Method for purifying Clostridium botulinum neurotoxin protein from which non-toxin proteins have been removed

Abstract

The present invention relates to a method for purifying Clostridium botulinum neurotoxin proteins from which non-toxin proteins have been removed.

Description

[Technical field]

[0001] The present invention relates to a method for purifying Clostridium botulinum neurotoxin proteins from which non-toxin proteins have been removed. [Background technology]

[0002] Botulinum toxin is a type of neurotoxin protein produced by bacteria such as Clostridium botulinum, which irreversibly attaches to presynaptic nerve terminals and inhibits the secretion of acetylcholine at the nerve junction, thereby inhibiting muscle contraction and exerting a secondary muscle relaxation effect. Due to this function, botulinum toxin has been used for therapeutic and cosmetic purposes since it was approved by the U.S. FDA in 1989 (Patent Documents 1, 2, etc.).

[0003] It is used therapeutically as an injection for neuromuscular disorders such as strabismus, torticollis, and facial spasms, and for cosmetic purposes to remove wrinkles, facial lines, and to treat square jaw, hyperhidrosis, and migraines. Although there have been reported cases of side effects such as dysphagia, voice change, dry mouth, and blurred vision, there have been no fatalities directly caused by botulinum toxin, and it is considered to be a very safe drug when used appropriately.

[0004] In particular, a pure neurotoxin protein obtained by removing non-toxic proteins from botulinum toxin is useful for indications requiring repeated administration, and therefore there is a demand for an improved purification method for isolating a botulinum neurotoxin protein that is stable and biologically active. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent Publication No. 10-2010-0107475 [Patent Document 2] Korean Patent Publication No. 10-2008-0049152 Summary of the Invention [Problem to be solved by the invention]

[0006] We provide an improved purification method for isolating stable, biologically active botulinum neurotoxin proteins. [Means for solving the problem]

[0007] An object of the present invention is to provide a method for purifying a Clostridium botulinum neurotoxin protein from which non-toxin proteins have been removed. Effect of the Invention

[0008] By using the purification method of the present invention, a highly purified botulinum neurotoxin protein can be obtained economically and efficiently. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the results of SDS-PAGE, i.e., sodium dodecylsulfate polyacrylamide gel electrophoresis, of purified 150 kD botulinum neurotoxin. [Diagram 2] FIG. 1 shows the results of HPLC, i.e., size exclusion chromatography (SEC-HPLC), of purified 150 kD botulinum neurotoxin. [Diagram 3]FIG. 1 shows the results of confirming the purity of the sample obtained by electrophoresis when a DEAE column containing a tertiary amine or a Q column containing a quaternary ammonium is used in the step of performing anion chromatography (HC: heavy chain, LC: light chain). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] These will be described in detail below. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0011] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0012] One aspect of the present invention is a method for purifying a Clostridium botulinum neurotoxin protein.

[0013] Specifically, the purification method may include the steps of (a) loading a solution containing Clostridium botulinum toxin protein separated from a culture medium onto a hydrophobic interaction column to capture the toxin and allow impurities to pass through; (b) separating the toxin captured in step (a) to obtain an eluate containing the toxin; (c) obtaining an uncomplexed form of neurotoxin protein from the toxin protein contained in the eluate obtained in step (b); (d) loading the solution containing the uncomplexed form of neurotoxin protein obtained in step (c) onto an anion exchange resin column packed with a resin containing a tertiary amine; and (e) separating the uncomplexed form of neurotoxin protein captured in step (d).

[0014] In the present invention, there may be no time interval between steps such as "(a), (b), (c), (d)..." and the steps may be performed simultaneously or with any interval such as a few seconds, minutes, or hours between steps.

[0015] In the present invention, "Clostridium botulinum toxin" is also called "botulinum toxin," and refers to a protein that is a type of protein derived from Clostridium botulinum, which irreversibly attaches to presynaptic nerve terminals and inhibits the secretion of acetylcholine at nerve junctions, thereby inhibiting muscle contraction and secondarily exerting a muscle relaxant effect.

[0016] Botulinum toxin proteins are divided into seven types, A through G, based on serological characteristics. Botulinum toxin type A is the most lethal known natural substance for humans, and in addition to serotype A, six generally immunologically distinct botulinum toxins are known, namely botulinum toxin serotypes B, C, D, E, F, and G. The different serotypes are distinguished by neutralization with type-specific antibodies and differ in the severity of the paralysis they cause and in the animal species they most affect.

