Method for purifying botulinum toxin

Abstract

The present invention relates to a method for purifying Botulinum Toxin (BTX), and more particularly, to a method for purifying Botulinum Toxin (BTX), which comprises the steps of: purifying Botulinum Toxin (BTX) by means of a column chromatography comprising the steps of: sequentially using cation exchange chromatography (Cation Chromatography, Cation Chromatography, Cation Chromatography, Cation Chromatography, Cation Chromatography); the method comprises the following steps: (1) carrying out CEX (Hydrophobic Interaction Chromatography), (2), (3), (4), (5), (6), (7), (8) and (8); according to the present invention, the botulinum toxin having excellent purity and activity is purified by a process comprising a step of purifying a botulinum toxin by using trypsin (Trypsin), a step of treating the botulinum toxin with trypsin (Trypsin) to activate the botulinum toxin, and a step of purifying the botulinum toxin by using trypsin (Trypsin), such that the botulinum toxin can be usefully used for producing the botulinum toxin.

Description

Technical Field

[0001] The present invention relates to a method for purifying botulinum toxin (BTX), and more particularly, to a method for purifying botulinum toxin type E, the method comprising the steps of sequentially purifying using cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), and mixed mode chromatography (cation exchange (phosphate) and affinity (calcium)), and further comprising the step of treating with trypsin to activate the botulinum toxin. Background Art

[0002] Botulinum toxin is a neurotoxin protein produced by bacteria such as Clostridium butyricum, Clostridium baratii, and Clostridium botulinum. Botulinum toxin blocks neuromuscular transmission and causes paralytic diseases in humans and animals. Seven different types of botulinum toxins, types A, B, C1, D, E, F, G, and H, have been identified, each type can be distinguished by type-specific antibodies, and the severity of paralysis caused by them and the animal species they affect are different from each other.

[0003] The molecular weight of the botulinum toxin protein molecule is about 150 kDa and is composed of a light chain of about 50 kDa conjugated with a heavy chain of about 100 kDa. However, the botulinum toxin released by Clostridium bacteria is released as a complex of a 150 kDa toxin and one or more non-toxin proteins. For example, the botulinum toxin is released in the form of complexes of 900 kDa, 500 kDa, and 300 kDa.

[0004] Although botulinum toxin can be very lethal to humans, in recent years, botulinum toxin has been developed for treating various symptoms including neuromuscular diseases characterized by excessive skeletal muscle activity. For example, Botox is a trademark for botulinum toxin A commercially developed by Allergan, Inc., which is used for the treatment of blepharospasm, strabismus, cervical dystonia, and improvement of glabellar (facial) wrinkles, and continuous research and development is being carried out on the appropriate uses of other serotypes for clinical applications.

[0005] Botulinum toxin for clinical use is usually isolated from cell cultures, in which case various purification methods are used.

[0006] Botulinum toxin for clinical use is purified in complexed form by a series of precipitation and tangential flow filtration steps [Schantz E J, et al, Properties and use of botulinum toxin and other microbial neurotoxins in medicine, Microbiol Rev 1992 March 56(1):80 - 99]. However, this method usually provides a relatively low yield of less than about 10%. Other methods use size exclusion, ion exchange, and / or affinity chromatography [Schmidt J J, et al, Anal Biochem 1986 July;156(1):213 - 219; Kannan K, et al, Mov Disord 2000;15(Suppl 2):20(2000); Wang YC, Dermatol Las FaciCosm Surg 2002;58(2002); and U.S. Patent No. 2003 / 0008367].

[0007] Another method is a method of recombinantly synthesizing either the heavy chain or the light chain of botulinum toxin alone, rather than the complete and biologically active botulinum toxin protein [Zhou L, et al, Biochemistry 1995;34(46):15175 - 81(1995); and Johnson S K, et al, Protein Expr and Purif 2003;32:1 - 9(2003)].

[0008] Newer methods include using hydrophobic interaction chromatography, mixed mode, and / or ion exchange chromatography to purify botulinum toxin as a complex (U.S. Patent Nos. 7452697 and 7354740).

[0009] However, there is still a need in the art for an improved purification method for isolating stable and biologically active intact botulinum toxin.

[0010] Accordingly, as a result of the inventors' dedicated efforts to develop a purification method for obtaining type E botulinum toxin with high purity and excellent activity, it was confirmed that type E botulinum toxin with very high purity and activity can be purified using a process of cation exchange chromatography, hydrophobic interaction chromatography, and mixed mode chromatography. In particular, it was confirmed that when an SP column is used as the cation exchange resin, a butyl column is used as the hydrophobic resin, and a CHT column is used as the mixed mode resin, type E botulinum toxin with a purity of 99% or more can be purified, thus completing the present invention. Summary of the Invention

[0011] Technical Problem

[0012] An object of the present invention is to provide a method for purifying botulinum toxin (BTX).

