TR201912107A2 - FAST AND EFFECTIVE PURIFICATION METHOD IN DERMAL FILLER PRODUCTION - Google Patents

Abstract

The invention is a purification method required for a dermal filler manufacturing process. The aim of the invention is to save a lot of time and to effectively remove the residual cross-linking agent in the formulation, in contrast to traditionally 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, 7 more washing times with PBS solution with an hour's interval, then filtered, the gels are brought to the desired particle size, to facilitate the extrusion of non-crosslinked HA It includes the steps of adding lidocaine HCl to the final mixture to provide anesthetic effect, adjusting the pH of the final mixture to 7, filling it into 1 ml syringes under vacuum, and performing steam sterilization on the filled syringes.

Description

DESCRIPTION FAST AND EFFICIENT PURIFICATION IN DERMAL FILLER PRODUCTION METHOD Technical Area This invention is produced by improving the purification process in the production of dermal fillers. significantly reducing the duration of the drug and can be relatively toxic relates to the effective removal of residual cross-linking agent (BDDE). Prior Art It offers different solutions for each region in humans and generally contains hyaluronic acid. dermal fillers for the facial area where aging is evident is used frequently. Hyaluronic acid (HA) based fillers, aesthetic They are the most common and popular dermal fillers used for this purpose. hyaluronic acid are substances normally found under the skin. Skin recline and face The defects in the contours can be corrected with fillers and more vivid and shiny. view can be obtained.

Dermal fillers based on hyaluronic acid (HA), usually more than 95% water and gel-like structures containing 0.5 to 3% HA. HA is interconnected by glycosidic bonds. repetitive disaccharide of bound (1,4)-glucuronic acid-ß (1,3)-N-acetylglucosamine It is a polysaccharide consisting of This disaccharide structure is common to all living organisms. is the same between This feature can be combined with protein-based fillers such as collagen. makes HA a biologically compatible molecule when compared to

The biggest disadvantage of HA when used as a dermatological filler is tissue rapidly by a family of enzymes called hyaluronidases. is the breakdown. Many chemicals are used to increase the life of HA in the tissue. modification has been made. The most preferred method is HA polymer chains. cross-link each other with the help of synthetic agents and increase enzymatic resistance. It is a winning method. With this method, dermal fillers can be filled in the tissue for 3 to 24 months. can maintain its effect throughout. Methacrylamide, hydrazide, carbodiimide, divinyl sulfone as cross-linking agents (DVS), diglycidyl ether used. Among them, the most commonly used agent is BDDE.

BDDE is biodegradable, to other ether-based cross-linking agents It is a less toxic substance. hydrogels over a long period of time Although it has been proven to be safe, the cross-linking agents used are reactive agents that can be cytotoxic and, in some cases, mutagenic. Therefore, the last BDDE concentration that may be present as a residue in the final product limited to < 2 ppm (two parts per million).

After cross-linking reaction to remove residual BDDE It is necessary to carry out adequate purification processes. Purification processes usually in distilled water or phosphate buffer solution. is carried out. The gels thrown into these solutions swell over time. the process of purification by expelling impurities present and unreacted is provided.

In some studies in the art, the saturation process is used to transfer cross-linked hydrogels to ethanol. It is also made by precipitating it in an alcohol solvent, such as In this case cross The bound gels settle to the bottom as a white solid in the environment. precipitation The process is repeated several times to ensure that the entire gel collapses. collapsed The solid is filtered through a liquid medium or decanted and dried. The residual BDDE and other impurities are removed from the mold environment in the liquid phase.

In the state of the art, Journal of Drug Delivery Science and “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” In the article, between pages 24-30, HA/BDDE cross-linked gels Purification processes were carried out and in distilled water for 2 days. the process has been continued.

In the state of the art, Deuk Yong Lee, Cheolbyung Cheon, Siwon By Son, Young-Zu Kim, Jin-Tae Kim, Ju-Woong Jang, and Seok-Soon Kim “Influence of Molecular Weight on Swelling and Published on 03/08/2015 HA and BDDE in the article “Elastic Modulus of Hyaluronic Acid Dermal Fillers” Hydrogels were obtained as a result of cross-linking reaction. in gel Phosphate buffer solution for 3 days before removing impurities and residual BDDE After that, the purification process was carried out in distilled water for 3 more days.