[0017] The molecular weight of the botulinum toxin protein molecule is about 150 kD in all seven of the known botulinum toxin serotypes. However, botulinum toxins are released by clostridial bacteria as complexes containing the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, botulinum toxin type A complexes are produced by clostridial bacteria as 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C are produced as 500 kD complexes, and botulinum toxin type D as 300 kD and 500 kD complexes. Botulinum toxin types E and F are produced as about 300 kD complexes. These complexes (i.e., those with a molecular weight greater than about 150 kD) are believed to contain the non-toxic hemagglutinin protein, as well as non-toxin and non-toxic non-hemagglutinin proteins.

[0018] The botulinum toxin protein includes a pure neurotoxin component of about 150 kD as well as a complex form of high molecular weight containing a non-toxin protein. Thus, the complex form may include a botulinum neurotoxin protein and at least one non-toxin hemagglutinin protein and / or at least one non-toxin non-hemagglutinin protein. Specifically, the botulinum toxin complex protein may be a complex of botulinum neurotoxin (BoNT), nontoxic nonhemagglutinin (NTNHA) and hemagglutinin (HA) proteins. The molecular weight of the complex may be greater than about 150 kD. For example, the complex form of botulinum toxin type A may have a molecular weight of about 900 kD, about 500 kD or about 300 kD.

[0019] In one embodiment, the botulinum toxin of the present invention may be a type A toxin.

[0020] The botulinum neurotoxin (BoNT) protein of the present invention, unless otherwise specified, refers to the botulinum neurotoxin protein that is not combined with any combination of non-toxin proteins such as non-toxic non-hemagglutinin, hemagglutinin, etc. This is also referred to as "botulinum neurotoxin protein in a non-complexed form," "pure neurotoxin protein," or "reduced botulinum neurotoxin protein from which non-toxin proteins have been removed."

[0021] In one embodiment, the botulinum neurotoxin protein of the present invention may be a protein having a molecular weight of about 50 kD to 150 kD. Specifically, the botulinum neurotoxin protein of the present invention includes all forms of a heavy chain (HC, heavy chain) of about 100 kD bound to a light chain (LC, light chain) of about 50 kD by a disulfide bond, and a single chain precursor protein of about 150 kD of the neurotoxin component.

[0022] In the present invention, "about" includes not only the exact numerical value following the term "about", but also a range that is approximately that numerical value or close to that numerical value. Considering the context in which the numerical value is used, it can be determined whether the numerical value is close to or approximately the specific numerical value mentioned. As an example, "about" indicates a range of -10% to +10% of the specified numerical value. As another example, "about" indicates a range of -5% to +5% of the specified numerical value. However, the present invention is not limited to these.

[0023] In any of the above-described embodiments, the Clostridium botulinum toxin protein of the present invention may be obtained from a culture of Clostridium botulinum, specifically, the protein may be isolated from a culture of Clostridium botulinum.

[0024] Therefore, the purification method of the present invention may further include, but is not limited to, a step of culturing Clostridium botulinum prior to the chromatography step for purification.

[0025] In the above-mentioned culturing step, the Clostridium botulinum strain is cultured in a suitable medium under suitable culture conditions known in the art. Specifically, the Clostridium botulinum strain is cultured in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, etc. under anaerobic conditions with temperature, pH, etc. being controlled.

[0026] For example, the culture is performed under anaerobic conditions at about 25° C. to 40° C., specifically, but not limited to, 27° C. to 40° C. The culture period is continued until a desired amount of toxic protein is produced, specifically, but not limited to, about 12 to 150 hours.

[0027] In any of the above embodiments, the solution containing the Clostridium botulinum toxin protein of the present invention may be precipitated prior to the chromatography step.

[0028] In one embodiment, the precipitation is, but is not limited to, acid precipitation. For example, the acid precipitation may include a process of adding an acid to a pH of 3.0 to 4.0, specifically, 3.3 to 3.5, more specifically, 3.4 to 3.5. For the acid precipitation, an acid solution known in the art, such as, but not limited to, sulfuric acid or hydrochloric acid, is used.

[0029] In any of the above-described embodiments, the solution containing the Clostridium botulinum toxin complex protein of the present invention may be filtered prior to the hydrophobic chromatography step, particularly after acid precipitation.

