Purification method for Clostridium botulinum toxin complex proteins

Abstract

The present invention relates to a method for purifying a Clostridium botulinum toxin complex protein.

Description

[Technical field]

[0001] The present invention relates to a method for purifying a Clostridium botulinum toxin complex protein. [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] On the other hand, botulinum toxin naturally forms a complex bound to several non-toxic proteins. [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 toxin complex proteins. [Means for solving the problem]

[0007] An object of the present invention is to provide a method for purifying a Clostridium botulinum toxin complex protein. Effect of the Invention

[0008] By using the purification method of the present invention, a highly purified botulinum toxin complex 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 botulinum toxin complex protein (900 kD) (NTNH: nontoxic nonhemagglutinin, HC: heavy chain of neurotoxin protein, LC: light chain of neurotoxin protein, HA33: hemagglutinin 33, HA17: hemagglutinin 17, HA50: hemagglutinin 50, HA20: hemagglutinin 20). [Diagram 2] FIG. 1 shows the results of HPLC, i.e., size exclusion chromatography (SEC-HPLC), of the purified 900 kD botulinum toxin complex. 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 toxin complex protein.

[0013] Specifically, the purification method may include the steps of (a) loading a solution containing Clostridium botulinum toxin complex protein separated from a culture medium onto a first hydrophobic interaction column to capture the toxin and allow impurities to pass through, (b) separating the toxin collected in step (a) to obtain an eluate containing the toxin, (c) loading the eluate obtained in step (b) onto a second hydrophobic interaction column to capture the toxin and allow impurities to pass through, (d) separating the toxin collected in step (c) to obtain an eluate containing the toxin, and (e) performing size exclusion chromatography using the eluate obtained 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 protein has a molecular weight of about 150 kD and is divided into seven types, A to 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 (NTNH) 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. According to the method for purifying the Clostridium botulinum toxin complex of the present invention, the various complexes described above can be purified.

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

[0020] In any of the above-mentioned embodiments, the botulinum toxin of the present invention may be in the form of a complex bound to a non-toxic protein. Specifically, the botulinum toxin complex protein of the present invention may be a complex of botulinum toxin (BoNT; Botulinum neurotoxin), nontoxic nonhemagglutinin (NTNH; nontoxic nonhemagglutinin) and hemagglutinin (HA; hemagglutinin) proteins. More specifically, the botulinum toxin complex protein of the present invention may be a complex of BoNT, NTNH, hemagglutinin 70 (HA70), hemagglutinin 33 (HA33) and hemagglutinin 17 (HA17). More specifically, the HA70 is divided into HA20 (hemagglutinin 20) and HA50 (hemagglutinin 50), and the BoNT is divided into a light chain (LC: Light chain) of about 50 kD and a heavy chain (HC: Heavy chain) of about 100 kD, but is not limited thereto.

[0021] In any of the above-mentioned embodiments, the botulinum toxin complex protein of the present invention may have a molecular weight of more than 150 kD, specifically, a molecular weight of about 250 kD to 1400 kD, more specifically, a molecular weight of 280 kD to 1300 kD, 300 kD to 1200 kD, 700 kD to 1100 kD, 800 kD to 1000 kD, or about 900 kD, but is not limited thereto.

[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 complex 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-described embodiments, the solution containing the Clostridium botulinum toxin complex protein of the present invention may be precipitated prior to the first hydrophobic 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 first 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 complex proteins onto a primary 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 primary hydrophobic interaction chromatography using a solution containing Clostridium botulinum toxin complex proteins.

[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 is selected from the group consisting of butyl sepharose high performance (HP), butyl sepharose fast flow (Butyl sepharose Fast Flow), phenyl sepharose high performance (HP) and phenyl sepharose fast flow (Phenyl sepharose Fast Flow) columns, but is not limited thereto, 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.

