WO2020129986A1 - ボツリヌス毒素の製造方法 - Google Patents
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- WO2020129986A1 WO2020129986A1 PCT/JP2019/049436 JP2019049436W WO2020129986A1 WO 2020129986 A1 WO2020129986 A1 WO 2020129986A1 JP 2019049436 W JP2019049436 W JP 2019049436W WO 2020129986 A1 WO2020129986 A1 WO 2020129986A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a method for producing botulinum toxin.
- Botulinum is an obligate anaerobic Gram-positive bacterium that forms spores and produces a neurotoxin (botulinum toxin) that causes systemic paralysis.
- the botulinum toxin produced from Clostridium botulinum forms a complex in which a nontoxic component is bound to the neurotoxic component NTX. This complex is classified into LL toxin (900 KDa), L toxin (500 KDa), and M toxin (300 KDa) depending on the difference in molecular weight.
- M toxin is NTX bound to NTX, which is a protein that is non-toxic and has no hemagglutination activity (non-toxic non-HA protein), and L toxin is bound to M toxin, which is a non-toxic HA protein that has hemagglutination activity.
- NTX a protein that is non-toxic and has no hemagglutination activity
- L toxin is bound to M toxin, which is a non-toxic HA protein that has hemagglutination activity.
- a toxin composed solely of NTX is called S toxin (150 KDa).
- the S toxin consisting of NTX alone can be isolated by subjecting the complex toxin to alkaline conditions to dissociate NTX and NTNH.
- Botulinum toxins are classified into serotypes A to G, and even among toxins of the same serotype, they are classified into several subtypes due to the difference in the structure of the toxin gene. For example, type A botulinum is classified into subtypes A1 to A5, and type A1 botulinum produces LL toxin, L toxin and M toxin, whereas type A2 botulinum produces only M toxin. .. Clostridium botulinum types B, C and D produce LL and M toxins. Clostridium botulinum E and F produce only M toxin, and Clostridium botulinum G produces only L toxin.
- Botulinum toxin is clinically applied using its neuroleptic effect. Examples of applications include post-stroke upper/lower extremity spasticity, dystonia, hemi-facial spasm, sequelae of cerebrovascular accidents, cosmetic surgery, and the like.
- botulinum toxin Due to such clinical utility, various methods for producing botulinum toxin from botulinum bacteria have been proposed.
- a method for producing a botulinum toxin a method of acid-precipitating a botulinum toxin by treating a culture medium of botulinum toxin with an acid has been used and improved since before.
- the method for producing botulinum toxin the method without acid precipitation as described above is also used.
- a step of culturing and fermenting Clostridium botulinum a step of removing cell debris present in the fermentation medium to collect a fermentation medium; a step of contacting the collected medium with an anion exchange medium
- a process for obtaining a botulinum neurotoxin complex of type A comprising capturing botulinum neurotoxin; and contacting an eluent eluted from an anion exchange medium with a cation exchange medium.
- a cell component bacterial cell component
- a nucleic acid component are co-precipitated in the acid precipitate, and nuclease digestion is performed on the acid precipitate, so that the nucleic acid
- the components are fragmented.
- the toxin is eluted from the bacterial cells by exposing the bacterial cells to acidic conditions by acid precipitation, and the toxin also precipitates together with the bacterial cell components and the nucleic acid components.
- the fragmented nucleic acid component and cell component are removed by the subsequent purification operation using chromatography or the like.
- the method using nucleic acid removal by anion exchange column chromatography also has a limitation in the yield of the toxin due to the adsorption of both the toxin and the nucleic acid on the anion exchange carrier, and the specific activity of the obtained toxin is also limited. Not at a satisfactory level.
- an object of the present invention is to provide a method for producing a botulinum toxin that is simple, has a high toxin yield, and can obtain a toxin with a high specific activity.
- the present inventor has conducted a nuclease treatment, which has been applied only to acid precipitates in which microbial components inevitably coexist with botulinum toxin, and removes microbial components from botulinum culture fermentation products. It was found that the botulinum toxin having a high toxin yield and a high specific activity can be produced by applying the mixture to the botulinum toxin mixture described above, without using acid precipitation or anion exchange chromatography for nucleic acid removal. The present invention has been completed by further studies based on this finding.
- Item 1 a step of producing a botulinum toxin from a botulinum toxin-producing bacterium in a medium to obtain a mixture a containing a botulinum toxin-derived bacterial cell component and a nucleic acid component and a botulinum toxin; (B) subjecting the mixture a to removal of the bacterial cell component to obtain a mixture b containing a nucleic acid component and a botulinum toxin; (C) a step of adding an endonuclease to the mixture b to obtain a mixture c containing a nucleic acid degradation product and a botulinum toxin, (D) subjecting the mixture c to removal of a nucleic acid degradation product to obtain a botulinum toxin isolated liquid d; A method for producing a botulinum toxin, comprising: Item 2.
- the method for producing a botulinum toxin according to Item 1 wherein the endonuclease is an endonuclease derived from Serratia marcescens.
- Item 3. Item 3. The method for producing a botulinum toxin according to Item 1 or 2, wherein the step (C) is performed under conditions of pH 5.8 to 6.5.
- Item 4. Item 4. The method for producing a botulinum toxin according to any one of Items 1 to 3, wherein in the step (C), the endonuclease is added multiple times.
- Item 5. Item 5.
- the purified product of the botulinum toxin complex is subjected to anion exchange chromatography under conditions where the non-toxic non-HA protein dissociates from the botulinum toxin complex to obtain a purified product of the botulinum toxin complex.
- Item 7. A method for producing a botulinum toxin according to Item 6, comprising a step.
- Item 8. The method for producing a botulinum toxin according to any one of Items 1 to 7, wherein at least the step (A) includes a pre-culturing step and a main culturing step, and at least the pre-culturing step is performed by static culturing.
- Item 10 The step (A) is (A1) a step of growing cells of the botulinum toxin-producing bacterium in a medium having a pH of 6.8 to 8.0, (A2) a step of fermenting the botulinum toxin-producing bacterium in a medium having a pH of 5.0 to 6.5; Item 10.
- Item 15. A purified botulinum toxin, which has a specific activity of 3.0 ⁇ 10 7 U/mg or more.
- Item 16. Item 15. A purified botulinum toxin produced by the method for producing a botulinum toxin according to any one of Items 1 to 14.
- a botulinum toxin that is simple, has a high toxin yield, and can obtain a toxin with a high specific activity.
- Example 1 shows a stained image of an electrophoresis gel showing purification of botulinum toxin in Example 1.
- 3 shows a stained image of an electrophoresis gel showing purification of botulinum toxin in Comparative Example 1.
- 3 shows a stained image of an electrophoresis gel showing purification of botulinum toxin in Example 2.
- 4 shows a stained image of an electrophoresis gel showing purification of botulinum toxin in Example 3.
- the method for producing a botulinum toxin of the present invention comprises (E) a step of subjecting the botulinum toxin isolated solution d to cation exchange chromatography to obtain a purified product of a botulinum toxin complex, or (F) the botulinum toxin complex.
- the purified toxin complex may be subjected to anion exchange chromatography under conditions where the nontoxic non-HA protein is released from the botulinum toxin complex to obtain a purified botulinum toxin complex.
- the botulinum toxin according to the present invention includes serotypes such as A, B, C, D, E, F, and G serotypes.
- Botulinum toxin also includes botulinum toxin complex and botulinum toxin non-complex.
- the botulinum toxin complex includes LL toxin (900 KDa), L toxin (500 KDa), and M toxin (300 KDa).
- the botulinum toxin non-complex refers to S toxin (150 KDa) consisting of NTX only.
- NTX has a double-chain fragment structure in which a 50 KDa light chain and a 100 KDa heavy chain are linked via a disulfide.
- Botulinum toxin is produced from the botulinum toxin-producing bacterium in a medium, and the botulinum toxin-derived bacterial cell component and nucleic acid component and botulinum toxin are contained. Mixture a is obtained.
- the natural botulinum toxin and the modified botulinum toxin are examples of the botulinum toxin produced in this step.
- Modified botulinum toxin means a botulinum toxin in which at least one of its amino acids has been deleted, modified, added, inserted or substituted as compared to a natural botulinum toxin.
- the modified botulinum toxin may be a recombinantly produced neurotoxin.
- the modified botulinum toxin may have at least one biological activity of a natural botulinum toxin, such as the ability to bind to a botulinum toxin receptor and the ability to inhibit neurotransmitter release from neurons.
- modified botulinum toxins include botulinum toxins that have a light chain from a botulinum toxin serotype (such as serotype A) and a heavy chain from a different botulinum toxin serotype (such as serotype B).
- a botulinum toxin serotype such as serotype A
- one type of botulinum toxin may be produced, or a plurality of types of botulinum toxin may be produced. From the viewpoint of purification efficiency and the like, it is preferable that one type of botulinum toxin is produced.
