KR20090091501A - Method of purifying botulinum toxin from clostridium botulinum - Google Patents

Abstract

A method for isolating botulinum toxin from Clostridium botulinum is provided to massively produce the botulinum toxin in a short time and improve the efficiency of botulinum toxin. A method for isolating botulinum toxin from Clostridium botulinum comprises: a first step of culturing the Clostridium botulinum; a second step of precipitating Clostridium botulinum and adding acid to precipitate again; and a step of performing liquid chlomatography of the precipitate and purifying the botulinum toxin.

Description

Method of purifying botulinum toxin from Clostridium botulinum}

The present invention relates to a method for purifying botulinum toxin from Clostridium botulinum , and more particularly, the first step of culturing the Clostridium botulinum, and the cultured Clostridium botulinum. Purification of the botulinum toxin by chromatography of the second and second precipitates comprising botulinum tonic acid precipitation and extraction with acetate followed by addition of acid to secondary acid precipitation. It relates to a method for purifying the botulinum toxin, characterized in that comprises a third step.

Clostridium strains that secrete neurotoxic toxins have been discovered since the 1890s and have been studied for toxins secreted by these strains for the past 70 years (Schant, EJ et al., Microbiol. Rev. 56; 80; 1992).

The botulinum toxin is divided into seven types, type A to G, depending on its serological characteristics. Each toxin has a toxin protein of about 150 KDa, which naturally consists of a complex of several non-toxic proteins. Medium complex (300kDa) consists of toxin protein and non-toxic-non-hemagglutinin protein.Large (450kDa) complex and Large-Large (900kDa) complex are intermediate complexes with hemagglutinin. It is in a combined form (Sugiyama, H., Microbiol. Rev., 44, 419; 1980). These non-toxic non-hemagglutinin proteins are known to function to protect toxins from low pH and various proteolytic enzymes in the intestine (Sugiyama, H., Microbiol. Rev., 44, 419; 1980).

The toxin is synthesized into one polypeptide having a molecular weight of about 150 kDa in the cell, and then cleaved at a position one third from the N terminal by the action of intracellular proteolytic enzymes or by an artificial enzymatic treatment such as trypsin. It is divided into light chain (L) (molecular weight; 50 kDa) and heavy chain (H: heavy chain) (molecular weight; 100 kDa).

The divided toxins are greatly increased in toxicity compared to single polypeptides. The two units are connected to each other by disulfide bonds, each having a different function. The heavy chain binds to the receptor of the target cell (Park. MK, et al., FEMS Microbiol. Lett. 72, 243; 1990) and reacts with the biofilm at low pH (pH4) to channel. (Mantecucco, C. et al., TIBS 18, 324; 1993), the light chain has pharmacological activity to impart permeability to cells using detergent or intracellular by electroporation or the like. Interferes with the secretion of neurotransmitters when injected (Poulain, B. et al., Proc. Natl. Acad. Sci. USA. 85, 4090; 1988).

The toxin inhibits exocytosis of acetylcholine at cholinergic presynapse of neuromuscular junctions, causing systemic asthenia. Treatment with trace amounts of toxins has been thought to be toxic, suggesting that these toxins may have some enzymatic activity (Simpson, L. L. et al., Ann. Rev. Pharmaeol. Toxicol. 26, 427; 1986).

Recently discovered, the toxin has metallopeptidase activity and its substrate is synaptobrevin, syntaxin, which is a unit protein of the exocytosis machinary complex. ), Synaptomal associated protein of 25KDa (SNAP25). Each type of toxin uses one of these three proteins as substrates, with types B, D, F, and G being synaptobrevin, types A and E cutting SNAP25, and type C syntaxin cutting at specific sites. Known (Binz, T. et al., J. Biol. Chem. 265, 9153; 1994).

The botulinum toxin is a toxin that can be used as four major bioterrorism weapons together with anthracis (Bacillus anthracis), pest (Yersinia pestis), smallpox virus because it is lethal to mass and easy to mass production. However, in the case of the A-type toxin of the botulinum toxin, when injected at a dose that does not affect the human body systemically, the local muscle of the injection site may be paralyzed. As described above, the muscles can be paralyzed locally, so it can be widely used as an anti-wrinkle remover, stiff hemiplegia and subparesis, and is a very useful toxin.

For the same reason, the demand for botulinum toxin is rapidly increasing, and in accordance with the demand, studies on the purification method of botulinum toxin have begun. In the conventional method of purifying botulinum toxin, ion exchange chromatography was used, and thus, a long time (about 10 to 14 days) was used, and thus, it was very difficult to apply industrially. In addition, since Clostridium botulinum is an anaerobic bacterium, it is difficult to mass-produce because there is a problem that the clostridial botulinum should be in an anaerobic facility at the time of fixation. In addition, the botulinum toxin, which is an active ingredient purified by the purification method, was not clearly identified and had a problem of including impurities.

