JP2011074025A - Method of purifying botulinus toxin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simpler method of purifying botulinus toxin suited in the purification on an industrial level without requiring a precipitation/centrifuging step. <P>SOLUTION: The method of purifying botulinus toxin comprises a combination of a film filtration method, an anion exchange chromatography, and a cation exchange chromatography. According to this purification method, a large quantity of high safety botulinus toxin can be very simply and effectively prepared in a short time from a culture medium of Clostridium botulinum. Further, elimination of the recovering operation of precipitates in the precipitation method/centrifugal separation enables providing complete automation facilities/in-line facilities of a production line. Furthermore, since no operator intervenes and no toxin solution is exposed to the environment, the security of the work safety health environment of operators, GMP conformity, and asepsis can be improved. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for purifying botulinum toxin. More specifically, the present invention relates to a method for purifying botulinum toxin characterized by combining a membrane filtration method and a chromatography method without performing precipitation or centrifugation.

Botulinum toxin produced by Clostridium botulinum , an anaerobic Gram-positive bacterium, is the most deadly toxin on earth, and so far serotypes A, B, C, D, E, F and G toxins from seven Clostridium botulinums and their properties have been elucidated. These are each identified by neutralizing antibodies specific for the serotype. Furthermore, among botulinum toxins of the same serotype, they are classified into several subtypes depending on the structure of the toxin gene. The botulinum toxin active center protein is a botulinum neurotoxin (NTX) having a molecular weight of about 150 KDa in all seven known botulinum toxin serotypes.

  When all botulinum toxins are produced from Clostridium botulinum, they are produced in the form of a complex in which nontoxic components having different molecular weights are bound to NTX having a molecular weight of about 150 KDa. Complex toxins are divided into molecular forms of LL toxin (900 KDa), L toxin (500 KDa), or M toxin (300 KDa) depending on the molecular weight. LL toxin and L toxin consist of NTX, HA protein and NTNH (protein which is non-toxic non-HA protein), and M toxin lacks HA protein. LL toxin, L toxin and M toxin are called botulinum toxin complex or progenitor toxin.

  Type A botulinum is classified into subtypes A1 to A5. For example, type A1 botulinum produces LL toxin, L toxin and M toxin, whereas type A2 botulinum produces only M toxin. Examples of A1-type Clostridium botulinum include Hall, 62A, 97A, NCTC887, NCTC7272, NCTC2916, and NCTC11199. Type A2 Clostridium botulinum was first identified in Japan in 1986 from patients with infantile botulism (Sakaguchi G., et al., Int. J. Food Microbiol., 11: 231-242, 1990: non-patented Reference 1). Clinical isolates of type A2 Clostridium botulinum are Kyoto-F, Chiba-H, Y-8036, 7I03-H, 7I05-H and KZ1828. In addition, Loch Maree strain is known as A3-type botulinum, 657Ba strain is known as A4-type botulinum, and H0 4244 0055, H0 4402 065, H0 4464 107, and H0 4068 0341 strains are known as A5-type botulinum.

  In other Clostridium botulinum, B, C and D botulinum produce LL and M toxins. Type E and F botulinum produce only M toxin, and type G botulinum produces only L toxin.

  Since the botulinum toxin complex dissociates NTX (also called S toxin) and NTNH under alkaline conditions, only 150 KDa NTX can be isolated using this property.

  Conventionally, botulinum toxin has been purified by several methods, such as acid precipitation that precipitates botulinum toxin complex and botulinum cells in culture, protamine precipitation that removes coexisting DNA and RNA, and ammonium sulfate salting-out for rough purification. After performing the precipitation method described above, it was a complicated method combining chromatographic methods such as ion exchange, metal or gel filtration.

