CN117402270A - Preparation method and application of sterile sodium hyaluronate - Google Patents
Preparation method and application of sterile sodium hyaluronate Download PDFInfo
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- CN117402270A CN117402270A CN202311704442.XA CN202311704442A CN117402270A CN 117402270 A CN117402270 A CN 117402270A CN 202311704442 A CN202311704442 A CN 202311704442A CN 117402270 A CN117402270 A CN 117402270A
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- 229920002385 Sodium hyaluronate Polymers 0.000 title claims abstract description 87
- 229940010747 sodium hyaluronate Drugs 0.000 title claims abstract description 87
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 title claims abstract description 87
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 29
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000001914 filtration Methods 0.000 claims abstract description 19
- 230000001954 sterilising effect Effects 0.000 claims abstract description 18
- 239000002537 cosmetic Substances 0.000 claims abstract description 8
- 238000004806 packaging method and process Methods 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 5
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 78
- 239000000243 solution Substances 0.000 claims description 59
- 238000006243 chemical reaction Methods 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 239000002245 particle Substances 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- 239000008215 water for injection Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 238000001179 sorption measurement Methods 0.000 claims description 5
- 238000001802 infusion Methods 0.000 claims description 4
- 238000005452 bending Methods 0.000 claims description 3
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000011045 prefiltration Methods 0.000 claims description 2
- 235000013402 health food Nutrition 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 5
- 235000013305 food Nutrition 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000002158 endotoxin Substances 0.000 description 37
- 238000010438 heat treatment Methods 0.000 description 19
- 230000000694 effects Effects 0.000 description 14
- 238000001514 detection method Methods 0.000 description 11
- 238000011013 endotoxin removal Methods 0.000 description 10
- 239000012085 test solution Substances 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 241000239218 Limulus Species 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 230000033001 locomotion Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010981 drying operation Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J4/00—Feed or outlet devices; Feed or outlet control devices
- B01J4/001—Feed or outlet devices as such, e.g. feeding tubes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J4/00—Feed or outlet devices; Feed or outlet control devices
- B01J4/001—Feed or outlet devices as such, e.g. feeding tubes
- B01J4/004—Sparger-type elements
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
Abstract
The invention belongs to the field of medicine/cosmetic raw materials, and particularly discloses a preparation method and application of sterile sodium hyaluronate. The preparation method comprises the following steps: dissolving non-sterile sodium hyaluronate raw material, pretreating by low-temperature alkaline method, adsorbing by active carbon, sterilizing, filtering, crystallizing, drying, and packaging to obtain sterile sodium hyaluronate product. The sodium hyaluronate sterile bulk drug prepared by the method has good quality and high stability, keeps stable molecular weight and increases use safety; the preparation method has simple process steps, is convenient to operate and suitable for industrial production, and the prepared sterile sodium hyaluronate can be widely applied to medicines, cosmetics or health-care foods.
Description
Technical Field
The invention belongs to the field of medicine/cosmetic raw materials, and particularly discloses a preparation method and application of sterile sodium hyaluronate.
Background
Sodium hyaluronate (SH or HA) is white fibrous or powdery solid, HAs strong hygroscopicity, is soluble in water, and is insoluble in organic solvents such as alcohol, ketone and diethyl ether. Its aqueous solution is negatively charged and has high viscoelasticity and osmotic pressure at high concentration. In addition, the aqueous sodium hyaluronate solution is a non-newtonian fluid with good viscoelasticity and flow deformation. Based on the special performance of sodium hyaluronate, sodium hyaluronate can be widely applied to medicines, cosmetics and health-care foods. The sodium hyaluronate is mainly applied to aspects of clinical treatment, diagnosis, cosmetic matrix, nutrition and health care products and the like. Commercial sodium hyaluronate is generally in the form of its sodium salt, with other metal salts such as zinc salts being rarely used. Sodium hyaluronate has various medicinal values and can be used as an effective ingredient and excipient in the pharmaceutical industry. The quality standard of the medicinal sodium hyaluronate is higher, and the requirements on the endotoxin content of microorganisms and bacteria are definite. In particular, sterile grade sodium hyaluronate requires the absence of viable microorganisms.
