Method for purifying clostridium botulinum neurotoxin protein

Abstract

The present invention relates to a method for purifying Clostridium botulinum toxin, comprising separating a non-toxin protein and an uncomplexed botulinum toxin protein, and performing a purification process of hydrophobic interaction and ion-exchange chromatography, thereby obtaining the uncomplexed botulinum toxin protein having high purity and increased yield. By using the botulinum purification method of the present invention, various industrial uses are possible and, in particular, botulinum toxin, which is in high demand in the pharmaceutical field, can be economically and highly-efficiently mass-produced, and thus can be effectively used in the industry.

Description

Method for purifying Clostridium botulinum neurotoxin protein

[0001] The present invention relates to a method for purifying Clostridium botulinum toxin, and more particularly, to a method for purifying Clostridium botulinum toxin, which can obtain a non-complexed form of botulinum toxin protein with high purity and increased yield by separating the non-complexed form of botulinum toxin protein into a non-toxin protein and a non-complexed form and performing a purification process of hydrophobic interaction and ion exchange chromatography.

[0002]

[0003] Botulinum toxin is a toxic protein produced by bacteria such as Clostridium butyricum, Clostridium baratii, and Clostridium botulinum. Botulinum toxin blocks neuromuscular transmission and causes neuroparalytic diseases in humans and animals.

[0004] However, despite the fact that botulinum toxin is a very lethal toxin to humans, it is a very safe drug that can have various therapeutic effects when used appropriately. Since its approval by the US FDA in 1989, it has been used not only as a treatment for neuromuscular disorders characterized by skeletal muscle hyperactivity, strabismus, torticollis, blepharospasm, hyperhidrosis, and migraine, but also as an injection for cosmetic purposes such as removing or improving wrinkles.

[0005] These botulinum toxins consist of a heavy chain of approximately 100 kD, joined to a light chain of approximately 50 kD, resulting in a protein molecular weight of approximately 150 kD. However, botulinum toxins released by Clostridium bacteria are released as complexes of the 150 kD toxin and one or more non-toxin proteins. For example, botulinum toxins are released as complexes of 900 kD, 500 kD, and 300 kD.

[0006] Accordingly, various botulinum toxin purification methods that remove non-toxin proteins are being developed, but existing purification methods do not yield high yields of pure toxin protein portions despite going through complex steps, and thus an improved purification method that isolates complete botulinum toxin using a simplified method is required.

[0007] Accordingly, the inventors of the present invention have made great efforts to develop a new method for purifying botulinum toxin, and as a result, have confirmed that botulinum toxin with high purity and increased yield can be produced by a method that improves and simplifies the multiple filtration and purification processes that are essential in conventional purification methods, thereby completing the present invention.

[0008]

[0009] An object of the present invention is to provide a method for purifying Clostridium botulinum toxin capable of obtaining a non-complexed form of botulinum toxin with high purity and increased yield.

[0010] Another object of the present invention is to provide a method for producing a crude Clostridium botulinum toxin solution comprising a non-complexed form of botulinum toxin protein produced by the above purification method.

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0012]

[0013] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0014]

[0015] To achieve the above object, the present invention provides a method for purifying Clostridium botulinum toxin, comprising the following steps:

[0016] (a) a step of first filtering a culture medium containing Clostridium botulinum neurotoxin protein;

[0017] (b) a step of dissociating the botulinum toxin protein in the culture solution filtered in step (a) into non-toxin proteins and non-complexed botulinum toxin proteins;

[0018] (c) a step of loading the botulinum toxin protein dissociated in step (b) onto a hydrophobic interaction column to capture the botulinum toxin protein in a non-complexed form and separate impurities;

[0019] (d) a second filtering step of the botulinum toxin protein captured in the step (c);

[0020] (e) A step of loading the botulinum toxin protein obtained in the above step (d) onto an ion exchange column to capture the non-complexed form of the botulinum toxin protein and further separate impurities.

[0021] According to another aspect of the present invention, the present invention provides a method for purifying Clostridium botulinum toxin, comprising the following steps:

[0022] (a) a step of dissociating the botulinum toxin protein contained in the filtrate obtained by first filtering a culture medium containing Clostridium botulinum neurotoxin protein into non-toxin proteins and non-complexed botulinum toxin proteins;

[0023] (b) a step of loading the botulinum toxin protein dissociated in step (a) onto a hydrophobic interaction column to capture the botulinum toxin protein in a non-complexed form and separate impurities;

[0024] (c) a second filtering step of the botulinum toxin protein captured in the step (b);

[0025] (d) A step of loading the botulinum toxin protein obtained in step (c) onto an ion exchange column to capture the non-complexed form of the botulinum toxin protein and further separate impurities.

[0026] According to a specific embodiment of the present invention, the culture medium containing the Clostridium botulinum neurotoxin protein is prepared through an acid precipitation and toxin elution step using a first buffer containing 300 to 500 mM sodium citrate and 100 to 300 mM sodium chloride at a pH of 4.5 to 6.5 in a culture medium of a Clostridium strain; and a second buffer containing 100 to 300 mM sodium citrate and 50 to 150 mM sodium chloride at a pH of 4.5 to 6.5.

[0027] According to a specific embodiment of the present invention, the first filtration or the second filtration is a vertical filter system (Tangential Flow Filtration, TFF) or a dialysis system.

[0028] According to a more specific embodiment of the present invention, the vertical filter system is at least one filtration method selected from the group consisting of ultrafiltration and diafiltration.