[0030] The filtration step may be carried out by a conventionally known process such as microfiltration, ultrafiltration, microfiltration, or depth filtration, and impurities may be removed in the filtration step.

[0031] In one embodiment, the device used for the filtration process includes a filter having a pore size of about 0.1 μm to 0.3 μm, specifically about 0.2 μm, but is not limited thereto.

[0032] In the purification method of the present invention, the steps of (a) loading a solution containing Clostridium botulinum toxin protein onto a hydrophobic interaction column to capture the toxin and allow impurities to pass through, and (b) separating the toxin captured in step (a) to obtain an eluate containing the toxin, are also referred to as, but not limited to, a step of performing hydrophobic interaction chromatography using a solution containing Clostridium botulinum toxin protein.

[0033] "Chromatography" in the present invention refers to any process in which the components of a mixture are separated by passing the mixture through a medium such that the components pass through the medium at different rates.

[0034] The chromatography of the present invention includes column chromatography, planar chromatography, thin layer chromatography, gas chromatography, liquid chromatography, fast protein liquid chromatography (FPLC), and high performance liquid chromatography (HPLC). In each step of the purification process of the present invention, an example of a chromatography process or device is disclosed, but these can be applied to all types of chromatography described above.

[0035] In the present invention, "hydrophobic chromatography" or "hydrophobic interaction chromatography" refers to a method for separating molecules based on the relative strength of hydrophobic interactions with a non-polar stationary phase. Hydrophobic chromatography uses the principle that the higher the salt concentration, the stronger the interaction of the substance to be separated from the non-polar stationary phase, and the lower the ionic strength or salt concentration of the buffer, the weaker the interaction. Thus, when a descending salt gradient is used, less hydrophobic substances elute first, and more hydrophobic substances elute later.

[0036] In one embodiment, the hydrophobic interaction column in step (a) of the purification method of the present invention may be a column having a ligand such as ether, isopropyl, butyl, octyl, or phenyl. For example, the hydrophobic interaction column in step (a) of the purification method of the present invention may be a butyl sepharose or phenyl sepharose column. Specifically, the hydrophobic interaction column may be a phenyl sepharose column. More specifically, the hydrophobic interaction column may be a butyl sepharose HP (butyl sepharose high performance) column. (trademark) , Butyl sepharose Fast Flow (trademark) , Phenyl Sepharose HP (Phenyl sepharose high performance) (trademark)and Phenyl sepharose Fast Flow (trademark) However, the present invention is not limited to these, and any column belonging to the hydrophobic interaction column may be used.

[0037] In any of the above-described embodiments, the purification method of the invention may further comprise the step of equilibrating the column with a buffer prior to step (a).

[0038] In the present invention, equilibration refers to a step of stabilizing the column with a buffer solution before injecting the protein to be purified into the column in order to prevent protein aggregation or loss of activity due to changes in the environment.

[0039] In the equilibration step prior to the step (a), conditions such as the flow rate of the buffer solution, temperature, time, and electrical conductivity may be appropriately adjusted.

[0040] For example, the buffer may be a phosphate buffer, a citrate buffer, or an acetate buffer. As an example, the column buffer may be a phosphate buffer, for example, sodium phosphate. As an example, the concentration of the column buffer may be adjusted to 5 mM to 100 mM, for example, 25 mM to 75 mM, or 40 mM to 60 mM.

[0041] For example, the flow rate is about 1.0 ml / min to 20.0 ml / min. For example, the electrical conductivity is about 170 to 220 mS / cm. However, the present invention is not limited to these.

[0042] In any of the above-mentioned Examples, step (a) of the purification method of the present invention is carried out under conditions of pH 4 to pH 8, but is not limited thereto.

[0043] In any of the above-mentioned examples, step (a) of the purification method of the present invention is carried out under conditions of electrical conductivity of about 170 to 220 mS / cm, but is not limited thereto.

[0044] In the steps (a) and / or (b), the conditions such as the flow rate, temperature, and time of flowing the solution may be appropriately adjusted.