The existing method for sterilizing sodium hyaluronate and removing endotoxin mainly comprises an alkaline method, for example, application number CN201910785756.4 discloses a method for removing endotoxin in oligomeric sodium hyaluronate, and sodium hydroxide solid and sodium chloride solid forms are directly added in the method, so that compared with the form of adding sodium hydroxide and sodium chloride solution, the method not only reduces the operation process of preparing the solution, but also reduces the volume occupied by the solution, and saves the ethanol consumption in the later precipitation. However, in the above invention, the molecular weight of the oligomeric sodium hyaluronate is limited to below 1 kilodaltons, and when the molecular weight of the sodium hyaluronate is greatly increased, particularly sodium hyaluronate with a molecular weight of above 100 kilodaltons, the use of sodium hydroxide with a high concentration of 0.5% -5% can cause the degradation of the sodium hyaluronate with a high molecular weight, so that the stability of the molecular weight cannot be maintained in the process of removing endotoxin, and the prepared sterile sodium hyaluronate cannot meet the use requirement.
Disclosure of Invention
In order to solve the problems, the invention discloses a preparation method and application of sterile sodium hyaluronate, which can keep the molecular weight stable under the low temperature and alkaline conditions and can obtain the sterile sodium hyaluronate with high molecular weight.
The technical scheme of the invention is as follows:
a method for preparing sterile sodium hyaluronate, comprising the following steps:
s1: dissolving non-sterile sodium hyaluronate powder in water to obtain sodium hyaluronate solution;
s2: adjusting the pH of the sodium hyaluronate solution to be alkaline at a temperature lower than 40 ℃ and stirring;
s3: adding activated carbon for activated carbon adsorption;
s4: filtering to remove active carbon;
s5: sterilizing and filtering by using a sterilizing filter;
s6: crystallizing the sterilized and filtered solution by using ethanol;
s7: dehydrating and drying the crystallized product to obtain a product;
s8: packaging the dried product;
step S2 is completed by the reaction vessel.
Further, the water in the step 1 is water for injection, the concentration of the sodium hyaluronate solution is 0.2-1.0% (w/w), and the molecular weight is 100-300 kilodaltons.
Further, in the step 2, the pH is 10.50-11.00, the temperature is 30-40 ℃, and the stirring treatment time is 3-4 hours; the amount of the activated carbon in the step 3 is 0.2-0.5% (w/w), and the adsorption time is 15-30 min.
Further, the sterilization filtration in step 5 uses two stages of sterilization filtration, including prefiltration using a 0.45 μm filter membrane and secondary filtration using a 0.2 μm filter membrane; the ethanol in the step 6 is ethanol with the mass percentage concentration of 95 percent after sterilization and filtration; the mass ratio of the solution to the ethanol is 1: 1.5-2.
Further, the step 6 and the step 7 are performed in a B-stage environment; the sub-packaging in the step 8 is carried out in the environment of the B+A environment.
Further, in step 2, the pH of the sodium hyaluronate solution was adjusted to 10.7 at the initial temperature of 40 ℃ and maintained at the stirring for 1 hour, and then cooled uniformly to 2 ℃ every 15 minutes until 30 ℃ and maintained for 45 minutes.
Further, solid sodium hydroxide particles are used for adjusting ph in the step 2, and nitrogen is introduced while adjusting ph.
Further, the reaction vessel adopted in the step 2 comprises a liquid outlet, a liquid inlet, a serpentine pipe, a ph adjusting port, a U-shaped pipe and a mixing tank;
the liquid outlet is arranged at one end, close to the bottom, of the side wall of the reaction container;
the liquid inlet is arranged at one end, close to the top, of the side wall of the reaction container;
the U-shaped pipe is arranged on the central axis of the bottom of the reaction container, and the bending part of the U-shaped pipe is fixedly connected with the bottom of the reaction container;
the mixing tank is arranged at one end of the coiled pipe far away from the U-shaped pipe;
the ph adjusting port is arranged in an extension section of one end of the U-shaped pipe far away from the coiled pipe and is fixed on the side wall of the reaction container, and an infusion port is reserved above the ph adjusting port;
the U-shaped pipe comprises a partition plate and a bubble generator;
the bubble generator is arranged at the joint of the U-shaped pipe and the bottom of the reaction vessel; the baffle plate is arranged at one end, close to the ph adjusting port, of the bubble generator;
further, the sterile sodium hyaluronate can be applied to the preparation of medicines, cosmetics or health-care foods.