- the botulinum toxin producing bacterium may be any bacterium that produces the above botulinum toxin, and examples thereof include Clostridium botulinum and its recombinants. More specifically, botulinum type A1 which produces LL toxin, L toxin and M toxin; botulinum type A2 which produces only toxin M; botulinum type B, C and D which produces LL toxin and M toxin Bacterium; E and F type botulinum producing only M toxin; and G type botulinum producing only L toxin.
- A2 type, E type and F type botulinum bacteria which produce only M toxin, more preferably A2 type botulinum bacteria.
- A2 type botulinum strain include infant botulinum-causing bacteria, and more specifically, Kyoyo-F, Chiba-H, Y-8036, 7I03-H, 7I05-H, KZ1828 and the like. And preferably Chiba-H.
- the botulinum toxin-producing bacterium is preferably a germinated body of botulinum bacterium preserved in the form of spores. As a result, the stability as a seed strain is improved and the toxin can be efficiently produced.
- the step (A) can include a pre-culture step and a main culture step.
- the pre-culture step can be preferably performed by static culture
- the main culture step can be preferably performed by stirring culture or static culture. More preferably, both the pre-culture step and the main culture step can be performed by static culture.
- the pre-culturing process is a process of expanding cells and the main culture process is a process of producing and culturing cells. It is preferable that the main culturing step includes (A1) a step of growing cells of the botulinum toxin-producing bacterium and (A2) a step of fermenting the botulinum toxin-producing bacterium in this order. In the step (A2), both elution (lysis) of the botulinum toxin and growth of the bacterium proceed.
- the pH of the medium in the step (A1) is preferably 6.8 to 8.0 from the viewpoint of promoting the growth of bacteria better.
- a medium adjusted to pH 7.0 to 8.0, preferably pH 7.1 to 8.0, more preferably pH 7.2 to 8.0, and still more preferably pH 7.2 to 7.8 is used. It is possible to prepare and use the medium to start the step (A1).
- the pH of the medium in the step (A2) is preferably 5.0 to 6.5 from the viewpoint of better elution (lysis) of botulinum toxin and growth of the bacteria.
- An example of the method of adjusting the pH in the step (A2) is a method of artificially lowering the pH by adding a pH adjuster to the medium in the step (A2).
- the operation of artificially lowering the pH can be performed after confirming that the growth of the bacteria in the step (A1) has reached the peak.
- Such a method of artificially lowering the pH is preferably performed in the case of stirring culture.
- Another example of the method of adjusting the pH in the step (A2) is a method of utilizing the phenomenon that the pH naturally decreases from the step (A1) without artificially controlling it.
- the adjustment utilizing the phenomenon that the pH naturally lowers can be preferably carried out in the case of static culture.
- the medium used in the step (A) is not particularly limited and is appropriately selected by those skilled in the art.
- the medium contains at least one of plant peptone and animal-derived peptone.
- Vegetable peptone is a degradation product of a protein obtained from a plant, and examples thereof include those derived from peas, soybeans, cotton seeds, wheat gluten, and the like, and peas derived from peas are preferable.
- Animal-derived peptone is a protein degradation product derived from animal tissue, and examples thereof include those derived from pigs, cattle, sheep and the like, and pig-derived peptone is preferable.
- the total content of plant peptone and animal-derived peptone in the medium used in the preculture step is, for example, 1 to 12 w/v%, preferably 2 to 10 w/v%, and is used in the main culture step.
- the total content of plant peptone and animal-derived peptone is preferably less than the total content of plant peptone and animal-derived peptone in the medium used in the preculture step, for example plant peptone and animal
- the total content of the derived peptone is 0.1 to 5 w/v%, preferably 1 to 3 w/v%.
- a unit "w/v%" refers to the mass-to-volume percentage in the 17th revision Japanese Pharmacopoeia.
- the medium may contain a carbon source and a medium reducing agent.
- carbon sources include monosaccharides (eg glucose, fructose etc.), disaccharides (eg maltose, sucrose etc.), oligosaccharides, polysaccharides (eg dextrin, cyclodextrin, starch etc.) or sugar alcohols (eg xylitol). , Sorbitol, erythritol, etc.), preferably monosaccharides, and more preferably glucose.
- the medium reducing agent include sodium thioglycolate, cysteine, sodium L-ascorbate and the like, and preferably sodium thioglycolate.
- the botulinum toxin produced by the botulinum toxin-producing bacterium coexists with the bacterial cell component and nucleic acid component derived from the botulinum toxin-producing bacterium in the medium at the end of step (A). Therefore, in the step (A), a mixture a containing a botulinum toxin and a bacterial cell component and a nucleic acid component derived from a botulinum toxin-producing bacterium is obtained. In addition to the bacterial cell component, nucleic acid component and botulinum toxin, the mixture a also contains a medium component.
- step (B) Removal of bacterial cell component
- the mixture a obtained in the step (A) is subjected to removal of the bacterial cell component to obtain a mixture b containing a nucleic acid component and a botulinum toxin.
- a method of precipitating the bacterial cell component and the nucleic acid component together with the botulinum toxin from the mixture a such as acid precipitation is not performed, and a method of previously removing the nucleic acid component is not performed. Therefore, the present invention does not require a complicated operation such as a precipitation treatment such as acid precipitation. Moreover, since acid precipitation is not performed, the loss of botulinum toxin is small, and an excellent yield can be achieved.
- nuclease is not added to the mixture a before the removal of the bacterial cell component in the step (B).
- the method for removing the bacterial cell components is not particularly limited, and specifically, the ability to separate bacterial cell components equal to or higher than the eluate when the mixture is filtered with a filter having a pore size of 0.22 ⁇ m or less is used. Any method can be used. That is, in the present invention, the removal accuracy in removing the bacterial cell component may be at least as high as that achieved by a filter having a pore diameter of 0.22 ⁇ m or less.
- More specific examples of the method for removing the bacterial cell component include, for example, filter filtration, centrifugation, and membrane filtration, and preferably filter filtration.
- the pore size of the filter used for filter filtration is, for example, 10 ⁇ m or less.
- the filter filtration may be performed in plural stages using filters having different pore sizes so that the pore size of the filter to be filtered is gradually reduced. In this case, the filter filtration can be carried out in 2 to 4 steps, preferably 3 to 4 steps.
- the pore size of the filter used in the final stage is preferably 0.22 ⁇ m or less from the viewpoint of more properly removing the bacterial cell component.
- the material of the filter used include cellulose, perlite, resin, diatomaceous earth, polyether sulfone, cellulose acetate, polyvinylidene fluoride and the like.
- step (B) What is removed in step (B) is mainly bacterial components.
- the nucleic acid component may be partially removed together with the bacterial cell component.
- bacterial cell components remain on the filter, and the eluate is collected as a mixture b.
- a mixture b containing the nucleic acid component and the botulinum toxin can be obtained.
- the mixture b may be obtained as an appropriately concentrated product.
- it may be obtained by further concentrating the eluate that has passed through the above filter.
- the concentration method is not particularly limited, and membrane concentration is preferable.
- the pore size (molecular weight cutoff) of the ultrafiltration membrane may be 20 to 40 KDa, preferably 25 to 35 KDa.
- the mixture b is free of bacterial components and can be obtained as a substantially clear liquid. More preferably, the mixture b can be obtained in a mode including a nucleic acid component and a botulinum toxin together with the above-mentioned buffer solution. Further, the mixture b may also contain the above-mentioned medium components.
- step (C) Endonuclease treatment
- an endonuclease is added to the mixture b to obtain a mixture c containing a nucleic acid degradation product and a botulinum toxin.
- the technique such as acid precipitation for precipitating the bacterial cell component and the nucleic acid component together with the botulinum toxin, which is often used in the production of botulinum toxin, is not performed.
- an endonuclease is added to the mixture b from which the bacterial cell component has been removed in advance.
- the nucleic acid component in the mixture b is fragmented to generate a nucleic acid decomposition product.
- the nucleic acid degradation product can be removed with excellent efficiency in the step (D) described below (the nucleic acid species can be removed so that the remaining nucleic acid species can be reduced). ) Is possible.
- the endonuclease since the bacterial cell component is removed in advance with the mixture b, the endonuclease easily acts on the nucleic acid component without being disturbed by the bacterial cell component. Therefore, it becomes possible to suppress the remaining undigested nucleic acid and efficiently fragment the nucleic acid. This point also contributes to the removal of the nucleic acid degradation product with excellent efficiency in the step (D) described below.
- an enzyme having an endonuclease activity can be used without particular limitation.
- the nucleic acid component serving as a substrate for endonuclease includes DNA and RNA, and preferably an enzyme capable of degrading both DNA and RNA.
- Specific endonucleases include Shewanella sp. The endonuclease derived from Staphylococcus aureus, the endonuclease derived from Serratia marcescens, and the endonuclease derived from Serratia are particularly preferred.
- endonuclease derived from Serratia include Benzonase (registered trademark of Merck K.G.A.A.R.A.), Denalase (registered trademark of Seerector GmbH), and Kaneka endonuclease (manufactured by Kaneka).
- Benzonase and denarase are preferred, and benzonase is more preferred.