Reactive and Functional Polymers, which is another known state of the art, Volume 109, published in December 2016, “Preparation and fracture process of high strength hyaluronic acid hydrogels cross-linked by ethylene glycol diglycidyl ether” between pages 42-51, hydrogels are made with HA and ethylene. It is obtained by the reaction of glycol diglycidyl ether (EGDE) crosslinker.

It was stated that the time applied for swelling and softening of the gel was 4 days.

In the United States patent document, cross-linked hyaluronic acid (HA)-based The purification of the hydrogels was carried out in distilled water for 7 days. is said to have been implemented.

In the European patent document number EP2988791A1 known in the art, the obtained Purification of cross-linked silk and HA hydrogels was done with phosphate for 8 days. It is mentioned that it is made in buffer solution.

It is mentioned in the European patent document EP2988791A1 known in the art. Hydrogels were obtained by using HA and BDDE crosslinker and The purification process was carried out in phosphate buffer solution for 185 hours. has been applied.

Purification processes in distilled water or phosphate buffer solution Since it takes a long time, it takes a lot of time during production.

Generally, in these processes, gels are placed in buffer solution in large pieces. BDDE and non-reacted BDDE and It is aimed to remove other impurities. Only in terms of time this situation Not only does it prolong the process too long, but at the same time the residual BDDE is sufficiently also carries the risk of not being removed.

It is carried out by precipitating in alcohol solvents such as ethanol. After the purification processes, the vacuum applied to completely remove the alcohol Drying processes that take longer under Drying Re-inflation by deteriorating the structural properties of the gels obtained after There are also problems during. Residual alcohol tests in final final product should be determined by analytical methods. The necessity of additional tool equipment is another is a disadvantage. In addition, the excess alcohol solvent used in the precipitation process causes costs.

Brief Description of the Invention The object of the invention is purification, which is the most time-consuming process in the production of dermal filler. shortening the process, saving time.

Another object of the invention is the BDDE residue in the dennal filling manufacturing process. significant reduction in concentration.

Another object of the invention is to remove impurities using a multiple wash-filter process. easily removed from the environment.

Detailed Description of the Invention The invention is a dermal filler production process, Preparation of 1% NaOH solution in deionized water, By slowly adding HA to the solution, ensuring that it dissolves, By adding BDDE to the HA mixture, it is ensured that it is mixed homogeneously. providing, The mixture was kept in a water bath at 40 °C for 4 hours. cross-linking reaction, to the dimensions of approximately 1-2 cm of the gel structure that hardens after the reaction. disintegrate in phosphate buffer solution (PBS) until pH: 6.8-7.4 neutralize with up to 0.1 M hydrochloric acid (HCl), Continuing neutralization until the pH of the solution is stable, filtering the gel structure with the help of a sieve, New PBS solution was added to the filtered gel and the gel was soaked for 5 hours. providing swelling, The size of the swollen gel is approximately 500-2000 with the help of homogenizer. micron reduction, Wash 7 more times with PBS solution at one hour intervals, then filtering, bringing the gels to the desired particle sizes, To facilitate the extrusion of non-crosslinked HA, lidocaine HCl is added to the final mixture to provide anesthetic effect, By adjusting the pH of the final mixture to T, it is poured into 1 ml syringes under vacuum. filling and performing steam sterilization process on filled syringes It contains the steps.

The process of the invention is the result of the cross-linking reaction of HA and BDDE. It is to obtain gels that allow it to be used as dermal filler. Easy Non-cross-linked HA is added to the final final formulation to ensure extrusion. Contains 0730 anesthetic agent lidocaine HCl.

Table 1. Required composition ratios to obtain cross-linked HA gel Active and Auxiliary Unit Formula Substances (%) Deionized water 80-95% The formulation indicated in Table 1 was repeated three times (Trial 1), and the analyzes were was repeated three times for each trial. Repeated analysis results The residual BDDE amounts are shown in Table 2 by taking the average. Supplement to results as obtained in the other trial (Trial 2) prepared with the same components and ratios. The results are also shown in Table 2.