[0013] Technical Solution

[0014] To achieve the above object, the present invention provides a method for purifying type E botulinum toxin, the method comprising:

[0015] Step (a), pretreating a culture solution of a type E botulinum toxin-producing strain;

[0016] Step (b), purifying the culture solution pretreated in step (a) using cation exchange chromatography (CEX);

[0017] Step (c), purifying the eluate purified in step (b) using hydrophobic interaction chromatography (HIC);

[0018] Step (d), activating the type E botulinum toxin by mixing the eluate purified in step (c) with trypsin; and

[0019] Step (e), purifying the trypsin reaction solution using mixed mode chromatography.

[0020] In the present invention, in step (a), the pretreatment may be ultrafiltration.

[0021] In the present invention, the ultrafiltration may be performed using a filtration membrane with a size of 10 kDa to 300 kDa.

[0022] In the present invention, the filter membrane may be a cassette type or a hollow fiber type filter membrane.

[0023] In the present invention, in step (b), the cation exchange chromatography column may be a column filled with a resin containing one or more functional groups selected from the group consisting of carboxymethyl (CM), sulfonylethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S).

[0024] In the present invention, the sulfopropyl (SP) column may be one or more selected from the group consisting of SP sepharose HP column, SP sepharose FF column, and Capto S.

[0025] In the present invention, in step (b), the culture medium pretreated in step (a) may be diluted with purified water or a buffer solution and then injected into the cation exchange chromatography column.

[0026] In the present invention, the buffer solution may be a sodium citrate buffer solution.

[0027] In the present invention, a fraction containing type E botulinum toxin can be obtained by injecting a sodium citrate buffer solution containing sodium chloride as an elution buffer into the column.

[0028] In the present invention, the elution buffer solution may be a 10 - 50 mM sodium citrate buffer solution with a pH of 4.0 - 6.0 and containing 0.5 - 1.5 M sodium chloride.

[0029] In the present invention, in step (c), the hydrophobic interaction chromatography column may be a column filled with a resin containing one or more functional groups selected from the group consisting of ether, isopropyl, butyl, octyl, and phenyl.

[0030] In the present invention, the butyl column may be one or more selected from the group consisting of Butylsepharose HP column, Capto butyl column, Capto phenyl column, and Phenyl HP column.

[0031] In the present invention, in step (c), the eluate purified in step (b) may be diluted with a buffer solution having a pH of 4.0 - 6.0 and then injected into the hydrophobic interaction chromatography column.

[0032] In the present invention, the buffer solution may be a 25 - 75 mM sodium phosphate and / or sodium citrate buffer solution with a pH of 4.0 - 6.0, and 3.5 - 4.5 M sodium chloride is added.

[0033] In the present invention, a fraction containing type E botulinum toxin can be obtained by injecting a 25 - 75 mM sodium phosphate buffer solution and / or sodium citrate buffer solution with a pH of 4.0 - 6.0 into the column.

[0034] In the present invention, after the step (c), a step (c') of pre - treating the eluate purified in the step (c) may further be included.

[0035] In the present invention, in the step (c'), the pre - treatment may be diafiltration and / or ultrafiltration.

[0036] In the present invention, the diafiltration and / or ultrafiltration may be carried out using a filtration membrane with a size of 10 kDa to 300 kDa.

[0037] In the present invention, the filtration membrane may be a cassette - type or hollow - fiber type filtration membrane.

[0038] In the present invention, the diafiltration may be carried out with a 25 - 75 mM sodium phosphate buffer solution with a pH of 4.0 - 6.0.

[0039] In the present invention, in the reaction in the step (d), the type E botulinum toxin in the eluate can be activated by reacting the eluate with trypsin at a temperature of 33 - 36 °C for 0.5 - 2 hours.

[0040] In the present invention, in the step (e), the mixed - mode chromatography column may be a column filled with a resin containing the functional group of calcium phosphate compound Ca 10 (PO4)6(OH)2 (ceramic hydroxyapatite (CHT)).

[0041] In the present invention, the CHT column may be type I CHT and / or CHT - XT.

[0042] In the present invention, in the step (e), the trypsin reaction solution of the step (d) can be diluted with purified water and then injected into the mixed - mode chromatography column.

[0043] In the present invention, a fraction containing type E botulinum toxin can be obtained by injecting a 5 - 50 mM sodium phosphate buffer solution with a pH of 4.0 - 6.0 and 1.5 - 3.0 M sodium chloride added into the column.

[0044] The present invention also provides a type E botulinum toxin prepared by the above method.

[0045] Effects of the Invention

[0046] According to the present invention, a type E botulinum toxin with excellent purity and activity can be purified, and thus it can be usefully used for the production of type E botulinum toxin.