[0045] For example, the column buffer may be a phosphate buffer, a citrate buffer, or an acetate buffer. As an example, the column buffer may be a phosphate buffer, for example, sodium phosphate. As an example, the concentration of the column buffer is adjusted to 5 mM to 100 mM, for example, 25 mM to 75 mM, or 40 mM to 60 mM. For example, the flow rate of the mobile phase is about 1.0 ml / min to 20.0 ml / min. However, it is not limited thereto.

[0046] In any of the above-described embodiments, in the step (b), a suitable elution solvent may be used to obtain an eluate containing the toxin protein.

[0047] In the step (b), a concentration gradient may be used, for example, a stepwise salt gradient or a continuous salt gradient may be used to elute the toxin complex protein.

[0048] The step (b) may include a process of decreasing the ionic strength or increasing the pH. In the elution step, for example, a reverse salt gradient may be started to decrease the salt concentration, so that the hydrophobic portion of the protein adsorbed to the stationary phase is desorbed by the mobile phase. Specifically, in the elution step, a descending concentration gradient of the buffer may be used. For example, a buffer having a concentration gradient of about 5.0 M to about 0.0 M, about 4.0 to about 0.0 M, about 3.5 M to about 0.0 M, or about 3.0 M to about 0.0 M may be used. As the buffer, for example, sodium sulfate (Na 2 SO 4 ), sodium chloride (NaCl), potassium chloride (KCl), ammonium acetate (NH 4 Specifically, a concentration gradient of sodium chloride (NaCl) is used in the elution step; however, the present invention is not limited to these, and any suitable concentration gradient may be selected from the range known in the art.

[0049] In any of the above-mentioned embodiments, the eluate obtained in step (b) of the purification method of the present invention may be a precipitated solution, for example, but not limited to, an acid precipitate.

[0050] The acid precipitation may include a process of adding an acid. Specifically, the acid precipitation may include adding ammonium sulfate to an eluent. More specifically, the acid precipitation may include adding ammonium sulfate to an eluent so that the final saturation degree is 30 to 50%. For example, the concentration of ammonium sulfate at the saturation degree is about 19.6 g / 100 ml to 31.3 g / 100 ml, but is not limited thereto.

[0051] The precipitated eluate may be subjected to further purification steps, such as filtration and / or centrifugation. The precipitate obtained may be redissolved for further purification steps. As an example, the precipitate is dissolved using a solution of pH 5.0-7.0, such as, but not limited to, a sodium phosphate buffer solution at a concentration of about 40-60 mM.

[0052] In the purification method of the present invention, the step (c) of obtaining a non-complexed form of neurotoxin protein from the toxin protein contained in the eluate obtained in the step (b) may dissociate the Clostridium botulinum toxin protein into a non-toxin protein and a pure neurotoxin protein.

[0053] The (c) step can be carried out by any conventional protein separation method to separate pure neurotoxin protein from non-toxin protein.

[0054] In one embodiment, step (c) involves UF diafiltration, pH titration, dialysis, precipitation, red blood cell processing, depth filtration (DF), microfiltration (MF), ultrafiltration (UF), sterile filtration, fractionation, HPLC and / or centrifugation, or any method for extracting proteins from other proteins, but any method that results in a pure neurotoxin protein may be used.

[0055] In any of the above-mentioned embodiments, the step (c) may be performed by dialysis. The dialysis may be performed by placing the eluate obtained in the step (b) in a permeable membrane and using a buffer solution under suitable temperature and / or pH conditions. The number of times of dialysis may be appropriately adjusted, for example, but is not limited to, three times.

[0056] For example, the temperature may be about 0° C. to 10° C., specifically about 2° C. to 8° C. For example, the dialysis may be performed with a buffer solution of pH 6 to 10. For example, the buffer solution may be a phosphate buffer solution, a citrate buffer solution, or an acetate buffer solution, specifically, sodium phosphate. The concentration of the buffer solution is appropriately adjusted, and is, for example, about 10 mM to 70 mM, but is not limited thereto.

[0057] In the purification method of the present invention, the step (d) of loading the solution containing the uncomplexed neurotoxin protein obtained in the step (c) onto an anion exchange resin column and the step (e) of separating the uncomplexed neurotoxin protein collected in the step (d) are also referred to as, but not limited to, a step of performing anion exchange chromatography using the solution containing the uncomplexed neurotoxin protein.

[0058] "Anion-exchange chromatography" in the present invention is a technique for separating negatively charged (or acidic) molecules according to their charge by binding them to a positively charged support; molecular homologues (acidic, basic and neutral) can be easily separated by this technique.