Compared with the prior art, the invention has the following beneficial effects:
firstly, after dissolving high molecular weight non-sterile sodium hyaluronate (raw materials), pretreating by a low-temperature alkaline method, adsorbing by activated carbon, sterilizing, filtering, crystallizing, drying and sub-packaging to obtain a sterile sodium hyaluronate product; the sterile raw material medicine of sodium hyaluronate prepared by the method has good quality and high stability, keeps the stability of molecular weight, obtains the sterile sodium hyaluronate with high molecular weight, and increases the use safety and application range; the preparation method has simple process steps, is convenient to operate and suitable for industrial production, and the prepared sterile sodium hyaluronate can be widely applied to medicines, cosmetics or health-care foods.
Secondly, the volume of the whole solution is reduced by adding solid sodium hydroxide particles, the scale of the whole preparation process can be controlled, the alkalinity of sodium hydroxide is similar to that of sodium hydroxide, the sodium hydroxide is completely ionized in water, the ionic solution has plasticizing effect on polymers with high molecular weight, nitrogen is introduced to protect the stability of the sodium hyaluronate with high molecular weight on the one hand, the molecular movement in the solution is increased to accelerate the dissolution of the sodium hydroxide particles, and the antioxidant capacity of the sodium hyaluronate is also improved.
Thirdly, the initial temperature is 40 ℃ to help dissolve solid sodium hydroxide particles, and the bacterial death speed is high at 40 ℃, so that high-level endotoxin removal can be maintained under the condition, the temperature is uniformly reduced, the temperature is reduced in the process, the ph floating caused by the decomposition of sodium hydroxide can be avoided, and the stability of the sodium hyaluronate solution can be maintained at low temperature.
Fourthly, sodium hydroxide particles are dissolved in advance through a coiled pipe, and are uniformly mixed to prevent local high alkaline solution from dissociating high molecular sodium hyaluronate, the U-shaped pipe and bubbles form liquid flow to drive sodium hydroxide to move and uniformly mix, the baffle is arranged to prevent bubbles from flowing backwards, new sodium hyaluronate solution is continuously injected in a circulating temperature change mode, a large amount of time is saved, and the temperature change in the solution is more uniform by changing the temperature of the bubbles.
Drawings
FIG. 1 is a graph showing kinematic viscosity trend at pH 10.0 in example 1;
FIG. 2 is a graph showing endotoxin tendency at pH 10.0 in example 1;
FIG. 3 is a graph showing the kinematic viscosity trend at pH 10.5 in example 2;
FIG. 4 is a graph showing endotoxin tendency at pH 10.5 in example 2;
FIG. 5 is a graph showing kinematic viscosity trend at pH 11.0 in example 3;
FIG. 6 is a graph showing endotoxin trend at pH 11.0 in example 3;
FIG. 7 is a graph showing kinematic viscosity trend at pH 11.5 in example 4;
FIG. 8 is a graph showing endotoxin tendency at pH 11.5 in example 4;
FIG. 9 is a schematic view of a reaction vessel used in example 7.
In the figure: 1. a liquid outlet; 2. a liquid inlet; 3. a serpentine tube; 4. a ph adjustment port; 5. a bubble generator; 6. a partition plate; 7. a U-shaped tube; 8. a mixing tank; 9. an infusion port; 10. a reaction vessel.
