WO2021025656A1 - Fast and effective purification method in dermal filler production - Google Patents
Fast and effective purification method in dermal filler production Download PDFInfo
- Publication number
- WO2021025656A1 WO2021025656A1 PCT/TR2020/050695 TR2020050695W WO2021025656A1 WO 2021025656 A1 WO2021025656 A1 WO 2021025656A1 TR 2020050695 W TR2020050695 W TR 2020050695W WO 2021025656 A1 WO2021025656 A1 WO 2021025656A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- solution
- gels
- gel
- bdde
- mixture
- Prior art date
Links
- 239000000945 filler Substances 0.000 title claims abstract description 22
- 230000002500 effect on skin Effects 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 238000000746 purification Methods 0.000 title abstract description 25
- 238000000034 method Methods 0.000 title abstract description 18
- 239000000499 gel Substances 0.000 claims abstract description 29
- 239000000203 mixture Substances 0.000 claims abstract description 16
- 239000002245 particle Substances 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 230000003444 anaesthetic effect Effects 0.000 claims abstract description 3
- 238000011049 filling Methods 0.000 claims abstract description 3
- 230000001954 sterilising effect Effects 0.000 claims abstract description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 3
- 238000005406 washing Methods 0.000 claims abstract description 3
- SHKUUQIDMUMQQK-UHFFFAOYSA-N 2-[4-(oxiran-2-ylmethoxy)butoxymethyl]oxirane Chemical compound C1OC1COCCCCOCC1CO1 SHKUUQIDMUMQQK-UHFFFAOYSA-N 0.000 claims description 31
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 239000008055 phosphate buffer solution Substances 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 238000004132 cross linking Methods 0.000 claims description 6
- 238000006386 neutralization reaction Methods 0.000 claims description 4
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 claims description 3
- 238000001125 extrusion Methods 0.000 claims description 3
- 229960004194 lidocaine Drugs 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 239000008240 homogeneous mixture Substances 0.000 claims description 2
- 239000003431 cross linking reagent Substances 0.000 abstract description 5
- 238000009472 formulation Methods 0.000 abstract description 2
- YECIFGHRMFEPJK-UHFFFAOYSA-N lidocaine hydrochloride monohydrate Chemical compound O.[Cl-].CC[NH+](CC)CC(=O)NC1=C(C)C=CC=C1C YECIFGHRMFEPJK-UHFFFAOYSA-N 0.000 abstract 1
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (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-carboxylic acid Chemical compound 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 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 28
- 229920002674 hyaluronan Polymers 0.000 description 28
- 229960003160 hyaluronic acid Drugs 0.000 description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 239000000017 hydrogel Substances 0.000 description 10
- 235000019441 ethanol Nutrition 0.000 description 7
- 239000012535 impurity Substances 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 5
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- UWFRVQVNYNPBEF-UHFFFAOYSA-N 1-(2,4-dimethylphenyl)propan-1-one Chemical compound CCC(=O)C1=CC=C(C)C=C1C UWFRVQVNYNPBEF-UHFFFAOYSA-N 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- KWOLFJPFCHCOCG-UHFFFAOYSA-N Acetophenone Chemical compound CC(=O)C1=CC=CC=C1 KWOLFJPFCHCOCG-UHFFFAOYSA-N 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 108010003272 Hyaluronate lyase Proteins 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 125000000600 disaccharide group Chemical group 0.000 description 2
- AFOSIXZFDONLBT-UHFFFAOYSA-N divinyl sulfone Chemical compound C=CS(=O)(=O)C=C AFOSIXZFDONLBT-UHFFFAOYSA-N 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000001815 facial effect Effects 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 229960003966 nicotinamide Drugs 0.000 description 2
- 235000005152 nicotinamide Nutrition 0.000 description 2
- 239000011570 nicotinamide Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000005456 alcohol based solvent Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- GYZLOYUZLJXAJU-UHFFFAOYSA-N diglycidyl ether Chemical compound C1OC1COCC1CO1 GYZLOYUZLJXAJU-UHFFFAOYSA-N 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 150000002118 epoxides Chemical class 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013020 final formulation Substances 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 229920001002 functional polymer Polymers 0.000 description 1
- 239000003193 general anesthetic agent Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229920013730 reactive polymer Polymers 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
Definitions
- the present invention relates to the reduction of production time significantly by improving the purification process in the production of dermal filler and the effective removal of the residual cross-linking agent (BDDE) which can be relatively toxic.
