Method for purifying botulinum toxin

Abstract

Disclosed is a method for purifying botulinum toxin, the method including: (a) pre-treating a culture medium solution containing botulinum toxin, (b) purifying the pre-treated botulinum toxin using cation exchange chromatography, (c) purifying the botulinum toxin using hydrophobic chromatography, and (d) purifying the botulinum toxin using anion exchange chromatography. The method can purify botulinum toxin having high purity and high activity, and thus can be used for the production of botulinum toxin.

Description

Background of the Invention Invention Field

[0001] This invention relates to a method for purifying botulinum toxin, and more specifically, to a purification method for obtaining high-purity and active botulinum toxin by simple methods of cation exchange chromatography, hydrophobic chromatography, and anion exchange chromatography.

[0002] Overview of related technologies

[0003] Botulinum toxin is a neurotoxin produced by bacteria such as *Clostridium butyricum*, *Clostridium baraffi*, and *Clostridium botulinum*. Botulinum toxin blocks neuromuscular transmission, causing neuroparalysis in humans and animals. In particular, type A botulinum toxin is known to be highly lethal in humans. Besides type A botulinum toxin, six other types—B, C1, D, E, F, G, and H—have been identified. Each type of botulinum toxin can be distinguished by corresponding type-specific antibodies. The severity of paralysis and the types of animals affected vary depending on the type of botulinum toxin.

[0004] The protein molecule of botulinum toxin has a molecular weight of approximately 150 kDa, comprising a light chain of approximately 50 kDa and a heavy chain of approximately 100 kDa conjugated to the light chain. However, botulinum toxin released from Clostridium botulinum is released as a complex of a 150 kDa toxin protein with at least one non-toxin protein. For example, botulinum toxin is released as complexes of 900 kDa, 500 kDa, and 300 kDa.

[0005] Botulinum toxin can be extremely deadly to humans, but recently developed forms are used to treat a variety of conditions, including neuromuscular disorders characterized by skeletal muscle hyperactivity. For example, It is a trademark of botulinum toxin A, commercially developed by Allergan for the relief or treatment of blepharospasm, strabismus, neck dystonia, and glabellar lines. Currently, research and development are underway for applications and clinical use of other serotypes.

[0006] Botulinum toxins used clinically are generally isolated from cell cultures. In this case, various purification methods are used.

[0007] For example, botulinum toxin is purified in complex form by a series of precipitation and tangential flow filtration steps. [See, e.g., Schantz E.J. et al., Microbiol. Rev. 1992 Mar;56(l):80-99]. However, this method generally provides a relatively low yield of less than about 10%. Other methods used include size exclusion, ion exchange and / or affinity chromatography [See, e.g., Schmidt J.J. et al., Anal. Biochem. 156:213, 1986; Kannan K. et al., Mov. Disord. 2000; 15(Suppl 2):20 (2000); Wang Y.C., Dermatol. Las. Fam. C, Dermatol. Faci. C, Dermatol. Las. Cosm. 2002:58, 2002; and U.S. Patent No. 2003 / 0008367].

[0008] Another method is to synthesize one of the heavy or light chains of botulinum toxin independently by recombinant means, rather than synthesizing the complete and biologically active botulinum toxin protein [See, e.g., Zhou L. et al., Biochemistry, 34(46): 15175 (1995); and Johnson S.K. et al., Protein Expr. and Purif. 2003.; 32: 1-9, (2003)]. However, these methods disadvantageously require an additional step of re-forming the complete and biologically active botulinum toxin protein.

[0009] A recent method involves the use of hydrophobic interaction chromatography, mixed mode and / or ion exchange chromatography to purify botulinum toxin as a complex (See, e.g.: U.S. Patent Nos. 7,452,697 and 7,354,740).

[0010] However, there remains a need in the art for an improved purification method for isolating stable and biologically active complete botulinum toxin. Thus, through extensive efforts to develop a purification method for isolating botulinum toxin of high purity and activity using a simplified method, the present inventors found that a botulinum toxin of high purity and activity can be produced using a simplified method of cation exchange chromatography, hydrophobic chromatography and cation exchange chromatography, in particular, botulinum toxin can be purified to a purity of 99% or more using a SP column as the cation exchange resin and a phenyl column as the hydrophobic resin and a Q column as the anion exchange resin. Based on this finding, the present invention has been completed.