  For example, as a purification method of NTX, the culture solution is precipitated and extracted sequentially with sulfuric acid, protamine and ammonium sulfate, and then the precipitate obtained by centrifugation is dissolved, and SP Sephadex C-50 (pH 3.8), Sephadex G- There is a method of Sakaguchi et al. (Infect. Immun. 12 (6), 1262-1270, 1975: Non-Patent Document 2) in which column chromatography is successively performed with 200 (pH 3.8) and DEAE Sephadex (pH 8.0). . In the method of Sakaguchi et al., It has been reported that LL toxin, L toxin, M toxin or NTX derived from botulinum A, B, C, D, E, F and G can be purified (Pharmac. Ther. 19,165-194). , 1983: Non-patent document 3).

  In addition, after the culture solution is precipitated sequentially with sulfuric acid, protoamine sulfate, and ammonium sulfate, the precipitate obtained by centrifugation is dissolved, and successively with DEAE Sephadex (pH6), DEAE Sephadex (pH7.9), and SP Sephadex (pH7). The column chromatography is performed on Pigalke Hans et al. (Special Table 2003-505343: Patent Document 1) or the botulinum toxin complex is recovered from DEAE Sephadex A-50 (pH 5.5) column and ammonium sulfate salted out. After dissolution of the precipitate, Johnson et al.'S method of performing column chromatography one after another with DEAE Sephadex A-50 (pH 7.9), SP Sephadex C-50 (pH 7.0), etc. Patent document 2) is known. Any of these methods is a complicated method of carrying out a precipitation method or centrifugation as a pretreatment for chromatography.

  When carrying out precipitation on an industrial scale, 1) the operation is complicated, 2) the separation is not sensitive and has low separation performance, 3) process control is difficult, and a slight difference in conditions greatly affects the degree of separation. 4) Difficult to change production scale (scale-up or scale-down), 5) low productivity, etc.

  Specifically, the problems with the prior art shown above are described: 1) It is difficult to guarantee that all botulinum cells have been destroyed / removed by acid precipitation, and 2) Protamine treatment mainly intended for nucleic acid removal. Therefore, it is difficult to set the reaction conditions, so it is impossible to remove nucleic acids with good reproducibility. 3) The remaining nucleic acids greatly affect the resolution of the chromatography process. 4) Combine multiple types of precipitation methods. Therefore, there are problems such as a long manufacturing period.

  In addition, in the centrifugation performed in conjunction with the precipitation method, 1) investment cost, 2) reduction in toxin recovery rate due to shear force, 3) processing time, 4) generation of aerosol containing botulinum toxin due to high speed rotation in centrifugation, 5 (6) It is difficult to provide workers with toxins during the sediment collection work, and (6) It is difficult to provide fully automated and fully inline facilities (Handbook of Process Chromatography Development, Manufacturing, Validation and Economics., Hagel) Elsevier Science & Technology, 2008: Non-Patent Document 4).

Special table 2003-505343 gazette Japanese National Patent Publication No. 11-507072

Sakaguchi G., et al., Int. J. Food Microbiol., 11: 231-242, 1990. Sakaguchi G., et al., Infect.Immun. 12 (6), 1262-1270, 1975. Sakaguchi G., Pharmac.Ther. 19,165-194, 1983 Handbook of Process Chromatography Development, Manufacturing, Validation and Economics., Hagel, Elsevier Science & Technology, 2008.

  Under such circumstances, the object of the present invention is to provide a simpler method for purifying botulinum toxin on an industrial scale, which reduces the number of steps by finding a separation technology that replaces the precipitation / centrifugation step as compared with the prior art. It is to provide.