[0047] Moreover, the type E botulinum toxin has the advantages that it takes effect on the injection day and lasts for up to 1 month, so it can be used for the preparation of biopharmaceuticals, tissue sutures, facial fixing lifting threads, lipolysis injection agents, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Results of analyzing botulinum toxin in the eluate after cation exchange chromatography by FPLC and SDS-PAGE, showing an embodiment of the present invention.

[0049] Figure 2 Results of analyzing botulinum toxin in the eluate after hydrophobic interaction chromatography by FPLC and SDS-PAGE, showing an embodiment of the present invention.

[0050] Figure 3 Results of analyzing the step of purification using trypsin by SDS-PAGE, showing an embodiment of the present invention.

[0051] Figure 4 Results of analyzing botulinum toxin in the eluate after mixed-mode chromatography by FPLC and SDS-PAGE, showing an embodiment of the present invention.

[0052] Figure 5 Results of analyzing the purity of botulinum toxin purified according to the present invention by SE-HPLC.

[0053] Figure 6 Results of analyzing samples of each step of purifying botulinum toxin according to the present invention by SDS-PAGE.

[0054] Figure 7 Results of analyzing the purity of botulinum toxin purified according to the present invention by RP-HPLC.

[0055] Figure 8 Results of confirming the HPLC purity of botulinum toxin purified under optimized conditions.

[0056] Figure 9 SDS-PAGE comparison results showing the botulinum toxin cleavage ability according to the source species of trypsin. Detailed implementation mode

[0057] Hereinafter, the present invention will be described in detail.

[0058] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs. Generally, the nomenclature used in this specification is well-known and commonly used in this technical field.

[0059] In the present invention, the term "botulinum toxin" refers to the neurotoxin produced by Clostridium botulinum, as well as botulinum toxin (or its light chain or heavy chain) prepared by recombination of non-Clostridium species. As used in this specification, botulinum toxin also includes botulinum toxin complexes (e.g., complexes of 300, 600, and 900 kDa) and pure botulinum toxin (e.g., about 150 kDa). The "pure botulinum toxin" is defined as botulinum toxin separated or substantially separated from other proteins including the proteins forming the botulinum toxin complex. The purity of the pure botulinum toxin can be 95% or more, preferably 99% or more.

[0060] In the present invention, the botulinum toxin-producing strain can be Clostridium botulinum or its variant, most preferably Clostridium botulinum type E, but not limited thereto. It will be apparent to those of ordinary skill in the art that any strain capable of producing botulinum toxin can be used.

[0061] Therefore, in one aspect, the present invention relates to a method for purifying botulinum toxin type E, the method comprising:

[0062] Step (a), pretreating the culture solution of the botulinum toxin type E-producing strain;

[0063] Step (b), purifying the culture solution pretreated in the step (a) by cation exchange chromatography (CEX);

[0064] Step (c), purifying the eluate purified in the step (b) by hydrophobic interaction chromatography (HIC);

[0065] Step (d), activating the botulinum toxin type E by mixing the eluate purified in the step (c) with trypsin; and

[0066] Step (e): Purify the trypsin reaction solution by Mixed Mode Chromatography.

[0067] In the present invention, in the step (a), the pretreatment may be ultrafiltration, but is not limited thereto.

[0068] In the present invention, the ultrafiltration may be carried out using a filtration membrane with a size of 10 kDa to 300 kDa, preferably 50 kDa to 100 kDa, but is not limited thereto.

[0069] In the present invention, the filtration membrane may be a Cassette type or Hallowfiber filtration membrane, but is not limited thereto.

[0070] In the present invention, in the step (b), the cation exchange chromatography column may be a column filled with a resin containing one or more functional groups selected from the group consisting of carboxymethyl (CM), sulfonylethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S), but is not limited thereto.

[0071] In particular, the sulfopropyl (SP) column can effectively remove impurities and maintain the high activity of botulinum toxin in the cation exchange chromatography. As the sulfopropyl (SP) column, an SP sepharose HP column, an SP sepharose FF column, Capto S, etc. can be used. Preferably, an SP sepharose HP column can be used, but is not limited thereto.

[0072] In an embodiment of the present invention, the SP sepharose HP column may be a column filled with a resin containing a sulfopropyl functional group, having a spherical shape, a 6% cross-linked agarose matrix form, a DBC of 55 mg / mL based on Ribonuclease A, and a particle size of 34 μm, but is not limited thereto.

[0073] In the present invention, in the step (b), when purifying using other types of chromatography (e.g., anion exchange chromatography, hydrophobic interaction chromatography, or mixed mode chromatography) and their columns other than cation exchange chromatography, the purification yield and purity of type E botulinum toxin may be lower than when purifying using the cation exchange chromatography and its column (e.g., sulfopropyl (SP) column) of the present invention.

[0074] In the present invention, in step (b), the culture solution pretreated in step (a) can be diluted with purified water or a buffer solution and then injected into a cation exchange chromatography column, but is not limited thereto.