[0011] Prior Art Documents

[0012] Patent document

[0013] US Patent Publication No. 2003 / 0008367

[0014] US Patent No. 7,452,697

[0015] US Patent No. 7,354,740

[0016] Non-patent document

[0017] Schantz EJ et al., Properties and use of botulinum toxin and other microbial neurotoxins in medicine, Microbiol. Rev. 1992 March 56(l):80-99

[0018] Schmidt JJ et al., Purification of type E botulinum neurotoxin by high-performance ion exchange chromatography, Anal.Biochem.1986July; 156(1):213-219

[0019] Kannan K. et al., Methods development for the biochemical assessment ofNeuroBloc(botulinum toxin type B), Mov.Disord.2000;15(Suppl 2):20

[0020] Wang Y.C., The preparation and quality of botulinum toxin type A for injection (BTXA) and its clinical use, Dermatol. Las. Faci. Cosm. Surg. 2002; 58 Zhou L. et al., Expression and purification of the light chain of botulinum neurotoxin A: A single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain, Biochemistry 1995; 34(46): 15175-81

[0021] Johnson S.K. et al., Scale-up of the fermentation and purification of the recombination heavy-chain fragment C of botulinum neurotoxin serotype F expressed in Pichia pastoris, Protein Expr. and Purif. 2003; 32: 1-9 SUMMARY

[0022] Therefore, the present application has been completed in view of the above problems, and an object of the present application is to provide a method for purifying botulinum toxin of high purity and activity using a simple method.

[0023] According to one aspect of the present application, the above and other objects can be accomplished by providing a method for purifying botulinum toxin, including: (a) pre-treating a culture medium solution containing botulinum toxin; (b) purifying the pre-treated botulinum toxin using cation exchange chromatography; (c) purifying the botulinum toxin using cation exchange chromatography, and (d) purifying the botulinum toxin using anion exchange chromatography.

[0024] EFFECTS OF THE INVENTION

[0025] The present application can improve the purity of purified botulinum toxin, and can maintain its activity using only a simple method including cation exchange chromatography, hydrophobic chromatography, and cation exchange chromatography, and thus, can be used for the production of botulinum toxin. BRIEF DESCRIPTION OF DRAWINGS

[0027] The above objects and other objects, features and advantages of the present application will become more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0028] Figure 1 shows the results of FPLC and SDS-PAGE analysis of botulinum toxin in the eluate after anion exchange chromatography according to one embodiment of the present application;

[0029] Figure 2 shows the results of FPLC and SDS-PAGE analysis of botulinum toxin in the eluate after hydrophobic chromatography according to one embodiment of the present application;

[0030] Figure 3 shows the results of FPLC and SDS-PAGE analysis of botulinum toxin in the eluate after anion exchange chromatography according to one embodiment of the present application;

[0031] Figure 4 shows the results of SDS-PAGE analysis for determining the purification effect of botulinum toxin according to the present application and commercially available botulinum toxin;

[0032] Figure 5 shows the results of determination of the purity of botulinum toxin purified according to the purification method of Korean Patent Application No. 10-2013-0092024;

[0033] Figure 6 shows the results of determination of the purity of botulinum toxin purified according to the purification method of U.S. Patent Application No. 11 / 932789; and

[0034] Figure 7 shows the results of determination of the purity of botulinum toxin purified according to the present application. DETAILED DESCRIPTION

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0037] In the present invention, it is found that when a culture solution of botulinum toxin is purified by using a series of processes of cation exchange chromatography, hydrophobic chromatography and anion exchange chromatography, a botulinum toxin of about 900 kDa can be isolated and purified in high purity and activity compared to a botulinum toxin prepared by a conventional method. In particular, the present invention finds that when a SP column is used as a cation exchange resin and a phenyl column is used as a hydrophobic resin and a Q column is used as an anion exchange resin, the botulinum toxin can be purified to a purity of 99% or more.