The present invention has been made to solve the above problems, the Clostridium botulinum ( Clostridium botulinum ) culturing step, and the acid-precipitating step of the botulinum toxin, comprising the first acidic precipitation of the incubated Clostridium botulinum and extracted with acetate, followed by a second acidic precipitation of the botulinum toxin It is an object of the present invention to provide a purification method capable of mass production of botulinum toxin only without an anaerobic facility, including purifying the botulinum toxin by chromatography of the precipitate containing the botulinum toxin.

A method for purifying botulinum toxin from Clostridium botulinum according to the present invention for achieving the above object is a first step of culturing Clostridium botulinum, the first acid precipitation of the cultured Clostridium botulinum and acetate And a third step of purifying the botulinum toxin by chromatography using a second step including extracting the acid and adding an acid to precipitate the second acid, and depositing the botulinum toxin according to the second step. It is characterized by.

Preferably, the culture medium is characterized in that it comprises peptone, yeast extract, glucose and brain heart infusion.

Preferably, the culture medium is characterized in that the concentration of yeast extract is 0.3 to 0.5%, the concentration of glucose is 0.5 to 1%.

Preferably, the culture in the culture step of 3 to 5 days and characterized in that the culture by a fermenter (fermentor).

Preferably, the acid precipitation step comprises the steps of precipitating the protein of Clostridium botulinum with sulfuric acid (H ₂ SO ₄ ); Extracting botulinum toxin from the protein precipitate precipitated in sulfuric acid with acetate; Precipitating the botulinum toxin using hydrochloric acid; and then, treating the precipitate by the hydrochloric acid precipitation step with DNase and RNase, and precipitating it with ethanol; And ammonium sulfate precipitation step of inducing crystallization by precipitating the precipitate by ethanol in ammonium sulfate ((NH ₄ ) ₂ SO ₄ ).

Preferably, the pH concentration of sulfuric acid in the sulfuric acid precipitation step is characterized in that 3.0 to 3.9.

Preferably, the acetate extraction step of the acetate is 0.04 to 0.09 molar concentration (M), the pH of the acetate is 6.3 to 6.5, the extraction time is characterized in that 1.5 to 2.5 hours.

Preferably, the hydrochloric acid in the hydrochloric acid precipitation step has a pH of 3.5 to 3.8, the hydrochloric acid is added 0.1N hydrochloric acid when the pH of the acetate extract is 5.0 to 6.5, the pH of the acetate extract is 5.0 to 3.7 Is characterized by adding 1N hydrochloric acid.

Preferably, DNase, RNase of the ethanol precipitation step is characterized in that the treatment time is 1.5 to 3 hours.

More preferably, the ethanol concentration of the ethanol precipitation step is 15 to 18%, characterized in that the treatment time of the ethanol is 1 to 24 hours.

Preferably, tris, potassium phosphate (K3HPO4), sodium phosphate (Na2PO4), sodium acetate (CH3COONa) may be used as the purification step buffer. More preferably, sodium acetate is used.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for purifying botulinum toxin from Clostridium botulinum, the step of culturing the Clostridium botulinum, the first acid precipitation of the cultured Clostridium botulinum and extraction with acetate followed by the addition of acid Purifying the botulinum toxin, including acid precipitation, clarification of the botulinum toxin and the precipitate containing the botulinum toxin by the acid precipitation step using liquid chromatography.

The culture medium includes peptone, yeast extract, glucose and brain heart infusion. Aerobic and breathable anaerobic bacterial culture medium is a nutrient-enhanced liquid medium that is tryptic (Trypticase) soy, supplemented peptone, brain heart infusion, Columbia, Brucella There is a medium. For anaerobic bacteria, thioglycollate, thiol, anaerobic brain heart infusion medium and the like are recommended.

The culture medium is a yeast extract concentration of 0.3 to 0.5%, the concentration of glucose is 0.5 to 1%. The concentration of glucose should be within the concentration range, but if it is below the concentration range, the bacteria are not cultivated because it is not nourishment to the Clostridium botulinum. In addition, when the concentration is greater than or equal to the concentration range, it is preferable that the concentration of the medium is increased so that Clostridium botulinum may be hemolytic and it is difficult to visually observe by glucose.

Incubate for 3 to 5 days in the culture medium and incubated in the fermentor. When the culture is less than 3 days, the Clostridium botulinum is not sufficiently cultured, and when cultured for 5 days or more, the dissolution and proteolysis of the bacteria occur excessively, thereby reducing the efficacy of the botulinum toxin. . In addition, since it is possible to secure a large number of the Clostridium botulinum, it is cultured in the fermenter rather than stagnant culture.