[0075] In the present invention, the E-type botulinum toxin can be bound to the cation exchange chromatography column by titrating the concentration and pH of the buffer solution, but is not limited thereto.

[0076] In the present invention, the buffer solution can be a sodium citrate buffer solution, but is not limited thereto.

[0077] In the present invention, a fraction containing the E-type botulinum toxin can be obtained by injecting a sodium citrate buffer solution containing sodium chloride as an elution buffer into the column, but is not limited thereto.

[0078] In the present invention, the elution buffer solution can be a 10-50 mM sodium citrate buffer solution with a pH of 4.0-6.0 and containing 0.5-1.5 M sodium chloride, but is not limited thereto.

[0079] In step (b) of the present invention, impurities can be removed and the E-type botulinum toxin in a complex form can be captured.

[0080] In the present invention, in step (c), the hydrophobic interaction chromatography column can be a column filled with a resin containing one or more functional groups selected from the group consisting of ether, isopropyl, butyl, octyl, and phenyl, but is not limited thereto.

[0081] In particular, a butyl column can effectively separate proteins in a non-complexed form. The butyl column can use a butyl sepharose HP column, a Capto butyl column, a Capto phenyl column, a phenyl HP column, etc. Preferably, a butyl sepharose HP column can be used, but is not limited thereto.

[0082] In an embodiment of the present invention, the Butylsepharose HP column may be a column filled with resin, and the resin has a 6% highly cross-linked agarose matrix, exhibiting a degree of substitution of 50 μmol of butyl groups / mL, and a bead size of 34 μm, but is not limited thereto.

[0083] In the present invention, in step (c), when purifying using other types of chromatography (such as cation exchange chromatography, anion exchange chromatography, or mixed mode chromatography) and their columns other than hydrophobic interaction chromatography, the purification yield and purity of type E botulinum toxin may be lower than those using the hydrophobic interaction chromatography and its column (such as a butyl column) of the present invention.

[0084] In the present invention, in step (c), the eluate purified in step (b) can be diluted with a buffer solution having a pH of 4.0 to 6.0 and then injected into a hydrophobic interaction chromatography column, but is not limited thereto.

[0085] In the present invention, the buffer solution may be a 25 - 75 mM sodium phosphate and / or sodium citrate buffer solution with a pH of 4.0 to 6.0, and 3.5 - 4.5 M sodium chloride is added, but is not limited thereto.

[0086] In the present invention, the binding of type E botulinum toxin to the hydrophobic interaction chromatography column can be achieved by titrating the concentration and pH of the buffer solution, but is not limited thereto.

[0087] In the present invention, a fraction containing type E botulinum toxin can be obtained by injecting a 25 - 75 mM sodium phosphate and / or sodium citrate buffer solution with a pH of 4.0 to 6.0 into the column, but is not limited thereto.

[0088] In step (c) of the present invention, impurities (75 kDa) can be further removed, but is not limited thereto.

[0089] In the present invention, after step (c), step (c') may further be included to pre-treat the eluate purified in step (c), but is not limited thereto.

[0090] In the present invention, in step (c'), the pre-treatment may be diafiltration and / or ultrafiltration, but is not limited thereto.

[0091] In the present invention, the filtration membrane may be a cassette type or a hollow fiber type filtration membrane, but is not limited thereto.

[0092] In the present invention, the diafiltration and / or ultrafiltration may be carried out using a filtration membrane with a size of 10 kDa to 300 kDa, preferably 30 kDa to 50 kDa, but is not limited thereto.

[0093] In the present invention, the diafiltration can be carried out with a 25 - 75 mM sodium phosphate buffer at a pH of 4.0 - 6.0, but is not limited thereto.

[0094] In step (d) of the present invention, when treating with trypsin at a temperature of 33 - 36 °C for 0.5 - 2 hours, the peptide bond (covalent bond) between the heavy chain (HC, 100 kDa) and the light chain (LC, 50 kDa) of the 150 kDa botulinum toxin type E protein is broken, thereby activating the botulinum toxin.

[0095] That is, in the present invention, in the reaction in step (d), the botulinum toxin type E in the eluate can be activated by reacting the eluate with trypsin at a temperature of 33 - 36 °C for 0.5 - 2 hours, but is not limited thereto.

[0096] In the present invention, the trypsin may be porcine-derived trypsin, but is not limited thereto.

[0097] In the present invention, in step (e), the mixed-mode chromatography column may be a column filled with a resin containing the functional group of calcium phosphate compound Ca 10 (PO4)6(OH)2, that is, ceramic hydroxyapatite (CHT), but is not limited thereto.

[0098] The CHT column can effectively remove other impurities and improve the purity of botulinum toxin with a molecular weight of up to ~300 kDa. The CHT column can use CHT type I, CHT-XT, etc. Preferably, the CHT-XT column can be used, but is not limited thereto.