[0038] Accordingly, in one aspect, the present invention relates to a method of purifying a botulinum toxin, the method comprising: (a) pre-treating a culture solution containing the botulinum toxin; (b) purifying the pre-treated botulinum toxin using cation exchange chromatography, (c) purifying the botulinum toxin using hydrophobic chromatography, and (d) purifying the botulinum toxin using anion exchange chromatography.

[0039] In the present invention, the cation exchange chromatography resin is preferably a SP column, the hydrophobic chromatography resin is preferably a phenyl column, and the anion exchange resin is preferably a Q column.

[0040] In the present invention, the SP column is a column packed with a material containing a sulfopropyl functional group, the phenyl column is a column packed with a material containing a phenyl group, and the Q column is a column packed with a material containing a quaternary ammonium (Q) functional group.

[0041] In step (a) of the present invention, the culture solution containing the botulinum toxin can be a culture solution of a botulinum strain obtained using a conventional method known in the art, and can also be obtained by culturing using a conventional culture medium for culturing, in particular, using a culture medium excluding animal-derived components, preferably, for example, a PYG medium (containing 3% of potato peptone, 1% of yeast extract and 1% of glucose).

[0042] The strain producing the botulinum toxin used in the present invention can be Clostridium botulinum or a variant thereof, and the most preferred strain is Clostridium botulinum type A, NCTC13319, but is not limited to the above-mentioned strain. It is obvious to those skilled in the art that any strain capable of producing botulinum toxin can be used.

[0043] In a specific embodiment, the pre-treatment of the culture solution in step (a) of the present invention can be acid precipitation or ultrafiltration of the culture solution sterilized (e.g., by depth filtration and / or sterilization filtration), but is not limited thereto.

[0044] The acid precipitation can be a precipitation using sulfuric acid or a precipitation using hydrochloric acid, but is not limited thereto. That is, the acid precipitation in step (a) of the present application can be an acid precipitation of the culture solution containing the botulinum toxin using an acid such as sulfuric acid in one embodiment or using hydrochloric acid in another embodiment, such that, after completion of the culture, the pH is adjusted to 3.0 to 4.5, preferably to 3.3 to 4.0, and most preferably to 3.4 to 3.6.

[0045] The ultrafiltration membrane can be a cassette type membrane or a hollow fiber membrane, but is not limited thereto. That is, in step (a) of the present application, the ultrafiltration can be an ultrafiltration using a membrane having a size of 50 kDa to 500 kDa, preferably 100 kDa to 300 kDa, to collect the culture solution containing the botulinum toxin.

[0046] Further, DNase, RNase, nuclease, and / or Benzonase can be optionally used in order to remove nucleic acids during the pre-treatment, but the present application is not limited thereto.

[0047] The present application can include additional purification in order to increase the purity of the resulting botulinum toxin. The purification in the present application is a step of additionally removing impurities from the acid precipitate, and can be performed by a conventional method such as known microfiltration, ultrafiltration, precision filtration, or depth filtration. In one embodiment of the present application, microfiltration can be performed using a hollow fiber membrane of 0.1 to 0.4 μm.

[0048] In step (b) of the present application, the cation exchange chromatography includes binding the pre-treated botulinum toxin dissolved in a buffer having a suitable concentration and pH to a cation exchange resin column, and then eluting using a buffer having an increased salt concentration. In the cation exchange chromatography, the non-complexed form of the botulinum toxin is removed.

[0049] In the method for purifying a botulinum toxin according to the present application, the column for the cation exchange chromatography is preferably a column packed with a resin having a functional group selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), and phosphate (P).

[0050] In the present application, the cation exchange chromatography can be performed using an SP column, preferably an SP Sepharose HP column, an SP Sepharose FF column, a Capto S column, or the like, and more preferably an SP Sepharose HP column, from the viewpoint of removing impurities and maintaining high activity of the botulinum toxin, but is not limited thereto.

[0051] In the present invention, the SP Sepharose HP column is a column filled with a resin having a sulfopropyl functional group, which has a 6% spherical, cross-linked sepharose matrix form, and has a DBC of 55 mg / mL based on RNase A, and a particle size of 34 μm.

[0052] The botulinum toxin in step (b) is dissolved in 10 to 30 mM of a sodium citrate buffer having a pH of 4.5 to 5.3, and then injected into the SP column.