The cultured Clostridium botulinum is subjected to an acid precipitation step because the botulinum toxin should be extracted. The acid precipitation step includes the step of precipitating the protein of Clostridium botulinum with sulfuric acid; Extracting botulinum toxin with acetate from the protein precipitate precipitated in sulfuric acid; Precipitating the botulinum toxin using hydrochloric acid; and then, treating the precipitate by the hydrochloric acid precipitation step with DNase, RNase, and precipitating it in ethanol; And ammonium sulfate precipitation step of inducing crystallization by precipitating the precipitate by ethanol in ammonium sulfate.

The acid precipitation step utilizes the principle that the protein is lowered by adding acid to Clostridium botulinum consisting of several proteins to reach the isoelectric point and precipitate. In a broad sense, acid precipitation also includes crystallization, and the precipitation method is a method of roughly separating a target object in a mixed state as compared to crystallization focused on pure purification. Usually, acid precipitation precipitates impurities that are structurally similar to the target substance.

The pH of sulfuric acid in the sulfuric acid precipitation step is preferably 3.3 to 3.8. The sulfuric acid precipitates a protein similar to the botulinum toxin. The recovery rate of the botulinum toxin increases as the pH is lower but affects the botulinum toxin itself when the pH becomes 3.3 or less. When the pH is 4.0 or more, the recovery rate of the botulinum toxin is lowered, so it is preferably made within the above range. The pH is most preferable because the recovery rate of the botulinum toxin is the highest when 3.5.

Since the precipitate precipitated by sulfuric acid in the sulfuric acid precipitation step includes all the proteins similar to the botulinum toxin, only the botulinum toxin should be separated and thus the acetate extraction step should be performed.

The acetate of the acetate extraction step is 0.04 to 0.09 molar concentration, the pH of the acetate is 6.3 to 6.5, the extraction time is 1.5 to 2.5 hours. The acetate has a similar polarity to the botulinum toxin and thus facilitates extraction of the toxin. If the molar concentration of the acetate is less than 0.04 is not extracted, if the amount is more than 0.09 it is preferable that the amount of unnecessary RNA in the extract is made within the above range. The extraction time is not properly extracted when the time is 1.5 hours or less, and when the process time is 2.5 or more, it is preferable that the extraction time is performed within the above time range because the titer is reduced and uneconomical. The pH concentration of the acetate is preferably within the range because the potency is high. When sodium chloride is added at the time of extraction with the acetate, since the titer is lowered, it is preferable not to add it. In addition, when calcium chloride is added, RNA removal rate is partially increased, but it is preferable not to include the botulinum toxin since the activity of the botulinum toxin decreases.

Since the acetate extract contains a botulinum toxin and a protein having a polarity similar to that of the botulinum toxin, the acetate extract must undergo a hydrochloric acid precipitation step of precipitating the protein with hydrochloric acid. The hydrochloric acid in the hydrochloric acid precipitation step has a pH of 3.5 to 3.8, the hydrochloric acid is added with 0.1N hydrochloric acid when the pH of the acetate extract is 5.0 to 6.5, 1N hydrochloric acid when the pH of the acetate extract is 5.0 to 3.7 Add. If done within the pH range botulinum toxin is 90% or more and the titer is high. In addition, since the RNA removal rate is increased, it is preferable to be made within the above range.

The hydrochloric acid is added with 0.1N hydrochloric acid when the pH of the acetate extract is 5.0 to 6.5, 1N hydrochloric acid is added when the pH of the acetate extract is 5.0 to 3.7, 1N hydrochloric acid is added regardless of the pH In this case, since the activity and recovery rate of the toxin are reduced, it is preferable to add as described above. In addition, the addition of hydrochloric acid at different concentrations according to pH, as described above, significantly reduces protein entanglement.

In the precipitate precipitated by hydrochloric acid, botulinum toxin, DNA, and RNA also exist together, so that the DNA and RNA must be treated with DNase, RNase, and only the toxin must be extracted to precipitate in ethanol. DNase and RNase in the ethanol precipitation step is 1.5 to 3 hours. It does not affect the throughput of the DNase and RNase, but if it is 1.5 hours or less, it is preferable to carry out within the above range because it is uneconomical if the DNA and RNA are not properly processed and 3 hours or more. In addition, by treating DNase and RNase in the ethanol precipitation step, nucleic acids present together with botulinum toxin in the fermentation medium can be removed.

