Method for purifying botulinum toxin complex with improved purification yield

Abstract

The present invention relates to a method for purifying a botulinum toxin with improved purification purity and yield When the method for purifying a botulinum toxin of the present invention is used, the botulinum toxin can be purified with a very high yield.

Description

[Technical Field]

[0001] This patent application claims priority to Korean Patent Application No. 10-2021-0096681, filed with the Korean Intellectual Property Office on July 22, 2021, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a method for purifying a botulinum toxin complex with improved purification purity and yield. [Background technology]

[0003] Botulinum toxin is a neurotoxin produced by the anaerobic bacterium Clostridium botulinum (C. botulinum). There are seven types (types A to G), of which only two types, botulinum types A and B, are currently purified and used medically. Botulinum toxin inhibits the release of the neurotransmitter acetylcholine, blocking muscle contraction signals and causing muscle relaxation. It induces nerve paralysis by blocking the release of acetylcholine, a neurotransmitter secreted at the presynaptic terminal of the neuromuscular junction. While fillers are medical devices that fill areas of skin where volume is lacking, botulinum toxin preparations are pharmaceutical products containing ingredients that reduce muscle use by preventing the release of neurotransmitters that cause muscle contraction.

[0004] Botulinum toxin is primarily used to suppress or remove wrinkles between the eyebrows and around the eyes, and is also used to treat upper limb spasticity after stroke, eyelid spasms, and equinus deformity, and its indications are gradually expanding.

[0005] All botulinum toxin serotypes block neurotransmission by similarly inhibiting the release of acetylcholine at the neuromuscular junction terminals of peripheral nerves. During the signal transduction process in normal neuromuscular junctions, the toxin binds to monosaccharides in cholinergic nerve terminals and then enters the cell, inhibiting the function of SNARE proteins. This inhibits the fusion of endoplasmic reticulum containing acetylcholine and the release of acetylcholine, thereby preventing neuromuscular transmission. Cholinergic synapses interrupted by botulinum toxin are replaced by the formation of new synapses. Furthermore, existing interrupted synapses gradually regenerate and regain their original function, lasting an average of 3 to 4 months.

[0006] The botulinum toxin complex can be obtained through a process of pretreatment and purification of C. botulinum cell cultures.

[0007] Various processes for purifying pretreated cell cultures to obtain botulinum toxin complexes are known. Previously, methods for purifying botulinum toxin using ion exchange chromatography and / or hydrophobic chromatography in sequence have been known (KR10-2020-0121245 A and KR10-2020-0121246 A). However, although these methods achieved relatively high purity, they suffered from low production yields. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have conducted extensive research to develop a method for purifying a botulinum toxin complex with high production purity and yield, and have discovered that a two-step purification process using a mixed-mode anion exchange resin and a mixed-mode cation exchange resin can significantly improve the purification yield of botulinum toxin, thereby completing the present invention.

[0009] Therefore, an object of the present invention is to provide a method for purifying a botulinum toxin complex with improved purification purity and yield. [Means for solving the problem]

[0010] According to one aspect of the present invention, there is provided a method for purifying a botulinum toxin complex, comprising the steps of: (a) preparing a culture broth of a botulinum toxin-producing strain; (b) purifying the prepared culture broth using a mixed-mode anion exchange resin as a first purification step; and (c) purifying the culture broth that has undergone the first purification step using a mixed-mode cation exchange resin as a second purification step.

[0011] The present inventors have conducted extensive research to develop a method for purifying a botulinum toxin complex with high production purity and yield, and have found that the purification yield of the botulinum toxin complex can be significantly improved by using a two-step purification process using a mixed-mode anion exchange resin and a mixed-mode cation exchange resin.

[0012] As used herein, the term "botulinum toxin" refers to a toxin that can be purified and obtained from a strain of Clostridium botulinum (C. botulinum). The term "botulinum toxin complex" refers to a complex substance in which a botulinum toxin is bound to one or more non-toxin proteins. The botulinum toxin produced by a C. botulinum strain has a size of approximately 150 kDa, and the botulinum toxin complex has a size of approximately 300 kDa to 900 kDa.