[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 about 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 embodiments, the step of obtaining an eluate comprising the toxin-complex protein in step (b) may comprise using a suitable elution solvent.

[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 ranging from 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 OAc), and specifically, sodium chloride (NaCl) is used, but the present invention is not limited to these, and any suitable solution 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 subjected to a precipitation treatment before the step (c) is performed. For example, the precipitation may be an acid precipitation.

[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 about 30 to 50%. For example, the concentration of ammonium sulfate at the saturation degree is about 19 g / 100 ml to 32 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 steps of (c) loading the eluate obtained in the step (b) onto a second hydrophobic interaction column to capture the toxins and pass the impurities, and (d) separating the toxins captured in the step (c) to obtain an eluate containing the toxins are also called the step of performing a second hydrophobic interaction chromatography using the eluate obtained from the first chromatography. Hydrophobic interaction chromatography is as described above.

[0053] In one embodiment, the secondary hydrophobic interaction column in step (c) 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 (c) of the purification method of the present invention may be a column having a ligand such as butyl-Sepharose. (trademark) or phenyl sepharose (trademark)The hydrophobic interaction column may be a column. Specifically, the hydrophobic interaction column may be a phenyl sepharose column. More specifically, the hydrophobic interaction column is selected from the group consisting of butyl sepharose high performance (HP), butyl sepharose fast flow (Fast Flow), phenyl sepharose high performance (HP) and phenyl sepharose fast flow (Fast Flow) columns, but is not limited thereto, and any column belonging to the hydrophobic interaction column may be used.

[0054] In any of the above-described embodiments, the purification method of the invention may further comprise the step of equilibrating the column prior to step (c).

[0055] The buffer may be, for example, 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.

[0056] The flow rate is, for example, about 1.0 ml / min to 20.0 ml / min. The electrical conductivity is, for example, about 130 to 170 mS / cm. However, the present invention is not limited to these values.

[0057] In any of the above-mentioned Examples, step (c) of the purification method of the present invention is carried out under conditions of pH 4 to pH 8, but is not limited thereto.

[0058] In any of the above-mentioned Examples, step (c) of the purification method of the present invention is carried out under conditions of an electric conductivity of 130 to 170 mS / cm, but is not limited thereto.

[0059] In the steps (c) and / or (d), the conditions such as the flow rate, temperature, and time of flowing the solution may be appropriately adjusted.

[0060] 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 about 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.

[0061] In any of the above embodiments, the step of obtaining an eluate comprising the toxin-complex protein in step (d) may comprise using a suitable elution solvent.

[0062] In the step (d), 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.

[0063] The step (d) 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 decreasing concentration gradient (descending gradient) of the buffer may be used. For example, a buffer having a concentration gradient ranging from about 2.5 M to about 0.0 M, about 2.3 M to about 0.0 M, about 2.1 M to about 0.0 M, or about 2.0 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 4OAc), and specifically, sodium chloride (NaCl) is used, but the present invention is not limited to these, and any suitable solution may be selected from the range known in the art.

[0064] In any of the above-mentioned embodiments, the eluate obtained in step (d) of the purification method of the present invention may be subjected to a precipitation treatment before the step (e) is performed. For example, the precipitation may be an acid precipitation.

[0065] 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 about 70 to 90%. For example, the concentration of ammonium sulfate at the saturation degree is about 47 g / 100 ml to 67 g / 100 ml, but is not limited thereto.

[0066] 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 10-30 mM.

[0067] In the purification method of the present invention, the components may be separated according to the size of the molecules by the (e) size exclusion chromatography (SEC) step. For example, the botulinum toxin complex protein having a specific size may be separated in the (e) step. In the present invention, "size exclusion chromatography (SEC)" means separating a mixture based on the speed (permeability) at which solutes of various sizes pass through a porous matrix. Size exclusion chromatography utilizes the principle that when a sample to be analyzed passes through a column filled with a porous stationary phase such as gel, matrix, or beads, large molecules that cannot pass through the holes of the column cannot enter the holes and pass through the column quickly through the surrounding empty space, while small molecules pass through the holes of the column while moving relatively slowly.