Ethanol of the ethanol precipitation step is 15 to 18% in concentration, the treatment time of the ethanol is 1 to 24 hours. The ethanol is to change the relative dielectric constant of the precipitate to precipitate the protein present in the precipitate. In the case of the concentration range and the treatment time, the titer is high and the recovery rate is good.

The precipitate by ethanol is precipitated in ammonium sulfate, so as to induce crystallization of ammonium sulfate. The ammonium sulphate precipitation step may be referred to as salting, in which salt is dissolved in water (such as ammonium sulphate) to the protein mixture to increase the ionic strength to precipitate the protein. If the target protein is precipitated predominantly at 60% saturation of ammonium sulphate, precipitate the protein other than the target at a concentration of ammonium sulphate below 60% saturation in advance and then remove ammonium sulphate ((NH ₄ ) ₂ SO ₄ ) is added to precipitate the desired protein which is collected by centrifugation. Salting out is often used as an initial means of purification.

Tris, potassium phosphate (K ₃ HPO ₄ ), sodium phosphate (Na ₂ PO ₄ ), sodium acetate (CH ₃ COONa) may be used as the buffer solution of the purification step, and sodium acetate is most preferred. Do.

The botulinum toxin purified by the above purification method becomes an active ingredient of the pharmaceutical composition. The botulinum toxin can be used for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscle. It can also be used in a variety of ways, including treatment of essential blepharospasm, strabismus and lateral facial spasms, treatment of cervical dystonia and treatment of glabellar (facial) wrinkles.

According to the method for purifying botulinum toxin from the Clostridium botulinum according to the present invention as described above, the step of culturing the Clostridium botulinum, the cultured Clostridium botulinum to at least one acid A step of purifying the botulinum toxin by sequentially treating the acid precipitation step of precipitating the botulinum toxin and the step of purifying the botulinum toxin by chromatography of the precipitate containing the botulinum toxin by the acid precipitation step. It works. In addition, by shortening the purification time it is possible to mass-produce the botulinum toxin and lower the cost of the mass production.

In addition, since the purification method according to the present invention does not have to be made in an anaerobic facility, there is an economic effect, and since botulinum toxin is clearly identified and identified by the purification method, there is an effect of increasing the efficacy of the toxin.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

< Example  One - Clostridium  Medium for Botulinum Culture>

1-1 Composition Determination of Culture Medium

Peptone medium, Brain Heart Infusion medium, Gesler F. Bohnel H. medium were prepared to determine the medium composition. After inoculating and incubating Clostridium botulinum in the prepared medium, the culture solution for each medium was injected into rats bred in a sterile state, and then the half dose LD 50 (Lethal dose 50) where 50% of the injected rats were killed was examined. 1 is shown.

Compositional Components and Half-Resistance by Media badge Peptone medium BHI badge Gessler F, Bohnel H. Badge Composition of Medium 2% peptone 1% yeast extract 1% N-Z amine 0.5% glucose (pH 7.4) 1% peptone 0.3% yeast extract 1% Brain Heart Infusion 0.5% glucose (pH 7.4) 1% peptone 0.3% yeast extract 1% N-Z amine 0.1% starch 0.5% NaCl 0.3% Sodium acetate 0.05% L-cysteine-HCl 0.5% glucose (pH 7.4) LD ₅₀ (U / ml) 1 × 10 ⁵ 2 × 10 ⁵ 8 × 10 ⁴

Cultured for 5 days under the same conditions, it was confirmed that the killing amount according to the botulinum toxin of BHI medium was more than two times higher than other conditions in the titer test. In addition, when confirmed in the culture solution, the peptone medium was confirmed that a lot of RNA, BHI medium was confirmed that almost no RNA.

1-2% determination of yeast extract in culture medium

In order to determine the amount of toxin according to the amount of yeast extract of the medium, different concentrations of medium were prepared, inoculated and inoculated with Clostridium botulinum to the medium, and the culture medium for each medium was bred under sterile conditions. Injected into rats, and examined for half lethal dose LD50. Shown in

Compositional Components and Half-Resistance by Media badge Yeast extract (0.3%) Yeast extract (0.5%) Yeast extract (1.0%) Ingredient 0.3% yeast extract 0.5% glucose (pH 7.4) 0.5% yeast extract 0.5% glucose (pH 7.4) 1% yeast extract 0.5% glucose (pH 7.4) LD ₅₀ (U / ml) 2.5 × 10 ⁵ 3 × 10 ⁵ 2 × 10 ⁵

Each medium was cultured for 5 days under the same conditions, and it was confirmed that the lethality according to botulinum toxin of the medium containing 0.5% of yeast extract in the titer test was 1.5 times higher than other conditions.

