An improved method for botulinum toxin purification using multimodal chromatography.

Abstract

The present invention relates to a method for purifying botulinum toxin, comprising the steps of purifying the toxin using multimode chromatography and purifying the toxin using cation exchange chromatography. When botulinum toxin is purified and produced by the purification method according to the present invention, botulinum toxin with a purity of 99% or more can be purified in high yield, making it useful for botulinum toxin production.

Description

[Technical Field]

[0001] The present invention relates to a method for purifying botulinum toxin, and more particularly to a method for purifying botulinum toxin comprising the steps of purifying the toxin using multimode chromatography and purifying the toxin using cation exchange chromatography. [Background technology]

[0002] Since the 1890s, a diverse range of Clostridium strains that secrete neurotoxic toxins have been discovered, and over the past 70 years, research has been conducted to elucidate the characteristics of the toxins secreted by these strains.

[0003] The neurotoxic toxins derived from the aforementioned Clostridium strains, i.e., botulinum toxin, are classified into seven types, A through G, based on their serological characteristics. Each toxin contains a toxic protein of approximately 150 kDa, but naturally consists of complexes bound to several non-toxic proteins. The intermediate (medium) complex (300 kDa) consists of the toxic protein and a non-toxic, non-hemagglutinin protein, while the large (450 kDa) and giant (large-large; 900 kDa) complexes are forms in which the intermediate complex is bound to hemagglutinin (Sugiyama, H, Microbiol Rev, 44:419, 1980). Such non-toxic, non-hemagglutinin proteins are known to protect the toxin from low pH and various protein hydrolases in the intestines.

[0004] The aforementioned toxin is synthesized intracellularly as a single polypeptide with a molecular weight of approximately 150 kDa. It is then cleaved at a position one-third of the way from the N-terminus by intracellular proteolytic enzymes or artificial enzymatic treatment such as trypsin, separating into two units: a light chain (L) (molecular weight: 50 kDa) and a heavy chain (H) (molecular weight: 100 kDa). The toxicity of the toxin after this separation is significantly increased compared to when it was a single polypeptide. The two units are linked by disulfide bonds and each has a different function. The heavy chain binds to receptors on target cells and reacts with biological membranes at low pH (pH 4) to form channels (Mantecucco, C et al., TIBS, 18:324, 1993). The light chain has pharmacological activity, such as imparting permeability to cells using detergents or inhibiting neurotransmitter secretion when introduced into cells by electroporation.

[0005] The aforementioned toxin inhibits acetylcholine exocytosis at the cholinergic presynapse of the neuromuscular junction, causing generalized paralysis. Because toxicity is observed even with minute amounts of the toxin, it has been thought that this toxin possesses some kind of enzymatic activity.

[0006] Recent findings have revealed that the toxins possess metalloprotease activity, and their substrates include synaptobrevin, syntaxin, and the 25kDa synaptosomal associated protein of 25kDa (SNAP25), which are unit proteins that make up the exocytosis machine complex. Each type of toxin uses one of these three proteins as a substrate, but it is known that types B, D, F, and G cleave synaptobrevin, types A and E cleave SNAP25, and type C cleaves syntaxin at specific sites.

[0007] In particular, botulinum toxin type A is known to be soluble in dilute aqueous solutions with a pH of 4.0 to 6.8. Stabilized non-toxic proteins are separated from the neurotoxin at a pH of approximately 7 or higher, resulting in a gradual loss of toxicity, but toxicity is known to decrease particularly with increasing pH and temperature.

[0008] Botulinum toxin, as mentioned above, is lethal to the human body in small amounts and is easy to mass-produce, making it a toxin that can be used as one of the four major bioterrorism weapons, along with anthrax (Bacillus anthracis), plague (Yersinia pestis), and smallpox virus. However, it has become clear that in the case of type A botulinum toxin, when injected in amounts below a level that does not affect the body systemically, it can paralyze the local muscles at the injection site. Based on these properties, it can be widely used as a wrinkle remover, a treatment for spastic hemiplegia and cerebral palsy, etc., and demand has surged, leading to active research into methods for producing botulinum toxin to meet this demand.

[0009] Botulinum toxin for clinical use is generally isolated from cell cultures, and a variety of purification methods are used in this process.

[0010] Conventional methods for producing botulinum toxin included acid precipitation, salting-out, and chromatography.

[0011] For example, Japanese Patent Publication No. 1994-192296 discloses a method for producing crystalline botulinum toxin type A by culturing a Clostridium botulinum strain, followed by acid precipitation, extraction, addition of a nucleic acid degradation agent, and crystallization steps. Furthermore, U.S. Patent No. 5,696,077 discloses a method for producing botulinum toxin type B by culturing a Clostridium botulinum strain and then following acid precipitation, extraction, ion exchange chromatography, gel filtration chromatography, and crystallization steps.

[0012] Meanwhile, Simpson et al. have produced botulinum toxin type A using gravity flow chromatography for the purification of botulinum neurotoxin, HPLC, a capture step using affinity resin, size exclusion chromatography, and ion (anion and cation) exchange chromatography including the use of two different ion exchange columns (Method in Enzymology, 165:76, 1988), and Wang et al. used precipitation and ion chromatography methods to purify botulinum toxin type A (Dermatol Las Faci Cosm Surg., 2002:58, 2002).

[0013] Furthermore, U.S. Patent No. 6,818,409 discloses the use of ion exchange and lactose columns for purifying botulinum toxin, and U.S. Patent No. 7,452,697 discloses a method for producing botulinum toxin type A using ion exchange chromatography and hydrophobic chromatography. Korean Patent Publication No. 2009-0091501 discloses a method for purifying botulinum toxin using acid precipitation and anion exchange chromatography, and U.S. Patent Publication No. 2013-0156756 discloses a method for purifying botulinum toxin using anion exchange chromatography and cation exchange chromatography.

[0014] Recently, there have been persistent reports that nucleic acids and animal-derived products that can be included as impurities in the botulinum toxin purification process can cause serious side effects, highlighting the need for methods to purify botulinum toxin to a higher purity. Currently, the botulinum toxin purification methods commonly used in this field only achieve a purity level of approximately 95 to 96%. However, despite the need for high-purity purification processes, even simple changes in the purification sequence significantly alter the purity and yield, making prediction difficult. Furthermore, even increasing the purity by about 1% drastically reduces the yield, making it economically inefficient. Therefore, developing methods for purifying high-purity botulinum toxin remains challenging.