[0059] In the purification method of the present invention, the resin used in the anion exchange chromatography in step (d) may be a weak anion exchange resin containing a secondary or tertiary amine as a functional group.

[0060] In one embodiment, the resin used in the anion exchange chromatography may be a resin containing a tertiary amine. Specifically, the resin may be a resin containing DEAE (DiEthylAminoEthyl), but is not limited thereto. In any of the above-mentioned embodiments, the anion exchange resin column of the present invention may be、D EAE Sepharose Fast Flow (DEAE Sepharose Fast Flow) (trademark) This is a column, Reni Not limited to this.

[0061] In any of the above-described embodiments, the purification method of the invention may further comprise the step of equilibrating the anion exchange resin with a buffer prior to step (d).

[0062] In the equilibration step prior to the step (d), conditions such as the flow rate of the buffer solution, temperature, time, and electrical conductivity may be appropriately adjusted.

[0063] For example, the column buffer may be a phosphate buffer, a citrate buffer, or an acetate buffer. Specifically, the column buffer may be a phosphate buffer, for example, sodium phosphate. For example, the concentration of the column buffer may be adjusted to 5 mM to 100 mM, for example, 5 mM to 35 mM, or 10 mM to 30 mM.

[0064] For example, the flow rate is 0.1 ml / min to 20.0 ml / min. For example, the electrical conductivity is about 0.1 to 10 mS / cm. However, the present invention is not limited to these.

[0065] In the steps (d) and / or (e), conditions such as the flow rate of the solution, temperature, time, and electrical conductivity may be appropriately adjusted.

[0066] For example, the buffer may be a phosphate buffer, a citrate buffer, or an acetate buffer. For example, the column buffer may be a phosphate buffer, for example, sodium phosphate. For example, the concentration of the column buffer may be adjusted to 5 mM to 100 mM, for example, 5 mM to 35 mM, or 10 mM to 30 mM.

[0067] For example, the flow rate is about 0.1 ml / min to 20 ml / min. For example, the electrical conductivity is about 0.1 to 10 mS / cm. However, the present invention is not limited to these.

[0068] In any of the above-mentioned Examples, step (d) of the purification method of the present invention is carried out under conditions of pH 4 to pH 10, but is not limited thereto.

[0069] In any of the above embodiments, step (e) may comprise altering the ionic strength or pH using a suitable elution medium.

[0070] A concentration gradient may be used in the step (e). For example, elution may be performed using a stepwise salt gradient or a continuous salt gradient. Specifically, in the elution step, an ascending concentration gradient of a buffer may be used to elute the protein bound to the column. For example, a buffer having a concentration gradient ranging from about 0.0 M to 3.0 M, about 0.0 M to 2.0 M, or about 0.0 M to 1.0 M may be used. In one embodiment, the buffer used in the elution step may be sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate, potassium phosphate, Tris, or the like. Specifically, a concentration gradient of sodium chloride (NaCl) is used in the elution step, but the present invention is not limited thereto, and any concentration gradient may be appropriately selected from the range known in the art.

[0071] In any of the above-mentioned embodiments, the purification method of the present invention does not include, as an essential component, a step of loading a solution containing a neurotoxin protein onto a cation exchange resin column, but is not limited thereto.

[0072] The neurotoxin protein separated by the purification method of the present invention has a high purity, specifically, but not limited to, a purity of about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 97.5% or more, or about 98% or more. EXAMPLES

[0073] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples.

[0074] Example 1 (Culture of Clostridium botulinum and isolation of toxin) Clostridium botulinum stored at -80℃ was thawed and placed in a seed culture and cultured at 37℃ under anaerobic conditions for 24 hours to increase the number of bacteria. The culture with the increased number of bacteria was placed in a main culture and cultured at 35℃ under anaerobic conditions for an additional 92-100 hours, after which the virus was inactivated by acid precipitation to a pH of 3.4. After precipitation, botulinum toxin was eluted with a buffer solution and then filtered using a sterilized 0.2μm filter to prevent contact with the outside.