[0029] More specifically, the ultrafiltration or diafiltration used in the present invention is performed using a membrane filter having a size of 10 to 300 kD, specifically 20 to 260 kD, more specifically 30 to 220 kD, 40 to 180 kD, even more specifically 50 to 150 kD, even more specifically 50 to 100 kD, and most specifically about 50 kD.

[0030] More specifically, the filtration used in the present invention is performed using a buffer solution containing at least one selected from the group consisting of purified water, sodium citrate, sodium chloride, and sodium phosphate.

[0031] According to a specific embodiment of the present invention, the first filtration step included in the purification method of the present invention is performed using purified water.

[0032] According to a specific embodiment of the present invention, the dissociation step included in the purification method of the present invention is performed with a buffer solution containing sodium phosphate at 10 to 100 mM, specifically 15 to 90 mM, more specifically 30 to 70 mM, still more specifically 40 to 60 mM, still more specifically 45 to 55 mM, and most specifically about 50 mM, and having a pH of 7.5 to 8.5, specifically 7.6 to 8.4, more specifically 8 to 8.5, more specifically 7.7 to 8.3, still more specifically 7.8 to 8.2, still more specifically 7.9 to 8.1, and most specifically 8.0.

[0033] According to a specific embodiment of the present invention, the hydrophobic interaction column is a column having one or more ligands selected from the group consisting of ether, isopropyl, butyl, octyl, and phenyl.

[0034] According to a specific embodiment of the present invention, the hydrophobic interaction column comprises sodium phosphate at a concentration of 10 to 100 mM, specifically 20 to 90 mM, more specifically 30 to 80 mM, even more specifically 40 to 70 mM, even more specifically 45 to 60 mM, even more specifically 45 to 55 mM, and most specifically about 50 mM; and a first buffer solution comprising sodium chloride of 1.5 to 2.5 M, specifically 1.6 to 2.4 M, more specifically 1.7 to 2.3 M, even more specifically 1.8 to 2.2 M, even more specifically 1.9 to 2.1 M, and most specifically about 2 M, and having a pH of 7.5 to 8.5, specifically 7.6 to 8.4, more specifically 8 to 8.5, more specifically 7.7 to 8.3, more specifically 7.8 to 8.2, even more specifically 7.9 to 8.1, and most specifically 8; and

[0035] A second buffer comprising sodium phosphate at a concentration of 10 to 100 mM, specifically 20 to 90 mM, more specifically 30 to 80 mM, more specifically 40 to 70 mM, more specifically 45 to 60 mM, more specifically 45 to 55 mM, and most specifically about 50 mM, and having a pH of 7.5 to 8.5, specifically 7.6 to 8.4, more specifically 8 to 8.5, more specifically 7.7 to 8.3, more specifically 7.8 to 8.2, more specifically 7.9 to 8.1, and most specifically 8.

[0036] According to a specific embodiment of the present invention, the hydrophobic interaction column of the present invention comprises, before adding the first and second buffers described above, 10 to 100 mM, specifically 20 to 90 mM, more specifically 30 to 80 mM, even more specifically 40 to 70 mM, even more specifically 45 to 60 mM, even more specifically 45 to 55 mM, and most specifically about 50 mM of sodium phosphate; And it may additionally comprise a step of adding a conductivity adjustment buffer comprising sodium chloride of 1.5 to 6.5 M, specifically 2 to 6 M, more specifically 2.5 to 5.5 M, even more specifically 3 to 5 M, even more specifically 3.5 to 4.5 M, and most specifically about 4 M, and having a pH of 7.5 to 8.5, specifically pH 7.6 to 8.4, more specifically pH 7.7 to 8.3, more specifically pH 7.8 to 8.2, even more specifically pH 7.9 to 8.1, and most specifically pH 8.

[0037] According to a specific embodiment of the present invention, the second filtration step included in the purification method of the present invention is performed with a buffer solution containing sodium phosphate at 10 to 100 mM, specifically 13 to 45 mM, more specifically 15 to 40 mM, even more specifically 17 to 35 mM, even more specifically 19 to 30 mM, even more specifically 20 to 25 mM, and most specifically 20 mM, and having a pH of 7.5 to 8.5, specifically 7.6 to 8.4, more specifically 7.7 to 8.3, even more specifically 7.8 to 8.2, even more specifically 7.9 to 8.1, and most specifically 8.0.

[0038] According to a specific embodiment of the present invention, the ion exchange column used in the present invention is an anion exchange resin column.

[0039] According to a more specific embodiment of the present invention, the anion exchange resin column is performed by a Q column.

[0040] The term “Q column” as used herein means a “strong-anion column” filled with quaternary ammonium resin, also referred to as Q-sephadex or mono-Q.

[0041] According to a specific embodiment of the present invention, the Q column that can be used in the present invention includes, but is not limited to, a Capto Q ImpRes column, a Toyopearl Super Q 650M column, a Q sepharose FF column, and a Q Sepharose High Performance (Q Sepharose HP) column, and any column known in the art as a strong-anion column containing quaternary ammonium can be used without limitation.