[0013] The purified botulinum toxin complex can be used for various medical purposes, including the prevention or improvement of frown lines and wrinkles around the eyes.

[0014] In one embodiment of the present invention, the botulinum toxin-producing strain of the present invention is Clostridium botulinum (C. botulinum). More specifically, Clostridium botulinum Type A (ATCC 19397) strain may be used, but is not limited thereto.

[0015] The present invention will be described in detail below step by step.

[0016] Step (a): Preparing a culture medium of a botulinum toxin-producing strain The step of preparing a culture medium of a botulinum toxin-producing strain can be carried out using various conventionally known pretreatment processes, and is not particularly limited.

[0017] For example, a culture solution before the purification process can be prepared by culturing a bacterial strain and then performing one or more precipitation, filtering, and re-dissolving steps. The culture can consist of a seed culture and a main culture, and the precipitation can be performed through an acid treatment step.

[0018] Various culture media known in the art and readily recognized by those skilled in the art as suitable for culturing Clostridium botulinum can be used for seed culture and main culture of the strain. A specific example is PYG medium, known to contain 2% peptone, 1% yeast extract, and 1% glucose. The culture medium can be sterilized before inoculation with the strain, for example, using an autoclave. The sterilized medium is preferably initialized and maintained under anaerobic conditions. For example, seed culture can be performed by inoculating the culture medium with the strain at a ratio of approximately 1 / 50 to 1 / 200, 1 / 50 to 1 / 150, or 1 / 80 to 1 / 120, or more specifically, at a ratio of approximately 1 / 100, but is not necessarily limited to these. Seed culture can be determined to have been completed when the entire medium becomes opaque when visually inspected.

[0019] As with seed culture, main culture is preferably carried out after sterilizing the culture medium. Main culture can be completed after culturing for about 60 to 75 hours, more specifically, 63 to 72 hours, after inoculation of the seed culture solution. The conditions for main culture can be appropriately adjusted according to the common technical knowledge of those skilled in the art depending on the culture environment.

[0020] In one embodiment of the present invention, step (a) of the present invention includes the following steps: (a-1) culturing a botulinum toxin in a culture medium; (a-2) a first precipitation step of adding sulfuric acid to cause a reaction; (a-3) an enzyme treatment step of adding benzonase to cause a reaction; (a-4) a second precipitation step of recovering the supernatant of the solution that has undergone the enzyme treatment step and adding phosphoric acid to cause a reaction; and (a-5) a redissolution step of redissolving the precipitate using a buffer solution.

[0021] In one embodiment of the present invention, the first precipitation step using sulfuric acid can be performed by titrating the culture solution after the completion of the main culture to about pH 3.5 using 5N sulfuric acid, confirming the presence of pale yellow suspended matter, and then leaving the solution at room temperature for half a day to one day.

[0022] In one embodiment of the present invention, the enzyme treatment step can be carried out by adding benzonase, and the benzonase treatment can be preceded by pretreatment with benzamidine hydrochloride hydrate. After the enzyme treatment, the reaction is continued for, for example, 2 to 10 hours, 3 to 8 hours, or 4 to 7 hours, and the enzyme-treated solution is recovered and centrifuged to collect only the supernatant, which can then be used for the next step, the second precipitation step.

[0023] According to one embodiment of the present invention, after the enzyme treatment, a second precipitation can be carried out using phosphoric acid. More specifically, phosphoric acid precipitation can be carried out by adding 10% phosphoric acid to a pH of about 3.5. After phosphoric acid precipitation is completed, the precipitation solution is left to stand for half a day to one day.

[0024] According to one embodiment of the present invention, the phosphate precipitate is centrifuged, the supernatant is removed, and only the pellets are recovered and redissolved in a buffer solution to prepare a culture medium.

[0025] In an embodiment of the present invention, after the step (a-5), a step of centrifuging to remove precipitates may be further included.

[0026] Step (b): The first purification step is to purify the prepared culture medium using a mixed-mode anion exchange resin. The first purification step, step (b) of the present invention, is a step of purifying the culture solution of the botulinum toxin-producing strain prepared through step (a) using a mixed-mode anion exchange resin.