- the optimum pH of the endonuclease derived from Serratia is higher than that of general endonucleases, and is specifically about 7.5 to 9.2. More specifically, the optimum pH of benzonase is 8.0 to 9.2, the optimum pH of denalase is 8.0 to 9.0, and the optimum pH of Kaneka endonuclease is 7.5 to 9.0. is there.
- the endonuclease is allowed to act under the conditions of preferably pH 5.8 to 6.5, more preferably pH 5.9 to 6.3. Is preferred. Also when using the above-mentioned preferred endonuclease, which is derived from Serratia bacterium, it is preferable that the endonuclease is allowed to act under the above-mentioned pH condition, although it largely deviates from the optimum pH. In the present invention, the nucleic acid component can be efficiently removed in the step (D) described below even if the endonuclease derived from Serratia is allowed to act under the condition of pH 5.8 to 6.5.
- the number of times to add the endonuclease to the mixture b in the step (C) is not particularly limited, and may be once or plural times.
- the endonuclease can be added 2 to 4 times, more preferably 2 to 3 times.
- the timing of each addition in multiple additions is not particularly limited, but the addition can be performed at intervals of, for example, 1 to 12 hours, preferably 2 to 9 hours, and more preferably 3 to 7 hours.
- the temperature at which the endonuclease acts is, for example, 25 to 40°C, preferably 28 to 38°C.
- the total time for the endonuclease treatment is, for example, 10 to 24 hours, preferably 12 to 20 hours.
- a series of steps (C) and (D) described below may be repeated a plurality of times. In this case, it is preferable that the endonuclease is added to the mixture b once in the step (C).
- the specific number of repetitions is, for example, 2 to 4 times, preferably 2 to 3 times.
- the present invention includes the step (A), the step (B), the step (C), the step (D), the step (C), and the step (D) in this order. It will be.
- a mixture c containing a nucleic acid degradation product and a botulinum toxin can be obtained.
- the mixture c may be obtained as a filtrate that has been subjected to a filtration treatment such as filter filtration, for the purpose of appropriately removing nucleic acids that have not been decomposed.
- the mixture c can be obtained in a mode including an endonuclease residue and a buffer together with a nucleic acid degradation product and a botulinum toxin.
- the mixture c can also subsequently contain the above-mentioned medium components.
- the mixture c is subjected to the removal of the nucleic acid degradation product to obtain the botulinum toxin isolated liquid d.
- the method for removing the nucleic acid degradation product is not particularly limited as long as it can separate the nucleic acid degradation product and the botulinum toxin, but an anion exchange carrier is not used.
- an anion exchange carrier is used, the yield of toxin is limited, and not only when the toxin and nucleic acid species are first captured and then the toxin is eluted by pH gradient elution, but only the nucleic acid species from the beginning.
- the anion exchange carrier is not used for removing the nucleic acid degradation product from the viewpoints of toxin yield, specific activity of toxin, workability and the like.
- a method capable of separating the nucleic acid decomposed product and the botulinum toxin can be used without particular limitation, as long as the anion exchange carrier is not used.
- removal by a membrane can be mentioned, and membrane filtration is preferable. This allows a low molecular weight substance such as a nucleic acid degradation product to permeate through the membrane and be separated from a botulinum toxin that does not permeate through the membrane.
- the pore size of the membrane used for removal by the membrane is, for example, 20 to 40 KDa, preferably 25 to 35 KDa as the molecular weight cutoff.
- a buffer solution capable of dissolving botulinum toxin such as a phosphate buffer solution or an acetate buffer solution.
- the botulinum toxin in the botulinum toxin isolate d obtained in the step (D) is contained in the form of M toxin. Be done.
- the botulinum toxin isolate d can be obtained as a clear liquid. More preferably, the botulinum toxin isolated solution d can be obtained in a mode containing the botulinum toxin together with the above-mentioned buffer solution.
- the botulinum toxin isolate d may also contain the above-mentioned medium components.
- the botulinum toxin isolated liquid d can be further subjected to any purification step capable of purifying botulinum toxin.
- the method for purifying the botulinum toxin is not particularly limited and can be appropriately selected by those skilled in the art according to the form of the botulinum toxin. From the viewpoint of obtaining excellent purity, purification by column chromatography can be mentioned, and ion exchange column chromatography is more preferable. The specific technique of ion exchange column chromatography can be appropriately determined depending on the pH dependency of the botulinum toxin.
- Botulinum toxin has the property of forming cations under acidic conditions, preferably pH 4.0 to 5.0, preferably about pH 4.0, and forming anions at pH 7.0 or higher, preferably about pH 7.5.
- the botulinum toxin complex stably exists as a complex under mildly acidic conditions, preferably pH 5.5 to 6.5, preferably about pH 6.0, and is increased by pH increase, for example, pH 7.3 to 8.0.
- purification of the botulinum toxin complex by the following step (E), or further purification of the botulinum toxin non-complex by the following step (F) is mentioned.
- step (E) Purification of botulinum toxin complex
- the botulinum toxin isolate liquid d is subjected to cation exchange chromatography to obtain a purified botulinum toxin complex. Since the botulinum toxin complex forms a cation under acidic conditions, purification by cation exchange chromatography is excellent in that the botulinum toxin complex can be stably captured and purified. Further, purification by cation exchange chromatography is also excellent in that it can be purified under pH conditions that can suppress dissociation of the botulinum toxin complex.
- Elution of the captured botulinum toxin is performed by using the same buffer as the buffer for equilibration, for example, 0.5 to 0.8 mol/L from the salt concentration of the buffer for equilibration. For example, it can be carried out by flowing under a gradient condition of up to 0.7 mol/L. Thereby, the purified botulinum toxin complex can be obtained as an eluate.
- the cation exchange carrier is not particularly limited, but examples thereof include SP Sepharose Fast Flow (GE Healthcare), CM Sepharose Fast Flow (GE Healthcare), S Sepharose Fast Flow (GE Healthcare), and SP Toyopearl (Tosoh). Commercially available cation exchange resins can be mentioned.
- the type of salt in the buffer solution is not limited and may be used so that the ionic strength has a concentration corresponding to the above-mentioned concentration of sodium chloride.
- the salt in the buffer solution includes sodium chloride.
- the botulinum toxin complex can be obtained by purification using cation exchange chromatography.
- the pH can be adjusted to 5.5 to 6.5, and specifically, pH 6.0, if necessary. If necessary, it can be obtained as having been subjected to treatments such as concentration and filtration.
- a specific method for concentration can be selected from the methods described in the step (B).
- Specific methods of filtration include methods used for the purpose of sterilization or final filtration, and examples include a method using a filter having a pore size of 0.15 to 0.3 ⁇ m, and a specific example of 0.22 ⁇ m.
- an additive such as a preservative may be further added before the filtration.
- step (F) Purification of botulinum toxin non-complex
- the purified product of the botulinum toxin complex is subjected to anion exchange chromatography under conditions where a non-toxic non-HA protein deviates from the botulinum toxin complex, and botulinum A purified product of non-toxin complex is obtained.
- the purified product of the botulinum toxin complex may be subjected to a condition in which a nontoxic non-HA protein deviates from the botulinum toxin complex in advance, before being subjected to anion exchange chromatography.
- the conditions under which the nontoxic non-HA protein dissociates from the botulinum toxin complex are preferably pH 7.3 to 8.5, more preferably pH 7.4 to 8.0, and a specific example is 7.5.
- the temperature is preferably 10° C. or lower.
- a phosphate buffer, Tris buffer, HEPES buffer adjusted to the pH and having a salt concentration of 0.1 mol/L or less, preferably 0.05 mol/L or less.
- the non-toxic non-HA protein can be dissociated by substituting the buffer solution such as, or the like, preferably a phosphate buffer solution.
- the pore size (molecular weight cut-off) of the ultrafiltration membrane is 5 to 30 KDa, preferably 8 to 20 KDa, more preferably 9 to 15 KDa.
- the anion exchange carrier is preliminarily equilibrated with a buffer solution adjusted to a condition (preferably pH 7.3 to 8.5, as a specific example, pH 7.5) at which the nontoxic non-HA protein dissociates from the botulinum toxin complex.
- a buffer solution adjusted to a condition (preferably pH 7.3 to 8.5, as a specific example, pH 7.5) at which the nontoxic non-HA protein dissociates from the botulinum toxin complex.
- the salt concentration of the buffer solution for equilibration is 0.1 mol/L or less, preferably 0.05 mol/L or less.
- the type of buffer solution is not particularly limited, and examples thereof include a phosphate buffer solution, a Tris buffer solution, and a HEPES buffer solution, and a phosphate buffer solution is preferable.
- anionization is performed depending on the pH of the contact environment. Trapped botulinum toxin (botulinum toxin non-complex).
- Elution of the captured botulinum toxin non-complex was carried out by using the same buffer as the equilibration buffer from the salt concentration of the equilibration buffer, for example, 0.1 to 0.5 mol/L, preferably 0.1. It can be performed under a gradient condition of 2 to 0.4 mol/L, and as a specific example, up to 0.3 mol/L. As a result, a purified botulinum toxin non-complex can be obtained as an eluate.