In the study named Experiment 2 indicated in Table 2, the purification process was carried out in PBS. The gels were made without breaking into small particles. Total cleaning time is in the range of 48-144 hours, and the final products are obtained by taking samples every day. has been made. BDDE analyzes of the obtained gels were performed and shown in Table 2.

Residual BDDE analysis Residual BDDE content in hydrogels determined by fluorescence spectrophotometer is being done. The epoxy compounds included in the BDDE are excitation at 370 nm. It can react with nicotinamide to produce fluorescence under Fluorescent density, with the epoxide content detectable at the emission wavelength of 430 nm. is directly proportional. In this direction, firstly, to obtain the standard graph, BDDE solutions were prepared and analyzes were made.

BDDE is diluted to different concentrations with ultrapure water. 20 µL diluted The BDDE solution is mixed with 10 µL of 0.125 M nicotinamide. Mixes 2 hours It was incubated in a water bath at 37 °C. Then add 100 µL % to the mixtures. acetophenone (dissolved in ethyl alcohol) and 100 µL of 1 M potassium hydroxide (KOH) was added and mixed in an ice bath. Then 500 µL of methanoic acid was added and the mixtures were incubated in a water bath at 60 °C for a further 5 min. has been made. The prepared standard solutions were analyzed using fluorospectrophotometry. standard chart was created.

The resulting hydrogels were incubated in hyaluronidase enzyme for 24 hours at 37 °C. and then the centrifugation process was carried out. Supernatant, 0.22 µm filtered through a pore-size membrane and measured by fluorescence spectrophotometer. has been analyzed.

Table 2. BDDE residual amounts after purification process Sample Purification Time Residue BDDE Trial 1 12 hours 0.13 Trial 2 2 days 15.43 Trial 2 3 days 9.77 Trial 2 4 days 5.18 Trial 2 5 days 3.03 Trial 2 6 days 1.14 In the analysis results, in the operation performed by reducing the particle size, Remaining BDDE amounts to 2 ppm7in, which is determined by the FDA as the limit limit. It is seen that it is well below (0,13 ppm). Particle of dernnal filling gel In the purification processes carried out without reducing the size (between 24-120 hours), this It is seen that the values are higher than 2 ppm. However, for 144 hours When the purification process is performed, the amount of BDDE falls below the desired level. is descending.

The surface areas of dermal filler gels, which are made into small pieces, are reduced. For this reason, water molecules enter into it faster and faster and cause it to swell and It can remove the impurities in it more easily. very effective and As a fast method, gels are broken down into small pieces during purification. It provides ease of production and time saving.

Claims (1)

REQUESTS 1. The invention is a dermal filler production process, 1% NaOH solution is prepared in deionized water, the solution is dissolved by slowly adding HA on it, BDDE is added to the HA mixture and mixed homogeneously, the mixture is heated at 40 EC for 4 hours. Carrying out the cross-linking reaction by keeping it in a water bath, disintegrating the gel structure after the reaction into approximately 1-2 cm71 dimensions and neutralizing it with 0.1 M hydrochloric acid (HCl) until pH: 6.8-7.4 in phosphate buffer solution (PBS). Reducing the size of the swollen gel to approximately 500-2000 microns with the help of a homogenizer, including the steps of neutralization until the pH of the solution is stable, filtering the gel structure with the help of a sieve, adding new PBS solution to the filtered gel, allowing the gel to swell for 5 hours, PBS Washing the gel with the desired parchment should be done 7 more times with an hourly solution and then filtered. Bringing it to the size of the flask, adding lidocaine HCl to the final mixture to facilitate the extrusion of non-cross-linked HA7, and adding lidocaine HCl to provide an anesthetic effect, filling the 1 ml syringes under vacuum by adjusting the lein of the final mixture to 7” and performing steam sterilization processes in the filled syringes. A dermal filler manufacturing process characterized by