- BDDE residual cross-linking agent
- Hyaluronic acid (HA) based fillers are the most common and popular dermal fillers used for aesthetic purposes. Hyaluronic acid substances are normally found under the skin. Skin aging and defects on facial contours can be eliminated with fillers and a more vivid bright appearance can be obtained.
- Dermal fillers based on hyaluronic acid are generally gel-like structures containing more than 95% water and 0.5 to 3% HA.
- HA is a polysaccharide composed of repetitive disaccharide units of (l,4)-gluronic acid-P(l,3)-N- acetylglucosamine, linked by glycosidic bonds. This disaccharide structure is the same among all living organisms. This feature makes HA a biocompatible molecule compared to protein-based fillers such as collagen.
- HA when used as a dermal filler is the rapid degradation by the hyaluronidase family of enzymes in the tissue. Many chemical modifications have been made to increase the half-life of HA in the tissue.
- the most preferred method is the crosslinking of HA polymer chains with the help of synthetic agents and providing more resistance towards enzymatic degradation. With this method, dermal fillers can maintain their effect in the tissue for 3 to 24 months.
- cross-linking agents methacrylamide, hydrazide, carbodiimide, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE) and poly (ethylene glycol) diglycidyl ether were used.
- the most widely used agent among these is BDDE.
- BDDE is a biodegradable substance with less toxicity than other ether-based crosslinking agents.
- hydrogels have been proven safe for a long time, the crosslinking agents used are reactive agents that can be cytotoxic and, in some cases, mutagenic. Therefore, the concentration of BDDE, which may be present as a residue in the final product, is limited by the FDA to ⁇ 2 ppm (two in a million).
- Purification processes are generally carried out in distilled water or phosphate buffer solution. The gels placed into these solutions swell over time and purification is provided by removing the impurities that are not reacted.
- purification is also performed by precipitation of cross-linked hydrogels in alcohol solvent such as ethanol.
- alcohol solvent such as ethanol.
- crosslinked gels settle to bottom of the vessels as white solids.
- the precipitation process is repeated several times, allowing the entire gel to settle completely.
- the precipitated solid is filtered or decanted from the liquid medium and dried. Residual BDDE and other impurities are removed from the medium in the liquid phase.
- US patent document US 20070224277 A 1 mentions that the purification of cross-linked hydrogels based on hyaluronic acid (HA) is carried out in distilled water for 7 days.
- HA hyaluronic acid
- hydrogels were obtained using HA and BDDE crosslinker and the purification process was carried out in phosphate buffer solution for 185 hours.
- the aim of the invention is to shorten the purification process, which is the most time-consuming process in the production of dermal fillers, thereby saving time.
- Another object of the invention is to significantly reduce the concentration of residual BDDE in the dermal filler production process.
- Another object of the invention is to easily remove impurities from the medium by using the multiple washing-filtering processes.
- the invention is the dermal filler production process
- a dermal filler production process comprising the steps of
- the process of the invention is to obtain gels that allow HA and BDDE to be used as dermal fillers as a result of the cross-linking reaction.
- the non-crosslinked HA was added to the final formulation (0.25-0.60%) to ensure easy extrusion.
- Total HA concentration is 20 mg / ml and contains lidocaine HC1, which is 0.25-0.30% anesthetic agent.
- the residual BDDE content found in hydrogels is determined by fluorescence spectrophotometry. Epoxy compounds in the content of BDDE can react with nicotinamide to produce fluorescence under excitation at 370 nm. The fluorescent intensity is directly proportional to the epoxide content, which can be detected at an emission wavelength of 430 nm. Accordingly, BDDE solutions were prepared and analyzes were carried out to obtain the standard graphic. BDDE is diluted to different concentrations with ultrapure water. 20 pL of diluted BDDE solution was mixed with 10 pL of 0.125 M nicotinamide. The mixtures were incubated in a water bath at 37 0 C for 2 hours.
