mixture of water and a solvent selected from (C1-C4) alkyl alcohols (methanol, isopropanol, acetone, etc.). DETAILED DESCRIPTION OF THE INVENTION

[0015] "Pharmaceutical, injectable or ophthalmic grade" means hyaluronic acid or its salts having a microbiological profile such that it meets the requirements set out in the pharmacopoeias for the corresponding purity.

[0016] The pharmaceutical, injectable or ophthalmic grade hyaluronic acid or salts thereof prepared by the method of the present invention preferably contain 10 3 It has the highest CFU / g and an endotoxin content of less than 0.5 EU / mg.

[0017] The hyaluronic acid salts are salts that are acceptable for pharmaceutical or cosmetic use or in medical devices such as sodium, potassium, lithium or quaternary ammonium salts, for example tetrabutylammonium, preferably the sodium salt.

[0018] Step a) is preferably carried out at a temperature in the range of 80°C to 100°C.

[0019] The solvent used in step a) is preferably formamide.

[0020] The solvent used for precipitation in step c) is preferably acetone.

[0021] The precipitate obtained in step c) is preferably washed with a solvent selected from isopropanol and methanol, preferably with methanol, and filtered.

[0022] The hyaluronic acid or salt thereof used in this method is preferably 10 3 ~10 6 It has a weight average molecular weight in the Dalton range.

[0023] The process according to the invention produces hyaluronic acid or its salts starting from a product with a lower microbiological purity (e.g. cosmetic grade) and results in a product suitable for injectable (mesodermal, subcutaneous or intra-articular), pharmaceutical or ophthalmic use.

[0024] The starting hyaluronic acid is typically cosmetic grade.

[0025] To increase the purity of the product, steps a), b), c) and d) of the process according to the invention can be repeated at least once.

[0026] The hyaluronic acid or salts thereof obtained by the process according to the invention have a microbiological profile compatible with use in pharmaceutical or dermocosmetic preparations or medical devices.

[0027] The hyaluronic acid obtained by the method according to the invention can be used in pharmaceutical or dermocosmetic preparations containing pharmaceutical, injectable or ophthalmic grade hyaluronic acid or its acceptable salts, in the dermocosmetic or pharmaceutical field, in medical devices or as a supplement, alone or together with at least one excipient and / or carrier acceptable for pharmaceutical or dermocosmetic use, or for use as a medical device. [Example]

[0028] method Measurement of endotoxin levels (LAL test) Endotoxin was quantified according to EU PHARMA method 0172018.20614, which corresponds to USP 41 NF 36 2018 paras. 85-161.

[0029] The cosmetic grade hyaluronic acid sodium salt used in the following experimental tests has an endotoxin level of 0.82 EU / mg as assessed by the above method.

[0030] Example 1: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 1500 kDa: (Step a) 95°C: 1 hour; Step b) 25°C: 19 hours 200ml of formamide was introduced into a 500ml three-necked flask, followed by 10g of cosmetic grade hyaluronic acid sodium salt with a molecular weight of 1500kDa.The mixture was adjusted to a temperature of 95°C and kept stirring at a constant temperature for 1 hour.Then, the temperature was lowered to 25°C, and the mixture was kept stirring at the same temperature for 19 hours.

[0031] Then, 2.6 g of sodium chloride dissolved in 14 ml of water for injection was added and left under stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0032] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0033] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.36EU / mg.

[0034] Example 2: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 300 kDa: (Step a) 95°C: 1 hour; Step b) 25°C: 19 hours 200ml of formamide was introduced into a 500ml three-necked flask, followed by 10g of cosmetic grade hyaluronic acid sodium salt with a molecular weight of 300kDa.The mixture was adjusted to a temperature of 95°C and kept stirring at a constant temperature for 1 hour.Then, the temperature was lowered to 25°C, and the mixture was kept stirring at the same temperature for 19 hours.

[0035] Then, 2.6 g of sodium chloride dissolved in 14 ml of water for injection was added and left under stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0036] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0037] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.27EU / mg.

[0038] Example 3: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 25 kDa: (Step a) 95°C: 1 hour; Step b) 25°C: 19 hours 200ml of formamide was introduced into a 500ml three-necked flask, followed by 20g of cosmetic grade hyaluronic acid sodium salt with a molecular weight of 25kDa.The mixture was adjusted to a temperature of 95°C and kept stirring at a constant temperature for 1 hour.Then, the temperature was lowered to 25°C, and the mixture was kept stirring at the same temperature for 19 hours.

[0039] Next, 4 g of sodium chloride dissolved in 20 ml of water for injection was added and left to stand with stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0040] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0041] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.38EU / mg.

[0042] Example 4: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 1500 kDa: (Step a) 95°C: 1 hour; Step b) 25°C: 43 hours 200ml of formamide was introduced into a 500ml three-necked flask, followed by 5g of cosmetic grade hyaluronic acid sodium salt with a molecular weight of 1500kDa.The mixture was adjusted to a temperature of 95°C and kept stirring at a constant temperature for 1 hour.Then, the temperature was lowered to 25°C, and the mixture was kept stirring at the same temperature for 43 hours.

[0043] Then, 2.6 g of sodium chloride dissolved in 28 ml of water for injection was added and left under stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0044] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0045] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.15EU / mg.