[0015] Against this technological backdrop, the inventors diligently strived to develop a method for purifying botulinum toxin that could yield botulinum in high yield while maintaining a purity of 99% or more. As a result, they discovered that by incorporating size exclusion chromatography into the purification step in a multimode or sequential manner, it is possible to purify botulinum toxin in a yield higher than that of conventional techniques, while maintaining a purity of 99% or more and a low aggregation degree of 0.5% or less, thus completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0016] The object of the present invention is to provide a method for purifying botulinum toxin with high purity and high yield. [Means for solving the problem]

[0017] To achieve the above objective, the present invention provides a method for purifying botulinum toxin, comprising the following steps: (a) Steps of purifying botulinum toxin from a sample containing botulinum toxin using anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC), and (b) Steps of purifying botulinum toxin using cation exchange chromatography.

Brief Description of Drawings

[0018] [Figure 1] This schematically shows the overall production process of botulinum toxin used in the examples of the present invention. The primary purification schematically shows the primary purification processes used in Examples 3-1 to 3-8 and 5-1, and the secondary purification shows the secondary purification process (CEX) used in Examples 4-1 and 5-1. In Example 3-7, the primary purification was performed sequentially with Multimodal (AEX + HIC) and SEC. [Figure 2] This shows the yield of the toxin obtained after purifying the toxin using various combinations of chromatography including the purification process of the present invention. [Figure 3] This shows the yield of the toxin obtained after purifying the toxin using various combinations of chromatography including the purification process of the present invention. [Figure 4] This is the result of confirming the presence or absence of impurities through SDS-PAGE after purifying the toxin using various combinations of chromatography including the purification process of the present invention. The red arrow indicates the impurity band. [Figure 5] This is the result of confirming the presence or absence of impurities through SDS-PAGE after purifying the toxin using various combinations of chromatography including the purification process of the present invention. The red arrow indicates the impurity band. Footnote 1) in Figure 5 is the botulinum toxin-containing peak described in the prior literature, and footnote 2) is the toxin-containing peak confirmed in this example. [Figure 6]The results show the purification of toxins using various combinations of chromatography, including the purification process of the present invention, followed by confirmation of purity, impurity peaks, and aggregation peaks via SEC-HPLC. [Figure 7] The results show the purification of toxins using various combinations of chromatography, including the purification process of the present invention, followed by confirmation of purity, impurity peaks, and aggregation peaks via SEC-HPLC. [Figure 8] The results show the purification of toxins using various combinations of chromatography, including the purification process of the present invention, followed by confirmation of purity, impurity peaks, and aggregation peaks via SEC-HPLC. [Figure 9] The results show the purification of toxins using various combinations of chromatography, including the purification process of the present invention, followed by confirmation of purity, impurity peaks, and aggregation peaks via SEC-HPLC. [Modes for carrying out the invention]

[0019] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by experts skilled in the art to which this invention pertains. The nomenclature and terminology used herein are generally well known and commonly used in the art.

[0020] In this invention, we have confirmed that when botulinum toxin culture solution is purified using multimode chromatography followed by cation exchange chromatography, botulinum toxin can be purified to a significantly higher purity compared to botulinum toxin purified by conventionally known methods.

[0021] Therefore, in one view, the present invention relates to a method for purifying botulinum toxin, comprising the following steps: (a) A step of purifying botulinum toxin from a sample containing botulinum toxin using anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC), and (b) A step of purifying botulinum toxin using cation exchange chromatography.

[0022] In this invention, the term "botulinum toxin" may include not only neurotoxins (NTXs) produced by Clostridium botulinum strains or their variants, but also all modified, recombinant, hybrid, and chimeric botulinum toxins. In this invention, recombinant botulinum toxin may have light and / or heavy chains produced by recombinant production by non-Clostridium species. In this invention, the botulinum toxin may include not only botulinum toxin complexes (i.e., 300, 600, and 900 kDa complexes) but also pure botulinum toxin (i.e., approximately 150 kDa neurotoxic molecules).

[0023] NTXs(7S), the main component of botulinum toxin, binds with non-toxic components in culture media or food to form large complexes (Oguma et al., "Structure and function of Clostridium botulinum ptogenitor toxin", J. Toxicology: Toxin Reviews, 18:17-34, 1999). Type A strains that produce botulinum toxin serotype A produce three forms of ptogenitor toxins: LL (19S, 900kDa), L (16S, 500kDa), and M (12S, 300kDa), all of which are considered fully activated. In contrast, type B, C, and D strains produce only two forms, L and M. Furthermore, while types E, F, and G produce only a single form of toxin, types E and F produce M toxin, and type G produces L toxin. M toxin consists of NTX (7S, 150kDa) and a non-toxic component (Non-Toxic-Non-HA, NTNH) that does not exhibit hemagglutinin (HA) activity.

[0024] In the present invention, the botulinum toxin may be selected from the group consisting of serotypes A, B, C, D, E, F, and G, but is not limited thereto.

[0025] In the present invention, the sample containing botulinum toxin in step (a) above is used without limitation to include any sample containing one or more botulinum toxins and impurities. For example, the sample may include, but is not limited to, a culture medium of Clostridium botulinum strains, its lysate, its concentrate or precipitate, or, as other examples, a process solution or result product of an initial or intermediate step that may occur in the process of the present invention or in the process of the other invention, and may also be a recombinant sample containing recombinant botulinum toxin.

[0026] In the present invention, the sample containing recombinant botulinum toxin in step (a) above is filtered and injected into a chromatography matrix. In the present invention, the filtration in step (a) above can be performed using a filter of 0.01 to 2 μm, preferably 0.05 to 1.5 μm, and more preferably 0.1 to 1 μm, but is not limited thereto. In the examples of the present invention, the sample was filtered with a 0.2 μm filter before injection into a column packed with chromatography resin, but is not limited thereto.

[0027] In the present invention, the anion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography in step (a) above can be performed in any order, or two or more of the chromatography can be performed simultaneously using a multimode chromatography resin.

[0028] In this invention, the term "multimodal" chromatography refers to a chromatographic method in which the separation of a target component, in this invention, botulinum toxin, is based on one or more types of interactions between the components of the stationary phase and the mobile phase. In this invention, "multimodal" can be used interchangeably with "mixed mode".

[0029] In the present invention, step (a) may be characterized by being carried out using a multimodal chromatography resin having two or more chromatographic functions, such as anion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography. For example, step (a) may be carried out using a resin having AEX and HIC functions, a resin having AEX and SEC functions, a resin having HIC and SEC functions, and / or a resin having all of AEX, HIC, and SEC functions.