- hydrogels were incubated in hyaluronidase enzyme at 37 0 C for 24 hours and then centrifugation was carried out. The supernatant was filtered through a 0.22 pm pore size membrane and analyzed by fluorescence spectrophotometer.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Materials For Medical Uses (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention is a purification method required for a dermal filler production process. The object of the invention is to save considerable time and to effectively remove the residual cross-linking agent in the formulation, in contrast to conventionally used purification methods. After the production of cross-linked dermal filler gels, the particle sizes of the gels are reduced to approximately 500-2000 microns with the help of a homogenizer, then washed 7 more times with PBS solution during one hour in each washing, then filtering, bringing the gels to the desired particle sizes, adding Lidocaine HCl to the final mixture to provide an anesthetic effect, adjusting the pH of the final mixture to 7, filling 1 ml syringes under vacuum and performing steam sterilization of the filled syringes.
Description
FAST AND EFFECTIVE PURIFICATION METHOD IN DERMAL
FILLER PRODUCTION
Technical Field
The present invention relates to the reduction of production time significantly by improving the purification process in the production of dermal filler and the effective removal of the residual cross-linking agent (BDDE) which can be relatively toxic.
Background Of The Invention Dermal fillers that offer different solutions for each region in humans and generally contain hyaluronic acid are frequently used for the facial area where aging is clearly seen. Hyaluronic acid (HA) based fillers are the most common and popular dermal fillers used for aesthetic purposes. Hyaluronic acid substances are normally found under the skin. Skin aging and defects on facial contours can be eliminated with fillers and a more vivid bright appearance can be obtained.
Dermal fillers based on hyaluronic acid (HA) are generally gel-like structures containing more than 95% water and 0.5 to 3% HA. HA is a polysaccharide composed of repetitive disaccharide units of (l,4)-gluronic acid-P(l,3)-N- acetylglucosamine, linked by glycosidic bonds. This disaccharide structure is the same among all living organisms. This feature makes HA a biocompatible molecule compared to protein-based fillers such as collagen.
The major disadvantage of HA when used as a dermal filler is the rapid degradation by the hyaluronidase family of enzymes in the tissue. Many chemical modifications have been made to increase the half-life of HA in the tissue. The most preferred method is the crosslinking of HA polymer chains with the help of synthetic agents
and providing more resistance towards enzymatic degradation. With this method, dermal fillers can maintain their effect in the tissue for 3 to 24 months.
As cross-linking agents, methacrylamide, hydrazide, carbodiimide, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE) and poly (ethylene glycol) diglycidyl ether were used. The most widely used agent among these is BDDE. BDDE is a biodegradable substance with less toxicity than other ether-based crosslinking agents. Although hydrogels have been proven safe for a long time, the crosslinking agents used are reactive agents that can be cytotoxic and, in some cases, mutagenic. Therefore, the concentration of BDDE, which may be present as a residue in the final product, is limited by the FDA to < 2 ppm (two in a million).
In order to remove residual BDDE, it is necessary to perform purification processes after the cross-linking reaction. Purification processes are generally carried out in distilled water or phosphate buffer solution. The gels placed into these solutions swell over time and purification is provided by removing the impurities that are not reacted.
In some studies in the technique, purification is also performed by precipitation of cross-linked hydrogels in alcohol solvent such as ethanol. In this case, crosslinked gels settle to bottom of the vessels as white solids. The precipitation process is repeated several times, allowing the entire gel to settle completely. The precipitated solid is filtered or decanted from the liquid medium and dried. Residual BDDE and other impurities are removed from the medium in the liquid phase.
In the state of the art, “Evaluation of in-vitro degradation rate of hyaluronic acid- based hydrogel cross-linked with 1,4-butanediol diglycidyl ether (BDDE) using RP-HPLC and UV-Vis spectroscopy”, published in Journal of Drug Delivery Science and Technology, Volume 29, October 2015 between pages 24-30, purification of HA / BDDE cross-linked gels was carried out and the process was continued in distilled water for 2 days.
In the state of the art, in the article titled “Influence of Molecular Weight on Swelling and Elastic Modulus of Hyaluronic Acid Dermal Fillers” published on 03/08/2015 by Deuk Yong Lee, Cheolbyung Cheon, Siwon Son, Young-Zu Kim, Jin-Tae Kim, Ju-Woong Jang, and Seok-Soon Kim, hydrogels were obtained as a result of the crosslinking reaction with HA and BDDE. In order to remove impurities and residual BDDE in the gel, purification was done for 3 days in phosphate buffer solution and then for 3 more days in distilled water.