- the separated non-toxic non-HA protein can also be captured by the carrier, but it can be separated from the botulinum toxin non-complex by a method that utilizes the fact that the elution timing differs depending on the salt concentration gradient of the mobile phase.
- the anion exchange carrier is not particularly limited, but weak in view of facilitating the elution of the botulinum toxin non-complex and facilitating the separation of the non-toxic non-HA protein dissociated from the botulinum toxin non-complex, It is preferably an anion exchange carrier.
- anion exchange carrier examples include commercially available anion exchange resins such as DEAE Sepharose Fast Flow (GE Healthcare), DEAE Toyopearl (Tosoh), Diaion WA10 (Mitsubishi Chemical), and Fractogel DEAE (Merck).
- the type of salt in the buffer solution is not limited and may be used so that the ionic strength has a concentration corresponding to the above-mentioned concentration of sodium chloride.
- the salt in the buffer solution includes sodium chloride.
- the botulinum toxin non-complex can be obtained by the purification using anion exchange chromatography.
- the botulinum toxin non-complex can also be obtained after being subjected to a treatment such as concentration and/or filtration, if necessary.
- a treatment such as concentration and/or filtration, if necessary.
- an additive such as a preservative may be further added.
- concentration and filtration can be selected from the methods described in the above step (E).
- the protein can be measured by an ultraviolet absorption measuring method (280 nm), a Lowry method, a BCA method or the like.
- an ultraviolet absorption measuring method 280 nm
- a Lowry method a BCA method or the like.
- U the LD50 value per 1 mL of the botulinum toxin solution
- the obtained measured value is the botulinum toxin. It can be derived by dividing by the amount of protein (mg) in 1 mL of the liquid.
- Botulinum toxin preparation The purified botulinum toxin obtained by the production method of the present invention is useful as an active ingredient of a botulinum preparation.
- Examples of the use of the botulinum preparation include all clinically applied uses, and examples thereof include post-stroke upper limb/lower limb spasticity, dystonia, hemi-facial spasm, sequelae of cerebrovascular accidents, and cosmetic surgery.
- the form of the botulinum formulation is not particularly limited, and may be liquid or solid.
- the solid botulinum formulation may be a freeze-dried, vacuum-dried or spray-dried product of the liquid formulation, which can be reconstituted as a liquid formulation by dissolving in saline or water at the time of use.
- Botulinum toxin was produced by the following procedure.
- a pre-culture step consisting of static culture 1 and static culture 2 and a main culture step consisting of a cell growth stage by stirring culture and a fermentation stage were carried out. That is, after the pre-culture step and the bacterial growth step of (A1) main culture step, the fermentation step of (A2) main culture step was performed.
- Bacto Proteose Peptone No. 3 (Butaton Peckton, Becton Dickinson) (final concentration 2 w/v%), yeast extract (final concentration 1 w/v%), sodium thioglycolate (final concentration 0.025 w/v%), and glucose (final concentration) 0.5 w/v%) containing 10 L of pH 7.3 medium
- yeast extract final concentration 1 w/v%)
- sodium thioglycolate final concentration 0.025 w/v%)
- glucose final concentration 0.5 w/v%) containing 10 L of pH 7.3 medium
- the pH of the medium was lowered to 5.8 by adding 1 mol/L HCl, and the fermentation step was performed.
- 1 mol/L HCl was added as needed, and the pH of the medium was maintained at 5.8 until the end.
- the time required for the main culture step was about 44 hours.
- the filtrate obtained by the membrane filtration is a 20 mmol/L phosphate buffer solution (pH 6.0) containing 0.1 mol/L NaCl and 20 mmol/L citric acid and a membrane having a pore size (molecular weight cut-off) of 30 KDa (Sartorius Slice Slice).
- the membrane was concentrated using Hydrosart 30 kD).
- the 0.9 L membrane concentrate thus obtained is also referred to as harvest liquid 11.
- a half amount of 0.9 L (0.45 L) was concentrated using a 10 mmol/L phosphate buffer solution (pH 6.0) and a membrane having a pore size (molecular weight cut-off) of 30 KDa (Sartorius Slice Hydrosart 30 kD manufactured by Sartorius). did.
- the membrane concentrate obtained by this is also referred to as a harvest liquid 12.
- the obtained membrane concentrate was subjected to membrane concentration using a 50 mmol/L sodium acetate (pH 4.2) containing 0.2 mol/L NaCl and a membrane having a pore size (fraction molecular weight) of 30 KDa (Sarton Slice Hydrosart 30 kD manufactured by Sartorius). ..
- the membrane concentrate obtained by these is also referred to as botulinum toxin isolate 1.
- Botulinum toxin isolate 1 was subjected to cation exchange chromatography.
- a cation exchange carrier SP-Sepharose-fast flow was used, and the carrier was packed in a column of 70 ⁇ 550 mm size for 30 cm.
- the column was equilibrated with 50 mmol/L sodium acetate (pH 4.2) containing 0.2 mol/L NaCl as the washing solution, botulinum toxin isolate 1 was applied, and 50 mmol/L containing 0.7 mol/L NaCl as the eluent. Elution was performed with sodium acetate (pH 4.2) up to 0.7 mol/L NaCl under a gradient condition of 8 column volumes.
- the flow rate was about 15 mL/min.
- the obtained eluate was subjected to membrane concentration using a 50 mmol/L acetate buffer (pH 6.0) containing 0.2 mol/L NaCl and a membrane having a pore size (molecular weight cut off) of 30 KDa (Sartorice Slice Hydrosart 30 kD manufactured by Sartorius).
- the obtained concentrated solution was further filtered using a filter having a pore size of 0.22 ⁇ m (bottle top filter manufactured by Corning, manufactured by PES) to obtain a purified botulinum toxin complex (M toxin).
- (F) Purification of botulinum toxin non-complex The obtained purified product of botulinum toxin complex (M toxin) was used as a membrane (Sartorius) with 10 mmol/L phosphate buffer (pH 7.5) and pore size (fraction molecular weight) of 10 KDa.
- the membrane was concentrated using a Sarton Slice 50 Hydrosart 10 kD (manufactured by Mfg. Co., and Pellicon XL Ultracell 10 kD manufactured by Millipore). Then, the membrane concentrate was subjected to anion exchange column chromatography.
- the anion exchange column carrier DEAE-Sepharose-fast flow, which is a weak anion exchange carrier, was used, and the carrier was packed in a column of 10 ⁇ 450 mm for 30 cm.
- the column was equilibrated using 10 mmol/L phosphate buffer (pH 7.5) as a washing solution, the membrane concentrate was applied, and 10 mmol/L phosphate buffer (pH 7.5) containing 0.3 mol/L NaCl was used as an eluent. ) was used for elution under a gradient condition of 10 column volumes up to 0.3 mol/L NaCl. The flow rate was approximately 1 mL/min.
- S toxin purified product 1 A purified product of non-toxin complex (S toxin) (hereinafter, also referred to as S toxin purified product 1) was obtained.
- Example 1 A purified botulinum toxin non-complex (S toxin) was obtained in the same manner as in Example 1 except that the above-mentioned steps (C) and (D) were replaced by the following method. Specifically, using the remaining half amount 0.45 L of the harvest liquid 11 (filter filtrate) obtained in the step (B) of Example 1, the following anion exchange chromatography, membrane concentration and filtration were performed. After that, the steps (E) and (F) of Example 1 were performed.
- Harvest liquid 11 was subjected to anion exchange chromatography.
- Qsepharose-fast flow was used as the anion exchange carrier, and the carrier was packed in a 140 ⁇ 500 mm column for 10 cm.
- the column was equilibrated using a 20 mmol/L phosphate buffer solution (pH 6.0) containing 0.1 mol/L NaCl and 20 mmol/L citric acid as a washing solution, and the harvest solution obtained in step (B) of Example 1 was used.
- 11 was applied, the same washing solution was flown at a flow rate of about 150 mL/min, and the toxin fraction was collected as a pass-through fraction without being adsorbed.
- the recovered solution was subjected to membrane concentration using a 50 mmol/L sodium acetate (pH 4.2) containing 0.2 mol/NaCl and a membrane (Sartocon Slice Hydrosart 30 kD) having a pore size (molecular weight cutoff) of 30 KDa.
- the obtained membrane concentrate is also referred to as botulinum toxin isolate 1'.
- the same steps (E) and (F) as in Example 1 were performed to obtain a purified product of a botulinum toxin non-complex (S toxin) (hereinafter, also referred to as S toxin purified product 1').
- S toxin botulinum toxin non-complex
- Example 1 Confirmation of Purification
- the harvest liquid 12, the botulinum toxin isolated liquid 1, and the S toxin purified product 1 were subjected to SDS-PAGE, and in Comparative Example 1, the harvest liquid 11 and the botulinum toxin alone were used.
- the separated liquid 1′ and the S toxin purified product 1′ were subjected to SDS-PAGE and subjected to CBB staining to confirm the purification of botulinum toxin.