Detailed Description
A method for preparing sterile sodium hyaluronate, comprising the following steps:
s1: adding water for injection into non-sterile sodium hyaluronate powder to dissolve into sodium hyaluronate solution, wherein the concentration of the sodium hyaluronate solution is 0.4% (w/w); the non-sterile sodium hyaluronate powder meets the sodium hyaluronate pharmaceutical excipient grade standard, and has a molecular weight of 220 kilodaltons;
s2: at the temperature of 30-40 ℃, using solid sodium hydroxide to adjust the pH of the sodium hyaluronate solution to 10.50-11.00, and stirring for 4 hours;
s3: adding activated carbon for activated carbon adsorption; the dosage of the activated carbon is 0.5% (w/w), and the activated carbon is adsorbed for 30min;
s4: filtering to remove active carbon;
s5: sterilizing and filtering by using a sterilizing filter; the sterilization filtration uses two stages of sterilization filtration, including prefiltering with a 0.45 μm filter membrane and secondary filtration with a 0.2 μm filter membrane;
s6: crystallizing the sterilized and filtered solution by using 95% ethanol; the mass ratio of the solution to the ethanol is 1:2; the crystallization operation is carried out in a B-stage environment;
s7: dehydrating and drying after crystallization to obtain a product; the drying operation is carried out in a B-stage environment;
s8: packaging the dried product; the dispensing operation is performed in the environment of b+a.
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents or instruments used in the examples of the present invention were not manufacturer-identified and were commercially available conventional reagent products. The experimental environment descriptions of the B-class environment, the B+A environment and the like are clean zone grades, and conform to the requirements of the pharmaceutical manufacturing practice (GMP).
Example 1
(1) Effect of different temperatures on product endotoxin at pH 10.0:
sodium hyaluronate powder was weighed, dissolved with water for injection to prepare a concentration of 0.4% (w/w), and added with a quantitative amount of bacterial endotoxin to prepare a test solution of bacterial endotoxin 1EU/ml to prepare 4kg.
And (3) transferring the sterilized 4 beakers with the number of 1-4 to a grade A air supply environment, adding 1kg into each beaker, heating each beaker respectively, heating the beakers to 60 ℃, heating the beakers to 70 ℃, heating the beakers to 80 ℃ and adjusting the pH value of the solution to 10.0.
The temperature and pH of the solution are kept, sampling detection is carried out for 4 hours, and the detection result of bacterial endotoxin (the sensitivity of limulus reagent is 0.5-0.015 EU/ml) is recorded.
(2) Effect of different temperatures on product kinematic viscosity at pH 10.0:
sodium hyaluronate powder was weighed and dissolved with water for injection to give a concentration of 0.4% (w/w).
And 6 beakers are numbered 1-6, 1kg of each beaker is added, each beaker is heated, the temperature of each beaker is respectively increased, the temperature of each beaker is increased to 25 ℃, the temperature of each beaker is increased to 30 ℃, the temperature of each beaker is increased to 35 ℃, the temperature of each beaker is increased to 40 ℃, the temperature of each beaker is increased to 45 ℃, the temperature of each beaker is increased to 50 ℃, and the pH of each beaker is adjusted to 10.0.
The solution temperature and pH were maintained and samples were taken 4 hours to detect and record the kinematic viscosity of the solution.
The experimental results are shown in figure 1: when the pH is 10.0, the kinematic viscosity of the test solution is kept unchanged at 25-40 ℃, and the kinematic viscosity is slightly reduced at 40-50 ℃; the solution was tested for bacterial endotoxin removal effect, only when > 60 ℃ and more pronounced with increasing temperature.
Example 2
(1) Effect of different temperatures on product endotoxin at pH 10.5:
sodium hyaluronate powder was weighed, dissolved with water for injection to prepare a concentration of 0.4% (w/w), and added with a quantitative amount of bacterial endotoxin to prepare a test solution of bacterial endotoxin 1EU/ml to prepare 4kg.
And (3) transferring the sterilized 4 beakers with the number of 1-4 to a grade A air supply environment, adding 1kg into each beaker, heating each beaker respectively, heating the beakers 1 to 30 ℃, heating the beakers 2 to 40 ℃, heating the beakers 3 to 50 ℃, heating the beakers 4 to 60 ℃, and regulating the pH of the solution to 10.5.
The temperature and pH of the solution are kept, sampling detection is carried out for 4 hours, and the detection result of bacterial endotoxin (the sensitivity of limulus reagent is 0.5-0.015 EU/ml) is recorded.
(2) Effect of different temperatures on product kinematic viscosity at pH 10.5:
sodium hyaluronate powder was weighed and dissolved with water for injection to give a concentration of 0.4% (w/w).