In another known state of the art, Reactive and Functional Polymers, Volume 109, published on December 2016, in the article between pages 42-51 "Preparation and fracture process of high strength hyaluronic acid hydrogels cross-linked by ethylene glycol diglycidyl ether" was obtained by reaction of HA and ethylene glycol diglycidyl ether (EGDE) crosslinker. It is stated that the applied time for swelling and purification of the gel is 4 days.
In another known state of the art, US patent document US 20070224277 A 1 mentions that the purification of cross-linked hydrogels based on hyaluronic acid (HA) is carried out in distilled water for 7 days.
In the European patent document EP2988791A1 known in the art, it is mentioned that the purification processes of the cross-linked silk and HA hydrogels are carried out in phosphate buffer solution for 8 days.
As mentioned in the European patent document EP2988791A1 known in the art, hydrogels were obtained using HA and BDDE crosslinker and the purification process was carried out in phosphate buffer solution for 185 hours.
Since the purification processes in distilled water or phosphate buffer solution have noticeably long durations, it takes considerably long time during production. Generally, these processes aim to remove the unreacted BDDE and other impurities by placing the gels in large pieces into the buffer solution and swelling the gel over
time. This not only lengthens the process in terms of time, but also carries the risk that the residual BDDE cannot be removed sufficiently.
After the purification processes carried out by settling in alcohol solvents such as ethanol, prolonged drying processes under vacuum for the complete removal of alcohol lead to extension of the process. Problems also arise when the structural properties of the gels obtained after drying are disturbed and re-swollen. Presence of residual alcohol in the final product should be determined by analytical methods. The necessity of additional tools or equipment is another disadvantage. In addition, the excess alcohol solvent used in the precipitation process causes additional costs.
Brief Description of the Invention
The aim of the invention is to shorten the purification process, which is the most time-consuming process in the production of dermal fillers, thereby saving time.
Another object of the invention is to significantly reduce the concentration of residual BDDE in the dermal filler production process.
Another object of the invention is to easily remove impurities from the medium by using the multiple washing-filtering processes.
Detailed Description of the Invention
The invention is the dermal filler production process,
A dermal filler production process comprising the steps of
- preparation of 1% NaOH solution in deionized water,
- gradually adding and dissolving HA in the solution, adding BDDE to HA mixture to obtain a homogeneous mixture,
- carrying out the crosslinking reaction by keeping the mixture in a water bath at 40°C for 4 hours,
- breaking up the gelled structure that is hardened after the reaction to sizes of approximately 1-2 cm and neutralization thereof with 0.1 M hydrochloric acid (HC1) in phosphate buffer solution (PBS) until the pH is 6.8-7.4,
- continuing neutralization until the pH of the solution is constant,
- filtering the gel structure by means of a sieve,
- adding new PBS solution to the filtered gel and ensuring that the gel swells for 5 hours, and characterized by the steps of
- reducing the size of the swollen gel to approximately 500-2000 microns by means of a homogenizer,
- washing with PBS solution 7 more times with an interval of one hour between the washes, and then filtering,
- bringing the gels to the desired particle sizes,
- adding non-crosslinked HA to the final mixture to facilitate extrusion, and adding lidocaine HC1 to the same to provide an anesthetic effect,
- adjusting the pH of the final mixture to 7, filling it into syringes of 1 ml under vacuum, and performing steam sterilization of the filled syringes.
The process of the invention is to obtain gels that allow HA and BDDE to be used as dermal fillers as a result of the cross-linking reaction. The non-crosslinked HA was added to the final formulation (0.25-0.60%) to ensure easy extrusion. Total HA concentration is 20 mg / ml and contains lidocaine HC1, which is 0.25-0.30% anesthetic agent.
Table 1. Composition ratios required to obtain cross-linked HA gel
The formulation specified in Table 1 was repeated three times (Trial 1), and the analyzes were repeated three times for each trial. The residual BDDE amounts are shown in Table 2 by taking the average of the repeated analysis results. In addition to the results, the results obtained in the other experiment (Trial 2) prepared with the same components and ratios are also shown in Table 2. In the study named Trial 2 mentioned in Table 2, the purification process was done without breaking the gels into small particles in PBS. Total purification time is in the range of 48-144 hours, and final products are obtained by taking samples every day. BDDE analyzes of the obtained gels were made and shown in Table 2.