[0099] In the present invention, in step (e), when purifying using other types of chromatography (such as cation exchange chromatography, anion exchange chromatography, or hydrophobic interaction chromatography) and their columns other than mixed-mode chromatography, the purification yield and purity of botulinum toxin type E may be lower than those of the mixed-mode chromatography and its column (such as the CHT column) of the present invention.

[0100] In the present invention, in the step (e), the trypsin reaction solution of step (d) can be diluted with purified water and then injected into a mixed-mode chromatography column, but it is not limited thereto.

[0101] In the present invention, dilution with the purified water can be used to reduce the buffer concentration of the trypsin reaction solution, so that botulinum neurotoxin type E binds to the mixed-mode chromatography column, but it is not limited thereto.

[0102] In the present invention, a fraction containing botulinum neurotoxin type E is obtained by injecting a 5-50 mM sodium phosphate buffer with a pH of 4.0-6.0 and containing 1.5-3 M sodium chloride into the column, but it is not limited thereto.

[0103] In the step (e) of the present invention, impurities (75 kDa) can be further removed and the purity of the botulinum toxin complex can be increased.

[0104] In the present invention, the mixed-mode chromatography step can use any mixed-mode chromatography process known in the art. Mixed-mode chromatography includes using a solid-phase chromatography support in the form of a resin, monolith, or membrane, which uses multiple chemical mechanisms to adsorb proteins or other solutes. In examples useful for the present invention, non-limitingly include chromatography supports that use a combination of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, thiophilic interaction, hydrogen bonding, π-π bonding, and metal affinity. In a specific embodiment, the mixed-mode chromatography process combines (1) anion exchange and hydrophobic interaction techniques; (2) cation exchange and hydrophobic interaction techniques; and / or (3) electrostatic and hydrophobic interaction techniques. In one embodiment, the mixed-mode chromatography step can be completed by using a column and a resin, for example, the adhere column and resin available from GE Healthcare Life Sciences. The adhere column is a multi-mode medium for intermediate purification and polishing of captured monoclonal antibodies. In a specific embodiment, the mixed-mode chromatography step can be performed in a flow-through mode. In yet another embodiment, the mixed-mode chromatography step can be performed in a bind-elute mode. In another embodiment, the mixed-mode chromatography step can be completed by using one or more of the following systems: MMC (Cytiva), HEA HyperCel TM (Pall Corporation), PPA HyperCel TM(Pall Corporation), MBI HyperCel TM (Pall Corporation), MEP HyperCel TM (Pall Corporation), Blue Trisacryl M (Pall Corporation), CFT TM Ceramic Fluoroapatite (Bio-Rad Laboratories, Inc.), CHT TM Ceramic Hydroxyapatite (Bio-Rad Laboratories, Inc.) and / or ABx TM (J.T. Baker). The specific method used in the mixed-mode chromatography step may depend on the specific column and resin used, and is generally provided by the manufacturer or well-known in the art.

[0105] In one embodiment of the present invention, in step (b), the cation exchange chromatography column may be a sulfopropyl (SP) column, in step (c), the hydrophobic interaction chromatography column may be a butyl column, and in step (e), the mixed-mode chromatography column may be a CHT-XT column, but is not limited thereto. When columns of different types from the above are used, the purification yield and purity of botulinum toxin type E may decrease.

[0106] The botulinum toxin purified by the method may be botulinum toxin type E with a purity of 99% or more. The purity of the botulinum toxin purified in this way may be higher than that of the botulinum toxin purified by the existing method.

[0107] Therefore, in another aspect of the present invention, it relates to a botulinum toxin type E prepared by the method.

[0108] The term "Fraction" used in the present invention refers to a group in which one or more impurities and at least one target molecule (e.g., botulinum toxin) contained together in a biopharmaceutical preparation are separated and collected separately after passing through a substance that binds to one or more impurities by a separation method that generally does not bind to the target molecule (i.e., flow-through) or is eluted after binding, including the target molecule.

[0109] The term "purification" used in the present invention refers to an operation of improving purity by removing mixed impurities from a certain substance. In this specification, purification refers to separating botulinum toxin produced when botulinum grows excessively and then dies from the culture solution of Clostridium botulinum, and refers to a process used as a method for improving purity during the production process of botulinum toxin.

[0110] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for exemplifying the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0111] Examples

[0112] Example 1

[0113] Pretreatment process of the culture solution of Clostridium botulinum type E toxin production strain - Ultra-Filtration (UF)

[0114] Installation in the TFF system 2 MiniCassette (50 kDa, 0.1 m 2 ). Measure the volume, pH, and conductivity of the culture recovery solution. Concentrate the culture recovery solution for the first time by ultrafiltration. Add purified water to the first concentrated process solution and perform a second concentration to recover the second concentrated process solution. Add purified water to the TFF system, recover the remaining residual process solution after recovery, and merge it with the second process concentrated solution. Confirm the amount, pH, and conductivity of the final combined process solution.