[0030] In the present invention, step (a) can be characterized by being performed using a multimodal resin having anion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography functions.

[0031] In the present invention, for example, step (a) may be characterized by further purifying the botulinum toxin-containing fraction, which has been purified using a multimodal resin having anion exchange chromatography and hydrophobic interaction chromatography functions, using size exclusion chromatography (SEC).

[0032] In the present invention, the anion exchange chromatography refers to a process of separating substances based on charge using an ion exchange resin containing a positively charged group such as the diethylaminoethyl group (DEAE). In the present invention, a variety of commercially available anion exchange chromatography resins may be used. For example, the resin used in the anion exchange chromatography may contain the diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups, and preferably TQ650, HQ, XQ, QXL, Capto, BigBeads resin, etc., may be used, but is not limited to these. More specific examples of the aforementioned anion exchange chromatography include, but are not limited to, *Protein Purification Methods, A Practical Approach*, Ed. Harris ELV, Angal S, IRL Press Oxford, England (1989); *Protein Purification*, Ed. Janson JC, Ryden L, VCH-Verlag, Weinheim, Germany (1989); *Process Scale Bioseparations for the Biopharmaceutical Industry*, Ed. Shukla AA, Etzel MR, Gadam S, CRC Press Taylor & Francis Group (2007), pages 188-196; *Protein Purification Handbook*, GE Healthcare 2007 (18-1132-29); and *Protein Purification, Principles, High Resolution Methods and Applications* (2nd Edition 1988), Ed. Janson JC and Ryden L.

[0033] In the present invention, the hydrophobic interaction chromatography is a method for separating target molecules based on their degree of hydrophobicity, characterized in that, for example, hydrophobic groups of target molecules such as phenyl, octyl, and butyl adhere to the HIC resin (stationary phase) through hydrophobic interaction. In the present invention, various commercially available hydrophobic interaction chromatography resins may be used. For example, the hydrophobic interaction chromatography resin may contain hydrophobic molecules selected from alkyl groups, aromatic groups, and ethers, more specifically, the alkyl groups may include lower alkyl groups such as n-propyl, isopropyl, n-butyl, isobutyl, and n-octyl, and the aromatic groups may include substituted or unsubstituted phenyl, but are not limited thereto. Furthermore, the hydrophobic interaction chromatography resin may contain metrics including agarose, Sepharose (GE Healthcare), polystyrene, divinylbenzene, and combinations thereof, but are not limited thereto.More specific examples include the hydrophobic interaction chromatography mentioned above, as seen in Protein Purification Methods, A Practical Approach, Ed. Harris ELV, Angal S, IRL Press Oxford, England (1989) page 224; Protein Purification, Ed. Janson JC, Ryden L, VCH-Verlag, Weinheim, Germany (1989) pages 207-226; Process Scale Bioseparations for the Biopharmaceutical Industry, Ed. Shukla AA, Etzel MR, Gadam S, CRC Press Taylor & Francis Group (2007), pages 197-206; Hydrophobic Interaction and Reversed Phase Chromatography, Principles and Merhods, GE Healthcare 2007 (11-0012-69); Protein Purification Handbook, GE Healthcare 2007 (18-1132-29); and Refer to, but are not limited to, the methods described in Protein Purification, Principles, High Resolution Methods and Applications (2nd Edition 1988), Ed. Janson JC and Ryden L, "Hydrophobic Interaction Chromatography," page 283, etc.

[0034] In the present invention, the size exclusion chromatography is known as molecular sieve chromatography and is a chromatography method that separates molecules based on their size and molecular weight. In the present invention, the size exclusion chromatography can be performed using a variety of size exclusion chromatography resins known in the art or columns containing them. For example, the size exclusion chromatography resin can generally be characterized by containing a polymer having fine porous beads, and the substances are separated according to the pore size of the beads. In the present invention, the polymer may be, for example, dextran, agarose, or polyacrylamide polymer, but is not limited to these. In the present invention, the size exclusion chromatography is approximately M r 10,000 to approximately M r 1,000,000, preferably about M r 100,000 or M r 500,000, most preferably about M r It can be characterized by having a cutoff value of 400,000. In the present invention, the cutoff value in size exclusion chromatography is a specific M r This means that molecules with a size greater than or equal to the specified value will be excluded.

[0035] In the present invention, a variety of buffers can be used in the multimode chromatography of step (a). For example, the buffers that can be used in the multimode chromatography of step (a) may include, but are not limited to, one or more selected from the group consisting of sodium phosphate, sodium chloride, potassium phosphate, sodium citarate, histidine, tris(hydroxymethyl)aminomethane (Tris), and bis-tris. In the present invention, the buffers for the multimode chromatography of step (a) may include equilibrium buffers, elution buffers, and wash buffers. In the present invention, the buffers for the multimode chromatography of step (a) may be selected and used at concentrations suitable for the application (equilibrium / elution or wash, etc.) or the type of buffer.

[0036] Furthermore, it is obvious that a variety of buffers known in the industry can be used as substitutes for each chromatography procedure.

[0037] In the present invention, the buffer solution may, if necessary, further contain one or more salts (e.g., NaCl), but is not limited thereto.

[0038] In the present invention, when using sodium phosphate buffer, it may be used at a concentration of 1 to 200 mM, preferably 10 to 150 mM, more preferably 25 to 100 mM, and most preferably 30 to 75 mM, but is not limited thereto.

[0039] In the present invention, the buffer solution may have a pH of 4 to 9, preferably 5 to 8, and more preferably 6 to 7.

[0040] In the present invention, in step (a) above, the botulinum toxin can be obtained by either eluting the FT (flow-through) from multimode chromatography into a fraction containing the botulinum toxin (flow-through mode), or by eluting and obtaining the fraction containing the botulinum toxin bound to the chromatographic resin (binding mode).

[0041] In the present invention, step (a) above is characterized by a flow-through mode in which the botulinum toxin is obtained in a fraction containing the botulinum toxin from the FT (flow-through) eluted from multimode chromatography. The terms "FT (flow-through)", "flow-through mode", or "flow-through purification", which are used interchangeably in the present invention, mean a separation method in which at least one target molecule (e.g., botulinum toxin) contained in a biopharmaceutical preparation along with one or more impurities passes through a substance that binds to one or more impurities, and the target molecule does not normally bind (i.e., flows through).