[0068] In one embodiment, the column used in the size exclusion chromatography of the present invention is a Superdex. (trademark) , Sephacryl (trademark) , super loin (trademark) , Sephadex (trademark) , Sepharose (trademark) Examples of such columns include polyacrylamide or silica-based columns, specifically Superose columns, more specifically Superose-6, but any column capable of separating the desired botulinum toxin complex protein may be used.

[0069] For example, a phosphate buffer, a citrate buffer, or an acetate buffer may be used as a column buffer for the size exclusion chromatography. 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 about 5 mM to 100 mM, for example, 5 mM to 50 mM, or 10 mM to 30 mM. For example, a buffer with a pH of 5 to 7 is used as the buffer. For example, the flow rate is about 0.001 ml / min to 10.0 ml / min, or about 0.05 ml / min to 0.5 ml / min. However, the present invention is not limited thereto.

[0070] In any of the above examples, the size exclusion chromatography step of the present invention separates botulinum toxin complex proteins ranging from 250 kD to 1400 kD, but is not limited thereto.

[0071] The botulinum toxin complex 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

[0072] 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.

[0073] 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.

[0074] Example 2 (Purification of Botulinum Toxin) 2-1. Primary 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.

[0075] 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 change (3.0M-0.0M sodium chloride gradient) in 50 mM sodium phosphate, pH 6.0 buffer at a flow rate of 5 ml / min and an electrical conductivity of 188 mS / cm.

[0076] 2-2. Ammonium sulfate precipitation Ammonium sulfate was added to the eluate collected in the method of Example 2-1 to a final saturation of 40% (24.3 g / 100 ml) to cause precipitation, 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 had no contact with the outside. The precipitate was dissolved in 50 mM sodium phosphate buffer (pH 6.0), and then a second hydrophobic interaction chromatography was performed.

[0077] 2-3. Secondary hydrophobic interaction chromatography The redissolution solution 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 Phenyl Sepharose hydrophobic interaction chromatography column.

[0078] Prior to injection of the supernatant, a column packed with Phenyl Sepharose resin was equilibrated with an equilibration buffer (50 mM sodium phosphate, 2 M sodium chloride, pH 6.0), and the toxin-containing buffer (50 mM sodium phosphate, 2 M sodium chloride buffer, pH 6.0) was then injected through the column at a flow rate of 5 ml / min and an electrical conductivity of 147 mS / cm. After equilibration, the injection step described above was performed, and the flow-through (FT) liquid was collected in a sterile container while the toxin complex bound to the hydrophobic interaction column material. A washing step was then performed, and after washing, the column was eluted using a salt step change (2.0 M-0.0 M sodium chloride gradient) decreasing with a buffer of 50 mM sodium phosphate, pH 6.0.

[0079] 2-4. Secondary ammonium sulfate precipitation and concentration Ammonium sulfate was added to the eluate collected in the method of Example 2-3 to a final saturation of 80% (56.1 g / 100 ml) to cause precipitation, and then centrifuged to obtain a precipitate. Even in this process, the toxin protein was in contact with the inner surface of the sterile container, and not with the outside. The precipitate was dissolved in 1 ml of 20 mM sodium phosphate buffer (pH 6.0), and then subjected to size exclusion chromatography.

[0080] 2-5. Size Exclusion Chromatography The resolubilized solution in Example 2-4 was centrifuged to separate the supernatant, and the separated supernatant was loaded onto an FPLC (fast protein liquid chromatography) connected to a Superose-6 size exclusion chromatography column. Prior to the loading, the column was equilibrated by passing an equilibration / elution buffer (20 mM sodium phosphate, pH 6.0) at a flow rate of 0.2 ml / min. After equilibration, 100 mL of 20 mM sodium phosphate buffer at pH 6.0 was passed through, and then fractions of the eluted peak were obtained in sequence to separate a sample containing a protein of about 900 kDa in size.