[0046] Example 5: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 300 kDa: (Step a) 95°C: 1 hour; Step b) 25°C: 43 hours) 200ml of formamide was introduced into a 500ml three-necked flask, followed by 10g of cosmetic grade hyaluronic acid sodium salt with a molecular weight of 300kDa.The mixture was adjusted to a temperature of 95°C and kept stirring at a constant temperature for 1 hour.Then, the temperature was lowered to 25°C, and the mixture was kept stirring at the same temperature for 43 hours.

[0047] Then, 2.6 g of sodium chloride dissolved in 28 ml of water for injection was added and left under stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0048] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0049] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.14EU / mg.

[0050] Example 6: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 25 kDa: (Step a) 95°C: 1 hour; Step b) 25°C: 43 hours 200ml of formamide was introduced into a 500ml three-necked flask, followed by 20g of cosmetic grade hyaluronic acid sodium salt with a molecular weight of 25kDa.The mixture was adjusted to a temperature of 95°C and kept stirring at a constant temperature for 1 hour.Then, the temperature was lowered to 25°C, and the mixture was kept stirring at the same temperature for 43 hours.

[0051] Then, 4 g of sodium chloride dissolved in 35 ml of water for injection was added and left under stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0052] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0053] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.15EU / mg.

[0054] Example 7: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 300 kDa: (Step a) 95°C: 43 hours; Step b) 25°C: 1 hour) 200ml of formamide was introduced into a 500ml three-necked flask, followed by 10g of cosmetic-grade hyaluronic acid sodium salt with a molecular weight of 300kDa. The mixture was adjusted to 95°C and maintained at a constant temperature for 43 hours under stirring. The temperature was then lowered to 25°C, and 2.6g of sodium chloride dissolved in 28ml of water for injection was added, and the mixture was left at the same temperature under stirring for 1 hour.

[0055] The product was isolated by precipitation in acetone followed by filtration.

[0056] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0057] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of <0.05EU / mg.

[0058] Example 8: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 25 kDa: (Step a) 95°C: 1 hour; Step b) 25°C: 43 hours 300ml of formamide was introduced into a 500ml three-necked flask, followed by 6g of cosmetic grade hyaluronic acid sodium salt with a molecular weight of 25kDa.The mixture was adjusted to a temperature of 95°C and kept stirring at a constant temperature for 1 hour.Then, the temperature was lowered to 25°C, and the mixture was kept stirring at the same temperature for 43 hours.

[0059] Next, 3 g of sodium chloride dissolved in 15 ml of water for injection was added and left to stand with stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0060] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0061] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.12EU / mg.

[0062] Example 9: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 300 kDa: (Step a) 95°C: 17 hours; Step b) 25°C: 1 hour 200 ml of formamide was added to a 500 ml three-necked flask, followed by 20 g of cosmetic-grade hyaluronic acid sodium salt with a molecular weight of 300 kDa. The mixture was then adjusted to 95°C and stirred at a constant temperature for 17 hours. The temperature was then lowered to 25°C.

[0063] Then, 2.6 g of sodium chloride dissolved in 28 ml of water for injection was added and left under stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0064] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0065] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.20EU / mg.

[0066] Example 10: Preparation of pharmaceutical grade hyaluronic acid sodium salt MW 300 kDa: Repeat the production process (step a) 95°C: 17 hours; step b) 25°C: 1 hour) 100 ml of formamide was introduced into a 500 ml three-necked flask, followed by the addition of 10 g of hyaluronic acid obtained in Example 9. The mixture was adjusted to 95°C and stirred at a constant temperature for 17 hours. The temperature was then lowered to 25°C, and 1.3 g of sodium chloride dissolved in 14 ml of water for injection was added, followed by stirring for about 1 hour. The product was isolated by precipitation in acetone and subsequent filtration.

[0067] The product was washed several times with methanol, each time by low-pressure filtration. The precipitate was dried at low pressure in a laminar flow hood at room temperature for about 16 hours.

[0068] A 5g sample of the product subjected to LAL testing was found to have an endotoxin content of 0.09EU / mg.

Claims

1. 1. A method for producing pharmaceutical, injectable or ophthalmic grade hyaluronic acid or a salt thereof, comprising the steps of: a) dissolving hyaluronic acid or a salt thereof in a solvent selected from dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and formamide at a temperature of 50 to 100°C, and stirring the solution at that temperature for 1 to 48 hours; b) adding aqueous sodium chloride solution, lowering the temperature to room temperature and keeping the solution under stirring for 1 hour to 48 hours; c) precipitating the hyaluronic acid or its salt by adding a solvent selected from a (C1-C4) alkyl alcohol or acetone; and d) removing impurities and by-products by subsequent washing in a mixture of water and a solvent selected from a (C1-C4) alkyl alcohol or acetone; A manufacturing method comprising:

2. Pharmaceutical, injectable or ophthalmic grade hyaluronic acid or its salts, up to 10 ３ 2. The method of claim 1, wherein the hyaluronic acid or salt thereof has an endotoxin content of less than CFU / g and 0.5 EU / mg.

3. 3. The method according to claim 1 or 2, wherein the temperature in step a) is in the range of 80°C to 100°C.

4. The method according to any one of claims 1 to 3, wherein the solvent used in step a) is formamide.

5. The process according to any one of claims 1 to 4, wherein the solvent used in step c) for precipitation is acetone.

6. The method of claim 1, wherein the solvent in step d) is selected from isopropanol and methanol.

7. The method of claim 6, wherein the solvent in step d) is methanol.

8. The method of any one of claims 1 to 7, wherein steps a), b), c) and d) are repeated at least once.