[0042] In the present invention, the cation exchange chromatography in step (b) above means a step of separating substances based on charge using an ion exchange resin containing a negative charge group. In the present invention, a variety of commercially available cation exchange chromatography resins may be used, for example, carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S), and preferably HS, XS, etc., may be used, but is not limited thereto. Other examples of cation exchange chromatography include, but are not limited to, *Protein Purification Methods, A Practical Approach*, Ed. Harris ELV, Angal S, IRL Press Oxford, England (1989); *Protein Purification*, Ed. Janson JC, Ryden L, VCH-Verlag, Weinheim, Germany (1989); *Process Scale Bioseparations for the Biopharmaceutical Industry*, Ed. Shukla AA, Etzel MR, Gadam S, CRC Press Taylor & Francis Group (2007), pages 188-196; *Protein Purification Handbook*, GE Healthcare 2007 (18-1132-29); and *Protein Purification, Principles, High Resolution Methods and Applications* (2nd Edition 1988), Ed. Janson JC and Ryden L.

[0043] In the present invention, the cation exchange chromatography in the step (b) can be characterized in that it is a binding mode in which the botulinum toxin binds to the resin and then elutes therefrom. The term "binding mode" used interchangeably in the present invention means a separation method in which at least one target molecule (e.g., botulinum toxin) contained in the sample binds to the resin and one or more impurities pass through (i.e., flow through) without binding to the resin.

[0044] In the present invention, the botulinum toxin can bind to the resin of the cation exchange chromatography in the step (b), and thus, the step of eluting the botulinum toxin from the resin may further be included. <​​​​​​​​​​​​​​​​​​​​​In the present invention, a variety of buffers can be used in the cation exchange chromatography of step (b) above. For example, the buffer that can be used in the cation exchange chromatography of step (b) above may include, but is not limited to, one or more selected from the group consisting of citric acid, sodium citrate, sodium acetate, acetic acid, sodium succinate, succinic acid, 2-(N-morpholino)ethanesulfonic acid (MES), potassium hydrogen phthalate, and hydroxyethyl piperazine ethanesulfonic acid (HEPES). In one embodiment of the present invention, a buffer was prepared by mixing trisodium citrate and citric acid, but is not limited to this.

[0047] In the present invention, the buffer for cation exchange chromatography in step (b) above may include an equilibrium buffer and an elution / wash buffer. In the present invention, the buffer for cation exchange chromatography in step (b) above can be selected and used at a suitable concentration depending on the application (equilibrium, elution, or washing, etc.) or the type of buffer.

[0048] In the present invention, the buffer solution may, if necessary, further contain one or more salts (e.g., NaCl), but is not limited thereto.

[0049] In the present invention, when sodium citrate buffer is used, it may be used at a concentration of 1 to 100 mM, preferably 5 to 50 mM, and more preferably 10 to 30 mM, but is not limited thereto.

[0050] In the present invention, the buffer solution may have a pH of 1.0 to 9.0, preferably 2.0 to 7.0, more preferably 3.0 to 6.0, and most preferably 4.0 to 5.0.

[0051] In the present invention, in addition to the chromatography-based purification steps described in (a) and (b) above, the present invention may further include steps that are typically included for the purification of botulinum toxin.

[0052] The present invention may further include the step of culturing a Clostridium botulinum strain. Culturing a Clostridium botulinum strain for the production of botulinum toxin can be done using conventional methods known in the art, and cultivation can be carried out using conventional culture media available for cultivation.

[0053] Non-limiting examples include the culture medium for Clostridium botulinum strains, which may contain casein hydrolysate, yeast extract, glucose, etc. Another example is that the culture medium for Clostridium botulinum strains may be characterized by not containing animal-derived products. In the present invention, the Clostridium botulinum strains may be characterized by being cultured at a temperature of 25 to 40°C for 90 to 200 hours, preferably 100 to 150 hours, but is not limited thereto.

[0054] The present invention may further include a filtration step. The present invention may be characterized in that the filtration step is performed in one or more steps prior to step (a), between steps (a) and (b), or after step (b). The present invention may be characterized in that the filtration step is performed before injecting the chromatographic resin into the packed column.

[0055] The present invention may be characterized by including a filtration step prior to step (a) above.

[0056] In the present invention, the filtration may be, but is not limited to, deep filtration (DF), microfiltration (MF), ultrafiltration (UF), sterile filtration, or a combination thereof. Through the filtration step of the present invention, it is possible to remove, concentrate, and replace buffers impurities contained in the sample, particularly nucleic acid impurities, HCD, cell debris, and endotoxins, etc., but is not limited to these functions.

[0057] In this invention, "depth filtration (DF)" refers to the removal of particles (e.g., impurities) from a solution using a series of filters with decreasing pore size, and the term "depth filter" as used in this invention achieves filtration within the depths of the filter material. The filter consists of any fibrous matrix that forms a complex, undulating labyrinth of fluid channels, and particle separation results from capture by or adsorption to the fibrous matrix. The deep filter medium most frequently used for bioprorosessing cell culture broths and other feedstocks consists of cellulose fibers, filter preparations such as DE, and positively charged resin binders. Unlike absolute filters, deep filter media retain particles through a porous medium, allowing retention of particles larger and smaller than the pore size. Particle retention can include both size exclusion and adsorption through hydrophobic, ionic, and other interactions. Commercially available deep filters include Millistak + Pod depth filter system, XOHC media (Millipore Coporation), Zeta Plus TMExamples include Depth Filter (3M Purification Inc.). In this invention, deep filtration can be performed by arranging and stacking two or more deep filters, in which case commercially available Millistak filters can be used. + Mini DOHC (Millipore Corporation) and XOHC (Millipore Corporation) filters can be used.

[0058] In the present invention, the deep filter generally has a nominal pore size of 0.01 to 20 μm, preferably 0.05 to 8 μm, and most preferably 0.1 to 6 μm. It can also include a porous deep filter medium having multiple graded layers for the removal of aggregated cellular biomass, including aggregated cell lysates and colloidal fine particles or smaller particles having a larger size than this, and for the removal of aggregated cellular biomass. Therefore, when deep filtration is used in the present invention, it is easy to remove impurities (e.g., nucleic acids), cell lysates, endotoxins, etc., contained in a sample containing botulinum toxin.

[0059] In this invention, "microfiltration (MF)" or "ultrafiltration (UF)" refers to a process of fractionating a target solute (e.g., botulinum toxin) from a mixed solution based on its size and structure through the pores of a membrane under constant pressure. For example, a 0.1 μm or 750 kDa molecular weight cutoff (MWCO) separation PS (polysulfone) membrane can be used to purify a solution under conditions of 5-40 psig and 4-60°C.