[0042] According to a specific embodiment of the present invention, the anion exchange column comprises sodium phosphate at a concentration of 10 to 50 mM, specifically 10 to 40 mM, more specifically 10 to 30 mM, even more specifically 10 to 25 mM, even more specifically 15 to 25 mM, and most specifically 20 mM; and a first buffer comprising sodium chloride at 1 to 50 mM, specifically 5 to 50 mM, more specifically 5 to 40 mM, even more specifically 5 to 30 mM, even more specifically 5 to 15 mM, even more specifically 7 to 13 mM, and most specifically 10 mM, and having a pH of 7.5 to 8.5, specifically 7.6 to 8.4, more specifically 7.7 to 8.3, even more specifically 7.8 to 8.2, even more specifically 7.9 to 8.1, and most specifically 8; and

[0043] Sodium phosphate at 10 to 50 mM, specifically 10 to 40 mM, more specifically 10 to 30 mM, even more specifically 10 to 25 mM, even more specifically 15 to 25 mM, and most specifically 20 mM; and sodium chloride at 50 to 100 mM, specifically 55 to 90 mM, more specifically 60 to 80 mM, even more specifically 65 to 75 mM, and most specifically 70 mM, and the second buffer having a pH of 7.5 to 8.5, specifically 7.6 to 8.4, more specifically 7.7 to 8.3, more specifically 7.8 to 8.2, even more specifically 7.9 to 8.1, and most specifically 8.

[0044] According to a specific embodiment of the present invention, the non-complexed form of the toxin protein has a molecular weight of 50 kD to 300 kD, specifically 100 kD to 200 kD, more specifically 120 kD to 180 kD, and most specifically about 150 kD.

[0045] According to a specific embodiment of the present invention, the Clostridium botulinum toxin protein is serotype A.

[0046] According to another aspect of the present invention, the present invention provides a method for producing a crude Clostridium botulinum toxin solution, comprising the step of filtering a sample containing a non-complexed form of botulinum toxin protein purified by the method of the present invention described above using a sterilizing filter.

[0047]

[0048] The features and advantages of the present invention are summarized as follows:

[0049] (a) The present invention provides an efficient method for purifying botulinum neurotoxin protein, which can separate botulinum toxin protein into non-toxin protein and non-complexed form, and obtain non-complexed form botulinum toxin protein with high purity and increased yield only through the purification process of hydrophobic interaction and ion exchange chromatography.

[0050] (b) In particular, the purification method of the present invention can be used for various industrial purposes including the pharmaceutical field, as it can be mass-produced economically and efficiently in the form of 150 kD pure toxin protein from botulinum toxin protein.

[0051] In addition, the effects of the present invention are not limited to the above-described effects, and should be understood to include all effects that can be inferred from the composition of the invention described in the detailed description or claims of the present invention.

[0052]

[0053] Figure 1a is a graph showing peaks according to the volume of the eluate as a result of purification through hydrophobic interaction chromatography from the botulinum toxin process solution separated after the first filtration and dissociation step in the purification method of the present invention.

[0054] Figure 1b is a drawing showing the SDS-PAGE result of the eluate purified through hydrophobic interaction chromatography from the botulinum toxin process solution separated after the first filtration and dissociation step in the purification method of the present invention.

[0055] Figure 2a is a graph showing peaks according to the volume of the eluate obtained by purifying the filtered process solution through anion exchange chromatography after the second filtration in the purification method of the present invention.

[0056] Figure 2b is a drawing showing the SDS-PAGE result of the eluate purified through anion exchange chromatography from the filtered process solution after the second filtration in the purification method of the present invention.

[0057] Figure 3a is a drawing showing the results of analyzing a non-complexed form of botulinum toxin protein according to Example 2 using size exclusion chromatography (SE-HPLC).

[0058] Figure 3b is a drawing showing the results of analyzing the non-complexed form of botulinum toxin protein according to Example 5 using size exclusion chromatography (SE-HPLC).

[0059] Figure 4 is a schematic diagram illustrating the purification method of the present invention.

[0060]

[0061] Hereinafter, the present invention will be described in detail.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0063]

[0064] The term “Clostridium botulinum toxin” in this specification may be used interchangeably with “Botulinum toxin” and refers to a toxic protein produced by the bacterium Clostridium Botulinum and its closely related species.

[0065] The term “culture solution” as used herein refers to a cultured product containing botulinum toxin or any solution that has been further processed using the cultured product as a starting material, which is a result of culturing cells capable of producing botulinum toxin, specifically Clostridium botulinum. Therefore, in this specification, “culture solution containing Clostridium botulinum neurotoxin protein” has the same meaning as “biological sample containing Clostridium botulinum neurotoxin protein.” The purification method of the present invention can be applied to a culture solution containing botulinum toxin. Therefore, in the purification method of the present invention, a step of culturing Clostridium botulinum may be further included prior to the chromatography step for purification, but is not limited thereto.

[0066] The above Clostridium botulinum strain can be cultured using a suitable medium and culture conditions known in the art. That is, the Clostridium botulinum strain can be cultured in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, etc., under anaerobic conditions while controlling temperature, pH, etc.

[0067] The term “acid precipitation” in this specification refers to a step of precipitating a target protein by adding acid to it.

[0068] The above acid precipitation occurs when an acid is added to a protein such as Clostridium botulinum toxin protein, which lowers the pH and causes the protein to reach its isoelectric point (pI), thereby causing it to aggregate and precipitate. That is, the acid precipitation of the Clostridium botulinum toxin can be performed using any type of conventional technique used in the art, including, but not limited to, a precipitation method using hydrochloric acid or sulfuric acid.