And 6 beakers are numbered 1-6, 1kg of each beaker is added, each beaker is heated, the temperature of each beaker is respectively increased, the temperature of each beaker is increased to 25 ℃, the temperature of each beaker is increased to 30 ℃, the temperature of each beaker is increased to 35 ℃, the temperature of each beaker is increased to 40 ℃, the temperature of each beaker is increased to 45 ℃, the temperature of each beaker is increased to 50 ℃, and the pH of each beaker is adjusted to 10.5.
The solution temperature and pH were maintained and samples were taken 4 hours to detect and record the kinematic viscosity of the solution.
The experimental results are shown in fig. 2, which show that: when the pH is 10.5, the kinematic viscosity of the test solution is basically maintained unchanged at 25-40 ℃, and the reduction is obvious when the relative pH is 10.0 at 40-50 ℃; the bacterial endotoxin removal effect of the solution was tested and only effective when > 30 ℃ and more pronounced with increasing temperature, and the temperature interval for endotoxin removal was decreasing with increasing pH.
Example 3
(1) Effect of different temperatures on product endotoxin at pH 11.0:
sodium hyaluronate powder was weighed, dissolved with water for injection to prepare a concentration of 0.4% (w/w), and added with a quantitative amount of bacterial endotoxin to prepare a test solution of bacterial endotoxin 1EU/ml to prepare 4kg.
And (3) transferring the sterilized 4 beakers with the number of 1-4 to a grade A air supply environment, adding 1kg into each beaker, heating each beaker respectively, heating the beakers to 25 ℃, heating the beakers to 30 ℃, heating the beakers to 35 ℃, heating the beakers to 40 ℃ and adjusting the pH value of the solution to 11.0.
The temperature and pH of the solution are kept, sampling detection is carried out for 4 hours, and the detection result of bacterial endotoxin (the sensitivity of limulus reagent is 0.5-0.015 EU/ml) is recorded.
(2) Effect of different temperatures on product kinematic viscosity at pH 11.0:
sodium hyaluronate powder was weighed and dissolved with water for injection to give a concentration of 0.4% (w/w).
And 6 beakers are numbered 1-6, 1kg of each beaker is added, each beaker is heated, the temperature of each beaker is respectively increased, the temperature of each beaker is increased to 25 ℃, the temperature of each beaker is increased to 30 ℃, the temperature of each beaker is increased to 35 ℃, the temperature of each beaker is increased to 40 ℃, the temperature of each beaker is increased to 45 ℃, the temperature of each beaker is increased to 50 ℃, and the pH of each beaker is adjusted to 11.0.
The solution temperature and pH were maintained and samples were taken 4 hours to detect and record the kinematic viscosity of the solution.
The experimental results are shown in fig. 3, which shows that: when the pH is 11.0, the kinematic viscosity of the test solution is basically maintained unchanged at 25-35 ℃, and the reduction is obvious at 40-50 ℃; the bacterial endotoxin removal effect of the solution was tested and the endotoxin removal temperature interval was further reduced with increasing pH.
Example 4
(1) Effect of different temperatures on product endotoxin at pH 11.5:
sodium hyaluronate powder was weighed, dissolved with water for injection to prepare a concentration of 0.4% (w/w), and added with a quantitative amount of bacterial endotoxin to prepare a test solution of bacterial endotoxin 1EU/ml to prepare 4kg.
And (3) transferring the sterilized 4 beakers with the number of 1-4 to a grade A air supply environment, adding 1kg into each beaker, heating each beaker respectively, heating the beakers to 25 ℃, heating the beakers to 30 ℃, heating the beakers to 35 ℃, heating the beakers to 40 ℃ and adjusting the pH value of the solution to 11.5.
The temperature and pH of the solution are kept, sampling detection is carried out for 4 hours, and the detection result of bacterial endotoxin (the sensitivity of limulus reagent is 0.5-0.015 EU/ml) is recorded.
(2) Effect of different temperatures on product kinematic viscosity at pH 11.5:
sodium hyaluronate powder was weighed and dissolved with water for injection to give a concentration of 0.4% (w/w).