[0060] Generally, microfiltration is a step that precedes ultrafiltration and is used to separate particles of 0.1 to 10 μm from a solution, which is typically 1 x 10⁻⁶. 5Microfiltration is used to separate polymers having a molecular weight of g / mol or more. Microfiltration is also used to remove sediments, protozoa, big bacteria, etc. In this invention, microfiltration can be easily used to remove polymers and cell disruptors. Generally, the microfiltration process is carried out using a pressure pump or vacuum pump at a speed of 0.1 to 5, preferably 1 to 3 m / s and a pressure of 50 to 600 kPa, preferably 100 to 400 kPa.

[0061] Ultrafiltration (UF) is used to separate particles of 0.01-0.1 μm from a solution, and is typically 1 x 10⁻⁶. 3 ~1x10 5 This corresponds to polymers having a molecular weight of Da. Ultrafiltration is used to remove proteins, endotoxins, viruses, silica, etc., but is not limited to these uses. When using an ultrafiltration separation membrane for MWCO 100~300K in this invention, impurities contained in a sample containing botulinum toxin can be removed, and the botulinum toxin can be concentrated.

[0062] "Sterile filtration" is a process that utilizes microfiltration or membrane filters to safely purify solutions containing target substances (e.g., biological agents, botulinum toxin, etc.) as an alternative to heating, radiation, and chemical treatment. To remove microorganisms that may be present in the solution, a microfilter with pores of 0.1 to 0.3 μm, preferably 0.15 to 0.25 μm, and most preferably 0.2 μm is used. For the removal and inactivation of viruses, a nanofilter with pores of 20 to 50 nm is used. Similarly, in the case of membrane filters, the purification process can be carried out using membrane filters with specific pores and composed of cellulose ester or PES (polyethersulfone) to remove microorganisms, viruses, etc.

[0063] In the present invention, the method for purifying botulinum toxin may further include a deep filtration step.

[0064] In the present invention, the deep filtration step may be characterized by being performed prior to step (a).

[0065] In the present invention, the deep filtration step is characterized by being performed by filtration using a deep filtration filter and a sterile filtration filter.

[0066] The present invention may further include a step of concentrating the filtered sample after the deep filtration step. The present invention may be characterized in that the concentration is carried out by ultrafiltration (UF). The concentration step can be carried out without limitation using a variety of concentration methods or concentration cassettes known in the art. The present invention may be characterized in that the concentration is 2 to 100 times, 5 to 70 times, 10 to 50 times, or 20 to 40 times concentrated from the filtered sample, but is not limited thereto.

[0067] In the present invention, the purification method may further include an ultrafiltration (UF) step.

[0068] In the present invention, the ultrafiltration step may be characterized by being performed prior to step (a).

[0069] In the present invention, the purification method may further include a constant-volume filtration step.

[0070] In the present invention, "Diafiltration (DF)" refers to a technique for removing or acquiring components (e.g., particles) contained in a target substance (solution), characterized by the use of a permeable filter that can separate components according to their molecular weight (molecular size), and is a technique for increasing the purity of a target substance. Diafiltration can be used interchangeably with diafiltration.

[0071] In the present invention, the constant-volume filtration step may be characterized by being performed by ultrafiltration. "Ultra / constant-volume filtration (UF / DF)" means performing constant-volume filtration using the aforementioned ultrafiltration (UF), but is not limited to this.

[0072] In the present invention, the filtration is characterized by being performed using a TFF (Tangential Flow Filtration) method.

[0073] In the present invention, the ultrafiltration step is characterized by being performed using TFF filtration (Tangential Flow Filtration).

[0074] In the present invention, the TFF filtration (Tangential Flow Filtration) is known as "Cross-flow filtration," and refers to a filtration method in which the flow of water and the sample permeating the membrane are configured at a right angle.

[0075] In the present invention, the filtration steps, for example, the deep filtration step and / or the ultrafiltration step, may each be repeated one or more times prior to step (a), but are not limited thereto.

[0076] The present invention may further include a filtration step after steps (a) and (b). Generally, the filtration step after steps (a) and (b) may be characterized by filtering for concentration or dilution to a concentration suitable for storage and use, but is not limited to such uses. For example, after the purification step in steps (a) and (b), the protein concentration may be 0.1 mg / mL to 5 mg / mL, preferably 0.5 mg / mL to 2.5 mg / mL, more preferably 1.0 mg / mL to 1.5 mg / mL, but is not limited thereto.

[0077] The present invention may be characterized by not including an acid precipitation step prior to step (a) above. The "acid precipitation step" means, for example, a step in which an acid such as sulfuric acid or phosphoric acid is added to precipitate the toxin.

[0078] In this invention, the "pH" of a solution is used to measure its acidity or alkalinity in relation to the ionization of a water sample. Water has a neutral pH, i.e., pH 7. Most pH readings are in the range of 0 to 14. Solutions with a higher [H+] concentration than water (pH less than 7) are acidic, and solutions with a lower [H+] concentration than water (pH greater than 7) are basic or alkaline. pH can be measured using a pH meter. The buffer pH can be adjusted using an acid or base such as HCl or NaOH.

[0079] The botulinum toxin purified by the method for purifying botulinum toxin comprising steps (a) and (b) of the present invention means pure botulinum toxin or botulinum toxin complex separated or substantially separated from other proteins and impurities that may be associated with the botulinum toxin when it is obtained from a culture or fermentation process.

[0080] The botulinum toxin purified by the method of the present invention exhibits a purity of 95% or more, preferably 97%, more preferably 98%, and most preferably 99% or more. In particular, in the examples of the present invention, it was confirmed that when the toxin is purified by performing multimode chromatography (flow-though mode) of the AEX+SEC+HIC combination followed by cation exchange chromatography (binding mode), or by performing multimode chromatography of the AEX+HIC combination followed by SEC and CEX sequentially, the toxin can be obtained in high yield while exhibiting an excellent purity of 99% or more.

[0081] In the present invention, a preferred method for purifying botulinum toxin is provided, (i) A step of filtering the sample containing botulinum toxin, (ii) A step of purifying botulinum toxin from the filtered sample using multimodal chromatography including anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC), (iii) The procedure may be characterized by including the step of purifying botulinum toxin from a sample purified by multimode chromatography using cation exchange chromatography.