[0075] Example 2 (Purification of Botulinum Toxin) 2-1.Hydrophobic interaction chromatography The filtrate containing the botulinum type A toxin of Example 1 was loaded onto a column packed with Phenyl Sepharose hydrophobic interaction chromatography resin. Prior to the loading, equilibration / washing was performed by flowing an equilibration / elution buffer (50 mM sodium phosphate, 3 M sodium chloride, pH 6.0) at a flow rate of 5 ml / min and an electric conductivity of 188 mS / cm.

[0076] The filtrate containing the botulinum type A toxin from Example 1 was loaded onto a Phenyl Sepharose column, and the botulinum toxin was then eluted from the column using a decreasing salt step (a sodium chloride concentration gradient of 3.0M-0.0M) with a 50 mM sodium phosphate, pH 6.0 buffer at a flow rate of 5 ml / min and an electrical conductivity of 188 mS / cm.

[0077] 2-2. Ammonium sulfate precipitation and dissociation of pure neurotoxin Ammonium sulfate was added to the eluate collected by the method of Example 2-1 to a final saturation of 40% (24.3 g / 100 ml) to precipitate, and then centrifuged to obtain a precipitate. In this process, the toxic protein was in contact with the inner surface of the sterile container and not with the outside. The precipitate was dissolved in 50 mM sodium phosphate buffer (pH 6.0), and then, in order to dissociate non-toxic protein and pure neurotoxin protein, the dissolved solution was placed in a permeable membrane as described above and dialyzed in 20 mM sodium phosphate buffer (pH 7.9) at 2 to 8°C. Dialysis was performed a total of three times.

[0078] 2-3. Anion exchange resin chromatography The dialysate obtained in Example 2-2 was centrifuged to separate the supernatant, and the separated supernatant was injected into an FPLC (Fast Protein Liquid Chromatography) connected to a DEAE Sepharose column. As a comparative example, anion exchange resin chromatography was performed using a Q column instead of the DEAE Sepharose column.

[0079] The specific experimental method is as follows: Before injecting the supernatant, a column packed with DEAE Sepharose resin was equilibrated with sodium phosphate buffer (20 mM, pH 7.9), and the toxin-containing sodium phosphate buffer (20 mM, pH 7.9) was then passed through the column. After equilibration, the supernatant was injected, and the flow-through (FT) liquid was collected in a sterile container while the neurotoxin was binding to the anion exchange column material. Then, a washing step was performed, in which a washing buffer (20 mM sodium phosphate, pH 7.9) was allowed to pass through the anion exchange column. The next step was an elution step, in which the botulinum toxin was eluted from the column using a 20 mM sodium phosphate, pH 7.9 buffer, followed by an ascending salt step change (0.0M-1.0M sodium chloride gradient). Anion exchange resin chromatography was performed under conditions of electrical conductivity 3 mS / cm and flow rate 3 ml / min.

[0080] Example 3 (Purity analysis of purified toxin) In order to purify the pure neurotoxin from which non-toxic proteins were removed, the purity of the sample obtained in the purification process by hydrophobic interaction chromatography and subsequent anion exchange resin chromatography was analyzed based on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE, FIG. 1) and size exclusion chromatography (SEC-HPLC, FIG. 2). Electrophoresis was performed using 4-20% polyacrylamide gel, which was stained with Coomassie blue after electrophoresis. As the mobile phase for SEC-HPLC, a 50 mM sodium phosphate solution containing 250 mM NaCl at pH 6.0 was used, and an HPLC column was connected to load 20 μg of the botulinum type A neurotoxin protein obtained in Example 2, and the flow rate was 0.4 mL / min for 60 minutes.

[0081] As a result, no impurity protein bands were observed other than the pure neurotoxin components, and only the pure neurotoxin components of 98 kD (heavy chain) and 52 kD (light chain) were observed (Figure 1). In addition, the purity of the botulinum neurotoxin was confirmed to be 99.86% (Figure 2).

[0082] On the other hand, when the purity of the samples obtained using a DEAE column or a Q Sepharose column in the anion chromatography step was analyzed by electrophoresis (Figure 3), when a Q column containing a quaternary ammonium salt was used, many impurity protein bands were observed in addition to the pure neurotoxin components.

[0083] Therefore, it was confirmed that the neurotoxin protein can be purified to a higher purity by using a DEAE column containing a tertiary amine than by using a Q column containing a quaternary ammonium salt.

[0084] These results suggest that the neurotoxin protein can be purified to a high purity by performing two steps of hydrophobic interaction chromatography and anion exchange chromatography using a DEAE column, with most of the impurities removed.