- the obtained stained gel is shown in FIG. 1 (Example 1) and FIG. 2 (Comparative Example 1).
- A2-NTX(H) shows a heavy chain of about 100 KDa
- A2-NTX(L) shows a light chain of about 50 KDa.
- the lane to the right of the marker represents the culture supernatant of the main culture solution, and the lane between the botulinum toxin isolate 1 and the purified S toxin 1 is from the left, the botulinum toxin complex ( M toxin), a toxin-containing solution after DEAE chromatography, an impurity solution 1 after DEAE chromatography, and an impurity solution 2 after DEAE chromatography.
- the lane to the right of the marker represents the culture supernatant of the main culture, and the lane between the botulinum toxin isolate 1'and the S toxin purified product 1'from the left is anion exchange chromatography.
- the impurity liquid 1 after the graphic, the impurity liquid 2 after the DEAE chromatography, the impurity liquid 3 after the DEAE chromatography, and the impurity liquid 4 after the DEAE chromatography are shown.
- Example 1 Purification efficiency by the nucleic acid removal operation was measured. Specifically, the purification efficiency in Example 1 is the same as that in the botulinum toxin isolate 1 obtained in step (D) with respect to the amount of nucleic acid in the harvest liquid 12 (membrane concentrate) obtained in step (B). The ratio (%) of the amount of nucleic acid was calculated and obtained by subtracting the ratio from 100%. The purification efficiency in Comparative Example 1 was calculated by calculating the ratio of the amount of nucleic acid in the botulinum toxin isolate 1'to the amount of nucleic acid in the harvest liquid 11 (filter filtrate) obtained in step (B), and from 100%, Obtained by subtracting the ratio.
- the amount of nucleic acid is the amount of DNA measured by ds DNA HS Assay Kit using a Qubit 3.0 fluorometer (made by Thermo Fisher) and the amount of RNA measured by RNA HS Assay Kit using the same device. It was derived as the total amount.
- the nucleic acid removal operation of Example 1 achieved significantly higher purification efficiency than the nucleic acid removal operation of Comparative Example 1.
- the botulinum toxin yield in Example 1 is the botulinum toxin isolated liquid obtained in step (D) with respect to the total amount of toxin in the harvest liquid 12 (membrane concentrate) obtained in step (B).
- the botulinum toxin yield in Comparative Example 1 was calculated as the ratio of the total toxin amount in 1; the botulinum toxin isolate 1 with respect to the total toxin amount in the harvest liquid 11 (filter filtrate) obtained in step (B). It was calculated as the ratio of the total amount of toxins in'.
- Example 1 As a result, the toxin yield by the nucleic acid removal operation in Example 1 was 53.18%, the toxin yield by the nucleic acid removal operation in Comparative Example 1 was 50.46%, and Example 1 was compared with Comparative Example 1. And the toxin yield was high.
- the titer in U/mL was derived as follows.
- the obtained purified product of S toxin was appropriately diluted, and 0.1 mL per mouse was inoculated through the tail vein of the mouse, and then the survival time of the mouse was confirmed.
- the LD50 value (U/mL) per mL of the purified product of S toxin was calculated from the regression line of the survival time verified beforehand and the LD50 value by intraperitoneal administration. Further, the protein mass (mg/mL) per 1 mL of the purified product of S toxin was measured by infrared absorption at 280 nm.
- the LD50 value (U/mL) per 1 mL of purified S toxin was divided by the protein mass (mL/mL) per 1 mL of purified S toxin to derive the specific activity (U/mg).
- the specific activity of the purified S toxin product 1 obtained in Example 1 was 8.21 ⁇ 10 7 U/mg, and the specific activity of the purified S toxin product 1′ obtained in Comparative Example 1 was 2.59 ⁇ . It was 10 7 U/mg, and Example 1 was able to obtain a purified product having a higher specific activity than Comparative Example 1. ..
- Example 2 a botulinum toxin was produced in the same manner as in Example 1 except that an animal-free medium was used in the botulinum toxin production step and the main modification was the use of denarase as an endonuclease. Specifically, botulinum toxin was produced by the following procedure.
- the filtrate obtained by the membrane filtration is a 20 mmol/L phosphate buffer solution (pH 6.0) containing 0.1 mol/L NaCl and 20 mmol/L citric acid and a membrane having a pore size (molecular weight cut-off) of 30 KDa (Sartorius Slice Slice).
- the membrane was concentrated using Hydrosart 30 kD). Further, the membrane was concentrated using a 10 mmol/L phosphate buffer (pH 6.0) and a membrane having a pore size (molecular weight cut-off) of 30 kDa (Sartorius Sarton Slice Hydrosart 30 kD).
- the membrane concentrate obtained by these is also referred to as a harvest liquid 22.
- the obtained membrane concentrate was used for about 20 minutes using a 50 mmol/L sodium acetate (pH 4.2) containing 0.2 mol/L NaCl and a membrane with a pore size (molecular weight cut-off) of 30 KDa (Sartorice Slice Hydrosart 30 kD manufactured by Sartorius). Membrane concentrated.
- the membrane concentrate obtained by these is also referred to as botulinum toxin isolate 2.
- Botulinum toxin isolate 2 was subjected to cation exchange chromatography in the same manner as in Example 1.
- the obtained eluate was subjected to membrane concentration using a 50 mmol/L acetate buffer (pH 6.0) containing 0.2 mol/L NaCl and a membrane having a pore size (molecular weight cut-off) of 30 KDa (Sartorius Slice Hydrosart 30 kD manufactured by Sartorius).
- the mixture was filtered using a 0.22 ⁇ m pore size filter (bottle top filter manufactured by Corning, manufactured by PES) to obtain a purified botulinum toxin complex (M toxin).
- the resulting eluate was subjected to membrane concentration using a 10 mmol/L phosphate buffer (pH 7.5) and a membrane having a pore size (molecular weight cut-off) of 10 KDa (Sartorius Sarton Slice 50 Hydrosart 10 kD), and botulinum toxin complex.
- a purified product of the body (M toxin) was obtained.
- the column was equilibrated with 10 mmol/L phosphate buffer (pH 7.5) as a washing solution, the purified product of botulinum toxin complex (M toxin) was applied, and 10 mmol/L containing 0.3 mol/L NaCl as an eluent.
- phosphate buffer pH 7.5
- elution was performed up to 0.3 mol/L NaCl under a gradient condition of 10 column volumes.
- the flow rate was approximately 1 mL/min.
- a preservative (L-arginine hydrochloride) was added to the obtained eluate so that the final concentration was 1 w/v%, and the mixture was filtered using a 0.22 ⁇ m pore size filter (Starlab PES syringe filter) and botulinum.
- a purified product of non-toxin complex (hereinafter, also referred to as S toxin purified product 3) was obtained.
- Example 2 Confirmation of Purification
- the harvest liquid 22, the botulinum toxin isolate 2 and the S toxin purified product 2 were subjected to SDS-PAGE, and in Example 3, the harvest liquid 32 and the botulinum toxin alone were used.
- the syneresis 3 and the purified S toxin 3 were subjected to SDS-PAGE and stained with CBB to confirm the purification of the botulinum toxin.
- the methods of SDS-PAGE and staining are the same as in Test Example 1.
- the obtained stained gel is shown in FIG. 3 (Example 2) and FIG. 4 (Example 3).
- A2-NTX(H) shows a heavy chain of about 100 KDa
- A2-NTX(L) shows a light chain of about 50 KDa.
- the lane between the botulinum toxin isolate 2 and the S toxin purified product 2 represents, from the left, a purified product of the botulinum toxin complex (M toxin) and a toxin-containing liquid after DEAE chromatography. ..
- the lane to the left of the harvest liquid 32 represents the culture supernatant of the main culture liquid, and the lane between the botulinum toxin isolate 3 and the purified S toxin 3 is from the left, after SP chromatography.
- a toxin-containing liquid, a purified product of a botulinum toxin complex (M toxin), a toxin-containing liquid after DEAE chromatography, an impurity liquid 1 after DEAE chromatography, and an impurity liquid 2 after DEAE chromatography are shown.
- botulinum toxin yield was measured by the nucleic acid removal procedure. Specifically, the yield of the botulinum toxin in Examples 2 and 3 was determined as follows: It was calculated as the ratio of the total amount of toxin in the toxin isolates 2 and 3.
- the toxin yield by the nucleic acid removal operation in Example 2 was 59.71%
- the toxin yield by the nucleic acid removal operation in Example 3 was 89.68%.