1-3 of culture medium Glucose  % Determined

In order to determine the amount of toxin according to the amount of glucose contained in the medium, different concentrations of medium were prepared, the medium was inoculated with Clostridium botulinum, and the medium was cultured in sterile rats. After injection, the half dose LD50 was investigated. Shown in

Compositional Components and Half-Resistance by Media badge Glucose (0.5%) Glucose (1%) Glucose (1.5%) Ingredient 0.5% yeast extract 0.5% glucose (pH 7.4) 0.5% yeast extract 1% glucose (pH 7.4) 0.5% yeast extract 1.5% glucose (pH 7.4) LD ₅₀ (U / ml) 3 × 10 ⁵ 3.5 × 10 ⁵ 2 × 10 ⁵

Each medium was cultured for 5 days under the same conditions to confirm that the lethality of botulinum toxin in the medium containing 1% glucose (Glucose) was at least 1.5 times higher than other conditions.

1-4 of Culture Medium pH  decision

In order to determine the amount of botulinum toxin according to the pH of the medium, media of different pHs were prepared using the medium conditions (0.5% yeast extract, 0.5% glucose) of Experimental Examples 1 to 3, and Clostridium botulinum was added to the medium. After inoculation and incubation, the culture medium was injected into rats bred in a sterile state, and then irradiated with a semi-numerical dose LD50. Shown in

Semi-numerical dose by pH of medium PH of medium pH 6.8 pH 7.4 pH 8.0 pH 8.5 LD ₅₀ (U / ml) 2.5 × 10 ⁵ 3.5 × 10 ⁵ 4 × 10 ⁵ 2 × 10 ⁵

Each medium was incubated for 5 days under the same conditions, and when tested for potency, the lethality of botulinum toxin in the medium having a pH of 8.0 was confirmed to be at least 1.5 to 2 times higher than other conditions.

1-5 Determine the culture days of the medium

In order to determine the amount of botulinum toxin according to the culture days, the conditions of 3 days, 4 days, 5 days, 6 days as the medium conditions (0.5% yeast extract, 0.5% glucose, pH 8.0) of Experimental Examples 1 to 4 The Clostridium botulinum was cultured in the medium. The culture medium for each medium was injected into rats bred in a sterile state and then irradiated with a lethal dose LD50. In addition, the amount of the protein extracted with the culture solution of the medium according to the culture days was examined, and shown in Table 5 together with the lethal dose.

Half dose of culture medium Incubation days 3 Day 4 Day 5 Day 6 Day LD ₅₀ (U / ml) 4 × 10 ⁵ 3 × 10 ⁵ 4 × 10 ⁵ 3 × 10 ⁵ After Acetate (Before Purification) Protein by Culture Day (mg / ml) 13.5 11 8.4 8.5

Looking at Table 5 , the lethal dose according to the botulinum toxin in the culture test under the same conditions and cultured under the same conditions was 1.5 times higher or similar to that of other media. In addition, when the lethal amount was similar, it was confirmed that the purity after purification was small because the amount of protein (protein) other than botulinum toxin was low.

1-6 Culture Method Decision

In order to determine the amount of botulinum toxin according to the culture method, the cloth was subjected to stagnant culture and fermenter culture conditions using the medium conditions (0.5% yeast extract, 0.5% glucose, pH 8.0) of Experimental Examples 1 to 4. Tridydum botulinum was incubated. The culture medium for each medium was injected into rats bred in a sterile state, and then irradiated with a semi-numerical dose LD50 is shown in Table 6.

Semi-numerical dose by culture method Culture method Stagnation Fermenter Culture LD ₅₀ (U / ml) 5 × 10 ⁴ 4 × 10 ⁵

Looking at Table 6 , it was confirmed that the fermenter (rmentor) culture in the potency test by the culture for 5 days under the same conditions at least 5 ~ 10 times higher than lethal culture according to the botulinum toxin.

< Example  2 - Mountain precipitation  Step>

2-1 Sulfuric Acid Precipitation

In Experimental Examples 1 to 7, the rats were cultured in a medium having the highest lethal culture condition (0.5% yeast extract, 0.5% glucose, cultured in an incubator for 5 days in a medium having a pH of 8.0) in the medium, and then Cultures of the medium were precipitated at different pH using sulfuric acid. Each of the precipitates were injected into rats bred in a sterile state to investigate the half lethal dose LD50 and are shown in Table 7 below.

Half dose of sulfuric acid by pH PH of sulfuric acid pH 2.5 pH 3.0 pH 3.5 pH 4.0 LD ₅₀ (U / ml) 1 × 10 ⁵ 3 × 10 ⁵ 4 × 10 ⁶ 2 × 10 ⁵

Referring to Table 7, the recovery rate of the botulinum toxin is higher as the pH is lower, but in the case of pH 2.5 and pH 3.0, it is confirmed that the titer is lowered by affecting the botulinum toxin itself, and the recovery rate of the botulinum toxin is lowered at pH 4.0. Eventually, it was confirmed that pH 3.5 recovered the highest titer.