Therefore, as a result of intensive studies in view of the above-mentioned problems, the present inventors have found a purification method of botulinum toxin, focusing on the membrane filtration method as a separation technique to replace the precipitation method / centrifugation, and the present invention. It came to be completed. The present invention provides a method for purifying botulinum toxin characterized by combining a membrane filtration method and a chromatography method from a botulinum culture solution containing a botulinum toxin complex. When the purification method of the present invention is applied, a large amount of highly stable botulinum toxin can be prepared in a short time and effectively in a very simple manner as compared with the prior art. The present invention is described in further detail below.
(1) A method for purifying botulinum toxin, comprising combining membrane filtration, anion exchange chromatography, and cation exchange chromatography.
(2) The purification method according to (1) above, comprising the following steps.
(a) a step of removing botulinum cells from the Clostridium botulinum culture solution by a membrane filtration method,
(b) developing the botulinum toxin solution from which the botulinum cells have been removed in the step (a) on an anion exchange chromatography column,
(c) developing the recovered fraction of the anion exchange chromatography column obtained in the step (b) on a cation exchange chromatography column under conditions such that the botulinum toxin component is adsorbed on the column; and
(d) washing away impurities from the cation exchange chromatography and then eluting the botulinum toxin.
(3) The purification method according to (1) or (2), wherein the membrane filtration method is a precision membrane filtration method.
(4) The purification method according to the above (3), wherein the membrane used in the precision membrane filtration method has a pore size of 0.22 μm or less.
(5) The purification according to (2), further including a step of performing rough membrane filtration with a membrane having a larger pore size than the membrane used for the precision membrane filtration before the membrane filtration method in the membrane filtration method. Method.
(6) The purification method according to (1) or (2) above, wherein the pH of the buffer in the anion exchange chromatography column is 4.5 to 6.8.
(7) The purification method according to (1) or (2) above, wherein the salt concentration of the buffer in the anion exchange chromatography column is 0 to 0.3 mol / L.
(8) In the anion exchange chromatography, when the botulinum toxin component is adsorbed on a column, the botulinum toxin adsorbed on the column is eluted with a gradient having a salt concentration of 0 to 2.0 mol / L. The purification method according to 2).
(9) before performing the step (b), further comprising the step of adjusting the botulinum toxin solution from which the botulinum cells have been removed in the step (a) to the conditions of the salt concentration and pH of the anion exchange chromatography. The purification method according to (2) above.
(10) The purification method according to (1) or (2) above, wherein the pH of the buffer in the cation exchange chromatography column is 4.0 to 4.5.
(11) The purification method according to (1) or (2) above, wherein the salt concentration of the buffer in the cation exchange chromatography column is 0 to 0.2 mol / L.
(12) Before performing the step (c), adjusting the recovered fraction of the anion exchange chromatography column obtained in the step (b) to the salt concentration and pH conditions of the cation exchange chromatography The purification method according to (2), further comprising:
(13) The purification method according to (1) or (2) above, wherein, in the step (d), the botulinum toxin adsorbed on the cation exchange chromatography is eluted with a gradient having a salt concentration of 0.2 to 0.7 mol / L.
(14) The purification method according to the above (2), wherein the botulinum toxin eluted in the step (d) is L toxin, LL toxin or M toxin.
(15) The method for purifying the botulinum toxin according to (1) or (2), further including the following steps after the steps (a) to (d).
(e) The cation exchange chromatography obtained in the step (d) under the condition that the botulinum toxin component is adsorbed on the column and the botulinum neurotoxin (NTX) and non-toxic non-HA protein (NTNH) are dissociated. Developing the elution fraction of the chromatography on a second anion exchange chromatography column; and
(f) washing away impurities from the second anion exchange chromatography and then eluting botulinum neurotoxin (NTX);
(16) The purification method according to any one of (1), (2), and (15) above, wherein the pH of the buffer in the second anion exchange chromatography column is 6.8 to 8.0.
(17) The purification method according to any one of (1), (2), and (15) above, wherein the salt concentration of the buffer in the second anion exchange chromatography column is 0 to 0.1 mol / L.
(18) In the step (f), the NTX adsorbed on the second anion exchange chromatography is eluted with a gradient having a salt concentration of 0 to 0.3 mol / L, according to the above (1), (2) or (15) The purification method according to any one of the above.
(19) Before performing the step (e), the elution fraction of the cation exchange chromatography obtained in the step (d) is adjusted to the salt concentration and pH conditions of the second anion exchange chromatography. The purification method according to (15), further comprising a step.
(20) The purification according to any one of (1) to (19), wherein the botulinum toxin is a botulinum toxin derived from botulinum type A, B, C, D, E, F, and G. Method.
(21) A botulinum toxin purified by the purification method according to any one of (1) to (20) above.
(22) The botulinum toxin according to (21), wherein the botulinum toxin is any one of LL toxin, L toxin, M toxin, and NTX.
(23) A drug containing the botulinum toxin according to (21) or (22) as an active ingredient.

