KR20200031333A - Process for producing porous biodegradable polymer microspheres with low endotoxin content - Google Patents

Abstract

The present invention relates to a method for producing porous biodegradable microparticles with reduced endotoxins, which can be used in injections for cosmetic fillers. A porous biodegradable microparticle composition produced through the present invention is an injection composition for cosmetic fillers, produced by combining 1) a surfactant in a dispersion, 2) weakly acidic water for water washing, and 3) gamma ray irradiation. The present invention has a remarkable effect of reducing endotoxins.

Description

Process for producing porous biodegradable polymer microspheres with low endotoxin content}

The present invention relates to a method for producing porous biodegradable polymer microparticles with reduced endotoxin content that can be used in injections for molding fillers.

The present invention is a porous biodegradable polymer microparticle as a micro-sized porous particle using a biodegradable polymer as a raw material for tissue repair at home and abroad, and interest in it is increasing. Due to the limitations of process development, only a very small portion is being industrialized.

As a biocompatible biomaterial material, porous biodegradable polymer scaffolds are widely used as templates for tissue repair. Materials of the biodegradable polymer support include polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), poly-ε-caprolactone (PCL), polyamino acid, polyanhydride, Polyorthoesters and copolymers thereof are known. However, until now, only PGA, PLA, PLGA, etc. have been approved as biodegradable polymers usable by the United States Food and Drug Administration (FDA) and are used as materials for porous polymer supports for the regeneration of human tissues in the body.

Among these biodegradable polymer scaffolds, micro-sized porous microspheres are harmless to the human body and have good tissue repair ability, so they are used in medicines, medical devices, cosmetics, etc., and research for application product development continues. . However, the porous biodegradable polymer microspheres have a high risk of exposure to the exothermic endotoxin at each manufacturing step.

The pyrogenic substance, although not yet elucidated in detail about its nature, refers to a substance that causes abnormal fever in the body, and to date, it has been known that Lipopolysaccharide, a cell membrane component of Gram-negative bacteria, is the main component. It is also called endotoxin.

Endotoxins themselves play an important role in the organization, stability and barrier function of the outer cell membrane, and many bacteriophage use endotoxins or general lipopolysaccharides as a specific method for finding their host bacteria. However, endotoxins are biomolecules that can be found in almost all aqueous solutions without additional precautions, and in humans and animals, endotoxins can cause potent misreactions of the immune system, such as tissue infection, hypotension, and pulse thrombosis. , Shock, immune failure, and sepsis.

Therefore, for pharmaceuticals, etc., especially for injectable products, the endotoxin level is required to be below a certain threshold (e.g., 0.91 EU / ml of immune serum globulin. This corresponds to 5 EU / kg body weight and time ( Dosage EU / kg * h); EU endotoxin units; US Food and Drug Administration (FDA); Guideline on Validation of LAL as End Product).

Endotoxin maintains a biologically stable state, such as maintaining activity even at high temperature, which is a condition where most proteins are denatured, and in order to efficiently destroy endotoxin, high temperature of 200 ° C or higher or at least decomposition by strong acid or strong base, UF ( There are physical methods such as separation by ultrafiltration (RO) or reverse osmosis (RO), and biochemical methods by reaction with polymixin B (Limulus Amebocyte Lysate).

Each of the pros and cons of the method of removing endotoxins used in the manufacture of such pharmaceuticals has the advantages and disadvantages of decomposition of biodegradable polymers when using high temperature and strong acids / bases, and separation by UF and RO is not suitable for polymer microparticles Also, the reaction with polymyxin B and LAL using a fixed resin is very difficult to separate only the porous polymer microparticles after removing endotoxin.

Particularly, the porous biodegradable polymer microparticles are difficult to apply physical methods due to the pores in the microparticles, and the reaction with polymyxin B is also difficult to remove residues after the reaction.

Patent Registration No. 10-1170363 Patent Registration No. 10-1442684

An object of the present invention is to provide a method for preparing a composition that can be used as an injection for a molding filler by effectively removing endotoxin from porous biodegradable microspheres.

In order to achieve the above object,

The present invention is a step of spinning a biodegradable polymer solution as a stock solution in a dispersion bath containing a nonionic surfactant,

Washing the polymer fine particles solidified in the dispersion bath,

Provided is a method of manufacturing porous biodegradable polymer microparticles having a low endotoxin content, comprising the steps of lyophilizing the always washed microparticles and treating them with gamma rays.

Porous biodegradable microparticles for injection molded fillers with reduced endotoxin produced by the present invention can expand the range of types of biodegradable polymers that can be directly applied to the human body, and also expand the application range applicable to the human body. can do.

Hereinafter, the present invention will be described in detail.

In the present invention, the porous biodegradable polymer microparticles have a dispersion bath containing a nonionic surfactant in the method for preparing microparticles, which is difficult to remove endotoxin due to the pores in the particle and the properties of the biodegradable polymer, and adjusts the pH of the washing liquid in the washing step And it proposes a method to effectively remove endotoxin by introducing a gamma ray irradiation process after ultrasonic treatment and freeze-drying of the particles.