[0115] Example 2

[0116] Purification process of Clostridium botulinum type E toxin

[0117] Example 2-1. Cation exchange chromatography

[0118] A column packed with SP-HP resin was installed onto a Fast Protein Liquid Chromatography (FPLC). The final process solution prepared in Example 1 was adjusted to pH 5.0 with 1N hydrochloric acid (HCl), and the process solution was injected into the column. After the injection was completed, the column was washed with a flowing equilibration / wash buffer (20 mM sodium citrate, pH 5.0). After washing, an equilibration / elution buffer (20 mM sodium citrate, 1 M sodium chloride, pH 5.0) was injected, and fractions in the range of 0 to 0.5 M sodium chloride were obtained in sequence. Each fraction was confirmed by SDS-PAGE, and samples for SDS-PAGE analysis were prepared according to the conditions shown in Table 1.

[0119] From the results of confirming each of the said fractions by SDS-PAGE, it was possible to confirm that botulinum toxin in the form of a complex purified to ~300 kDa was present in the corresponding fraction Figure 1 ).

[0120] Table 1

[0121] Sample preparation conditions for SDS-PAGE analysis

[0122]

[0123] Example 2-2. Hydrophobic interaction chromatography

[0124] A column packed with Butyl-HP resin was installed onto a Fast Protein Liquid Chromatography (FPLC). The fractions eluted from the SP-HP column of Example 2-1 were titrated to pH 5.0 with 1N sodium hydroxide. 50 mM sodium citrate, 4M sodium chloride, and pH 5.0 buffer were added to the SP-HP process eluate to bring the conductivity to 170-180 mS / cm. The eluate added with the buffer was injected into the column. After the injection was completed, the column was washed with flowing equilibration / wash buffer (50 mM sodium citrate, 2.5M sodium chloride, pH 5.0). After the washing, equilibration / elution buffer (50 mM sodium citrate, pH 5.0) was injected, and fractions in the range of 0-2.5M sodium chloride were obtained in sequence ( Figure 2 ).

[0125] By confirming the results of each fraction through SDS-PAGE, it was confirmed that many impurities (75 kDa) were removed, and botulinum toxin in the form of a ~300 kDa complex was purified in the corresponding fraction ( Figure 2 ).

[0126] Example 2-3. Ultra-Filtration / Dia-Filtration (UF / DF)

[0127] A Sartocon Slice 200 (30 kDa, 0.02 m 2 ) was installed in the TFF system. The amount of the eluate eluted from the Butyl-HP column was confirmed, and the pH and conductivity were measured. The eluate obtained from the second hydrophobic interaction chromatography was diafiltered (DF) at least 1000-fold with 50 mM sodium phosphate, pH 5.8 (hereinafter referred to as DF buffer). The UV absorbance, pH, and conductivity of the final process liquid were measured. The final process liquid was filtered through a 0.2 μm filter and stored at a temperature of 4°C.

[0128] Example 2-4. Trypsin Treatment

[0129] The final process solution of Example 2-3 was reacted with Trypsin (Roche, 06369880) at a temperature of 35 °C for 1 hour, and then the Trypsin reaction solution was recovered. The Trypsin reaction solution was diluted with cooled purified water at a ratio of 1:4. The amount, pH, and conductivity of the diluted reaction solution were confirmed. After confirming the pH, it was titrated with 1N sodium hydroxide to pH 5.8, and then confirmed by SDS-PAGE respectively ( Figure 3 ).

[0130] As a result, it was confirmed that the density of the 150 kDa neurotoxin protein was 5% or less, and it was divided into a heavy chain (HC, 100 kDa) and a light chain (LC, 50 kDa) ( Figure 3 ).

[0131] Example 2-5. Mixed-mode chromatography

[0132] A column packed with CHT-XT resin was installed in Fast Protein Liquid Chromatography (FPLC). The Trypsin reaction solution of Example 2-5 was injected into the column. After the injection was completed, the column was washed with a balance / wash buffer (10 mM Sodium phosphate, pH 5.8). After washing, a balance / elution buffer (10 mM Sodium phosphate, 2 M Sodium chloride, pH 5.8) was injected, and fractions in the range of 0-2.0 M Sodium chloride were obtained in sequence.

[0133] By confirming the results of each fraction by SDS-PAGE, it was possible to confirm that many impurities (75 kDa) were removed, and botulinum toxin in the form of a complex of ~300 kDa was purified in the corresponding fraction ( Figure 4 ).