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Abstract
Description
項1.(A)培地中でボツリヌス毒素産生菌からボツリヌス毒素を産生させ、前記ボツリヌス毒素由来の菌体成分及び核酸成分とボツリヌス毒素とを含む混合物aを得る工程と、
(B)前記混合物aを前記菌体成分の除去に供し、核酸成分とボツリヌス毒素とを含む混合物bを得る工程と、
(C)前記混合物bにエンドヌクレアーゼを添加し、核酸分解物とボツリヌス毒素とを含む混合物cを得る工程と、
(D)前記混合物cを核酸分解物の除去に供し、ボツリヌス毒素単離液dを得る工程と、
を含む、ボツリヌス毒素の製造方法。
項2. 前記(C)工程において、前記エンドヌクレアーゼがセラチア菌Serratia marcescens由来のエンドヌクレアーゼである、項1に記載のボツリヌス毒素の製造方法。
項3. 前記(C)工程を、pH5.8~6.5の条件下で行う、項1又は2に記載のボツリヌス毒素の製造方法。
項4. 前記(C)工程において、前記エンドヌクレアーゼを複数回添加する、項1~3のいずれかに記載のボツリヌス毒素の製造方法。
項5. 前記(D)工程において、前記核酸分解物の除去が膜による除去を含む、項1~4のいずれかに記載のボツリヌス毒素の製造方法。
項6. さらに、(E)前記ボツリヌス毒素単離液dを、陽イオン交換クロマトグラフィーに供し、ボツリヌス毒素複合体の精製物を得る工程を含む、項1~5のいずれかに記載のボツリヌス毒素の製造方法。
項7. さらに、(F)前記ボツリヌス毒素複合体の精製物を、前記ボツリヌス毒素複合体から無毒性非HAタンパク質が乖離する条件下で陰イオン交換クロマトグラフィーに供し、ボツリヌス毒素非複合体の精製物を得る工程を含む、項6に記載のボツリヌス毒素の製造方法。
項8. 少なくとも前記(A)工程が前培養工程と本培養工程とを含み、少なくとも前記前培養工程を静置培養で行う、項1~7のいずれかに記載のボツリヌス毒素の製造方法。
項9. 前記前培養工程と前記本培養工程との両方を静置培養で行う、項1~8に記載のボツリヌス毒素の製造方法。
項10. 前記(A)工程が、
(A1)pH6.8~8.0の培地中で前記ボツリヌス毒素産生菌の菌体増殖を行う工程と、
(A2)pH5.0~6.5の培地中で前記ボツリヌス毒素産生菌の発酵を行う工程と、
をこの順番で含む、項8又は9に記載のボツリヌス毒素の製造方法。
項11. 前記(A)工程において、前記ボツリヌス毒素産生菌が、芽胞の形態で保存された菌体の発芽体である、項1~10のいずれかに記載のボツリヌス毒素の製造方法。
項12. 前記(B)工程において、前記菌体成分の除去がフィルターろ過を含む、項1~11のいずれかに記載のボツリヌス毒素の製造方法。
項13. 前記(F)工程において、前記条件がpH7.3~8.5である、項7~12のいずれかに記載のボツリヌス毒素の製造方法。
項14. 前記(F)工程において、陰イオン交換クロマトグラフィーが弱陰イオン交換クロマトグラフィーである、項7~13のいずれかに記載のボツリヌス毒素の製造方法。
項15. 比活性が3.0×107U/mg以上であることを特徴とする、精製ボツリヌス毒素。
項16. 項1~14のいずれかに記載のボツリヌス毒素の製造方法によって製造される、精製ボツリヌス毒素。
(A)工程では、培地中でボツリヌス毒素産生菌からボツリヌス毒素を産生させ、前記ボツリヌス毒素由来の菌体成分及び核酸成分とボツリヌス毒素とを含む混合物aを得る。
(B)工程では、(A)工程で得られた混合物aを前記菌体成分の除去に供し、核酸成分とボツリヌス毒素とを含む混合物bを得る。本発明では、例えば酸沈殿のような混合物aからボツリヌス毒素とともに菌体成分と核酸成分とを沈殿させる手法は行わず、先に核酸成分を除去する手法も行わない。このため、本発明は、酸沈殿等の沈殿処理のような煩雑な操作が不要となる。また、酸沈殿を行わないため、ボツリヌス毒素のロスが少なく、優れた収率を達成することができる。さらに、ボツリヌス毒素が強い酸性条件に晒されないため、ボツリヌス毒素の毒素活性を良好に保つことができる。なお、本発明では、(B)工程による菌体成分の除去を行う前に、混合物aにヌクレアーゼを加えることも行わない。
(C)工程では、混合物bにエンドヌクレアーゼを添加し、核酸分解物とボツリヌス毒素とを含む混合物cを得る。上述のとおり、本発明では、ボツリヌス毒素の製造でよく行われる、ボツリヌス毒素とともに菌体成分と核酸成分とを沈殿させる酸沈殿等の手法は行わない。まず、核酸成分を除去するため、菌体成分が予め除去された混合物bに対してエンドヌクレアーゼを添加する。エンドヌクレアーゼの作用によって、混合物b中の核酸成分が断片化され、核酸分解物を生じる。これによって、陰イオン交換カラムクロマトグラフィーを用いなくとも、後述の(D)工程において優れた効率で核酸分解物を除去すること(残存する核酸種がより少なくなるように核酸種が除去されること)が可能になる。また、本発明では、混合物bで予め菌体成分が除去されているため、菌体成分に邪魔されることなくエンドヌクレアーゼが核酸成分に対して作用しやすい。従って未消化核酸の残存を抑制して効率的に断片化することも可能となる。この点も、後述の(D)工程において優れた効率で核酸分解物を除去することに寄与する。
工程(D)では、混合物cを核酸分解物の除去に供し、ボツリヌス毒素単離液dを得る。核酸分解物の除去の手法としては、核酸分解物とボツリヌス毒素とを分離できる手段であれば特に限定されないが、陰イオン交換担体は用いない。陰イオン交換担体を用いた場合には、毒素収率に限界があり、最初に毒素と核酸種との両方を捕捉した後にpHグラジエント溶離で毒素を溶出させる場合のみならず、最初から核酸種のみを捕捉して毒素を捕捉せずに溶離させる場合であっても、毒素の回収が不十分となる。また、陰イオン交換担体を用いると、比活性が十分な毒素を得ることもできない。さらに、陰イオン交換担体を用いると、作業員による作業時間が長く、コンタミネーションの機会も増える。従って、本発明では、毒素収率、毒素の比活性、作業性等の観点から、核酸分解物の除去に陰イオン交換担体は用いない。
(E)工程では、ボツリヌス毒素単離液dを、陽イオン交換クロマトグラフィーに供し、ボツリヌス毒素複合体の精製物を得る。ボツリヌス毒素複合体は、酸性条件で陽イオンを形成するため、陽イオン交換クロマトグラフィーによる精製は、ボツリヌス毒素複合体を安定的に捕捉し精製できる点で優れている。また、陽イオン交換クロマトグラフィーによる精製は、ボツリヌス毒素複合体の乖離を抑制できるpH条件で精製できる点でも優れている。
(F)工程では、ボツリヌス毒素複合体の精製物を、ボツリヌス毒素複合体から無毒性非HAタンパク質が乖離する条件下で陰イオン交換クロマトグラフィーに供し、ボツリヌス毒素非複合体の精製物を得る。
本発明の方法で得られるボツリヌス毒素の精製体(精製ボツリヌス毒素とも記載する)は、比活性に優れる。具体的な比活性としては、例えば3.0×107U/mg以上、好ましくは4.0×107U/mg以上、より好ましくは5.0×107U/mg以上、更に好ましくは6.0×107U/mg以上、一層好ましくは7.0×107U/mg以上、より一層好ましくは8.0×107U/mg以上、特に好ましくは9.0×107U/mg以上、最も好ましくは10.0×107U/mg以上が挙げられる。
本発明の製造方法によって得られる精製ボツリヌス毒素は、ボツリヌス製剤の有効成分として有用である。ボツリヌス製剤の用途としては、臨床応用されているあらゆる用途が挙げられ、例えば、脳卒中後上肢/下肢痙縮、ジストニア、半側顔面痙攣、脳血管障害後後遺症、美容整形などが挙げられる。
[1]ボツリヌス毒素の製造
以下の手順で、ボツリヌス毒素を製造した。
(A)ボツリヌス菌の産生
静置培養1及び静置培養2からなる前培養工程と、撹拌培養による菌体増殖段階及び発酵段階からなる本培養工程とを行った。つまり、前培養工程と(A1)本培養工程の菌体増殖段階を行った後、(A2)本培養工程の発酵段階を行った。
上記本培養工程によって得られた培養物を、以下のフィルターと膜を用いて、粗ろ過及び膜濃縮した。フィルターろ過は、本培養工程によって得られた培養物を、フィルター1[3M社製ゼータプラスフィルター(05SP)/1020cm2;孔径1.0~10μm相当]、フィルター2[3M社製ゼータプラスフィルターマキシマイザー(60SP03A)/1020cm2;孔径0.1~1.0μm相当]、及びフィルター3[ザルトリウス社製ザルトポア2(孔径0.45μm+0.2μm)/0.1m2]の順に通すフィルターろ過により行った。膜ろ過で得られたろ液を0.1mol/LNaClと20mmol/Lクエン酸とを含む20mmol/Lリン酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮した。これによって得られた0.9Lの膜濃縮液をハーベスト液11とも記載する。続いて0.9Lの半量(0.45L)を、10mmol/Lリン酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮した。これによって得られた膜濃縮液を、ハーベスト液12とも記載する。