And 6 beakers are numbered 1-6, 1kg of each beaker is added, each beaker is heated, the temperature of each beaker is respectively increased, the temperature of each beaker is increased to 25 ℃, the temperature of each beaker is increased to 30 ℃, the temperature of each beaker is increased to 35 ℃, the temperature of each beaker is increased to 40 ℃, the temperature of each beaker is increased to 45 ℃, the temperature of each beaker is increased to 50 ℃, and the pH of each beaker is adjusted to 11.5.
The solution temperature and pH were maintained and samples were taken 4 hours to detect and record the kinematic viscosity of the solution.
The experimental results are shown in fig. 4, which show that: when the pH is 11.5, the kinematic viscosity of the test solution is reduced at 25-35 ℃ and is reduced more obviously at 40-50 ℃; the bacterial endotoxin removal effect of the solution was tested and the endotoxin removal temperature interval was further reduced with increasing pH.
From the above experimental data of examples 1 to 4, it can be demonstrated that the endotoxin removal effect is better with increasing pH and the temperature interval is lower. The higher the pH, the faster the kinematic viscosity of the product is degraded, and the higher the temperature, the faster the degradation speed is. In order to keep the product kinematic viscosity not degraded or slightly degraded, keep the stability of the molecular weight of the product and remove endotoxin, according to the trend chart of examples 1-4, the pH range is determined to be 10.50-11.00, and the temperature range is determined to be 30-40 ℃.
Example 5
As can be seen from examples 1-4, when the pH is in the range of 10.5-11.0 and the temperature is in the range of 30-40 ℃, endotoxin can be removed while the high molecular weight sodium hyaluronate is not dissociated, and the stability of the high molecular weight sodium hyaluronate in alkaline environment is further improved for better protection.
Step S2, at the temperature of 35 ℃, using solid sodium hydroxide to adjust the pH of the sodium hyaluronate solution to 10.7, simultaneously introducing nitrogen at the speed of 15ml/min, and stirring for 4 hours;
the detection molecular weight of the product is 207 ten thousand daltons, the endotoxin of the product is less than 0.005, and the product is sterile.
On the one hand, nitrogen is introduced to protect the stability of high molecular sodium hyaluronate, and the molecular motion in the solution is increased to accelerate the dissolution of sodium hydroxide particles, so that the antioxidant capacity of sodium hyaluronate can be improved.
Example 6
Example 5 nitrogen gas was introduced during sterilization to increase the stability of the sodium hyaluronate of the polymer and the controllability of the scale of the preparation process. To further optimize the bactericidal effect and the stability of sodium hyaluronate.
In the step 2, the pH of the sodium hyaluronate solution is regulated to 10.7 by using solid sodium hydroxide at the temperature of 40 ℃, nitrogen is introduced, the temperature is kept for 1 hour by stirring, and then the temperature is uniformly reduced to 2 ℃ every 15 minutes until the temperature reaches 30 ℃ and kept for 45 minutes.
The detection molecular weight of the product is 213 ten thousand daltons, the endotoxin of the product is less than 0.005, and the product is sterile.
The initial temperature is 40 ℃ to help regulate ph of dissolved solid sodium hydroxide particles, and at 40 ℃, bacterial death speed is high, high-level endotoxin removal can be maintained under the condition, then the temperature is uniformly reduced, the temperature is reduced in the process, ph floating caused by decomposition of sodium hydroxide can be avoided, and the stability of the sodium hyaluronate solution can be maintained at low temperature.
Example 7
In example 6, the stability of the sodium hyaluronate solution was increased while endotoxin was efficiently removed by the gradient temperature change, and further improved to enhance the applicability of the overall preparation process.
