KR100312638B1 - Method for manufacturing high purity hyaluronic acid - Google Patents

Abstract

PURPOSE: Provided is a method for manufacturing and purifying high purity hyaluronic acid having 0.01% or less of protein content and 0.02% or less of nucleic acid content. CONSTITUTION: The method for manufacturing and purifying high purity hyaluronic acid is characterized by the steps of: adding a diatomite or an activated carbon to a culture solution containing hyaluronic acid produced from microbes; and removing nucleic acids and proteins from the hyaluronic acid solution by using Duolite A7 resin and hydroxyapatite resin.

Description

[Name of invention]

Method for producing high purity hyaluronic acid

Detailed description of the invention

The present invention relates to a method for preparing and purifying hyaluronic acid produced from microorganisms.

Hyaluronic acid is a high molecular weight linear polysaccharide with a molecular weight of 50,000 to 13 million, in which glucuronic acid and N-acetyl glucosamine are repeatedly linked by 1-3 bonds and 1-4 bonds. Extracted from biological tissues such as chicken crust, cow's eye, and umbilical cord, but this method has many disadvantages. First, the production yield is low, and second, impurities such as chondroitin sulfate and glycosamino glycan sulfate are mixed. Third, the manufacturing and purification process is expensive.

On the other hand, according to Swan's paper Arch, Opthal., 88, 544 (1970), the production method of hyaluronic acid by microorganisms is first, the production cost is low, second, the production yield is high, and third, the produced hyaluronic acid is injected. It has been reported to have various advantages such as low reactivity, fourth, high average molecular weight, and fifth, almost no impurities such as proteins and nucleic acids.

Since hyaluronic acid is found in the vitreous fluid of the eye and the synovial fluid of the joints of mammals, it is necessary to inject the human body as a substitute for treating the pathology of the eyes and joints. Therefore, hyaluronic acid should be obtained as high as possible. It is very important. It is known that the protein content of the high purity hyaluronic acid preparation used as a pharmaceutical should be 0.01% or less and the nucleic acid content of 0.02% or less.

Therefore, it is an object of the present invention to provide a method for purifying a hyaluronic acid product produced from a microorganism so that the protein content is 0.01% or less and the nucleic acid content is 0.02% or less.

In one preferred embodiment, the present invention purifies the hyaluronic acid-containing culture solution produced from microorganisms through ethanol precipitation and appropriate resins to produce hyaluronic acid, which is free of impurities (nucleic acid and protein), free of microorganisms and can be used for medical purposes. Provide a method.

In a more preferred embodiment, the present invention produces only hyaluronic acid without the production capacity of streptolysin, is non-hemolytic, inhibits hyaluronidase (hyaluronic acid degrading enzyme), increases the production capacity of hyaluronic acid, short incubation time, Hyaluronic acid produced by Streptococcus equine CH124 (muted strain KFCC-10839, deposited on August 22, 1994) with mutant strain producing hyaluronic acid with relatively high molecular weight was removed by adding activated carbon and diatomaceous earth. Next, a method of preparing high purity hyaluronic acid is provided by removing nucleic acids and proteins contained in a hyaluronic acid solution using a diolite A7 resin and a hydroxyapatite resin.

The present invention is described in detail as follows. Variant strain Streptococcus equiqui CH124 frozen at −70 ° C. was inoculated into two 250 ml flasks containing 50 ml of Brain heart infusion medium and incubated for 14 hours in a rotary shaker. 100 ml of this culture was inoculated into a 5 L fermenter with a fermentation medium of 2 L and cultured. At this time, the fermentation medium is glucose 80g / l, yeast extract 10g / l, soybean meal extract 35g / l, potassium diphosphate 10g / l, potassium monophosphate 10g / l, magnesium sulfate 0.7g / l, ammonium sulfate 0.6g / l, sulfuric acid Manganese 0.6 g / l and the like. In the fermentation conditions, the pH of the culture medium was 7.0, the culture temperature was 35-37 ° C., the aeration amount was 0.5-2.0vvm, the stirring speed was 300-1,400 rpm, and the incubation time was 12-16 hours.

When cultured by the above method, the cell concentration after the culture was 0D _600nm = 8.0-10.0, the concentration of hyaluronic acid was about 5-6g / ℓ, the average molecular weight was 3 million to 5 million.