[0081] Example 3 (Purity analysis of purified toxin) In order to purify a highly pure complex neurotoxin, the purity of the sample obtained in the two purification steps by hydrophobic interaction chromatography and size exclusion chromatography in Example 2 was analyzed by 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 toxin protein obtained in Example 2, and the flow rate was 0.4 mL / min for 60 minutes.

[0082] As a result, no impurity protein bands were observed other than the botulinum complex neurotoxin components (Figure 1). The purity of the botulinum toxin was also confirmed to be 99.97% (Figure 2). Therefore, by using the above purification method, botulinum toxin complex proteins (approximately 900 kD), such as botulinum toxin type A, can be obtained with high purity and without impurities.

[0083] These results show that by using the method and system described in this specification, botulinum complex proteins can be separated, purified and produced with high purity.

[0084] 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 complex proteins isolated from the culture medium is (a) loading a primary 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) loading the eluate from step (b) onto a second hydrophobic interaction column to capture the toxins and pass on the impurities; (d) separating the toxins collected in the (c) step to obtain an eluate containing the toxins; (e) performing size exclusion chromatography using the eluate obtained in step (d).

2. 2. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, further comprising culturing a Clostridium botulinum strain prior to step (a).

3. The method for purifying a Clostridium botulinum toxin complex protein according to claim 2, further comprising the step of acid precipitation of the culture medium of the strain.

4. The method for purifying a Clostridium botulinum toxin complex protein according to claim 3, wherein the acid precipitation comprises the step of adding an acid to a pH of 3.0 to 4.

0.

5. 4. The method for purifying a Clostridium botulinum toxin complex protein of claim 3, further comprising the step of filtering the acid precipitated solution.

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

7. 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.

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

9. The method according to claim 1, further comprising a step of subjecting the eluate obtained in the step (b) to acid precipitation between the steps (b) and (c). A method for purifying botulinum toxin complex proteins.

10. The method for purifying a Clostridium botulinum toxin complex protein according to claim 9, wherein the acid precipitation step comprises adding ammonium sulfate to the eluent to a final saturation level of 30-50%.

11. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the step (c) is carried out under conditions of pH 4 to pH 8 and electrical conductivity of 130 to 170 mS / cm.

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

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

14. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, further comprising a step of acid precipitation of the eluate obtained in step (d) between steps (d) and (e).

15. The method for purifying a Clostridium botulinum toxin complex protein according to claim 14, wherein the acid precipitation step comprises adding ammonium sulfate to the eluent to a final saturation level of 70-90%.

16. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the primary hydrophobic interaction column or the secondary hydrophobic interaction column is selected from the group consisting of Butyl Sepharose (trademark) and Phenyl Sepharose (trademark) columns.

17. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the step (e) includes a process of isolating and obtaining a protein having a molecular weight of 250 kD to 1400 kD.

18. 2. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the size exclusion chromatography in step (e) is performed using a Superdex (trademark), Sephacryl (trademark), Superose (trademark), Sephadex, Sepharose (trademark), polyacrylamide or silica-based column.

19. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the size exclusion chromatography in the step (e) is carried out under conditions of pH 5 to pH 7.

20. 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.

21. 2. The method for purifying a Clostridium botulinum toxin complex protein according to claim 1, wherein the purity of the Clostridium botulinum toxin complex protein purified by said method is 98% or more.

22. The method for purifying a Clostridium botulinum toxin complex protein according to any one of claims 1 to 21, wherein the Clostridium botulinum toxin complex protein is a complex of botulinum toxin (BoNT; Botulinum neurotoxin), nontoxic nonhemagglutinin (NTNH; nontoxic nonhemagglutinin) and hemagglutinin (HA) proteins.

Images