[0053] The botulinum toxin in step (b) is eluted with 10 to 30 mM of a sodium citrate buffer supplemented with 0.5 to 1.5 M of sodium chloride and having a pH of 4.5 to 5.5, but is not limited thereto.

[0054] In step (c) of the present invention, the hydrophobic chromatography includes binding a solution containing the botulinum toxin dissolved in a buffer eluted through the cation exchange chromatography of step (b) to a hydrophobic chromatography column, the buffer having a suitable concentration and a pH that binds the solution to the hydrophobic chromatography column, and then eluting with a buffer having a reduced salt concentration. In the hydrophobic chromatography, the botulinum toxin in a non-complexed form is further removed.

[0055] In the method for purifying the botulinum toxin of the present invention, the column for the hydrophobic chromatography is a column filled with a resin having a functional group selected from the group consisting of an ethyl ether, an isopropyl, a butyl, an octyl, and a phenyl, but is not limited thereto.

[0056] In the present invention, in order to effectively separate the non-complexed protein, the hydrophobic chromatography can be performed using a phenyl column, preferably a phenyl sepharose HP column, a phenyl sepharose FF (fast flow) column, a Capto phenyl column, or the like, and more preferably a phenyl sepharose HP column, but is not limited thereto.

[0057] The phenyl sepharose HP column used in the present invention is a column filled with a resin having a 6% highly cross-linked sepharose matrix form and having a degree of substitution of 25 μmol of phenyl / mL and a particle size of 34 μm.

[0058] In step (c), the botulinum toxin is dissolved in 30 to 70 mM of a sodium phosphate buffer supplemented with 1.8 to 2.2 M of sodium chloride and having a pH of 5.7 to 6.7, and can be injected into the phenyl column, but is not limited thereto.

[0059] In step (c), the botulinum toxin is eluted with 30 to 70 mM of a sodium phosphate buffer having a pH of 5.7 to 6.7, but is not limited thereto.

[0060] In step (d) of the present application, the anion exchange chromatography includes binding botulinum toxin dissolved in a buffer eluted through hydrophobic chromatography to an anion exchange chromatography column, the buffer having a suitable concentration and pH for the botulinum toxin to bind to the column, and then eluting using a buffer having an increased salt concentration. In anion exchange chromatography, other impurities are removed and the purity of the 900 kDa botulinum toxin is increased.

[0061] In the method for purifying botulinum toxin of the present application, the column for anion exchange chromatography is a column filled with a resin having a functional group selected from the group consisting of diethylaminoethyl (DEAE), tetraaminoethyl (QAE), and quaternary ammonium (Q), but is not limited thereto.

[0062] In the present application, in order to remove other impurities and particularly increase the purity of the 900 kDa botulinum toxin, anion exchange chromatography can be performed using a Q column, preferably a Capto Q ImpRes column, a Toyopearl Super Q 650M column, a Q Sepharose FF column, a Q Sepharose High Performance (Q Sepharose HP) column, etc., and more preferably a Capto Q ImpRes column.

[0063] The Capto Q ImpRes column used in the present application is a column filled with a resin having a high-flow agarose matrix including a functional group of quaternary ammonium (Q), a total ion capacity based on BSA of 0.15 to 0.18 mmol(Cl-) / mL, and a particle size of 40 μm.

[0064] The botulinum toxin in step (d) is dissolved in a 40 to 60 mM sodium phosphate buffer having a pH of 5.7 to 6.7, and then injected into a Q column, but is not limited thereto.

[0065] In step (d), the botulinum toxin can be eluted with a 40 to 60 mM sodium citrate buffer having a pH of 5.7 to 6.7 supplemented with 0.8 to 1.2 mM sodium chloride, but is not limited thereto.

[0066] The botulinum toxin purified by the above-described method can be a type A botulinum toxin having a purity of at least 99%, and the purified botulinum toxin can have a higher purity than botulinum toxin purified by a conventional method.

[0067] The botulinum toxin can be derived from a type A botulinum, NCTC13319, but is not limited thereto.

[0068] As used herein, the term "fraction" refers to a group of penetrating substances containing at least one target molecule, which is separated and collected by a separation method, in which a biological agent containing the at least one target molecule (such as botulinum toxin) and one or more impurities, the at least one target molecule penetrates the substance bound to one or more impurities, and the target molecule is not generally bound to the substance (i.e., flows through the substance) or is bound to the substance and then eluted.