Sodium chloride was added to the culture broth to obtain 1.5 M sodium chloride solution to remove the cells and remove the impurities of the hyaluronic acid culture, and 0.2-0.8%, preferably 0.4% of activated carbon (Cheil Carbon) and 0.5-2.0 %, Preferably 2.0% of diatomaceous earth (Samson Corporation, No. 200) was added and mixed for at least 1 hour, and filtered using a filter press (Filter press) using a filter (Advantec, NA-050). The filtrate and ethanol were mixed in a 1: 1.5 ratio using a Y tube to precipitate hyaluronic acid. The hyaluronic acid precipitated was dried with sterile nitrogen gas, dissolved in 1.5M sodium chloride solution, 0.1% activated carbon and 0.5% diatomaceous earth were added, and the hyaluronic acid was completely dissolved and filtered using a filter (NA-050). The filtrate and ethanol were mixed at a ratio of 1: 1.5 using a Y tube to precipitate hyaluronic acid, and the precipitated hyaluronic acid was dried with sterile nitrogen gas.

The dried hyaluronic acid was dissolved in distilled water for the resin work, and sodium chloride was added to have a conductivity of 40-50 mS and pH 4.0-8.0. This solution was passed through a Duolite A7 (Diamond Shamrock) resin. Resin treatment conditions were flow rate 2SV, pH 7.0, hyaluronic acid concentration 2-4g / ℓ, throughput was less than 7RV. The solution passed through the resin was treated with 1.5 times ethanol to precipitate. The precipitated hyaluronic acid was dissolved in distilled water at about 1-3g / L, and 0.2% of hydroxyapatite (Hydroxyapatite, Bio-rad) resin was added and reacted for 2 hours. The resin in the solution was removed by a filter, followed by sterilization filtration using a 0.2 µm cellulose acetate filter (Cellulose acetate filter, Sartorius). The solution was made into 1M sodium chloride solution, added to 1.5 times ethanol solution to precipitate, and washed with reagent-grade ethanol to remove sodium chloride. The precipitated hyaluronic acid was dried over sterile nitrogen gas.

Streptococcus aquio CH124 was cultured to obtain 5-6 g of hyaluronic acid per liter of fermentation broth, and 1.5-2.5 g of final hyaluronic acid was obtained through the above purification process (purification yield: about 30-40%), and the molecular weight of the final hyaluronic acid product was It was about 3 million to 4 million.

Summary of the purification process of hyaluronic acid produced from microorganisms is as follows.

(1) Streptococcus Equi CH124 Microbial Growth

(2) dissolution of sodium chloride, activated carbon and diatomaceous earth

(3) Filter filtration (filter press filtration using NA-050 filter)

(4) Ethanol Precipitation and Drying of Hyaluronic Acid Solution

(5) dissolution of sodium chloride, activated carbon and diatomaceous earth addition

(6) Filter Filtration (using NA-050 filter)

(7) Ethanol Precipitation and Drying of Hyaluronic Acid Solution

(8) Sodium chloride added to adjust conductivity

(9) passed through the zeolite A7 resin

(10) Ethanol Precipitation and Drying of Hyaluronic Acid Solution

(11) hydroxyapatite resin addition reaction

(12) Resin removal and sterilization filtration (0.2 μm cellulose acetate filter)

(13) Ethanol precipitation of hyaluronic acid solution

(14) ethanol washing and drying

In another aspect of the invention, the hyaluronic acid product of the invention is prepared from microorganisms that are hyaluronidase-negative or hyaluronidase inhibited, and contain little protein or nucleic acid and are considered unnecessary contaminants when used in medicine. Is different from the commercially available hyaluronic acid in that it does not contain chondroitin sulfate.

In the case of hyaluronic acid, the analytical method of Bilter and Muir, Anal. Biochem. 4. Carbazol modified by 330 (1962) was used, and the cell concentration was measured at 0D _{600 nm} . Proteins were measured by Bio-rad's Micro-assay. This method can detect up to low protein concentrations of 25 μg / ml or less. In addition, the nucleic acid was measured at 0D _260nm . The molecular weight was measured at the same time by a viscometer method and high performance liquid chromatography (High Performance Liquid Chromatography). The method by viscosity is described in Fasman's article Crit. Rev. [η] = 0.036xMW ^0.76 presented in in Biochem., 8, 225 (1980), and the method by high performance liquid chromatography was based on the method of Pharmacia, Sweden.