[0085] Thus, the purification method can be used to obtain only the pure neurotoxin portion (about 150 kD) of botulinum toxin type A from which non-toxin proteins have been removed, with high purity, demonstrating that the method and system described herein can be used to efficiently produce a pure neurotoxin from which non-toxin proteins have been removed, with high potency and purity.

[0086] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A solution containing Clostridium botulinum toxin protein isolated from the culture medium, (a) loading onto a hydrophobic interaction column to capture the toxins and pass impurities; (b) separating the toxins collected in the (a) step to obtain an eluate containing the toxins; (c) obtaining the neurotoxin protein in an uncomplexed form from the toxin protein contained in the eluate obtained in (b); (d) loading the solution containing the uncomplexed neurotoxin protein obtained in step (c) onto an anion exchange resin column packed with a resin containing a tertiary amine; (e) isolating the uncomplexed form of the neurotoxin protein collected in step (d).

2. The method for purifying Clostridium botulinum neurotoxin protein in uncomplexed form according to claim 1, wherein the anion exchange resin column is a DEAE Sepharose Fast Flow™ column.

3. 2. The method for purifying the uncomplexed form of Clostridium botulinum neurotoxin protein according to claim 1, further comprising culturing a Clostridium botulinum strain prior to the step (a).

4. The method for purifying the uncomplexed form of Clostridium botulinum neurotoxin protein according to claim 3, further comprising the step of acid precipitation of the culture broth of said strain.

5. The method for purifying a non-complexed Clostridium botulinum neurotoxin protein according to claim 4, wherein the acid precipitation comprises adding an acid to a pH of 3.0 to 4.

0.

6. 5. The method for purifying a Clostridium botulinum toxin complex protein of claim 4, further comprising the step of filtering the acid precipitated solution.

7. 2. The method for purifying Clostridium botulinum neurotoxin protein in an uncomplexed form according to claim 1, wherein the hydrophobic interaction column is selected from the group consisting of Butyl Sepharose High Performance™, Butyl Sepharose Fast Flow™, Phenyl Sepharose High Performance™ and Phenyl Sepharose Fast Flow™ columns.

8. The method for purifying a non-complexed Clostridium botulinum neurotoxin protein according to claim 1, wherein the step (a) is carried out under conditions of pH 4 to pH 8 and conductivity of 170 to 220 mS / cm.

9. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the column buffer in step (a) is a phosphate buffer.

10. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the column buffer in step (b) is a phosphate buffer.

11. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1 , wherein the step (b) uses a concentration gradient.

12. The method for purifying the uncomplexed Clostridium botulinum neurotoxin protein according to claim 1, further comprising a step of acid precipitation of the eluate obtained in step (b) between steps (b) and (c).

13. The method for purifying a Clostridium botulinum neurotoxin protein according to claim 12, wherein the acid precipitation step comprises adding ammonium sulfate to the eluent to a final saturation level of 30-50%.

14. 2. The uncomplexed form of claim 1, wherein the step (c) dissociates the Clostridium botulinum toxin protein into non-toxin protein and pure neurotoxin protein. A method for purifying Clostridium botulinum neurotoxin protein.

15. The method for purifying a Clostridium botulinum neurotoxin protein in an uncomplexed form according to claim 1, wherein the step (c) comprises at least one selected from the group consisting of UF diafiltration, pH titration, dialysis and precipitation.

16. 2. The method for purifying a Clostridium botulinum neurotoxin protein in an uncomplexed form according to claim 1, wherein the step (c) is dialysis three times with a sodium phosphate buffer.

17. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the column buffer in step (e) is a phosphate buffer.

18. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1 , wherein the step (e) uses a concentration gradient.

19. The method for purifying a non-complexed Clostridium botulinum neurotoxin protein according to claim 1, wherein the purified Clostridium botulinum neurotoxin protein has a purity of 98% or more.

20. 2. The method for purifying Clostridium botulinum neurotoxin protein in an uncomplexed form according to claim 1, characterized in that the method does not include a step of loading a solution containing the neurotoxin protein onto a cation exchange resin column.

21. The method for purifying the uncomplexed form of Clostridium botulinum neurotoxin protein according to claim 1, wherein the uncomplexed form of neurotoxin protein has a molecular weight of 50 kD to 150 kD.

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