[0082] In the present invention, a more preferred method for purifying botulinum toxin is provided, (i) A step of deep filtration of the sample containing botulinum toxin, (ii) The step of ultrafiltration the deep-filtered sample, (iii) A step of purifying botulinum toxin from the filtered sample using multimodal chromatography including anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC), (iv) The procedure may be characterized by including a step of purifying botulinum toxin from a sample purified by multimode chromatography using cation exchange chromatography.

[0083] In the present invention, another preferred method for purifying botulinum toxin is provided, (i) A step of filtering the sample containing botulinum toxin, (ii) A step of purifying botulinum toxin from the filtered sample using multimodal chromatography, including anion exchange chromatography (AEX) and hydrophobic interaction chromatography (HIC), (iii) A step of purifying botulinum toxin from a sample purified by multimode chromatography using size-exclusion chromatography (SEC), (iv) The procedure may be characterized by including a step of purifying botulinum toxin from a sample purified by size exclusion chromatography using cation exchange chromatography.

[0084] In the present invention, a more preferred method for purifying botulinum toxin is provided, (i) A step of deep filtration of the sample containing botulinum toxin, (ii) The step of ultrafiltration the deep-filtered sample, (iii) A step of purifying botulinum toxin from the filtered sample using multimodal chromatography including anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC), (iv) The method may be characterized by including a step of purifying botulinum toxin from a sample purified by multimode chromatography using cation exchange chromatography.

[0085] In another aspect, the present invention relates to a composition comprising botulinum toxin purified by the purification method of the present invention.

[0086] In the present invention, the composition containing the botulinum toxin may be characterized by having a purity of about 95% or more, preferably about 97%, more preferably about 98%, and most preferably about 99% or more.

[0087] In the present invention, the composition containing the botulinum toxin may be characterized by having about 2% or less, preferably about 1.5% or less, more preferably about 1% or less, and even more preferably about 0.5% or less or about 0.4% or less of toxin-related impurities.

[0088] In the present invention, the composition containing the botulinum toxin may be characterized by exhibiting an agglutination peak at a concentration of about 2% or less, preferably about 1.5% or less, more preferably about 1% or less, and even more preferably about 0.5% or less. [Examples]

[0089] The present invention will be described in more detail below through examples. These examples are merely illustrative and it will be obvious to those ordinary in the art that the scope of the present invention is not limited by these examples.

[0090] Example 1: Main culture of Clostridium botulinum strain Clostridium botulinum strains for botulinum toxin production were prepared by inoculating Clostridium botulinum type A strains into 10 L of a medium containing 2% casein hydrolysate, 1% yeast extract, and 1% glucose, and culturing at 35°C for 120 hours.

[0091] Example 2: Filtration (deep filtration and ultra / constant volume (UF / DF) filtration) The culture medium was filtered by connecting a deep filtration filter and a sterile filtration filter in series, maintaining a flow rate of 300 mL / min and a pressure of 0.35 bar or less, to remove insoluble impurities and sterilize the solution. The filters used are as follows: 3M Deep Filtration Filter TM Zeta Plus TM Encapsulated System Filter Capsule E1020FSA Sterile filtration filters: Sartopore 2MidiCaps

[0092] The culture medium, obtained by deep-filtration and sterile filtration, was concentrated 30-fold in a 100 kDa MWCO cassette. The concentrated filtrate was then diluted 2-fold with 50 mM sodium phosphate pH 6.5, and then diluted again 2-fold in a 100 kDa MWCO cassette. This ultrafiltration process was repeated seven times, with buffer replacement (constant volume filtration). Ultrafiltration was performed using the Tangential Flow Filtration (TFF) method.

[0093] Example 3: Primary Purification To develop a superior purification process, primary purification steps were performed using each of the following chromatography methods. Before primary purification, it was confirmed that the UV278nm and conductivity baselines of the equipment (FPLC) were stabilized.

[0094] Example 3-1: Multimodal resin (AEX+HIC+SEC, Capto Core400) - FT mode A column packed with Capto Core 400 resin was equilibrated with equilibrium buffer. After buffer replacement was completed via ultrafiltration, the sample was filtered through a 0.2 μm filter and injected at a linear velocity of 60–150 cm / h. Subsequently, equilibrium buffer was injected at a rate of 5 column volumes (CV) or more, and the portion that did not bind to the resin and eluted via flow-through was collected. Wash buffer was then flushed at a rate of 2 CV or more to remove (elute) impurities bound to the column. The buffers used are as follows: -Equilibration / elution buffer: 50mM sodium phosphate pH6.5 -Washing buffer: 50mM sodium phosphate pH6.5, 1M NaCl

[0095] Example 3-2: AEX resin (TOYOPEARL Super Q 650M) - Binding Mode A column packed with TOYOPEARL Q resin was equilibrated with equilibrium buffer. After completing buffer replacement via ultrafiltration, the sample was filtered through a 0.2 μm filter and injected at 60–150 cm / h, injecting at least 5 column volumes (CV) of equilibrium buffer. Wash buffer was passed through at least 20 CVs with a 0–100% gradient to sequentially elute the toxins bound to the column. The section in which only toxins were eluted and no impurities were identified and collected. The buffers used are as follows: -Equilibration / wash buffer: 50mM sodium phosphate, pH6.5 -Elution buffer: 50mM sodium phosphate pH6.5, 1M NaCl

[0096] Example 3-3: Multimodal resin (CEX+HIC, Capto MMC) - FT mode A column packed with Capto MMC resin was equilibrated with equilibrium buffer. After buffer replacement was completed by ultrafiltration, the sample was diluted with 50 mM sodium citrate pH 4.0 to pH 4.5. The process sample was injected at 60-150 cm / h, followed by injection of equilibrium buffer at least 5 column volumes (CV). The portion that did not bind to the resin and eluted via flow-through was collected. Impurities bound to the column were removed (eluted) by running wash buffer at a 0-100% gradient at least 10 CV. The buffers used are as follows: -Equilibration / elution buffer: 25mM sodium acetate pH4.5 -Washing buffer: 25mM sodium phosphate pH6.5, 1M NaCl