[0069] The term "process solution" as used herein refers to a mixture containing the acid precipitation washing and extraction product obtained by subjecting a culture solution of botulinum toxin to acid precipitation and toxin extraction processes. By applying the purification method of the present invention, non-complexed botulinum toxin can be obtained from this process solution.

[0070] The term “filtration” in this specification means a step in which a substance added to a process solution is filtered prior to a purification step and the volume of the process solution is reduced, and includes both “first filtration” and “second filtration” steps.

[0071] The above filtration can utilize various means for removing impurities, and specifically, microfiltration, ultrafiltration (UF), precision filtration, depth filtration, and diafiltration (DF) can be used.

[0072] Ultrafiltration (UF), also known as "UF," is a membrane separation process that selectively separates fine particles by creating a pressure gradient. This ultrafiltration method can filter and separate fine chlorine particles that are difficult to remove through conventional filtration.

[0073] Diafiltration (DF), also referred to as "DF," refers to the process of adding water or buffer to an extract during the concentration process. Diafiltration simultaneously involves diffusion and convective mass transfer, driven by two driving forces: a concentration gradient and a transmembrane pressure gradient. It can be an effective method for selectively removing lower molecular weight substances from a mixture.

[0074] Additionally, the above filtration may be accompanied by a buffer exchange process as needed, and the “filtration step” may include up to the “buffer exchange step.”

[0075] The term “dissociation” in this specification refers to the process of separating a complexed toxic protein into a pure toxic protein, which is a non-complexed toxic protein.

[0076] The term “hydrophobic interaction column” in this specification means a column (separation tube) used in hydrophobic interaction chromatography.

[0077] The term “ion exchange resin column” in this specification means a column (separation tube) used in cation or anion exchange chromatography.

[0078] The term “chromatography” in this specification refers to a technique for separating a mixture, in which various substances present in the mixture are moved through a mobile phase on a stationary phase (stationary phase), and the separation is achieved by the difference in movement speed resulting from the difference in the degree of interaction of each substance with the stationary phase.

[0079] The term “hydrophobic interaction chromatography” in this specification may be referred to as “hydrophobic chromatography” and means an experimental technique for separating each substance (e.g., protein) included in a mixture for separation based on the difference in the degree of hydrophobicity of the substances.

[0080] The above hydrophobic chromatography utilizes a non-polar substance as the stationary phase, and when separating proteins, their separation is achieved by utilizing the reversible interaction between the protein and the hydrophobic surface of the chromatography resin. This reversible binding between the protein and the hydrophobic surface is controlled by the salt concentration. Under high salt conditions, the protein binds to the resin, and under low salt conditions, the protein is eluted. Therefore, each protein can be separated by utilizing the difference in elution rate due to the difference in hydrophobicity according to the downward salt concentration gradient.

[0081] The term “ion exchange chromatography” in this specification refers to a type of liquid chromatography, a technique for qualitatively and quantitatively analyzing ionic substances in a solution.

[0082] The above ion exchange chromatography is a type of liquid chromatography that utilizes an ion exchange reaction using an ion exchanger as a stationary phase, and can be used to separate amino acids, proteins, enzymes, and metal ions, etc. If the resin is a cation, it can be “anion exchange chromatography,” and if the resin is an anion, it can be “cation exchange chromatography.”

[0083] The above anion exchange chromatography may be a type of ion exchange chromatography that separates ions or target substances having negative polarity by fixing them with a stationary resin having positive polarity.

[0084] The term “non-complexed botulinum toxin protein” or “pure toxin protein” as used herein means a toxin protein of about 150 kDa isolated by removing non-toxin proteins (NTNHA, HA, etc.) from a complexed botulinum toxin protein derived from Clostridium botulinum, and is a purified toxin protein having a purity of 95% or more, preferably 99% or more, according to analytical standards. These terms are used interchangeably herein.

[0085] The pure toxin of about 150 kD can be obtained by isolating a non-toxin protein, such as hemagglutinin protein or non-toxic non-hemagglutinin protein (NTNH), from a naturally occurring botulinum toxin complex (e.g., 900 kD).

[0086] Specifically, the molecular weight of the botulinum toxin protein molecule (pure toxin) in all seven known botulinum toxin serotypes is approximately 150 kD. The botulinum toxin protein molecule (pure toxin) can be divided into three domains according to its function: a protease domain, a translocation domain, and a receptor binding domain. The protease domain may be referred to as the light chain (LC), the translocation domain as the N-terminal heavy chain (HN), and the receptor binding domain as the C-terminal heavy chain (HC). In addition, in order for botulinum toxin to take effect in the body, it must first bind to the receptor of the target nerve cell (neuron), and the serotypes of botulinum toxin identified so far are known to bind to SV2 (synaptic vesicle 2 protein) or synaptotagmin, which are membrane proteins of neurons.

[0087] Additionally, when botulinum toxin is produced by Clostridial bacteria, the 150 kD botulinum toxin protein molecule is released in the form of a complex with related non-toxin proteins. Thus, botulinum toxin type serotype A complexes can be produced by Clostridial bacteria as 900 kD, 500 kD, and 300 kD types. Botulinum toxin types B and C can be produced as 500 kD complexes, botulinum toxin type D can be produced as 300 kD and 500 kD complexes, botulinum toxin types E and F can be produced as about 300 kD complexes, botulinum toxin type G can be produced as about 500 kD complexes, and botulinum toxin type FA (formerly known as type H or type FA) can be produced as about 300 kD to 900 kD complexes. That is, it can be seen that the complex with a molecular weight greater than about 150 kD contains a non-toxic hemagglutinin protein and a non-toxic and non-toxic non-hemagglutinin protein.