[0069] As used herein, the term "purification" refers to an operation to improve purity by removing coexisting impurities from a certain substance, and in the present specification, purification refers to the separation of botulinum toxin produced when overgrown botulinum bacteria die from the culture of botulinum bacteria, and means a process to improve purity during botulinum toxin production. EMBODIMENT

[0070] Hereinafter, the present application will be described in more detail with reference to a plurality of embodiments. However, it will be obvious to those skilled in the art that these embodiments provided are only for the purpose of clarifying the present application, and should not be interpreted as limiting the scope of the present application.

[0071] Example 1: Preparation of samples and experimental materials

[0072] 1-1, Sample preparation

[0073] The botulinum bacteria strain used in the present application is Clostridium botulinum type A, NCTC13319, and the strain is first inoculated into 500 mL of PYG medium (potato peptone 3%, yeast extract 1%, glucose 1%) and cultured for 12 to 24 hours at 34±1℃ under anaerobic conditions in a ReadytoProcess WAVE 25 incubator. After the culture, when the growth of the strain reaches the logarithmic phase, 100 mL of the strain is inoculated into 5 L of PYG medium and cultured for 40 to 72 hours under anaerobic conditions in a ReadytoProcess WAVE 25 incubator. The culture solution is sterilized using a sterilizing filter, and only the culture solution is recovered. The culture solution is titrated to pH 3.5 using 3N sulfuric acid, a precipitate is observed, and then the resulting product is stored in a refrigerated state for 16 hours or more.

[0074] 1-2, Preparation of experimental materials

[0075] The experimental materials used in the present application are as follows: pure water (ultra-pure water or water having a quality equal to or higher than ultra-pure water), butyl sepharose HP (GE Healthcare, 175432), Q-sepharose-HP (GE Healthcare, 171014), phenyl sepharose HP (GE Healthcare, 171082), Q-sepharose-FF (GE Healthcare, 170510), SP-sepharose-FF (GE Healthcare, 170729), DEAE-FF (GE Healthcare, 170709), SP-sepharose-HP (GE Healthcare, 171087), Capto Q ImpRes column (GE Healthcare, 175470), citric acid (Merck, 1.37002.5000), anhydrous trisodium citrate (Merck, 1.37042.5000), sodium dihydrogen phosphate (Merck, 1.06349.1000), disodium hydrogen phosphate (Merck, 1.06585), and sodium chloride (Merck, 1.37017.5000).

[0076] Example 2: Purification of botulinum toxin

[0077] 2-1, Microfiltration

[0078] The microfiltration device, AKTA flux 6 (GE Healthcare), was opened and a 0.2 pm hollow fiber was connected to the microfiltration device. 5 L of distilled water was added to the microfiltration device and the device and hollow fiber were washed twice at a TMP of 0.3. The 5 L of botulinum toxin culture fluid sulfate precipitate prepared in Example 1-1 was injected into the microfiltration device and concentrated to 1 L in two steps at a TMP of 0.3, 2 L of DW was added to the concentrate, and after 2 L of DW was added to the concentrate, it was further concentrated from 3 L to 1 L for 5 times. 500 mL of 200 mM sodium citrate (pH 5.5) was added and extraction was performed for 1 hour and 30 minutes by circulation. The extract was recovered through the permeate line of the microfiltration device, and a 300 kDa cut-off hollow fiber was connected to the microfiltration device. The extract was added to the microfiltration device and concentrated to 500 mL at a TMP of 0.3 bar, recovered, and stored at 4°C.

[0079] 2-2, Cation exchange chromatography

[0080] The SP-HP resin was loaded in a HiScale 50 / 20 column (GE Healthcare, 28964445) to a height of 8 to 12 cm and then installed in an AKTA purification system. The column was equilibrated via a flow equilibration / wash buffer (20 mM sodium citrate, pH 4.8). The sample prepared in Example 2-1 was pH adjusted to 4.8 with 1 M citric acid and diluted 5-fold in 2,000 mL of distilled water. The sample was injected at 15 mL / min. After injection of the sample, the column was washed with 380 mL of equilibration / wash buffer (20 mM sodium citrate, pH 4.8). After washing, the equilibration / elution buffer was injected according to step (1) 5 CV, 30% gradient (linear gradient) and step (2) 3 CV, 100% gradient (step gradient) and a total of 31 fractions of 50 mL were obtained in sequence (see Table 1). A total of 31 fractions were obtained in sequence and each fraction was identified by SDS-PAGE.