[0027] In one embodiment of the present invention, the multi-functional anion exchange resin of the present invention has multi-functional selectivity due to the combination of hydrophobic interaction and ionic interaction.

[0028] In one embodiment of the present invention, the multifunctional anion exchange resin of the present invention may be selected from the group consisting of TOYOPEARL NH2-750F, HyperCel STAR AX Resin, or POROS XQ. More specifically, TOYOPEARL NH2-750F may be used. The above-mentioned specific examples are provided to facilitate easy implementation of the present invention, and it will be obvious to those skilled in the art that multifunctional anion exchange resins having similar or equivalent physical properties may also be used.

[0029] This first purification step can be performed by loading the culture medium prepared in step (a) onto a column packed with a mixed-mode anion exchange resin. After loading, washing and elution can be performed using conventional methods.

[0030] Step (c): The second purification step is to purify the culture solution that has undergone the first purification step using a mixed-mode cation exchange resin. The second purification step of step (c) of the present invention is a step of secondarily purifying the purified solution obtained by the first purification step (b) using a mixed-mode cation exchange resin.

[0031] In one embodiment of the present invention, the multi-functional cation exchange resin of the present invention has multi-functional selectivity due to the combination of hydrophobic interaction and ionic interaction.

[0032] In one embodiment of the present invention, the multi-functional cation exchange resin of the present invention may be selected from the group consisting of TOYOPEARL Sulfate-650F, TOYOPEARL MX-Trp-650M, Eshmuno CPS, or POROS XS. More specifically, TOYOPEARL Sulfate-650F may be used. The above-mentioned specific examples are provided to facilitate easy implementation of the present invention, and it will be apparent to those skilled in the art that multi-functional anion exchange resins having similar or equivalent physical properties may also be used.

[0033] This second purification step can be performed by loading the purified solution obtained through step (b) into a column packed with a mixed-mode cation exchange resin. After loading, washing and elution can be performed using conventional methods.

[0034] The present invention is characterized in that a mixed-mode anion exchange resin is used in the first purification step, followed by a mixed-mode cation exchange resin in the second purification step.

[0035] When purifying a botulinum toxin complex, it was confirmed that a process using a general anion exchange resin, a cation exchange resin, etc. in sequence rather than a multi-functional exchange resin, showed a significantly lower purification yield than using the two-step process according to one embodiment of the present invention. In addition, it was confirmed that a three-step purification process using an additional hydrophobic exchange resin also showed a significantly lower purification yield than the purification process of the present invention. [Effects of the Invention]

[0036] The features and advantages of the present invention can be summarized as follows: (a) The present invention provides a method for purifying a botulinum toxin complex. (b) When the method for purifying a botulinum toxin complex of the present invention is used, the botulinum toxin complex can be purified with a very high yield. [Brief explanation of the drawings]

[0037] [Figure 1] The analysis results of the product obtained after the first purification step using a mixed-mode anion exchange resin are shown. [Figure 2] 1 shows the results of SDS-PAGE analysis of the product obtained after the first purification step using a mixed-mode anion exchange resin. [Figure 3] 1 shows the analytical results of the product obtained after the second purification step using a mixed-mode cation exchange resin. [Figure 4] 1 shows the results of SDS-PAGE analysis of the product obtained after the second purification step using a mixed-mode cation exchange resin. [Figure 5] 1 shows the results of SDS-PAGE analysis of the stock solution of the botulinum toxin complex used in the present invention. [Figure 6] 1 shows the results of SE-HPLC analysis of the stock solution of the botulinum toxin complex used in the present invention. [Figure 7] 1 is a graph showing the results of a comparative experiment on purification yields. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are provided solely for the purpose of illustrating the present invention in more detail, and that the scope of the present invention is not limited by these examples.

[0039] Example Example 1: Seed culture 1-1. Medium preparation The medium used for seed culture was prepared by adding 2% peptone (Sigma, 29185), 1% yeast extract (Merck, 1.03753), and 1% glucose (Merck, 1.37048). The medium was sterilized using an autoclave. The sterilized medium was placed in an anaerobic incubator and maintained at 36°C under anaerobic conditions.