[0088] The term “botulinum toxin protein” as used herein refers to a high molecular weight complex form comprising a pure toxin component of about 150 kDa as well as non-toxin proteins. That is, the complexed form may comprise a botulinum toxin protein; and one or more non-toxin hemagglutinin proteins; and / or one or more non-toxin non-hemagglutinin proteins.

[0089] The above botulinum toxin protein may have a molecular weight greater than about 150 kD. For example, a complexed form of botulinum toxin type A may have a molecular weight of about 900 kD, about 500 kD, or about 300 kD.

[0090] Additionally, the above botulinum toxin protein refers to a botulinum toxin protein that is not combined with any combined non-toxin proteins, such as non-toxic non-hemagglutinin, hemagglutinin, etc., unless otherwise indicated. It may also be referred to as “non-complexed botulinum toxin protein,” “pure toxin protein,” or “botulinum toxin protein with non-toxin proteins removed (reduced).”

[0091] The term “purity” as used herein means the percentage (%) of the target protein (e.g., non-complexed botulinum toxin protein) in the purified protein composition relative to the total protein or total solid component, which can be measured by analytical methods such as SDS-PAGE, HPLC, ELISA, or immunoblot analysis.

[0092] Specifically, the purity of the non-complexed toxin protein measured herein is at least 95%, more specifically at least 97%, and most specifically at least 99%.

[0093] The term “serotype A” in this specification means type A among the forms classified according to the serological characteristics of the botulinum toxin protein.

[0094] Serotype A is the most lethal known natural agent to humans, and seven other serotypes, B, C, D, E, F, G, and H, have been identified. Different serotypes can be identified by neutralization by type-specific antibodies, and the severity of paralysis caused by each serotype and the animal species most affected may differ.

[0095] The term “sterilizing filter” as used herein refers to a filter capable of removing mold, bacteria, etc. Specifically, any type of sterilizing filter that does not affect the yield or purity of the active ingredient (toxin), such as by adsorption or decomposition of the active ingredient, may be used.

[0096]

[0097] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0098]

[0099] Example 1. Preparation of samples and experimental materials

[0100] 1-1. Culture of botulinum toxin type A

[0101] The botulinum strain used in the present invention is Clostridium botulinum type A, NCTC13319. The strain was first inoculated into 500 mL of PYG medium (3% potato peptone, 1% yeast extract, 1% glucose) and then cultured at 34 ± 1 °C for 12 to 24 hours in a ReadytoProcess WAVE 25 incubator under anaerobic conditions. After culture, when the strain was in the log phase, 100 mL was inoculated into 5 L of PYG medium and cultured for 40 to 72 hours in a ReadytoProcess WAVE 25 incubator under anaerobic conditions. The culture solution was sterilized using a sterilizing filter and only the culture solution was recovered.

[0102]

[0103] 1-2. Acid precipitation and toxin release from culture medium

[0104] The above culture medium containing botulinum toxin type A was adjusted to pH 3.5 using 3N sulfuric acid and precipitated.

[0105] Afterwards, the precipitate containing the botulinum toxin type A was washed with purified water, and then the botulinum toxin type A protein was eluted with buffer A (400 mM sodium citrate, pH 5.5, 200 mM sodium chloride) and buffer B (200 mM sodium citrate, pH 5.5, 100 mM sodium chloride).

[0106]

[0107] Example 2. Purification of botulinum toxin I

[0108] 2-1. First filtration and dissociation

[0109] The process solution of Example 1 was concentrated using a vertical filter system (Tangential Flow Filtration, TFF) using a 50 kD membrane filter to obtain a high yield, and diafiltration (DF) was performed with a buffer solution (50 mM sodium phosphate, pH 8.0).

[0110] By performing the above filtration, not only was the sulfuric acid remaining after washing in the above examples 1-2 additionally removed, but the entire volume of the culture solution was concentrated to simplify the process time, and by performing diafiltration with a buffer (50 mM sodium phosphate, pH 8.0), the botulinum toxin protein was separated into non-toxin protein and 150 kD pure toxin protein.

[0111]

[0112] 2-2. Hydrophobic interaction chromatography (first purification)

[0113] Hydrophobic interaction chromatography (Phenyl-HP) was performed on the botulinum toxin process solution separated by the method of Example 2-1 above.

[0114] Specifically, a sample was prepared by adding a conductivity adjustment buffer (50 mM sodium phosphate, pH 8.0, 4 M sodium chloride) to add salt to the process solution, and then hydrophobic interaction chromatography (Phenyl-HP) was performed with the first buffer (Buffer A: 50 mM sodium phosphate, pH 8.0, 2 M sodium chloride) and the second buffer (Buffer B: 50 mM sodium phosphate, pH 8.0) to separate the botulinum toxin protein and impurities.

[0115] The results of hydrophobic interaction chromatography performed under the above conditions are as shown in Figs. 1a and 1b.

[0116] Specifically, the notations described in each drawing mean the following:

[0117] In Fig. 1a, the X-axis is volume (mL), the Y-axis is UV value, and in the SDS-PAGE in Fig. 1b, M is a marker (Protein Size Marker), LS is a sample before column treatment (Loading sample), FT is a column flow-through, W is a washing, E is an elution, and Strip is a high-concentration salt elution. In addition, NTNH is non-toxic non-hemagglutinin, Hc is heavy chain, Lc is light chain, and HA is hemagglutinin.