終濃度が1mmol/LとなるMgCl2と、終濃度が20U/mLとなるベンゾナーゼ(メルク・カー・ゲー・アー・アー登録商標)とをハーベスト液12へ添加し、pH6.0の条件下で撹拌しながら30℃で約15時間反応させた。ベンゾナーゼの添加回数は1回であった。その後、エンドヌクレアーゼ処理物を、3M社製Zeta Plus Encapsulated Filterを用いてフィルターろ過した。
得られたろ液を、10mmol/Lリン酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮し、核酸分解物を除去した。なお、この膜による核酸分解物の除去には、タンジェンシャルフローろ過を用いた。これによって膜濃縮液を得た。
得られた膜濃縮液を、0.2mol/LNaClを含む50mmol/L酢酸ナトリウム(pH4.2)び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮した。これらによって得られた膜濃縮液をボツリヌス毒素単離液1とも記載する。
ボツリヌス毒素単離液1を、陽イオン交換クロマトグラフィーに供した。陽イオン交換担体としてはSP-Sepharose-fast flowを用い、70×550mmサイズのカラムに担体を30cm充填した。洗浄液として0.2mol/LNaClを含む50mmol/L酢酸ナトリウム(pH4.2)を用いてカラムを平衡化し、ボツリヌス毒素単離液1をアプライし、溶出液として0.7mol/LNaClを含む50mmol/L酢酸ナトリウム(pH4.2)を用い、0.7mol/LNaClまで8カラムボリュームのグラジエント条件で溶出させた。流速は約15mL/分であった。得られた溶出液を、0.2mol/LNaClを含む50mmol/L酢酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮し、得られた濃縮液をさらに、孔径0.22μmフィルター(コーニング社製ボトルトップフィルター、PES製)を用いてろ過し、ボツリヌス毒素複合体(M毒素)の精製物を得た。
得られたボツリヌス毒素複合体(M毒素)の精製物を、10mmol/Lリン酸緩衝液(pH7.5)及び孔径(分画分子量)10KDaの膜(ザルトリウス社製Sartocon Slice 50 Hydrosart 10kD、及びミリポア社製ペリコンXLウルトラセル10kD)を用いて膜濃縮した。その後、膜濃縮物を、陰イオン交換カラムクロマトグラフィーに供した。陰イオン交換カラム担体としては、弱陰イオン交換担体であるDEAE-Sepharose-fast flowを用い、10×450mmのカラムに担体を30cm充填した。洗浄液として10mmol/Lリン酸緩衝液(pH7.5)を用いてカラムを平衡化し、膜濃縮物をアプライし、溶出液として0.3mol/LNaClを含む10mmol/Lリン酸緩衝液(pH7.5)を用い、0.3mol/LNaClまで10カラムボリュームのグラジエント条件で溶出させた。流速は約1mL/分であった。得られた溶出液を孔径0.22μmフィルター(Starlab社製PES製シリンジフィルター)を用いてろ過し、保存剤(L-アルギニン塩酸塩)を終濃度が1w/v%になるよう添加し、ボツリヌス毒素非複合体(S毒素)の精製物(以下、S毒素精製物1とも記載する)を得た。
上記(C)工程及び(D)工程を、以下の方法に置き換えたことを除いて、実施例1と同様にしてボツリヌス毒素非複合体(S毒素)の精製物を得た。具体的には、実施例1の(B)工程で得られたハーベスト液11(フィルターろ液)の残りの半量0.45Lを用いて、以下の陰イオン交換クロマトグラフィー、膜濃縮及びろ過を行い、その後、実施例1の(E)工程及び(F)工程を行った。
実施例1については、ハーベスト液12、ボツリヌス毒素単離液1、及びS毒素精製物1をそれぞれSDS-PAGE展開し、比較例1については、ハーベスト液11、ボツリヌス毒素単離液1’、及びS毒素精製物1’をSDS-PAGE展開し、それぞれCBB染色を行い、ボツリヌス毒素の精製を確認した。得られた染色ゲルを図1(実施例1)及び図2(比較例1)に示す。図中、A2-NTX(H)は約100KDaの重鎖を示し、A2-NTX(L)は約50KDaの軽鎖を示す。
核酸除去操作による精製効率を測定した。具体的には、実施例1における精製効率は、(B)工程で得られたハーベスト液12(膜濃縮液)中の核酸量に対する(D)工程で得られたボツリヌス毒素単離液1中の核酸量の比率(%)を算出し、100%から当該比率を減じて得た。比較例1における精製効率は、(B)工程で得られたハーベスト液11(フィルターろ液)中の核酸量に対するボツリヌス毒素単離液1’中の核酸量の比率を算出し、100%から当該比率を減じて得た。
核酸除去操作によるボツリヌス毒素収率を測定した。具体的には、実施例1におけるボツリヌス毒素収率は、(B)工程で得られたハーベスト液12(膜濃縮液)中の総毒素量に対する(D)工程で得られたボツリヌス毒素単離液1中の総毒素量の比率として算出し;比較例1におけるボツリヌス毒素収率は、(B)工程で得られたハーベスト液11(フィルターろ液)中の総毒素量に対するボツリヌス毒素単離液1’中の総毒素量の比率として算出した。
以下のようにして、単位U/mLとする力価を導出した。
得られたS毒素精製物を適宜希釈し、マウスの尾静脈より、1匹あたり0.1mLを接種した後、マウスの生存時間を確認した。あらかじめ検証した生存時間と腹腔内投与によるLD50値との回帰直線より、S毒素精製物1mL当たりのLD50値(U/mL)を算出した。また、280nmの赤外線吸収からS毒素精製物1mL当たりのタンパク質質量(mg/mL)を測定した。S毒素精製物1mL当たりのLD50値(U/mL)をS毒素精製物1mL当たりのタンパク質質量(mg/mL)で除して、比活性(U/mg)を導出した。
[1]ボツリヌス毒素の製造
本実施例では、ボツリヌス毒素産生工程でアニマルフリーの培地を用い、エンドヌクレアーゼとしてデナラーゼを用いた点を主な変更点としたことを除いて、実施例1と同様にしてボツリヌス毒素を製造した。具体的には、以下の手順でボツリヌス毒素を製造した。
前培養工程及び本培養工程における培地として、Vegetable Peptone No.1(植物ペプトン、サーモフィッシャー社)を用いたこと、前培養工程(静置培養1及び静置培養2)における培養量を半量にし、静置培養1の培養物約10mLを静置培養2の培地に播種したことを除き、実施例1と同様にしてボツリヌス菌を産生した。
上記本培養工程によって得られた培養物を、以下のフィルターと膜を用いて、粗ろ過及び膜濃縮した。フィルターろ過は、本培養工程によって得られた培養物を、フィルター1[3M社製ゼータプラスフィルター(05SP)/1020cm2;孔径1.0~10μm相当]、フィルター2[3M社製ゼータプラスフィルターマキシマイザー(60SP03A)/1020cm2;孔径0.1~1.0μm相当]、及びフィルター3[ザルトリウス社製ザルトポア2(孔径0.45μm+0.2μm)/0.1m2]の順に通すフィルターろ過により行った。膜ろ過で得られたろ液を0.1mol/LNaClと20mmol/Lクエン酸とを含む20mmol/Lリン酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮した。さらに、10mmol/Lリン酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮した。これらによって得られた膜濃縮液を、ハーベスト液22とも記載する。
エンドヌクレアーゼとしてデナラーゼ(シーレクターゲーエムベーハー登録商標)を用い、反応時間を約19時間としたことを除いて、実施例1と同様にしてエンドヌクレアーゼ処理及びろ過を行い、ろ液を得た。
得られたろ液を、10mmol/Lリン酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて約1時間膜ろ過し、核酸分解物を除去した。なお、この膜による核酸分解物の除去には、タンジェンシャルフローろ過を用いた。これによって膜濃縮液を得た。得られた膜濃縮液を、0.2mol/LNaClを含む50mmol/L酢酸ナトリウム(pH4.2)び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて約20分間膜濃縮した。これらによって得られた膜濃縮液をボツリヌス毒素単離液2とも記載する。
ボツリヌス毒素単離液2を、実施例1と同様にして陽イオン交換クロマトグラフィーに供した。得られた溶出液を、0.2mol/L NaClを含む50mmol/L酢酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮し、さらに、孔径0.22μmフィルター(コーニング社製ボトルトップフィルター、PES製)を用いてろ過し、ボツリヌス毒素複合体(M毒素)の精製物を得た。