2-2 Acetate Extraction

2-2-1 Acetate Concentration in Acetate Extraction Step

Extraction was performed using acetate to extract botulinum toxin from the precipitated protein of Clostridium botulinum which was subjected to sulfuric acid precipitation step by sulfuric acid. To determine the amount of botulinum toxin according to the concentration of acetate, acetates of 0.05M, 0.1M and 0.15M concentrations were prepared and extracted at respective concentrations. The extracts extracted at the respective concentrations were injected into rats bred in a sterile state to examine the semi-numerical dose LD50.

In addition, the extract extracted at each concentration was subjected to the ethanol precipitation step, and the precipitates were injected into rats bred in a sterile state to examine the semi-numerical dose LD50. The results of the investigation are shown in Table 8 below.

Stepwise half dose according to acetate concentration 0.05M 0.1M 0.15M Mouse LD ₅₀ after acetate treatment at each concentration (U / ml) 1 × 10 ⁶ 2 × 10 ⁶ 1.5 × 10 ⁶ After acid precipitation for each concentration (after treatment with EtOH) Mouse LD ₅₀ (U / Protein-mg) 4 × 10 ⁶ 2 × 10 ⁶ 1.5 × 10 ⁶

Referring to Table 8, when the acetate concentration is 0.1M, the titer is about twice as good as the other concentrations, but after the acid precipitation process, the titer comes out to be similar to the titer of 0.05M, and the toxin recovery rate per protein-mg is 0.05M. Found to be the highest at.

2-2-2 Extraction Time in Acetate Extraction Step

The precipitated protein of Clostridium botulinum, which had been subjected to sulfuric acid precipitation by sulfuric acid, was extracted using acetate. To determine the amount of botulinum toxin according to the extraction time of the acetate, it was extracted with acetate for 1 hour, 2 hours and 24 hours. The extracts extracted at the respective concentrations were injected into rats bred in a sterile state and irradiated with a semi-numerical dose LD50 is shown in Table 9.

Half dose according to acetate extraction reaction time Extraction time 1 hr 2 hr 24 hr Mouse LD ₅₀ (U / ml) after acetate treatment by reaction time 1.5 × 10 ⁶ 2 × 10 ⁶ 1 × 10 ⁶

Looking at the Table 9, it was confirmed that the titer was the highest when extracted with the acetate for 2 hours, and when the extraction time was performed for 24 hours, the titer was reduced by 1.5 to 2 times compared to 2 hours.

2-2-3 Acetate in Acetate Extraction pH

To determine the amount of botulinum toxin extracted according to the pH of the acetate, an acetate of pH 5.3, pH 6.0, and pH 6.5 was prepared, and the extract extracted for each pH was injected into sterile rats to investigate the semi-numerical dose.

The lethal amount of acetate at pH 5.3 was 3 × 10 ⁵ U / ml, and the lethal amount of acetate at pH 6.0 was 2 × 10 ⁶ U / ml. In addition, the lethal amount of acetate having a pH of 6.5 was 4 × 10 ⁵ U / ml.

It was confirmed that the pH.6.5 of the acetate was about 5 to 10 times higher than the pH 5.3, and about 4 to 5 times higher than the pH 6.0.

2-2-4 Sodium Chloride in Acetate Extraction Addition

To determine the amount of botulinum toxin depending on whether sodium chloride (NaCl) was added when extracted with acetate, sodium chloride at 0.05 M, 0.1 M and 0.15 M concentrations was prepared and extracted at each concentration. The extracts extracted at the respective concentrations were injected into rats bred in a sterile state to examine the half lethal dose LD50.

The lethal amount of acetate having 0.05M sodium chloride was 1 × 10 ⁶ U / ml, and the lethal amount of the extract extracted with acetate having 0.1M sodium chloride was 1 × 10 ⁶ U / ml. In addition, the lethal amount of the extract extracted with 1M sodium chloride acetate was 7 × 10 ⁵ U / ㎖, the lethal amount of the extract extracted with acetate containing no sodium chloride was 2 × 10 ⁶ U / ㎖.

Looking at the results, it was confirmed that the case of not containing sodium chloride is 2 to 3 times higher in titer than other conditions.