[0118] Through the above primary purification, it was possible to confirm the elution range containing pure toxin protein of 150 kD (heavy chain and light chain) of botulinum toxin type A with some impurities removed.

[0119]

[0120] 2-3. Second filtration

[0121] The process solution, which completed the first purification, was subjected to diafiltration (DF) with a buffer solution (20 mM sodium phosphate, pH 8.0) to remove the salt contained therein.

[0122] As a result, only a process solution containing pure botulinum toxin type A 150 kD toxin protein was obtained.

[0123]

[0124] 2-4. Anion exchange chromatography (second purification)

[0125] Anion exchange chromatography (Capto Q ImpRes) was performed on the process solution filtered using the method of Example 2-3 above.

[0126] Specifically, anion exchange chromatography was performed with the first buffer (buffer A: 20 mM sodium phosphate, pH 8.0, 10 mM sodium chloride) and the second buffer (buffer B: 20 ​​mM sodium phosphate, pH 8.0, 70 mM sodium chloride) to further separate the 150 kD pure toxin protein and impurities.

[0127] The results of anion exchange chromatography performed under the above conditions are shown in Figs. 2a and 2b. The notations described in each drawing are the same as in Figs. 1a and 1b.

[0128] Through the above secondary purification, it was possible to confirm the elution range containing pure toxin protein of 150 kD (heavy chain and light chain) of high-purity botulinum toxin type A with a large amount of impurities removed.

[0129]

[0130] 2-5. Formulation

[0131] The process solution purified by the method of Example 2-4 was further filtered using a 0.2 μm sterilizing filter to remove insoluble substances and contaminants, and the filtered final product was manufactured into a pure botulinum toxin type A 150 kD toxin protein stock solution formulation.

[0132]

[0133] Example 3. Purification of botulinum toxin II

[0134] 3-1. First filtration

[0135] The process solution of Example 1 was concentrated using a vertical filter system (Tangential Flow Filtration, TFF) using a 50 kD membrane filter to achieve high yield, and diafiltration (DF) was performed with purified water.

[0136] By performing the above filtration, not only was the sulfuric acid remaining after washing in Example 1-2 additionally removed, but the entire volume of the culture solution was concentrated so as to simplify the process time.

[0137]

[0138] 3-2. Dissociation

[0139] To separate the botulinum toxin protein into non-toxin protein and 150 kD pure toxin protein, 1 M sodium phosphate was directly added to the first filtered process solution to make a concentration of approximately 50 mM, and 1 N sodium hydroxide was titrated to make a pH of 8.0. After stirring at 100 rpm for 1 hour at room temperature, the solution was stored under refrigerated conditions at 0 to 10°C for 12 hours.

[0140]

[0141] 3-3. Hydrophobic interaction chromatography (first purification)

[0142] The same hydrophobic interaction chromatography (Phenyl-HP) as in Example 2-2 was performed on the process solution from which the botulinum toxin protein was separated using the method of Example 3-2.

[0143]

[0144] 3-4. Second filtration

[0145] The process solution for which the above first purification was completed was subjected to a second filtration in the same manner as in Example 2-3.

[0146]

[0147] 3-5. Anion exchange chromatography (second purification)

[0148] Anion exchange chromatography identical to Example 2-4 was performed on the process solution filtered by the method of Example 3-4.

[0149]

[0150] 3-6. Formulation

[0151] The process solution purified by the method of Example 3-5 was further filtered using a 0.2 μm sterilizing filter to remove insoluble substances and contaminants, and the filtered final product was manufactured into a pure toxin protein stock solution formulation of botulinum toxin type A 150 kD.

[0152]

[0153] Example 4. Purification of botulinum toxin III

[0154] 4-1. First filtration

[0155] The process solution of Example 1 was concentrated using a vertical filter system (Tangential Flow Filtration, TFF) using a 100 kD membrane filter, and diafiltration (DF) was performed with purified water.

[0156] By performing the above filtration, not only was the sulfuric acid remaining after washing in Example 1-2 additionally removed, but the entire volume of the culture solution was concentrated so as to simplify the process time.

[0157]

[0158] 4-2. Dissociation

[0159] Dissociation, identical to that of Example 3-2, was performed from the first filtered process solution using the method of Example 4-1.

[0160]

[0161] 4-3. Hydrophobic interaction chromatography (first purification)

[0162] The same hydrophobic interaction chromatography (Phenyl-HP) as in Example 2-2 was performed on the process solution from which the botulinum toxin protein was separated using the method of Example 4-2.

[0163]

[0164] 4-4. Second filtration

[0165] The process solution for which the above first purification was completed was subjected to a second filtration in the same manner as in Example 2-3.

[0166]

[0167] 4-5. Anion exchange chromatography (second purification)

[0168] Anion exchange chromatography identical to Example 2-4 was performed on the process solution filtered by the method of Example 4-4.

[0169]

[0170] 4-6. Formulation

[0171] The process solution purified by the method of the above Example 4-5 was further filtered using a 0.2 μm sterilizing filter to remove insoluble substances and contaminants, and the filtered final product was manufactured into a pure toxin protein stock solution formulation of botulinum toxin type A 150 kD.