ボツリヌス毒素複合体(M毒素)の精製物から、実施例1と同様にして、ボツリヌス毒素非複合体(S毒素)の精製物(以下、S毒素精製物2とも記載する)を得た。
本実施例では、ボツリヌス毒素産生工程の本培養工程で静置培養を行い、エンドヌクレアーゼの添加を2回行ったことを主な変更点としたことを除いて、実施例1と同様にしてボツリヌス毒素を製造した。具体的には、以下の手順でボツリヌス毒素を製造した。
シード菌体を0.4mL播種したこと、静置培養1における培養量を10mLにしたこと、静置培養2における培養量を250mLにしたこと、静置培養1の培養物約10mLを静置培養2の培地に播種したことを除いて、実施例1と同様にして前培養工程(静置培養1及び静置培養2)を行った。本培養工程の菌体増殖段階では、Bacto Proteose Peptone No.3(ブタペプトン、ベクトン・ディッキンソン社製)(終濃度2w/v%)、酵母エキス(終濃度1w/v%)、チオグリコール酸ナトリウム(終濃度0.025w/v%)、及びグルコース(終濃度1.0w/v%)を含む、pH7.3の培地10Lに、静置培養2の培養物約150mLを播種し、密封条件下、35℃で静置培養し、pH調整を行うことなく静置培養のまま自然に発酵段階に移行させた。本培養工程に要した時間は、約44時間であり、培養終了時の培地のpHは約6.0であった。
上記本培養工程によって得られた培養物について、0.1mol/LNaClと20mmol/Lクエン酸とを含む20mmol/Lリン酸緩衝液(pH6.0)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮しなかった点を除いて、実施例1と同様にして、ハーベスト液12に対応するハーベスト液32を得た。
終濃度が1mmol/LとなるMgCl2と、終濃度が50U/mLとなるベンゾナーゼ(1回目添加分)とをハーベスト液32を加え、pH6.0の条件下で撹拌しながら30℃で約5時間反応させた。その後、ベンゾナーゼ(2回目添加分)を更に50U/mL添加することで、1回目と2回目とのベンゾナーゼの合計量が100U/mLとなるようにし、更に約12時間(合計約13時間)反応させた。その後、エンドヌクレアーゼ処理物を、3M社製Zeta Plus Encapsulated Filterを用いてフィルターろ過した。
得られたろ液を、0.2mol/L NaClを含む50mmol/L酢酸緩衝液(pH4.2)及び孔径(分画分子量)30KDaの膜(ザルトリウス社製Sartocon Slice Hydrosart 30kD)を用いて膜濃縮し、核酸分解物を除去した。なお、この膜による核酸分解物の除去には、タンジェンシャルフローろ過を用いた。これによって得られた膜濃縮液をボツリヌス毒素単離液3とも記載する。
得られたボツリヌス毒素単離液3を、実施例1と同様にして陽イオン交換クロマトグラフィーに供した。溶出液として0.7mol/LNaClを含む50mmol/L酢酸ナトリウム(pH4.2)を用い、0.7mol/LNaClまで8カラムボリュームのグラジエント条件で溶出させた。得られた溶出液を、10mmol/Lリン酸塩緩衝液(pH7.5)及び孔径(分画分子量)10KDaの膜(ザルトリウス社製Sartocon Slice 50 Hydrosart 10kD)を用いて膜濃縮し、ボツリヌス毒素複合体(M毒素)の精製物を得た。
得られたボツリヌス毒素複合体(M毒素)の精製物を、10mmol/Lリン酸塩緩衝液(pH7.5)及び孔径(分画分子量)10KDaの膜(ミリポア社製ペリコンXLウルトラセル10kD)を用いて膜濃縮し、陰イオン交換カラムクロマトグラフィーに供した。陰イオン交換カラム担体としては、弱陰イオン交換担体であるDEAE-Sepharose-fast flowを用い、10×450mmのカラムに担体を30cm充填した。洗浄液として10mmol/Lリン酸緩衝液(pH7.5)を用いてカラムを平衡化し、ボツリヌス毒素複合体(M毒素)の精製物をアプライし、溶出液として0.3mol/LNaClを含む10mmol/Lリン酸緩衝液(pH7.5)を用い、0.3mol/LNaClまで10カラムボリュームのグラジエント条件で溶出させた。流速は約1mL/分であった。得られた溶出液に保存剤(L-アルギニン塩酸塩)を終濃度が1w/v%になるよう添加し、孔径0.22μmフィルター(Starlab社製PES製シリンジフィルター)を用いてろ過し、ボツリヌス毒素非複合体(S毒素)の精製物(以下、S毒素精製物3とも記載する)を得た。
実施例2については、ハーベスト液22、ボツリヌス毒素単離液2、及びS毒素精製物2をそれぞれSDS-PAGE展開し、実施例3については、ハーベスト液32、ボツリヌス毒素単離液3、及びS毒素精製物3をSDS-PAGE展開し、それぞれCBB染色を行い、ボツリヌス毒素の精製を確認した。SDS-PAGE及び染色の方法は、試験例1と同様である。得られた染色ゲルを図3(実施例2)及び図4(実施例3)に示す。図中、A2-NTX(H)は約100KDaの重鎖を示し、A2-NTX(L)は約50KDaの軽鎖を示す。
核酸除去操作によるボツリヌス毒素収率を測定した。具体的には、実施例2,3におけるボツリヌス毒素収率は、(B)工程で得られたハーベスト液22,32(膜濃縮液)中の総毒素量に対する(D)工程で得られたボツリヌス毒素単離液2,3中の総毒素量の比率として算出した。
タンパク質含量をBCA法で測定したことを除いて、試験例1と同様に、得られたボツリヌス毒素(S毒素精製物2,3)の比活性を測定した。その結果、実施例2で得られたS毒素精製物2の比活性は10.06×107U/mgであり、実施例3で得られたS毒素精製物3の比活性は6.46×107U/mgであった。
Claims (16)
- (A)培地中でボツリヌス毒素産生菌からボツリヌス毒素を産生させ、前記ボツリヌス毒素由来の菌体成分及び核酸成分とボツリヌス毒素とを含む混合物aを得る工程と、
(B)前記混合物aを前記菌体成分の除去に供し、核酸成分とボツリヌス毒素とを含む混合物bを得る工程と、
(C)前記混合物bにエンドヌクレアーゼを添加し、核酸分解物とボツリヌス毒素とを含む混合物cを得る工程と、
(D)前記混合物cを核酸分解物の除去に供し、ボツリヌス毒素単離液dを得る工程と、
を含む、ボツリヌス毒素の製造方法。 - 前記(C)工程において、前記エンドヌクレアーゼがセラチア菌Serratia marcescens由来のエンドヌクレアーゼである、請求項1に記載のボツリヌス毒素の製造方法。
- 前記(C)工程を、pH5.8~6.5の条件下で行う、請求項1又は2に記載のボツリヌス毒素の製造方法。
- 前記(C)工程において、前記エンドヌクレアーゼを複数回添加する、請求項1~3のいずれかに記載のボツリヌス毒素の製造方法。
- 前記(D)工程において、前記核酸分解物の除去が膜による除去を含む、請求項1~4のいずれかに記載のボツリヌス毒素の製造方法。
- さらに、(E)前記ボツリヌス毒素単離液dを、陽イオン交換クロマトグラフィーに供し、ボツリヌス毒素複合体の精製物を得る工程を含む、請求項1~5のいずれかに記載のボツリヌス毒素の製造方法。
- さらに、(F)前記ボツリヌス毒素複合体の精製物を、前記ボツリヌス毒素複合体から無毒性非HAタンパク質が乖離する条件下で陰イオン交換クロマトグラフィーに供し、ボツリヌス毒素非複合体の精製物を得る工程を含む、請求項6に記載のボツリヌス毒素の製造方法。
- 少なくとも前記(A)工程が前培養工程と本培養工程とを含み、少なくとも前記前培養工程を静置培養で行う、請求項1~7のいずれかに記載のボツリヌス毒素の製造方法。
- 前記前培養工程と前記本培養工程との両方を静置培養で行う、請求項1~8に記載のボツリヌス毒素の製造方法。
- 前記(A)工程が、
(A1)pH6.8~8.0の培地中で前記ボツリヌス毒素産生菌の菌体増殖を行う工程と、
(A2)pH5.0~6.5の培地中で前記ボツリヌス毒素産生菌の発酵を行う工程と、
をこの順番で含む、請求項8又は9に記載のボツリヌス毒素の製造方法。 - 前記(A)工程において、前記ボツリヌス毒素産生菌が、芽胞の形態で保存された菌体の発芽体である、請求項1~10のいずれかに記載のボツリヌス毒素の製造方法。
- 前記(B)工程において、前記菌体成分の除去がフィルターろ過を含む、請求項1~11のいずれかに記載のボツリヌス毒素の製造方法。
- 前記(F)工程において、前記条件がpH7.3~8.5である、請求項7~12のいずれかに記載のボツリヌス毒素の製造方法。
- 前記(F)工程において、陰イオン交換クロマトグラフィーが弱陰イオン交換クロマトグラフィーである、請求項7~13のいずれかに記載のボツリヌス毒素の製造方法。
- 比活性が3.0×107U/mg以上であることを特徴とする、精製ボツリヌス毒素。
- 請求項1~14のいずれかに記載のボツリヌス毒素の製造方法によって製造される、精製ボツリヌス毒素。
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