Specifically, in order to prepare the biodegradable polymer microparticles of the present invention, a biodegradable polymer solution is prepared by dissolving a biodegradable polymer raw material in dichloromethane containing a pore-forming agent. The prepared solution is sprayed through a porous permeable membrane in a spray bath in which cationic surfactant, anionic surfactant, and nonionic surfactant are dissolved. For the dryness of the prepared biodegradable polymer microparticles, water with a pH of 4 to 6 is included in a spray bath and stirred to form particles, and after the particle formation is completed, the spray bath is washed with water with a pH of 4 to 6. In the water washing step, the microparticle solution containing weakly acidic water having a pH of 4 to 6 is sonicated so that no surfactant remains. Freeze-dried particles are lyophilized to make porous microparticles, and they can be stored in a container to irradiate gamma-rays, thereby significantly reducing endotoxins existing outside the microparticles.

It provides a non-limiting example of a method for producing porous biodegradable polymer microparticles for injection molding molding with reduced endotoxin according to the present invention.

< Example 1 ~ 5> Preparation of porous microparticles for tissue repair

Step 1: Mother liquor  Ready

A polymer solution was prepared by dissolving polycaprolactone (PCL) having an average molecular weight of 110,000 in a methylene chloride (MC) solution containing porogen.

Step 2: Continuous phase  Preparation of dispersion

A surfactant was dissolved in water to prepare a dispersion.

Example 1 PVA (polyvinyl alcohol) Example 2 Polysorbate 20 (polysorbate) Example 3 Sodium lauryl sulfate Example 4 Polyoxyethylene lauryl ester (polyoxyethylene lauryl ester) Example 5 Lauramidopropyl betaine

Step 3: into the container Mother liquor  And Continuous phase  Fill dispersion

The polymer solution of step 1 was filled in a part of the container where the space was separated by a porous permeable membrane, and the continuous phase dispersion of step 2 was filled in another part.

Step 4: Form porous microparticles

While stirring the continuous phase dispersion liquid, pressure was applied to the mother liquid through nitrogen gas injection, and the polymer solution was sprayed into the continuous phase dispersion bath in the form of porous microparticles through the pores of the porous permeable membrane.

Step 5: Organic solvent  Removal and cleaning

The organic solvent that has passed into the continuous phase dispersion was evaporated, water was added with a pH of 4-6, stirred to dry the fine particles, and then water was added with a pH of 4-6 to remove the surfactant by ultrasonic treatment, filtered and lyophilized. .

Step 6: Gamma ray  Research

Gamma-ray was irradiated to the lyophilized microparticles to prepare porous biodegradable polymer microparticles.

< Experimental example  1>

The endotoxin content of the fine particles of Examples 1 to 5 was measured and is shown in Table 2 below. The test method was performed according to the endotoxin test method in the general test method of the Korean Pharmacopoeia.

division Example 1 Example 2 Example 3 Example 4 Example 5 (EU / mL) 1.02 124.5 103.01 25.7 304.2

As a result of measuring the endotoxin content of the fine particles of Examples 1 to 5, the endotoxin content of Example 1 was most reduced.

< Comparative example 1>

In the washing process of step 5 of the process of Example 1, neutral water of pH 7 was used instead of weakly acidic water of pH 4 to 6, and microparticles were prepared without γ-ray irradiation after lyophilization.

< Experimental example  2>

The endotoxin content of the fine particles of Example 1 and Comparative Example 1 was measured and is shown in Table 1 below. The test method was performed according to the endotoxin test method in the general test method of the Korean Pharmacopoeia.

division Example Comparative example (EU / mL) 1.02 11,103

As shown in Table 3, Example 1 showed an endotoxin content of 1 / 10,000 level compared to Comparative Example 1, and it was confirmed that the process of the Example was effective.

Claims (6)

Spraying a biodegradable polymer solution into a dispersion bath containing a nonionic surfactant,
Washing the coagulated biodegradable polymer microparticles in the dispersion bath,
A method of manufacturing a porous biodegradable polymer microparticle with low endotoxin content, comprising lyophilizing the washed microparticles and irradiating with gamma ray. According to claim 1,
In the dispersing step, the dispersion bath is a method for producing a porous biodegradable polymer microparticle having a low endotoxin content, characterized in that a dispersion containing polyvinyl alcohol, which is a nonionic surfactant, is used. According to claim 2,
In the dispersing step, the dispersion bath is a nonionic surfactant, and polyvinyl alcohol is a method of manufacturing a porous biodegradable polymer microparticle with low endotoxin content, characterized in that the content is 3 to 6%. According to claim 1,
In the step of washing the coagulated fine particles in the dispersion bath after the dispersing step, the washing solution is a method for producing porous biodegradable polymer microparticles with low endotoxin content, characterized by using weakly acidic water having a pH of 4 to 6. According to claim 4,
In the step of washing the coagulated fine particles in the dispersion bath after the dispersing step, ultrasonic treatment of weakly acidic water and fine particles having a pH of 4 to 6 removes endotoxins and nonionic surfactants. Method for producing biodegradable polymer microparticles. According to claim 1,
The gamma ray has a wavelength range of 10 ^-11 ~ 10 ^-14 μm, characterized in that the production method of the porous biodegradable polymer microparticles with low endotoxin content.