[0081] Table 1

[0082] Column resin SP Sepharose HP Column volume 190 mL Binding and wash buffer 20 mM sodium citrate / 0.5 M sodium chloride, pH 4.8 Elution buffer 20 mM sodium citrate / 1 M sodium chloride, pH 4.8

[0083] The results are shown in Figure 1 . From fractions 10 to 21 of the total of 31 fractions, HA33 was detected and a 900 kDa complex form of botulinum toxin was purified from the corresponding fractions.

[0084] 2-3, Hydrophobic chromatography

[0085] The Phenyl-HP resin was loaded in a HiScale 26 / 20 column (GE Healthcare, 28964514) to a height of 7 to 11 cm and then installed in an AKTA purification system. The column was equilibrated via a flow equilibration / wash buffer (50 mM sodium phosphate, 2 M sodium chloride, pH 6.2). A total of 600 mL of fractions 10 to 21 eluted from the SP Sepharose HP column of Example 2-2 were collected and the pH was adjusted to 6.2 with 1 M sodium phosphate dibasic solution. The sample was injected at 8 mL / min with 600 mL of 50 mM sodium phosphate and 4 M sodium chloride added at pH 6.2. After injection, the column was washed with 94 mL of equilibration / wash buffer (50 mM sodium phosphate, 2 M sodium chloride, pH 6.2). After washing, the equilibration / elution buffer was injected according to step (1) 10 CV, 100% gradient (linear gradient) and step (2) 3 CV, 100% gradient (step gradient) and a total of 27 fractions of 23 mL were obtained in sequence and each fraction was identified by SDS-PAGE.

[0086] Table 2

[0087] Column resin Phenyl Sepharose HP Column volume 47 mL Binding and wash buffer 50 mM sodium phosphate / 2 M sodium chloride, pH 6.2 Elution buffer 50 mM sodium phosphate, pH 6.2

[0088] The results are as follows Figure 2 As shown, a botulinum toxin complex of 900 kDa was detected in fractions 16 to 23 out of a total of 27 fractions.

[0089] 2-4, Anion exchange chromatography

[0090] Capto-Q ImpRes resin was loaded into an Omnifit 6.6 / 25 column (Diba, 006EZ0625AA) at a stack height of 8 to 12 cm and then installed in an AKTA purification system. A total of 168 mL fractions eluted from the phenyl agarose HP column of Examples 2-3 were dialyzed (DF) 100-fold using a dialysis membrane (Spectra / Por, 132680) with 50 mM sodium phosphate (pH 6.2) buffer. The column was equilibrated with flow equilibration / wash buffer (50 mM sodium phosphate, pH 6.2). The samples prepared in Examples 2-3 were injected at a rate of 1 mL / min. After injection, the column was washed with 1 mL of equilibration / wash buffer (50 mM sodium phosphate, pH 6.2). After washing, equilibration / elution buffer was injected according to the following step gradients: (1) 2CV, 15% step gradient; (2) 2CV, 20% step gradient; (3) 2CV, 30% step gradient; and (4) 2CV, 100% step gradient, followed by elution. A total of 29 1 mL fractions were obtained sequentially, and each fraction was identified by SDS-PAGE.

[0091] Table 3

[0092] Column resin Capto Q IPRUS Column volume 3.6 mL Binding and wash buffer 50 mM sodium phosphate, pH 6.2 Elution buffer 50 mM sodium phosphate / 1 M sodium chloride, pH 6.2

[0093] The results are as follows Figure 3 As shown, among the 29 fractions in total, fractions 7 to 10 contained 900 kDa botulinum toxin complex without impurities (red circles).