As shown in fig. 5, the reaction vessel 10 comprises a liquid outlet 1, a liquid inlet 2, a serpentine pipe 3, a ph adjusting port 4, a U-shaped pipe 7 and a mixing tank 8;
the liquid outlet 1 is arranged at one end, close to the bottom, of the side wall of the reaction container 10;
the liquid inlet 2 is arranged at one end, close to the top, of the side wall of the reaction vessel 10;
the U-shaped pipe 7 is arranged on the central axis of the bottom of the reaction vessel 10, and the bending part of the U-shaped pipe 7 is fixedly connected with the bottom of the reaction vessel 10;
the coiled pipe 3 is arranged at one end of the U-shaped pipe 7 and communicated with the U-shaped pipe 7, and the mixing tank 8 is arranged at one end of the coiled pipe 3 far away from the U-shaped pipe 7;
the ph adjusting port 4 is arranged in an extension section of one end of the U-shaped pipe 7 far away from the coiled pipe 3 and is fixed on the side wall of the reaction container 10, and an infusion port 9 is reserved above the ph adjusting port 4;
the U-shaped pipe 7 comprises a baffle 6 and a bubble generator 5;
the bubble generator 5 is arranged at the joint of the U-shaped pipe 7 and the bottom of the reaction vessel 10; the baffle plate 6 is arranged at one end, close to the ph adjusting port 4, of the bubble generator 5;
when the device works, firstly, sodium hyaluronate solution is injected from a liquid inlet 2, when the sodium hyaluronate solution is full of three fourths of the volume of a reaction container 10, solid sodium hydroxide particles are put into a ph adjusting port 4, meanwhile, a bubble generator 5 is opened to be filled with nitrogen, under the action of water flow, the sodium hydroxide particles enter a U-shaped pipe 7 along with the flowing of liquid and move along with the bubbles in the direction of the coiled pipe 3 due to the movement of the bubbles to the direction of the coiled pipe 3, sodium hydroxide completely dissolves in the solution and is uniformly mixed in the process, and the mixing speed of the solution is quickened again in the rising process of the bubbles in the coiled pipe 3, so that the solution in the mixing tank 8 tends to be more uniform; the bubble generator 5 is also provided with a temperature control device, the temperature of bubbles can be adjusted, the temperature is kept for 20 minutes at 40 ℃ initially, then the temperature is uniformly reduced to 30 ℃ within 20 minutes, the temperature is kept for 20 minutes, 1 hour is a cycle, after the operation is carried out for 4 hours, the liquid inlet 2 continues to be fed, the liquid feeding speed is the speed at which the reaction vessel 10 can be filled for 4 hours, sodium hydroxide particles are continuously added, and ph change is monitored in real time.
The detection molecular weight of the product is 215 ten thousand, the endotoxin of the product is less than 0.005, and the product is sterile.
Note that: the method for detecting endotoxin is a gel limit method, which is a method for detecting or quantifying bacterial endotoxin produced by gram-negative bacteria by using a limulus reagent to determine whether the limit of bacterial endotoxin in a sample meets a predetermined specification.
Sodium hydroxide particles are dissolved in advance through a coiled pipe 3 and uniformly mixed to prevent local high alkaline solution from dissociating high molecular sodium hyaluronate, the U-shaped pipe 7 and bubbles form liquid flow to drive sodium hydroxide to move and uniformly mix, the baffle 6 is arranged to prevent bubbles from flowing backwards, new sodium hyaluronate solution is continuously injected in a circulating temperature change mode, the circulating preparation can be realized, a large amount of time is saved, and the temperature change in the solution is more uniform by changing the temperature of the bubbles.
The foregoing is a description of only a limited number of preferred embodiments of the invention, which are described in greater detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. A method for preparing sterile sodium hyaluronate, which is characterized by comprising the following steps:
s1: dissolving non-sterile sodium hyaluronate powder in water to obtain sodium hyaluronate solution;
s2: adjusting the pH of the sodium hyaluronate solution to be alkaline at a temperature lower than 40 ℃ and stirring;
s3: adding activated carbon for activated carbon adsorption;
s4: filtering to remove active carbon;
s5: sterilizing and filtering by using a sterilizing filter;
s6: crystallizing the sterilized and filtered solution by using ethanol;
s7: dehydrating and drying the crystallized product to obtain a product;
s8: packaging the dried product;
step 2 is completed by the reaction vessel (10).
2. The method for preparing sterile sodium hyaluronate according to claim 1, wherein the water in the step 1 is water for injection, the concentration of the sodium hyaluronate solution is 0.2-1.0% (w/w), and the molecular weight is 100-300 kilodaltons.