[0172]

[0173] Example 5. Purification of botulinum toxin IV

[0174] 5-1. First filtration

[0175] The process solution of Example 1 was concentrated using a vertical filter system (Tangential Flow Filtration, TFF) using a 100 kD membrane filter, and diafiltration (DF) was performed with purified water.

[0176]

[0177] 5-2. Enzyme treatment (Nuclease) and second filtration

[0178] The process solution obtained by the method of Example 5-1 was subjected to enzyme treatment with benzonase at room temperature for 6 to 16 hours to remove residual nucleic acids, and then diafiltration (DF) was performed with a buffer solution (50 mM sodium citrate, pH 6.0, 25 mM sodium chloride).

[0179] Example 5 attempted to perform cation exchange chromatography, unlike the purification processes of Examples 2, 3, and 4. Before performing cation exchange chromatography, enzyme treatment was performed to facilitate the attachment of proteins to the cation resin, and filtration was additionally performed to remove the enzyme after the reaction.

[0180]

[0181] 5-3. Cation exchange chromatography (first purification)

[0182] Cation exchange chromatography (SP-HP) was performed on the process solution filtered using the method of Example 5-2 above.

[0183] Specifically, botulinum toxin protein and impurities were separated by performing cation exchange chromatography (SP-HP) with a first buffer (buffer A: 20 mM sodium citrate / citric acid, pH 4.8) and a second buffer (buffer B: 20 ​​mM sodium citrate / citric acid, pH 4.8, 1 M sodium chloride). The yield at this time was 55.31 mg.

[0184]

[0185] 5-4. Dissociation

[0186] In order to separate the botulinum toxin protein into non-toxin protein and 150 kD toxin protein, the first filtered process solution was subjected to a dissociation step by dialysis, 1 M sodium phosphate was added to make it about 50 mM, and 1 N sodium hydroxide was titrated to make it pH 8.0. Afterwards, it was stirred at 100 rpm at room temperature for 1 hour and stored under refrigerated conditions at 0-10°C for 12 hours.

[0187]

[0188] 5-5. Hydrophobic interaction chromatography (second purification)

[0189] Hydrophobic interaction chromatography (Phenyl-HP) was performed on the process solution separated by the method of Example 5-4 above.

[0190] Specifically, botulinum toxin protein and impurities were separated by performing hydrophobic interaction chromatography (Phenyl-HP) with a first buffer (buffer A: 50 mM sodium phosphate, pH 8.0, 2 M sodium chloride) and a second buffer (buffer B: 50 mM sodium phosphate, pH 8.0). The yield at this time was 3.84 mg.

[0191]

[0192] 5-6. Third filtration

[0193] The purified process solution was subjected to diafiltration (DF) with a buffer solution (20 mM sodium phosphate, pH 8.0) to remove the salt used in the purification.

[0194]

[0195] 5-7. Anion exchange chromatography (third purification)

[0196] Anion exchange chromatography (Capto Q ImpRes) was performed on the process solution filtered using the method of Example 5-6 above.

[0197] Specifically, by performing anion exchange chromatography with a first buffer (buffer A: 20 mM sodium phosphate, pH 8.0, 10 mM sodium chloride) and a second buffer (buffer B: 20 ​​mM sodium phosphate, pH 8.0, 70 mM sodium chloride), the 150 kD pure toxin protein and impurities were additionally separated. The yield at this time was 1.02 mg.

[0198]

[0199] 5-8. Formulation

[0200] The process solution purified by the method of the above Examples 5-7 was further filtered using a 0.2 μm sterilizing filter to remove insoluble substances and contaminants, and the filtered final product was manufactured into a pure toxin protein stock solution formulation of botulinum toxin type A 150 kD.

[0201]

[0202] Example 6. Analysis of purified botulinum toxin

[0203] The samples obtained through the purification process of each of the above examples of botulinum toxin were analyzed through size exclusion chromatography (SE-HPLC).

[0204] Specifically, size exclusion chromatography was performed under the following conditions: mobile phase: 20 mM sodium phosphate, pH 8.0, 150 mM sodium chloride; flow rate: 0.5 mL / min; method: isocratic elution, A 100%, 60 min.

[0205] As a result, as shown in Fig. 3a for the purification process of Example 2, a 150 kD pure toxin protein was confirmed, and the purity was 99.91%. As shown in Fig. 3b for the purification process of Example 5, a 150 kD pure toxin protein was confirmed, and the purity was 99.75%.

[0206] On the other hand, in both the purification processes of Examples 2 and 5, only trace amounts of impurities (about 0.2%) were detected.

[0207] That is, when comparing the purification processes according to Examples 2 and 5, it was confirmed that a 150 kD pure toxin protein with the same high purity of about 99% or more could be obtained despite the process steps being simplified.

[0208]

[0209] In addition, the yields for each purification process according to Examples 2, 3, and 4 were confirmed, and Table 1 below was completed. At this time, the “Eluate amount” value in Table 1 was calculated based on the UV (278 nm) absorbance value, and was performed based on 5 L of culture medium.

[0210] Process Example 2 Example 3 Example 4 Membrane size 50kD 50kD 100kD First filtration and dissociation OXX Dissociation (titration) XOO Hydrophobic interaction chromatography 21.21mg 27.42mg 15.84mg Second filtration OOO Anion chromatography 7.39mg 4.98mg 3.48mg

[0211]

[0212] As confirmed in Table 1, in the final anion exchange chromatography purified product, 7.39 mg of 150 kD pure toxin protein was obtained in Example 2, 4.98 mg in Example 3, 3.48 mg in Example 4, and 1.02 mg in Example 5, and it was confirmed that a high yield of 150 kD pure toxin protein could be obtained when the purification process according to Example 2 was performed.