2-2-5 Calcium Chloride in Acetate Extraction Addition

To determine the amount of botulinum toxin depending on the addition of calcium chloride (CaCl ₂ ) upon extraction with acetate, calcium chloride at 0.15M, 0.6M and 1M concentrations was prepared and extracted at each concentration. The extracts extracted at the respective concentrations were injected into rats bred in a sterile state to examine the half lethal dose.

The lethal amount of acetate having 0.15M of calcium chloride was 1 × 10 ⁶ U / mL, and the lethal amount of the extract extracted with acetate having a concentration of 0.6M of calcium chloride was 5 × 10 ⁵ U / mL. In addition, the lethal amount of the extract extracted with 1M calcium chloride acetate was 2 × 10 ⁵ U / ㎖, and the lethal amount of the extract extracted with acetate containing no calcium chloride was 2 × 10 ⁶ U / ㎖.

When the calcium chloride is included, the RNA removal rate is partially increased, but the toxicity is reduced by about 2 to 10 times.

< Example  3-hydrochloric acid precipitation step>

3-1 Hydrochloric Acid in Hydrochloric Acid Precipitation pH  Amount of Toxins According to Concentration of Hydrochloric Acid and Hydrochloric Acid>

Hydrochloric acid of pH 2.5, pH 3.0, pH 3.5 and pH 3.7 to determine the amount of botulinum toxin extracted according to the pH of the hydrochloric acid in the hydrochloric acid precipitation step of precipitating the botulinum toxin using the hydrochloric acid acetate extract of the acetate extraction step Prepare the pH by The precipitate precipitated as was injected into rats bred in a sterile state to investigate the mortality is shown in Table 10 below.

pH 2.5 pH 3.0 pH 3.5 pH 3.7 Mouse LD ₅₀ after HCl treatment (U / ml) 3 × 10 ⁶ 3 × 10 ⁶ 5 × 10 ⁶ 5 × 10 ⁶

Referring to Table 10, the pH of the hydrochloric acid at pH 3.7 was confirmed that the botulinum toxin is more than 90% and the titer is 1.5 times higher than other conditions. In addition, referring to Figure 3, it was also confirmed that the case of precipitation with hydrochloric acid having a pH of 3.7 is higher RNA removal rate than other conditions.

In addition, the method of adding 0.1 N HCl at pH 6.5 to pH 5.0 and 1 N hydrochloric acid at pH 5.0 to pH 3.7 is more active or toxin compared to adding only 1 N hydrochloric acid (pH 6.5 to pH 3.7). (Toxin) recovery showed similar results, but the precipitated form, ie, protein entanglement was significantly reduced.

< Example  4 - RNase Of DNase  Processing>

4-1 Ethanol Precipitation RNase According to throughput and processing time RNA Removal rate

In treating the precipitate by the hydrochloric acid precipitation step, 50ug / ml, 100ug / ml, and 200ug / ml were prepared to determine the removal rate of DNA and RNA according to the throughput of the DNase and RNase. It was. The removal rate of DNA and RNA according to the amount of DNase and RNase was examined, but there was no significant difference.

In order to determine the removal rate of DNA and RNA according to the treatment time of DNase, RNase, the DNase and RNase were treated for 1 hour, 2 hours and 3 hours. Referring to Figure 4, the DNase, RNase was not significantly different when treated for 2 hours, 3 hours, but DNA, RNA was mostly decomposed (less bands during electrophoresis), DNA, RNA at 1 hour treatment It was confirmed that a lot remained (a lot of bands during electrophoresis).

Example  5-Ethanol Precipitation

5-1 Ethanol Concentration in Ethanol Precipitation

Treatment of the precipitate by the hydrochloric acid precipitation step DNase, RNase, and to determine the amount of toxin according to the concentration of ethanol in the ethanol precipitation step of precipitating in ethanol (%) 12%, 15% , 18% and 21% ethanol were prepared. The precipitates precipitated at the respective concentrations were injected into rats bred in a sterile state to investigate the lethality and are shown in Table 11 below.

EtOH concentration 12% 15% 18% 21% LD ₅₀ (U / Protein-mg) 1 × 10 ⁶ 4 × 10 ⁶ 3 × 10 ⁶ 2 × 10 ⁶

Referring to Table 11, when the ethanol concentration is 15 and 18%, the titer is better than the concentration of 12 and 21%, but there is no significant difference between the two. However , 15% of the total protein recovered was less because it was confirmed that the best.

5-2 Amount of Toxins Depending on Treatment Time in Ethanol Precipitation

In order to determine the amount of toxin according to the treatment time of the ethanol, the treatment time of ethanol was treated with 1 hour and 24 hours. The amount of mortality was examined by injecting the precipitate according to each treatment time into rats bred in a sterile state. When the ethanol was treated for 1 hour, the lethal dose was 8 × 10 ⁶ U / ml, and when treated for 24 hours, the lethal dose was 2 × 10 ⁷ U / ml. As a result, it was confirmed that the titer was about 3 times higher in the case of treatment for 24 hours than in the case of treatment for 1 hour.