[0094] Example 3: Comparison of Standard Product and Purified Product

[0095] The purified botulinum toxin fractions 7 to 10 from Example 2 and commercially available botulinum toxin C-BoNT / A1 (catalog number 3102, miprolab) were diluted to a concentration of 1 mg / mL in 50 mM sodium phosphate buffer (pH 6.2). Samples for loading were prepared under the reducing and non-reducing conditions shown in Table 3. The samples were subjected to electrophoresis in 10-well Novex wedge-shaped disks containing 4 to 20% cis-glycine (Invitrogen, NP04200BOX), with approximately 30 mL of Instant Blue staining reagent added, and the samples were stained on a shaker for 60 minutes. The staining reagent was completely removed, and the samples were washed 5 or more times on a shaker with approximately 30 mL of purified water added for 30 minutes. When the background was sufficiently removed and bands were detectable, the gel was analyzed using an image analyzer.

[0096] Table 4

[0097]

[0098]

[0099] As a result, Figure 4 As shown, the botulinum toxin purified by the purification method of the present invention was identified at the same position as commercially available botulinum toxin, indicating that the purification method of the present invention can accurately purify only the target protein of interest.

[0100] Example 4: Comparison of the activities of the standard product and purified product of the botulinum toxin of the present invention.

[0101] The activity of the botulinum toxin purified by Example 2 was compared with that of commercially available botulinum toxin Botoxin (Allergan, 100 units).

[0102] Based on the concentration of each sample, each sample was first diluted 2... × 10 5 Up to 3 × 10 5 The sample was diluted 1.45 mL in 4.4 mL of physiological saline. Ten groups were diluted in the same manner, and 0.1 mL of the sample was injected intraperitoneally into 10 female ICR mice (18 to 22 g) in each group. Lethality was assessed after 3 days. The LD50 was obtained through probabilistic statistical analysis.

[0103] The results showed that the activity of 1 ng of botulinum toxin purified by the present invention and the botulinum toxin from Allergan was 40.73 units and 25.32 units, respectively, and the purification method according to the present invention can be carried out without reducing the activity of botulinum toxin.

[0104] Example 5, Comparison of purity obtained by purification method of other company

[0105] The purity of botulinum toxin purified according to the purification method described in Korean Patent Application No. 10-2013-0092024 filed by Allergan, the leading manufacturer of botulinum toxin, was analyzed. As a result, as shown in FIG. 1, in addition to the main peak, a smaller impurity was detected, and the 900 kDa protein was not effectively separated from the 150 kDa, 300 kDa, or 500 kDa protein. Figure 5

[0106] In addition, the purity of botulinum toxin purified according to the purification method of U.S. Patent Application No. 11 / 932789 filed by Allergan was analyzed. As a result, in addition to the main peak, a larger impurity was detected, as shown in FIG. 2. This means that an undesired precipitate was formed, and it can be expected that the purification method affects the protein structure. Figure 6

[0107] On the other hand, as can be seen from the results of Figure 7 , the present application can specifically purify only about 900 kDa of toxin, and the modification of the toxin during the purification process is also minimized.

[0108] Under the conditions shown in Table 5, the purity of the purified botulinum toxin was analyzed by HPLC.

[0109] Table 5

[0110] Item Condition Column PROTEIN KW-804 Size 8 mm x 300 mm Stationary phase Silica gel (7 μm) for chromatography R Temperature 25℃ Mobile phase 50 mM sodium phosphate, pH 6.0, 0.15 M sodium chloride Flow rate 1.0 mL / min Run time 30 minutes Detector UV detector, 278 nm Injection volume 20 μL

[0111] Example 6, Comparison of three-step chromatography in previous patents and purification method of the present application

[0112] The purity and titer of purified botulinum toxin were compared between the purification method of the resin for purifying botulinum toxin disclosed in the previous patents (U.S. Patent Publication No. 2019-0201505 and U.S. Patent No. 7,452,697) and the purification method using a different type of resin in the present application (Table 6).

[0113] The purity was detected by HPLC according to the method of Table 5, and the titer was calculated using CombiStats by Probit statistical analysis based on the results of the number of 3-day dead mice at a protein concentration of 1 mg / mL in a mouse test.

[0114] Table 6

[0115]

[0116] ​​The type of column and purification conditions used in the experimental examples and comparative examples are shown in Tables 7 to 8, respectively.