  According to the present invention, compared with the prior art involving precipitation method / centrifugation, the membrane filtration method and the chromatographic method are combined to make the botulinum culture solution very easily, in a short time and effectively stable. It became possible to prepare a large amount of high botulinum toxin. In addition, by eliminating the sediment collection work in the precipitation method and centrifugation, it has become possible to provide fully automated equipment and in-line equipment for the production line. In addition, since the intervention of workers is eliminated and the toxin solution is not exposed to the environment, it is possible to improve the worker's occupational safety and health environment, GMP compliance, and ensuring sterility.

  The Clostridium botulinum to which the method of the present invention is applied is an A to G type bacterium.

The botulinum toxin purification method combining the membrane filtration method, anion exchange chromatography and cation exchange chromatography of the present invention specifically comprises the following steps.
(a) a step of removing botulinum cells from the Clostridium botulinum culture solution by a membrane filtration method,
(b) developing the botulinum toxin solution from which the botulinum cells have been removed in the step (a) on an anion exchange chromatography column,
(c) developing the recovered fraction of the anion exchange chromatography column obtained in the step (b) on a cation exchange chromatography column under conditions such that the botulinum toxin component is adsorbed on the column; and
(d) washing away impurities from the cation exchange chromatography and then eluting the botulinum toxin.

  Clostridium botulinum progenitor toxins., 21-34, Lewis GE., 1981, Academic Press, New York). As an example, it is obtained by a method of culturing at 30 ° C. for 4 days in a PYG medium, and the medium and culture conditions to be used are appropriately selected depending on the purpose. As another culture method, a culture solution prepared by a modified method of Siegel, L.S. et al. (Appl. Microbiol. Vol. 38, No. 4: 606-611., 1979) can also be used.

  According to the purification method of the present invention, first, as a step (a), the obtained Clostridium botulinum culture solution is filtered with a filter for microfiltration to remove botulinum cells. There is no particular restriction on the mode of filtration, and for example, a dead end filtration method, a tangential flow method, or the like can be employed. The microfiltration membrane has low protein adsorption, and for example, commercially available filtration membranes such as cellulose acetate, hydrophilic polyethersulfone, and hydrophilic polyvinylidene fluoride (PVDF) can be used. Further, for the purpose of reducing the load on the microfiltration membrane, the membrane may be filtered with a membrane having a relatively large pore size before the microfiltration. The membrane pore size is not particularly limited, but the membrane pore size of the final filtration membrane is 0.1 to 0.22 μm, preferably 0.2 μm, and removal of botulinum cells should be ensured by a filter integrity test. I must.

  Before the botulinum toxin complex solution containing contaminants from which the botulinum cells obtained in step (a) have been removed is developed on the anion exchange chromatography column in step (b), the botulinum toxin complex solution (b) ) Adjust the salt concentration and pH of the anion exchange chromatography in the process. In this adjustment, for example, concentration and buffer exchange are performed with an industrial tangential flow ultrafiltration apparatus to adjust protein concentration, salt concentration, and pH. The membrane pore diameter of the ultrafiltration membrane may be 10 to 100 KDa.