Residual BDDE Analysis
The residual BDDE content found in hydrogels is determined by fluorescence spectrophotometry. Epoxy compounds in the content of BDDE can react with nicotinamide to produce fluorescence under excitation at 370 nm. The fluorescent intensity is directly proportional to the epoxide content, which can be detected at an
emission wavelength of 430 nm. Accordingly, BDDE solutions were prepared and analyzes were carried out to obtain the standard graphic. BDDE is diluted to different concentrations with ultrapure water. 20 pL of diluted BDDE solution was mixed with 10 pL of 0.125 M nicotinamide. The mixtures were incubated in a water bath at 37 0 C for 2 hours. Then, 100 pL of 15% acetophenone (dissolved in ethyl alcohol) and 100 pL of 1 M potassium hydroxide (KOH) were added to the mixtures and mixed in an ice bath. Then 500 pL of methanoic acid was added and the mixtures were incubated for 5 more minutes at 600 C in a water bath. The standard graphic was prepared using fluoro spectrophotometry.
The obtained hydrogels were incubated in hyaluronidase enzyme at 37 0 C for 24 hours and then centrifugation was carried out. The supernatant was filtered through a 0.22 pm pore size membrane and analyzed by fluorescence spectrophotometer.
Table 2. Amount of residual BDDE after purification process
In the results of the analysis, it is seen that the residual BDDE amounts in the purification process by reducing the particle size are well below the 2 ppm determined by the FDA as the limit (0.13 ppm). It is seen that these values are higher than 2 ppm in the purification processes of the dermal filler gel without decreasing the particle size (in the range of 24-120 hours). However, when 144 hours of purification is done, the amount of BDDE drops below the desired level.
Since the surface areas of the dermal filler gels, which are broken into small pieces, are getting smaller, they enter the water molecules faster and faster and swell and remove the impurities inside. As a very effective and fast method, breaking the gels into small pieces during purification provides ease of production and time saving.
Claims
1. A dermal filler production process comprising the steps of
- preparation of 1% NaOH solution in deionized water,
- gradually adding and dissolving HA in the solution,
- adding BDDE to HA mixture to obtain a homogeneous mixture,
- carrying out the crosslinking reaction by keeping the mixture in a water bath at 40°C for 4 hours,
- breaking up the gelled structure that is hardened after the reaction to sizes of approximately 1-2 cm and neutralization thereof with 0.1 M hydrochloric acid (HC1) in phosphate buffer solution (PBS) until the pH is 6.8-7.4,
- continuing neutralization until the pH of the solution is constant,
- filtering the gel structure by means of a sieve,
- adding new PBS solution to the filtered gel and ensuring that the gel swells for 5 hours, and characterized by the steps of
- reducing the size of the swollen gel to approximately 500-2000 microns by means of a homogenizer,
- washing with PBS solution 7 more times with an interval of one hour between the washes, and then filtering,
- bringing the gels to the desired particle sizes,
- adding non-crosslinked HA to the final mixture to facilitate extrusion, and adding lidocaine HC1 to the same to provide an anesthetic effect,
adjusting the pH of the final mixture to 7, filling it into syringes of 1 ml under vacuum, and - performing steam sterilization of the filled syringes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP20849479.9A EP4010040A4 (en) | 2019-08-08 | 2020-08-07 | Fast and effective purification method in dermal filler production |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TR2019/12107 | 2019-08-08 | ||
| TR2019/12107A TR201912107A2 (en) | 2019-08-08 | 2019-08-08 | FAST AND EFFECTIVE PURIFICATION METHOD IN DERMAL FILLER PRODUCTION |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2021025656A1 true WO2021025656A1 (en) | 2021-02-11 |
Family
ID=74504138
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/TR2020/050695 WO2021025656A1 (en) | 2019-08-08 | 2020-08-07 | Fast and effective purification method in dermal filler production |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP4010040A4 (en) |