Example 1

Various cell removal methods were tested. The centrifugation method of the cell removal method is a method of removing the cells by centrifuging the fermentation broth. Ethanol precipitation is a method in which a fermentation broth is precipitated with ethanol to remove cells, hyaluronic acid is obtained, and then dried and dissolved. Filtration removes the cells by filtering the fermentation broth (using NA-050 filter). The results are recorded in Table 1. Among the cell removal methods, nucleic acids and proteins that were impurities were best removed.

Example 2

In order to effectively remove impurities and cells, ethanol precipitation was used after filtration, and activated carbon and diatomaceous earth were used to analyze hyaluronic acid recovery and the content of nucleic acid and protein in hyaluronic acid solution. In the first filtration, the concentration of diatomaceous earth was 1.0% and the experiment was performed while changing the concentration of activated carbon. As can be seen in Table 2, the higher the content of activated carbon during primary filtration, the better the removal of nucleic acids and proteins, but the yield of hyaluronic acid was lowered. In the second filtration, the resultant was filtered at 0.1% activated carbon concentration and 1.0% diatomaceous earth concentration. In the second filtration, the higher the amount of activated carbon in the first filtration, the better the nucleic acid and protein were removed.

Activated carbon concentration 0.1%

In the first filtration, the concentration of activated carbon was 0.4% and the experiment was performed while changing the concentration of diatomaceous earth. As can be seen in Table 3, the higher the diatomaceous earth content in the first filtration, the more likely the nucleic acid and protein were removed, but there was no significant difference. In the second filtration, the filtrate was filtered at 0.1% activated carbon and 1.0% diatomaceous earth. In the second filtration, the higher the diatomaceous earth content in the first filtration due to the effect of the first filtration, the better the nucleic acid and protein were removed during the second filtration.

Diatomaceous Earth Concentration 1.0%

Example 3

Among the impurities contained in the hyaluronic acid product, the protein was almost removed during the ethanol precipitation process, but the nucleic acid was not completely removed but included to some extent even after repeated ethanol precipitation several times. Therefore, experiments were carried out using a duolite A7 resin to intensively remove the nucleic acid in the hyaluronic acid solution.

As a result of measuring the nucleic acid removal rate by conductivity during the optimization experiment of the diolite A7 resin, it was as shown in Table 4. The diaolite A7 resin was passed through the hyaluronic acid solution having a nucleic acid content of 0.15%, a concentration of hyaluronic acid at 3.0 g / L, and a pH of 7.0 with varying conductivity. As a result, the conductivity was the best in nucleic acid removal with 65% removal rate near 40mS.

The diaolite A7 resin was passed through a hyaluronic acid solution having a nucleic acid content of 0.15%, a concentration of hyaluronic acid at 3.0 g / L, and a conductivity of 50 mS with varying pH. As a result, nucleic acid removal was best with a nucleic acid removal rate of 65% near pH 7.0 (Table 5).

Example 4

While varying the hydroxyapatite resin concentration, it was added to a hyaluronic acid solution (nucleic acid content 0.05%, hyaluronic acid concentration 2.0g / l, pH 7.0, conductivity 0.2mS), reacted at 30 ° C for 2 hours, and then filtered to remove the resin. As a result of measuring the content, it was shown in Table 6. The addition of hydroxyapatite concentration of 0.2% or more, preferably 0.2 to 1.0%, almost eliminated the nucleic acid.

Example 5

As a result of examining the content of nucleic acid and protein contained in hyaluronic acid during the purification process using the above purification method was as shown in Table 7. After the treatment of the diolite A7 resin during the purification step, almost no protein was removed, and after treatment with the hydroxyapatite resin, impurities and nucleic acids and proteins were almost removed.

Claims (5)

Hyaluronic acid is prepared by adding activated carbon and diatomaceous earth to a culture solution containing hyaluronic acid produced from a microorganism, and removing nucleic acids and proteins from the resulting hyaluronic acid solution using a diolite A7 resin and a hydroxyapatite resin. How to. The method of claim 1, wherein 0.2-0.8% activated carbon and 0.5-2.0% diatomaceous earth are added to remove the cells. The method of claim 1, wherein the hyaluronic acid solution is adjusted to a conductivity of 40-50 mS, pH 4.0-8.0, and passed through the diolite A7 resin to remove the nucleic acid. The method of claim 1 wherein 0.2-1.0% hydroxyapatite resin is added to remove the nucleic acid. The method of claim 1, wherein the culture is produced from Streptococcus equ CH124.