[0097] Examples 3-4: Multimodal Resin (AEX+HIC, Capto Adhere) - FT Mode A column packed with CaptoAdhere resin was equilibrated with equilibrium buffer. After completing buffer replacement via ultrafiltration, the sample was filtered through a 0.2 μm filter and injected at 60–150 cm / h. Equilibrium buffer was injected in volumes of 5 or more columns (CV). The portion that did not bind to the resin and eluted via flow-through was collected. Impurities bound to the column were removed (eluted) by running wash buffer through the column in a 0–100% gradient for volumes of 10 or more. The buffers used are as follows: -Equilibration / elution buffer: 50mM sodium phosphate pH6.5 -Washing buffer: 50mM sodium phosphate pH6.5, 1M NaCl

[0098] Example 3-5: AEX resin (TOYOPEARL Super Q 650M) - FT mode A column packed with TOYOPEARL Q resin was equilibrated with equilibrium buffer. After completing buffer replacement via ultrafiltration, the sample was filtered through a 0.2 μm filter and injected at 60–150 cm / h. At least 5 column volumes (CV) of equilibrium buffer were injected. The portion that did not bind to the resin and eluted via flow-through was collected. Impurities bound to the column were removed (eluted) by running wash buffer through the column at a 0–100% gradient for at least 10 CV. The buffers used are as follows: -Equilibration / elution buffer: 50mMsodium phosphate, 10mS / cm pH6.5 -Washing buffer: 50mM sodium phosphate pH6.5, 1M NaCl

[0099] Example 3-6: HIC resin (Butyl SEPHAROSE HP) - Binding Mode A column packed with Butyl SEPHAROSE HP resin was equilibrated with equilibrium buffer. After buffer replacement was completed by ultrafiltration, NaCl was added to the sample to make 3M NaCl, which was then diluted 5-fold with equilibrium buffer and filtered through a 0.2 μm filter. The sample was injected into the column at 60-150 cm / h, and then equilibrium buffer was injected at least 5 column volumes (CV). Wash buffer was flowed at a 0-100% gradient at least 10 CV to sequentially elute the toxins bound to the column. The section containing the fewest impurities during the toxin elution was identified and collected. The buffers used are as follows: -Equilibration / washing buffer: 50mM sodium phosphate pH6.5, 3M NaCl -Elution buffer: 50mM sodium phosphate pH6.5

[0100] Examples 3-7: Sequential execution of Multimodal resin (AEX+HIC, Capto Adhere)-FT mode+SEC resin (Superdex 75pg) 3-7-1: Multimodal resin (AEX+HIC, Capto Adhere) A column packed with Capto Adhere resin was equilibrated with equilibrium buffer. After completing buffer replacement via ultrafiltration, the sample was filtered through a 0.2 μm filter and injected at 60–150 cm / h. Equilibrium buffer was injected at a rate of 5 column volumes (CV) or more. The portion that did not bind to the resin and eluted via flow-through was collected. Wash buffer was flushed at a rate of 0–100% gradient for 10 CV or more to remove (elute) impurities bound to the column. The buffers used are as follows: -Equilibration / elution buffer: 50mM sodium phosphate pH6.5 -Washing buffer: 50mM sodium phosphate pH6.5, 1M NaCl

[0101] 3-7-2:SEC resin (Superdex 75pg) The eluate recovered using multimodal resin (AEX+HIC, Capto Adhere) was filtered through a 0.2 μm filter. A column packed with Superdex 75 pg resin was then equilibrated with 50 mM sodium phosphate pH 6.5 and injected at a flow rate of 30 cm / h. After injection was complete, equilibrium / elution buffer was injected, and injection continued in 5 mL fractions until the UV value decreased to baseline. Subsequently, fractions containing toxins were collected. The buffers used are as follows: - Equilibration / elution buffer: 50mM sodium phosphate pH6.5

[0102] Example 3-8: SEC resin (Superdex 200pg) After completing buffer replacement via ultrafiltration, the sample was filtered through a 0.2 μm filter. A column packed with Superdex 200 pg resin was then equilibrated with 50 mM sodium phosphate pH 6.5 and injected at a flow rate of 30 cm / h. After injection, equilibrium / elution buffer was injected into 5 mL fractions until the UV value decreased to baseline. The fractions containing toxins were then collected. The buffers used are as follows: -Equilibration / elution buffer: 50mM sodium phosphate pH6.5

[0103] Example 4-1: Secondary purification (CEX, TOYOPEARL SP-650S) - Binding mode The samples purified under the conditions of Examples 3-1 to 3-8 were diluted with 50 mM sodium citrate at pH 4.0 to a pH of 4.5. Equilibration was performed by flowing equilibrium buffer through a column packed with TOYOPEARL SP-650S resin. After injecting the diluted solution at 60-150 cm / h, equilibrium buffer was injected at least 5 column volumes (CV). Wash buffer was flowed at a 0-100% gradient at least 10 CV to sequentially elute and recover the toxins bound to the column. The buffers used are as follows: -Equilibration / wash buffer: 20 mM sodium citrate pH4.5 -Elution buffer: 20 mM sodium citrate pH4.5, 1M NaCl

[0104] Example 5: Comparison with the latest purification process To compare its effectiveness with the latest botulinum toxin purification process, the purification process described in Korean Patent No. 10-2512757, registered in 2023, was used as a comparison group.

[0105] Example 5-1: Primary purification (AEX, TOYOPEARL NH2-750F) - Binding mode After completing buffer replacement via ultrafiltration, the sample was filtered through a 0.2 μm filter and injected at 46 cm / h into a column packed with TOYOPEARL NH2-750F resin. Following injection, the UV value and conductivity value were equilibrated by washing with 50 mM sodium phosphate buffer pH 6.5 for 5 CV. After washing, fractions were collected in 2 mL increments while eluting at 60% using 50 mM sodium phosphate buffer pH 6.5 and 1 M sodium chloride buffer, and pooling was performed based on the SDS-PAGE results. The buffers used are as follows: -Equilibration / washing buffer: 50mM sodium phosphate pH6.5 -Elution buffer: 50mM sodium phosphate, 1M NaCl pH6.5

[0106] Example 5-2: CEX (TOYOPEARL Sulfate-650F) - Binding Mode The eluate recovered in Example 5-1 was adjusted to pH 5.5 ± 0.1 and conductivity 7.0 ± 0.5 mS / cm by adding 25 mM citrate buffer pH 5.5. The solution was filtered through a 0.22 μm bottle-top filter to remove impurities. The filtered toxin solution was injected at a linear velocity of 46 cm / hr into a column packed with Sulfate-650F, a cation exchange resin. After injection, the column was washed to 4.5 CV with 25 mM citrate buffer pH 5.5 to equilibrate UV and conductivity. After washing, fractions were collected in 2 mL increments while eluting with 25 mM citrate buffer pH 5.5 and 14% 1 M sodium chloride buffer, and pooled based on SDS-PAGE results. The buffers used are as follows: -Equilibration / wash buffer: 25mM sodium citrate pH5.5 -Elution buffer: 25mM sodium citrate, 1M NaCl pH5.5

[0107] Example 6: Filtration and Storage The concentration of the purified botulinum toxin protein was measured, and if it was 1.5 mg / mL or higher, it was diluted with 20 mM sodium citrate pH 4.5 buffer to a concentration of 1.0 mg / mL to 3.0 mg / mL, and then filtered through a 0.2 μm filter. The purified toxin was aliquoted and stored in a -70°C freezer.