5-3 Ammonium Sulfate Precipitation

The precipitate by ethanol of Example 5-2 was precipitated in ammonium sulfate ((NH 4) ₂ SO ₄ ) to induce crystallization of botulinum toxin.

Example  6-tablet

6-1 Determination of Use Buffer in Purification

The precipitate containing the botulinum toxin obtained by the acid precipitation step was purified using liquid chromatography. Solubility of the toxin using Tris, calcium phosphate (K ₃ PO ₄ ), sodium phosphate (Na ₂ PO ₄ ) sodium acetate (CH ₃ COONa) to determine the buffer solution for the purification Was investigated. When sodium acetate was used as a buffer, the solubility of botulinum toxin was small but high compared to other conditions.

In addition, it was shown in Table 12 to investigate the lethality according to toxin after dissolving in each buffer solution.

Tris Potassium phosphate Sodium phosphate Sodium acetate LD ₅₀ (U / ml) 5 × 10 ⁶ 8 × 10 ⁶ 1 × 10 ⁶ 1 × 10 ⁷ A _260/280 1.01 1.00 1.00 1.00 A ₂₆₀ 0.092 0.083 0.089 0.103 A ₂₈₀ 0.092 0.083 0.089 0.103

Looking at Table 12, it can be seen that the highest lethal dose when sodium acetate was selected as the buffer solution.

Although the present invention has been described with reference to the examples, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

Purification method of the botulinum toxin of the present invention is capable of mass production of the botulinum toxin by shortening the purification time and has an effect of lowering the cost by the mass production, and there is no need to be made in an anaerobic facility, so there is an economic effect. Since the botulinum toxin is clearly separated and identified by the purification method, the efficacy of the toxin can be increased. The botulinum toxin purified by the above purification method may be used as an active ingredient of the pharmaceutical composition, for the treatment of neuromuscular disorders, essential blepharospasm, treatment of the glabellar (facial) wrinkles, and the like.

1 is a process chart showing a process for purifying the botulinum toxin;

Figure 2 shows the comparison of the inclusion of the nucleic acid in the acid precipitation process in brainheart infusion medium and peptone medium by electrophoresis results;

3 is a diagram showing the results of electrophoresis by pH of HCl precipitation in the acid precipitation step;

4 is a view showing the results of electrophoresis by DNase, RNase treatment time; And

5 is a view showing the results by purification step (SDS-PAGE).

Claims (11)

A first step of culturing Clostridium botulinum; Primary acid precipitation of the Clostridium botulinum cultured in the first step; After extraction with acetate; A second step of precipitating by adding acid; And And a third step of purifying the botulinum toxin by performing liquid chromatography on the precipitate of the second step. The method of purifying botulinum toxin from Clostridium botulinum, characterized in that it comprises a. The method of claim 1, wherein the culture medium of the first step comprises brain heart infusion, peptone, yeast extract, and glucose. The method of claim 2, wherein the culture medium is a yeast extract concentration of 0.3 to 0.5%, the concentration of glucose is 0.5 to 1%, the culture is cultured by a fermenter (fermentor) for 3 to 5 days How to. The method of claim 1, wherein the second acid precipitation is sulfuric acid, and the second acid precipitation is hydrochloric acid. The method of claim 1, wherein the acetate used in the acetate extraction of the second step is 0.04 to 0.09 M, pH is 6.3 to 6.5, and the extraction time is 1.5 to 2.5 hours. The method of claim 4, wherein the sulfuric acid has a pH concentration of 3.0 to 3.9. The method of claim 4, wherein when precipitation with the hydrochloric acid, 0.1N hydrochloric acid is added when the pH of the acetate extract is 5.0 ~ 6.5, 1N hydrochloric acid is added when the pH of the acetate extract is 3.7 ~ 5.0, PH of the hydrochloric acid added is 3.5 to 3.8. According to claim 1, wherein the second step is treated with DNase, RNase to the precipitate by the hydrochloric acid, and then precipitated in ethanol; Precipitating the precipitate by ethanol in ammonium sulfate to induce crystallization; characterized in that it further comprises. According to claim 8, DNase, RNase treatment time of the ethanol precipitation step is characterized in that the 1.5 to 3 hours. The method of claim 9, wherein the ethanol concentration of the ethanol precipitation step is 15 to 18%, the process time of the ethanol is characterized in that 1 to 24 hours. The method of claim 1, wherein the third step is characterized in that using sodium acetate solution as a buffer solution.

Images