[0117] Table 7

[0118] Comparative Example 1 (butyl-HP→Q-HP→phenyl-HP)

[0119] Column Butyl-HP (diameter: 0.66 cm, height: 12 cm) Column volume 4.11 mL Binding and wash buffer 50 mM sodium phosphate / 2 M sodium chloride, pH 6.2 Elution buffer 50 mM sodium phosphate, pH 6.2 Elution condition 2 to 0 M sodium chloride 10 CV, 0 M sodium chloride 5 CV

[0120]

[0121] Column Phenyl-HP (diameter: 0.66 cm, height: 12 cm) Column volume 4.11 mL Binding and wash buffer 50 mM sodium phosphate / 2 M sodium chloride, pH 6.2 Elution buffer 50 mM sodium phosphate, pH 6.2 Elution condition 2 to 0 M sodium chloride 10 CV, 0 M sodium chloride 5 CV

[0122] Table 8 Comparative Example 2 (Q-FF(FT)→butyl-HP→SP-HP)

[0123]

[0124]

[0125] As can be seen from Table 9, the botulinum toxin purified according to the experimental examples (purification method of the present application) resulted in a high purity of 99.4%, and the purity and titer of the botulinum toxin purified according to Comparative Example 1 were both lower than those of the experimental examples. Comparative Example 2 had very low purity and titer. Therefore, these results indicate that the botulinum toxin purified by the method of the present application has significantly higher purity and titer than the botulinum toxin purified by the conventional method.

[0126] Table 9

[0127]

[0128] While particular configurations of the present application have been described in detail herein, it will be appreciated by those skilled in the art that the application provides many preferred embodiments and that the present application should not be construed as limited to the preferred embodiments set forth herein. Therefore, the spirit and scope of the present application are defined by the appended claims and their equivalents.

Claims

1. A method for purifying 900 kDa compound botulinum toxin type A, the method comprising: (a) Acid precipitation or ultrafiltration of a culture medium solution containing 900 kDa of compound botulinum toxin type A; (b) Purification of pretreated 900 kDa complex botulinum toxin type A by microfiltration; (c) The pretreated 900 kDa complex botulinum toxin type A was purified from the product of step (b) by cation exchange chromatography using an SP agarose HP column; (d) The 900 kDa complex botulinum toxin type A was purified from the product of step (c) by hydrophobic chromatography using a phenyl agarose HP column; (e) The 900 kDa compound botulinum toxin type A was purified from the product of step (d) by dialysis filtration; as well as (f) The 900 kDa compound botulinum toxin type A was purified from the product of step (e) by anion exchange chromatography using a Capto-Q ImpRes column.

2. The method according to claim 1, wherein the 900 kDa compound botulinum toxin type A in step (c) is dissolved in 10 to 30 mM sodium citrate buffer at pH 4.5 to 5.5 and then injected into the SP agarose HP column.

3. The method of claim 1, wherein the 900 kDa complex botulinum toxin type A in step (c) is eluted using a 30 to 70 mM sodium citrate buffer at a pH of 4.5 to 5.5, wherein the sodium citrate buffer is supplemented with 0.5 to 1.5 M sodium chloride.

4. The method according to claim 1, wherein the 900 kDa compound botulinum toxin type A in step (d) is dissolved in 30 to 70 mM sodium phosphate buffer at pH 5.7 to 6.7 and then injected into the phenyl agarose HP column.

5. The method of claim 1, wherein the 900 kDa complex botulinum toxin type A in step (d) is eluted using a 30 to 70 mM sodium phosphate buffer at a pH of 5.7 to 6.

7.

6. The method of claim 1, wherein the 900 kDa compound botulinum toxin type A from step (f) is dissolved in 40 to 60 mM sodium phosphate buffer at pH 5.7 to 6.7 and then injected into the Capto-Q ImpRes column.

7. The method of claim 1, wherein the 900 kDa complex botulinum toxin type A in step (f) is eluted using a 40 to 60 mM sodium phosphate buffer at a pH of 5.7 to 6.7, wherein the sodium phosphate buffer is supplemented with 0.8 to 1.2 mM sodium chloride.

8. The method according to claim 1, wherein the purified 900 kDa compound botulinum toxin type A has a purity of 99% or higher.

Images