| TR (1) | TR201912107A2 (en) |
| WO (1) | WO2021025656A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130203696A1 (en) * | 2011-06-03 | 2013-08-08 | Allergan, Inc. | Dermal filler compositions for fine line treatment |
| WO2019002369A1 (en) * | 2017-06-28 | 2019-01-03 | Nestlé Skin Health Sa | Glycosaminoglycan hydrogel with grafted dextran or cyclodextrin |
-
2019
- 2019-08-08 TR TR2019/12107A patent/TR201912107A2/en unknown
-
2020
- 2020-08-07 WO PCT/TR2020/050695 patent/WO2021025656A1/en unknown
- 2020-08-07 EP EP20849479.9A patent/EP4010040A4/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130203696A1 (en) * | 2011-06-03 | 2013-08-08 | Allergan, Inc. | Dermal filler compositions for fine line treatment |
| WO2019002369A1 (en) * | 2017-06-28 | 2019-01-03 | Nestlé Skin Health Sa | Glycosaminoglycan hydrogel with grafted dextran or cyclodextrin |
Non-Patent Citations (1)
| Title |
|---|
| See also references of EP4010040A4 * |
Also Published As
| Publication number | Publication date |
|---|---|
| EP4010040A4 (en) | 2023-06-14 |
| EP4010040A1 (en) | 2022-06-15 |
| TR201912107A2 (en) | 2021-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Maiti et al. | Physical and self–crosslinking mechanism and characterization of chitosan-gelatin-oxidized guar gum hydrogel | |
| Montembault et al. | Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium | |
| Kang et al. | Photocrosslinked methacrylated carboxymethyl chitin hydrogels with tunable degradation and mechanical behavior | |
| Yang et al. | Construction and characterization of Mesona chinensis polysaccharide-chitosan hydrogels, role of chitosan deacetylation degree | |
| US20190046429A1 (en) | Dermal filler composed of macroporous chitosan microbeads and cross-linked hyaluronic acid | |
| EP2199308B1 (en) | Swellable crosslinked hyaluronic acid powder and method for producing the same | |
| Andac et al. | Poly (hydroxyethyl methacrylate)‐based macroporous hydrogels with disulfide cross‐linker | |
| Tan et al. | Synthesis and characterization of injectable photocrosslinking poly (ethylene glycol) diacrylate based hydrogels | |
| CN105111512A (en) | Chemical crosslinking type glucan hydrogel and preparation method thereof | |
| EP1951761B1 (en) | New derivatives of hyaluronic acid, their preparation process and their uses | |
| US12102735B2 (en) | Biocompatible hydrogel, process for producing same, and use thereof in a mechanical viscosupplementation system | |
| CN108478875B (en) | Preparation method and application of crosslinked hyaluronic acid gel microspheres | |
| Kim et al. | Stretchable and self-healable hyaluronate-based hydrogels for three-dimensional bioprinting | |
| Zhao et al. | Gamma ray-induced synthesis of hyaluronic acid/chondroitin sulfate-based hydrogels for biomedical applications | |
| CN111732741A (en) | Method for crosslinking hyaluronic acid and polylysine, composite crosslinked product obtained by method and application of composite crosslinked product | |
| CN107118360A (en) | A kind of soybean protein isolate base natural polymer hydrogel and preparation method thereof | |
| CN111574733A (en) | Preparation method of high-transparency ultraviolet shielding film | |
| CN112851988B (en) | Preparation method of sodium hyaluronate gel | |
| CN116903884A (en) | A kind of hyaluronic acid-polyglutamic acid hydrogel and preparation method thereof | |
| AU2004303599B2 (en) | Compositions of semi-interpenetrating polymer network | |
| Vo et al. | Fabrication and Characterization of Gelatin/Chitosan Hydrogel Membranes | |
| CN106999626B (en) | Biocompatible compositions and methods of preparation | |
| WO2021025656A1 (en) | Fast and effective purification method in dermal filler production | |
| CN115671388A (en) | Performance-adjustable silk protein injectable microsphere gel and preparation method thereof | |
| CN111499889A (en) | Chondroitin sulfate magnesium hyaluronic acid hydrogel, preparation method and application thereof, and gel product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20849479 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| ENP | Entry into the national phase |
Ref document number: 2020849479 Country of ref document: EP Effective date: 20220309 |