[0108] Example 7: Results Example 7-1: Amount of toxin obtained The yield of botulinum toxin obtained through various primary purification steps and secondary purification steps using CEX was confirmed. As shown in Figures 2 and 3, when secondary purification (CEX) was performed after primary purification (Multimodal, AEX+HIC+SEC) as in Example 3-1, a high yield of 29.25% was observed. Furthermore, it was confirmed that a high yield of 22.47% was also observed when multimode chromatography (AEX+HIC) and SEC were performed sequentially in the primary purification.

[0109] The commonly known industry method of performing CEX after AEX (binding mode) showed a low yield of 12.47%, while other primary purification chromatography methods yielded different results: multimode chromatography (CEX+HIC: 27.29% and AEX+HIC: 27.74%), AEX-FT (32.74%), SEC alone (17.77%), or HIC (19.06%).

[0110] The latest purification process, Korean Patent No. 10-2512757, showed a slightly higher yield, but as described later, it was observed to exhibit a very low purity value below the standard, and was confirmed to be an unsuitable process regardless of the yield. Therefore, it was confirmed that the yield of botulinum toxin in the purification process of the present invention is similar to or higher than that of various recently developed purification processes.

[0111] Example 7-2: Purity confirmation of botulinum toxin (SDS-PAGE) SDS-PAGE was performed on each sample to confirm the absence of impurities in the purified botulinum toxin samples. As shown in Figures 4 and 5, impurity bands were visually confirmed in the remaining primary purification steps, excluding the primary purification steps of Example 3-1 (multimode, AEX+HIC+SEC), Example 3-6 (HIC-Butyl), and Example 3-7 (multimode (AEX+HIC) followed by SEC). In relation to the latest purification technology, the toxin band itself was not detected in the peaks described in prior literature, while impurity bands were visually confirmed in the peaks containing the toxin.

[0112] Example 7-3: Confirmation of botulinum toxin purity (SEC-HPLC) and degree of aggregation. For more accurate purity verification, SEC-HPLC was performed using a TSK G-4000 to confirm the toxin purity of the final purified sample. As a result, as shown in Figures 6 to 9, when performing primary purification (multimode, AEX+HIC+SEC) and secondary purification of CEX in Example 3-1, a remarkably high purity of 99.72% was observed, and when confirming primary purification (multimode (AEX+HIC) followed by SEC) and secondary purification of CEX in Example 3-7, a remarkably high purity of 99.19% was observed. In particular, the purification techniques including the primary steps in Examples 3-1 and 3-7 showed very low impurity content and aggregation degree compared to other steps, confirming that they are botulinum toxin purification techniques with remarkably high purity and stability compared to conventional techniques.

[0113] In the other examples 3-2 to 3-6, the purity was approximately 94-96%, which is lower than in Example 3-1, and the impurity peaks were 1-3%. The degree of aggregation was also at least 8 times higher, confirming that there were stability problems. On the other hand, when using the purification process from prior literature (Example 5), it was confirmed that the purity was very low, at approximately 69%.

[0114] As mentioned earlier, given the potent toxicity of botulinum toxin, the purity and stability of the purified toxin are crucial factors. However, raising the purity to nearly 99% inevitably leads to a sharp decline in yield. Therefore, most research has focused on increasing yield at a purity of 95%.

[0115] The results of the present invention mean that, exceeding the limitations of the prior art, when using a purification technique that includes multimode chromatography including SEC or a primary purification step of sequentially performing multimode chromatography and SEC, and a secondary purification step of CEX, botulinum toxin can be obtained with a remarkably high purity of 99% or more, excellent stability, and a yield higher than that of the prior art. [Industrial applicability]

[0116] The present invention provides a method for purifying botulinum toxin that yields higher yields than commonly used purification methods in the art, while achieving ultra-high purity of approximately 99% or more. Therefore, it can be usefully utilized in the production and manufacture of botulinum toxin.

[0117] Having described in detail certain aspects of the present invention, it will be clear to anyone with ordinary skill in the art that such specific descriptions are merely preferred modes of implementation and do not limit the scope of the invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The following steps are included in the purification method for botulinum toxin: (a) A step of purifying botulinum toxin from a sample containing botulinum toxin using anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC), and (b) A step of purifying botulinum toxin using cation exchange chromatography.

2. The method for purifying botulinum toxin according to claim 1, characterized in that step (a) is performed using a multimodal resin having anion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography functions.

3. The method for purifying botulinum toxin according to claim 1, characterized in that step (a) is performed by purifying a fraction containing botulinum toxin purified using a multimodal resin having anion exchange chromatography and hydrophobic interaction chromatography functions, using size exclusion chromatography.

4. The method for purifying botulinum toxin according to claim 2 or 3, characterized in that in the multimode chromatography of step (a) above, the botulinum toxin passes through without binding to the multimode chromatography resin.

5. The method for purifying botulinum toxin according to claim 1, characterized in that in the cation exchange chromatography of step (b) above, the botulinum toxin is eluted after binding to the cation exchange resin.

6. The method for purifying botulinum toxin according to claim 1, characterized in that the sample containing botulinum toxin in step (a) is a culture medium, lysate, or precipitate of a Clostridium botulinum strain.

7. The method for purifying botulinum toxin according to claim 1, further comprising the filtration step.

8. The method for purifying botulinum toxin according to claim 1, further comprising the following steps before step (a): (i) The step of deep filtration of the sample containing botulinum toxin and (ii) The step of ultrafiltration the deep-filtered sample.

9. The method for purifying botulinum toxin according to claim 8, characterized in that step (ii) is performed by a TFF filtration method.

10. The method for purifying botulinum toxin according to claim 1, characterized in that the purity of the final purified botulinum toxin is 99% or higher.

11. A composition comprising botulinum toxin purified by the purification method described in claim 1.

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