[0134] Example 3

[0135] SDS-PAGE of type E botulinum toxin for each purification step was compared

[0136] Fractions containing the purified botulinum toxin in Example 2 were collected, and the company used the Clostridium botulinum type E complex toxin (Metabiologics, Inc. Lot: E010821-01) product as a research reference standard for commercially available botulinum toxin. They were diluted to a concentration of 1 mg / mL with 10 mM sodium phosphate buffer at pH 5.8 and divided into reduced and non-reduced conditions as shown in Table 1 to prepare samples for SDS-PAGE. The samples were electrophoresed in a Novex WedgeWell 8-16% Tris-Glycine, 10-well (Invitrogen, XP08160BOX), about 30 mL of Instant Blue stain reagent was poured in, and it was placed on a shaker and stained for 60 minutes. Then the staining reagent was completely removed and about 30 mL of purified water was poured in, and the washing process was repeated more than 5 times on the shaker. When the dye was sufficiently removed and the bands could be confirmed, the gel was analyzed using an Image Analyzer.

[0137] As a result, the process of purifying the neurotoxin protein in each step was confirmed by SDS-PAGE ( Figure 6 ), and the production yield (Yield) of each step was confirmed (Table 2). It can be seen that the purification method of the present invention can purify only the accurate neurotoxin protein.

[0138] Table 2

[0139] Yield of each purification step of the toxin protein

[0140]

[0141]

[0142] Example 4

[0143] Activity of the purified botulinum toxin product of the present invention

[0144] The specific titer value of the stock solution was evaluated by a potency test using animals with the purified botulinum toxin in Example 3. 0.1 mL was intraperitoneally injected into each group of 10 female mice weighing 18-22 g, and then the lethality rate (LD 50)。The specific efficacy value for the number of deaths measured over 3 days was calculated by probability analysis using Combi-stats as a statistical program. The experiment was repeated 3 times, and the average specific efficacy value for the 3 repetitions was evaluated to be 4.5×10 7 U / mg.

[0145] As shown in Table 3, it was confirmed that the purification method of the present invention can purify botulinum toxin without weakening its activity.

[0146] Table 3

[0147] Activity of the purified botulinum toxin

[0148] Test Potency (U / mg) Test 1 <![CDATA[4.5×10 7 > Test 2 <![CDATA[4.4×10 7 > Test 3 <![CDATA[4.6×10 7 <!-- 10 -->]]> Average <![CDATA[4.5×10 7 >

[0149] Example 5

[0150] Confirmation of the purity of the purification process

[0151] The purity of the purified botulinum toxin was analyzed by SE-HPLC and RP-HPLC using the conditions in Tables 4 and 5. The SE-HPLC results are as Figure 5 shown, and it was confirmed that the purity at a retention time (RT) of 15.9 was 99.61%. Also, the RP-HPLC results are as Figure 7 shown, and 2 main peaks were confirmed, and no impurity peaks were confirmed. It was confirmed that the peak with a retention time of 15.6 was pure botulinum toxin, and the peak with a retention time of 17.2 was the complex component NTNH.

[0152] Table 4 SE-HPLC analysis conditions

[0153]

[0154]

[0155] Table 5

[0156]

[0157] Example 6

[0158] In Process Control (IPC) analysis results of the purified botulinum toxin

[0159] To determine the purity of the products purified using the first SP-HP column, the second Butyl-HP column, and the third CHT-XT column according to the results of Example 2, analysis was performed by HPLC (Table 4). As a result, it was confirmed that the purity of botulinum toxin was 99.61% ( Figure 5 and Figure 8 ).

[0160] Example 7

[0161] Comparison of the cleavage ability of botulinum toxin according to the source species of trypsin

[0162] As is well known, when using trypsin from animal sources other than bovine trypsin, the effect of activator purification of botulinum toxin is poor. Therefore, the present inventors compared and analyzed the case of using porcine trypsin and the case of using bovine trypsin (Sigma, T1426). Specifically, the present inventors reacted porcine trypsin and bovine trypsin at a temperature of 33 to 36 °C for 0.5 to 2 hours, and then added a trypsin inhibitor (in the same ratio as the amount of trypsin added to botulinum toxin) to terminate the enzyme reaction, and analyzed the corresponding samples by SDS-PAGE.

[0163] As a result, it was confirmed that there was no significant difference in the cleavage ability of botulinum toxin between the case of using porcine trypsin and the case of using bovine trypsin compared under the above conditions ( Figure 9 ).

Claims

1. A purification method for type E botulinum toxin, characterized in that, It includes the following steps: Step (a), pre-treating the culture solution of Clostridium botulinum type E toxin-producing strain; Step (b), purifying the pre-treated culture solution in step (a) by cation exchange chromatography; Step (c), purifying the eluate purified in step (b) by hydrophobic interaction chromatography; Step (d), activating the Clostridium botulinum type E toxin by mixing the eluate purified in step (c) with trypsin; And Step (e), purifying the trypsin reaction solution by mixed-mode chromatography.

2. The purification method of type E botulinum toxin according to claim 1, characterized in that, In step (a), the pre-treatment is ultrafiltration.

3. The purification method of type E botulinum toxin according to claim 2, characterized in that, The ultrafiltration is carried out using a filtration membrane with a size of 10 kDa to 300 kDa.