3. The preparation method of sterile sodium hyaluronate according to claim 1, wherein in the step 2, the pH is 10.50-11.00, the temperature is 30-40 ℃, and the stirring treatment time is 3-4 hours; the amount of the activated carbon in the step 3 is 0.2-0.5% (w/w), and the adsorption time is 15-30 min.
4. The method for preparing sterile sodium hyaluronate according to claim 1, wherein the sterilization filtration in step 5 uses two-stage sterilization filtration including prefiltration using a 0.45 μm filter and secondary filtration using a 0.2 μm filter; the ethanol in the step 6 is ethanol with the mass percentage concentration of 95 percent after sterilization and filtration; the mass ratio of the solution to the ethanol is 1: 1.5-2.
5. The method for preparing sterile sodium hyaluronate according to claim 1, wherein step 6 and step 7 are performed in a class B environment; the sub-packaging in the step 8 is carried out in the environment of the B+A environment.
6. A process for the preparation of sterile sodium hyaluronate according to claim 3, characterized in that in step 2 the pH of the sodium hyaluronate solution is adjusted to 10.7 at an initial temperature of 40 ℃, the temperature is maintained for 1 hour with stirring, after which the temperature is uniformly reduced by 2 ℃ every 15 minutes up to 30 ℃ and maintained for 45 minutes.
7. The method of claim 6, wherein solid sodium hydroxide particles are used for adjusting ph in step 2.
8. The method for preparing sterile sodium hyaluronate according to claim 7, wherein ph is adjusted while introducing nitrogen gas; the nitrogen flow rate was 15ml/min.
9. The preparation method of the sterile sodium hyaluronate according to claim 8, wherein the reaction vessel (10) adopted in the step 2 comprises a liquid outlet (1), a liquid inlet (2), a serpentine pipe (3), a ph adjusting port (4), a U-shaped pipe (7) and a mixing tank (8);
the liquid outlet (1) is arranged at one end, close to the bottom, of the side wall of the reaction container (10);
the liquid inlet (2) is arranged at one end, close to the top, of the side wall of the reaction container (10);
the U-shaped pipe (7) is arranged on the central axis of the bottom of the reaction vessel (10), and the bending part of the U-shaped pipe (7) is fixedly connected with the bottom of the reaction vessel (10);
the mixing tank (8) is arranged at one end of the coiled pipe (3) far away from the U-shaped pipe (7);
the ph adjusting port (4) is arranged in an extension section of one end, far away from the coiled pipe (3), of the U-shaped pipe (7), and is fixed on the side wall of the reaction container (10), and an infusion port (9) is reserved above the ph adjusting port (4);
the U-shaped pipe (7) comprises a baffle plate (6) and a bubble generator (5);
the bubble generator (5) is arranged at the joint of the U-shaped pipe (7) and the bottom of the reaction vessel (10); the baffle (6) is arranged at one end near the bubble generator (5) and close to the ph adjusting opening (4).
10. The use of sterile sodium hyaluronate prepared by the process for preparing sterile sodium hyaluronate according to any one of claims 1-9, characterized in that it is used in the preparation of a pharmaceutical, cosmetic or health food.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203223906U (en) * | 2013-01-29 | 2013-10-02 | 北京源深节能技术有限责任公司 | Boiler system and boiler water-supply system with U-shaped flue |
| CN105985994A (en) * | 2015-03-11 | 2016-10-05 | 上海其胜生物制剂有限公司 | Method for preparing sodium hyaluronate by means of salt-free and organic solvent-free purification technology |
| CN112940148A (en) * | 2021-04-06 | 2021-06-11 | 山东众山生物科技有限公司 | Hyaluronic acid purification method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203223906U (en) * | 2013-01-29 | 2013-10-02 | 北京源深节能技术有限责任公司 | Boiler system and boiler water-supply system with U-shaped flue |
| CN105985994A (en) * | 2015-03-11 | 2016-10-05 | 上海其胜生物制剂有限公司 | Method for preparing sodium hyaluronate by means of salt-free and organic solvent-free purification technology |
| CN112940148A (en) * | 2021-04-06 | 2021-06-11 | 山东众山生物科技有限公司 | Hyaluronic acid purification method |
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