[0213] These results suggest that by simultaneously performing the first filtration and dissociation steps as in Example 2 to separate the non-toxin protein and the 150 kD pure toxin protein, and then performing the purification process of hydrophobic interaction chromatography and ion exchange chromatography, it is possible to maintain high purity while reducing the process time and purify the 150 kD pure toxin protein with an increased yield.

[0214]

[0215] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for purifying Clostridium botulinum toxin comprising the following steps: (a) a step of first filtering a culture medium containing Clostridium botulinum neurotoxin protein; (b) a step of dissociating the botulinum toxin protein in the culture solution filtered in step (a) into non-toxin proteins and non-complexed botulinum toxin proteins; (c) a step of loading the botulinum toxin protein dissociated in step (b) onto a hydrophobic interaction column to capture the botulinum toxin protein in a non-complexed form and separate impurities; (d) a second filtering step of the botulinum toxin protein captured in the step (c); (e) A step of loading the botulinum toxin protein obtained in the above step (d) onto an ion exchange column to capture the non-complexed form of the botulinum toxin protein and further separate impurities.

2. A method for purifying Clostridium botulinum toxin comprising the following steps: (a) a step of dissociating the botulinum toxin protein contained in the filtrate obtained by first filtering a culture medium containing Clostridium botulinum neurotoxin protein into non-toxin proteins and non-complexed botulinum toxin proteins; (b) a step of loading the botulinum toxin protein dissociated in step (a) onto a hydrophobic interaction column to capture the botulinum toxin protein in a non-complexed form and separate impurities; (c) a second filtering step of the botulinum toxin protein captured in the step (b); (d) A step of loading the botulinum toxin protein obtained in step (c) onto an ion exchange column to capture the non-complexed form of the botulinum toxin protein and further separate impurities.

3. In paragraph 1 or 2, A method characterized in that the culture medium containing the Clostridium botulinum neurotoxin protein is prepared through an acid precipitation and toxin elution step using a first buffer containing 300 to 500 mM sodium citrate and 100 to 300 mM sodium chloride at a pH of 4.5 to 6.5 in a culture medium of a Clostridium strain; and a second buffer containing 100 to 300 mM sodium citrate and 50 to 150 mM sodium chloride at a pH of 4.5 to 6.

5.

4. In paragraph 1 or 2, A method characterized in that the first or second filtration is a vertical filter system (Tangential Flow Filtration, TFF) or a dialysis system.

5. In paragraph 4, A method characterized in that the above vertical filter system (Tangential Flow Filtration, TFF) is at least one filtration method selected from the group consisting of ultrafiltration and diafiltration.

6. In paragraph 5, A method characterized in that the above ultrafiltration or diafiltration is performed using a membrane filter having a size of 10 to 300 kD.

7. In paragraph 5, A method characterized in that the above filtration is performed using a buffer solution containing at least one selected from the group consisting of purified water, sodium citrate, sodium chloride, and sodium phosphate.

8. In paragraph 1 or 2, A method characterized in that the first filtration is performed using purified water.

9. In paragraph 1 or 2, A method characterized in that the above dissociation is performed using a buffer solution containing 10 to 100 mM sodium phosphate and having a pH of 7.5 to 8.

5.

10. In paragraph 1 or 2, A method characterized in that the hydrophobic interaction column is a column having one or more ligands selected from the group consisting of ether, isopropyl, butyl, octyl, and phenyl.

11. In paragraph 1 or 2, The hydrophobic interaction column comprises a first buffer solution containing 10 to 100 mM sodium phosphate and 1.5 to 2.5 M sodium chloride and having a pH of 7.5 to 8.5; and A method characterized in that it is performed by a second buffer solution containing 10 to 100 mM sodium phosphate and having a pH of 7.5 to 8.

5.

12. In paragraph 1 or 2, A method characterized in that the above ion exchange column is an anion exchange resin column.

13. In paragraph 12, A method characterized in that the above anion exchange resin column is performed by a Q column.

14. In paragraph 13, The method is characterized in that the above Q column is one Q column selected from the group consisting of a Capto Q ImpRes column, a Toyopearl Super Q 650M column, a Q sepharose FF column, and a Q Sepharose High Performance (Q Sepharose HP).

15. In paragraph 12, The anion exchange column comprises a first buffer solution containing 10 to 50 mM sodium phosphate and 1 to 50 mM sodium chloride, and having a pH of 7.5 to 8.5; and A method characterized in that it is performed by a second buffer solution containing 10 to 50 mM sodium phosphate and 50 to 100 mM sodium chloride and having a pH of 7.5 to 8.

5.

16. In paragraph 1 or 2, A method characterized in that the non-complexed form of the toxic protein has a molecular weight of 50 kD to 300 kD.

17. In paragraph 1 or 2, A method characterized in that the Clostridium botulinum toxin protein is serotype A.

18. In paragraph 1 or 2, A method characterized in that the purified botulinum toxin protein has a purity of 95% or more of non-complex toxin protein.

19. A method for producing a crude Clostridium botulinum toxin solution, comprising a step of filtering a sample containing a non-complexed form of botulinum toxin protein purified by the method of paragraph 1 or 2 using a sterilizing filter.

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