4. The purification method of type E botulinum toxin according to claim 3, characterized in that, The filtration membrane is a cassette-type or hollow fiber-type filtration membrane.

5. The purification method of type E botulinum toxin according to claim 1, characterized in that, In step (b), the cation exchange chromatography column is a column filled with a resin containing one or more functional groups selected from the group consisting of carboxymethyl, sulfonethyl, sulfopropyl, phosphate ester, and sulfonate ester.

6. The purification method of type E botulinum toxin according to claim 5, characterized in that, The sulfopropyl column is one or more selected from the group consisting of sulfopropyl agarose gel HP column, sulfopropyl agarose gel FF column, and Capto S.

7. The purification method of type E botulinum toxin according to claim 1, characterized in that, In step (b), the pre-treated culture solution in step (a) is diluted with purified water or buffer solution and then injected into the cation exchange chromatography column.

8. The purification method of type E botulinum toxin according to claim 7, characterized in that, The buffer solution is sodium citrate buffer solution.

9. The purification method of type E botulinum toxin according to claim 7, characterized in that, By injecting the sodium citrate buffer solution containing sodium chloride as the elution buffer solution into the column, a fraction containing Clostridium botulinum type E toxin is obtained.

10. The purification method of type E botulinum toxin according to claim 9, characterized in that, The elution buffer solution is 10 - 50 mM sodium citrate buffer solution with a pH of 4.0 - 6.0 and added with 0.5 - 1.5 M sodium chloride.

11. The purification method of type E botulinum toxin according to claim 1, wherein In step (c), the hydrophobic interaction chromatography column is a column filled with a resin containing one or more functional groups selected from the group consisting of ether, isopropyl, butyl, octyl, and phenyl.

12. The purification method of type E botulinum toxin according to claim 11, wherein The butyl column is one or more selected from the group consisting of butyl agarose gel HP column, Capto butyl column, Capto phenyl column, and phenyl HP column.

13. The purification method of type E botulinum toxin according to claim 1, characterized in that, In step (c), the eluate purified in step (b) is diluted with a buffer solution with a pH of 4.0 - 6.0 and then injected into the hydrophobic interaction chromatography column.

14. The purification method of type E botulinum toxin according to claim 13, characterized in that, The buffer solution is 25 - 75 mM sodium phosphate and / or sodium citrate buffer solution with a pH of 4.0 - 6.0 and added with 3.5 - 4.5 M sodium chloride.

15. The purification method of type E botulinum toxin according to claim 13, characterized in that, By injecting the 25 - 75 mM sodium phosphate buffer solution and / or sodium citrate buffer solution with a pH of 4.0 - 6.0 into the column, a fraction containing Clostridium botulinum type E toxin is obtained.

16. The purification method of type E botulinum toxin according to claim 1, characterized in that, After step (c), it further includes step (c'), pre-treating the eluate purified in step (c).

17. The purification method of type E botulinum toxin according to claim 16, characterized in that, In step (c'), the pre-treatment is diafiltration and / or ultrafiltration.

18. The purification method of type E botulinum toxin according to claim 17, wherein, The diafiltration and / or ultrafiltration is carried out using a filtration membrane with a size of 10 kDa to 300 kDa.

19. The purification method of type E botulinum toxin according to claim 18, wherein, The filtration membrane is a cassette-type or hollow fiber-type filtration membrane.

20. The purification method of type E botulinum toxin according to claim 17, characterized in that, The diafiltration is carried out using a 5 - 50 mM sodium phosphate buffer solution with a pH of 4.0 - 6.

0.

21. The purification method of type E botulinum toxin according to claim 1, characterized in that, In the reaction in step (d), the type E botulinum toxin in the eluate is activated by reacting the eluate with trypsin at a temperature of 33 to 36 °C for 0.5 to 2 hours.

22. The purification method of type E botulinum toxin according to claim 1, characterized in that, In the step (e), the mixed-mode chromatography column is a column filled with a resin containing the functional group of calcium phosphate compound Ca 10 (PO4)6(OH)2, namely ceramic hydroxyapatite.

23. The purification method of type E botulinum toxin according to claim 22, characterized in that, The ceramic hydroxyapatite column is type I ceramic hydroxyapatite and / or ceramic hydroxyapatite-XT.

24. The purification method of type E botulinum toxin according to claim 1, characterized in that, In step (e), the trypsin reaction solution from step (d) is diluted with purified water and then injected into a mixed-mode chromatography column.

25. The purification method of type E botulinum toxin according to claim 24, wherein A fraction containing type E botulinum toxin is obtained by injecting a 5 to 50 mM sodium phosphate buffer with a pH of 4.0 to 6.0 containing 1.5 to 3.0 M sodium chloride into the column.

26. An E-type botulinum toxin, characterized in that, It is prepared by the method for purifying type E botulinum toxin according to any one of claims 1 to 25.

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