[0040] 1-2. Inoculation and culture Clostridium botulinum type A strain was transferred into the anaerobic incubator using a pass box maintained at 36±1°C under anaerobic conditions (O2, 1.0% or less). After the strain thawed in the anaerobic incubator, the bacteria were inoculated into the medium at a ratio of 1 / 100. After 24 hours of static incubation at 36±1°C under anaerobic conditions, the medium was deemed to have become a seed culture when the entire medium became opaque when visually inspected.

[0041] Example 2: Main culture 2-1. Medium preparation The medium used for the main culture was prepared by adding 2% peptone (Sigma 29185), 1% yeast extract (Merck 1.03753), and 1% glucose (Merck 1.37084). Prior to the main culture, the sensors used in the fermenter were calibrated, and a turbidity sensor, pH sensor, and pO2 sensor were attached to the fermenter. The prepared main culture medium was placed in the fermenter and sterilized at 121°C for 20 minutes. After sterilization, the medium was maintained under anaerobic conditions (pO2 below 5.0%).

[0042] 2-2.Main culture The seed culture was removed from the anaerobic incubator. The inoculation line was opened and the seed culture was inoculated into the incubator. After the start of main cultivation, the main cultivation was completed after 72 hours of cultivation.

[0043] Example 3: Sulfuric acid precipitation After the main culture was completed, the fermenter was titrated to pH 3.5 using 5N sulfuric acid. After the pH was adjusted to 3.5±0.1, the fermenter was left at room temperature for 12 hours.

[0044] Example 4: Enzyme treatment 4-1.pH titration The supernatant of the sulfuric acid precipitate was removed, and after the supernatant was removed, the sulfuric acid precipitate was titrated to pH 6.0±0.1 by adding 5N sodium hydroxide solution (Merck, 1.06482) at 36°C and 100 rpm.

[0045] 4-2.Enzyme treatment Benzamidine hydrochloride hydrate (Sigma, B6506) was added to a concentration of 10 mM / L. Benzonase (Merck, 1.01697) was added to a concentration of 500,000 U / L. The mixture was allowed to react at 36°C and 100 rpm for 5 hours.

[0046] After the reaction, the recovered enzyme-treated solution was centrifuged at 13,000×g for 20 minutes at 4° C., and the supernatant was collected and the pellet was removed.

[0047] Example 5: Phosphoric Acid Precipitation The pellet was removed from the enzyme-treated supernatant, and 10% phosphoric acid (AppliChem, 147143) was added to the supernatant to titrate to pH 3.5. After phosphoric acid precipitation was completed, the solution was left at room temperature for 12 hours.

[0048] Example 6: Toxin Reconstitution After leaving the phosphate precipitate, the solution was centrifuged at 13,000 x g for 20 minutes at 4°C, the supernatant was discarded, and the pellet was collected. The pellet was redissolved in 50 mM sodium phosphate buffer (pH 6.5). The redissolved toxin solution was centrifuged at 13,000 x g for 20 minutes at 4°C, the supernatant was collected, and the pellet was discarded.

[0049] Example 7: Primary purification UV 278nm The conductivity baseline was checked for stabilization. The reconstituted toxin solution was filtered through a 0.22 μm bottle-top filter to remove foreign matter. The filtered toxin solution was loaded onto a column packed with NH2-750F, a multimodal anion exchange resin, at a linear velocity of 46 cm / hr. After loading, the column was washed with 500 mL or more of 50 mM sodium phosphate buffer, pH 6.5. After washing, the column was eluted with 50 mM sodium phosphate buffer, pH 6.5, and 1 M sodium chloride buffer at 60%, and 5 mL fractions were collected. The 5 mL fractions eluted at 60% were collected and pooled based on the SDS-PAGE results.

[0050] Example 8: Secondary purification UV 278nm The conductivity baseline was checked for stabilization. 25 mM citrate buffer, pH 5.5, was added to the primary purified pool to adjust the pH to 5.5 ± 0.1 and the conductivity to 7.0 ± 0.5 mS / cm. The solution was filtered through a 0.22 μm bottle-top filter to remove impurities. The filtered toxin solution was loaded onto a column packed with multimodal cation exchange resin, Sulfate-650F, at a linear velocity of 46 cm / hr. After loading, the column was washed with 25 mM citrate buffer, pH 5.5, for a volume of at least 500 mL. After washing, the column was eluted with 25 mM citrate buffer, pH 5.5, and 1 M sodium chloride buffer at 14%, and 3 mL fractions were collected and pooled based on the SDS-PAGE results.

[0051] Example 9: Filtration and aliquoting The secondary purified pooled solution was filtered using a 0.2 μm Syringe filter. After filtration, the secondary purified pooled solution was divided into small portions and stored at -70°C or below.

[0052] Example 10: Purified product protein band analysis SDS-PAGE analysis samples were prepared under the reducing conditions shown in the table below. The analysis samples were electrophoresed on Novex 4-12% Bis-Tris gels (Invitrogen, NP0321BOX) using MES SDS Running buffer (Invitrogen, NP0002). After electrophoresis, the gels were stained with Coomassie Blue stain reagent (Instant Blue, expedeon, ISB1L) for 1 hour and then thoroughly washed with triple-distilled water. After washing, the gels were analyzed using an image analyzer (ATTO, WSE-6100).

[0053] [Table 1]

[0054] As a result of the analysis, it was confirmed that no impurity proteins were observed other than the seven protein bands known to constitute the botulinum toxin type A complex (see Figure 5).

[0055] Example 11: Purified product purity analysis The SE-HPLC analysis was carried out under the following conditions.

[0056] [Table 2]

[0057] As a result of the analysis, a single peak of 100% purity was confirmed with a retention time of approximately 7.7 minutes (approximately 900 kDa) (see Figure 6).

[0058] Example 12: Yield analysis through protein quantification BCA assay (Thermo Scientific, 23227) was performed under the conditions shown in the table below, and yield comparison was performed through protein quantification.

[0059] [Table 3]

[0060] The methods described in Korean Application No. 2020-044716 (KR 10-2020-0121245 A) and Korean Application No. 2020-044717 (KR 10-2020-0121246 A) were set as controls to compare the yield of the final product.

[0061] [Table 4]

[0062] As a result, it was confirmed that the yield was improved by about 44 times compared to Comparative Example 1 and by about 7 times compared to Comparative Example 2.

Claims

1. (a) preparing a culture of a botulinum toxin-producing strain; (b) a first purification step of purifying the prepared culture solution using a mixed-mode anion exchange resin; (c) a second purification step of purifying the culture solution that has undergone the first purification step using a mixed-mode cation exchange resin; The method for purifying a botulinum toxin complex is characterized in that the multi-functional anion exchange resin and the multi-functional cation exchange resin have multi-functional selectivity resulting from a combination of hydrophobic interaction and ionic interaction.

2. 2. The method for purifying a botulinum toxin complex according to claim 1, wherein the botulinum toxin-producing strain is C. botulinum Type A.

3. 2. The method for purifying a botulinum toxin complex according to claim 1, wherein the multifunctional anion exchange resin is selected from the group consisting of TOYOPEARL® NH2-750F, HyperCel® STAR AX Resin, or POROS® XQ.

4. 2. The method for purifying a botulinum toxin complex according to claim 1, wherein the multi-functional cation exchange resin is selected from the group consisting of TOYOPEARL® Sulfate-650F, TOYOPEARL® MX-Trp-650M, Eshmuno® CPS, or POROS® XS.

5. 2. The method for purifying a botulinum toxin complex according to claim 1, wherein step (a) comprises the following steps: (a-1) culturing a botulinum toxin in a culture medium; (a-2) a first precipitation step in which sulfuric acid is added and reacted; (a-3) an enzyme treatment step in which benzonase is added and reacted; (a-4) a second precipitation step in which the supernatant of the solution that has been subjected to the enzyme treatment step is recovered and phosphoric acid is added to cause a reaction; and (a-5) A redissolution step in which the precipitate is redissolved using a buffer solution.

6. The method for purifying a botulinum toxin complex according to claim 5, further comprising the step of centrifuging to remove precipitates after step (a-5).

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