  Next, as the step (b), the prepared botulinum toxin complex solution is developed on an anion exchange resin equilibrated with a buffer solution or the like to adsorb and remove nucleic acids and other impurities. The buffer solution is not particularly limited as long as the salt concentration is 0.3 mol / L or less and the pH is in the range of 4.5 to 6.8, preferably the salt concentration is 0.1 mol / L and the pH is 6.0. A citrate / phosphate mixed buffer is a suitable example. The botulinum toxin complex may be either adsorbed or non-adsorbed as long as the nucleic acid can be adsorbed and removed. Preferably, the nucleic acid is removed by adsorption and the botulinum toxin complex is non-adsorbed. When the botulinum toxin complex is adsorbed to the column, it is eluted with the same buffer with a salt concentration with a gradient of 0 to 2.0 mol / L. The anion exchange resin used is DEAE Sepharose Fast Flow (GE Healthcare), Q Sepharose Fast Flow (GE Healthcare), CAPTO-Q (GE Healthcare), DEAE Toyopearl (Tosoh), QAE Toyopearl (Tosoh), etc. Commercially available anion exchange resins can be used. The type of salt is not limited, and the salt may be used at a concentration corresponding to sodium chloride having the above-mentioned concentration.

  Before the botulinum toxin complex solution obtained in step (b) is developed in the cation exchange chromatography of step (c), the botulinum toxin complex solution is subjected to the salt concentration and pH conditions of the cation exchange chromatography. Adjust to. In this adjustment, for example, concentration and buffer exchange are performed with an industrial tangential flow ultrafiltration apparatus to adjust protein concentration, salt concentration, and pH.

  Next, as step (c), the botulinum toxin complex solution obtained in step (b) is developed on a cation exchange resin to adsorb the botulinum toxin complex. The cation exchange resin is previously equilibrated with a buffer solution such as an acetate buffer solution having a salt concentration of 0.2 mol / L or less and a pH of 4.0 to 4.5, preferably a salt concentration of 0.2 mol / L and a pH of 4.2. deep. The buffer solution to be used is not particularly limited to the acetate buffer solution, as long as the salt concentration is 0.2 mol / L or less and the pH is in the range of 4.0 to 4.5. As long as the botulinum toxin complex can be adsorbed, the contaminating protein may be adsorbed or non-adsorbed. Preferably, the botulinum toxin complex is adsorbed and contaminating proteins are non-adsorbed. After washing with the buffer solution, the botulinum toxin complex is eluted and fractionated with a linear gradient using the same buffer solution with a salt concentration of 0.2 to 0.7 mol / L. Cation exchange resins used are commercially available cation exchanges such as SP Sepharose Fast Flow (GE Healthcare), CM Sepharose Fast Flow (GE Healthcare), S Sepharose Fast Flow (GE Healthcare), SP Toyopearl (Tosoh) Resin can be used. The type of salt is not limited, and the salt may be used at a concentration corresponding to sodium chloride having the above-mentioned concentration.

  By the above method, contaminating proteins are removed, and highly purified botulinum toxin complex (L toxin, LL toxin, M toxin) is obtained. Furthermore, when a higher purity botulinum toxin complex is required, further chromatography, for example, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc., or a combination thereof may be used. Can be highly purified.

In order to obtain NTX from the obtained botulinum toxin complex solution, the following steps must be further performed to dissociate and remove non-toxic non-HA protein (NTNH) from the botulinum toxin complex.
(e) The cation exchange chromatography obtained in the step (d) under the condition that the botulinum toxin component is adsorbed on the column and the botulinum neurotoxin (NTX) and non-toxic non-HA protein (NTNH) are dissociated. Developing the elution fraction of the chromatography on a second anion exchange chromatography column; and
(f) washing away impurities from the second anion exchange chromatography and then eluting botulinum neurotoxin (NTX);

  (e) As a step, the botulinum toxin complex solution is developed on an anion exchange resin to adsorb NTX. The anion exchange resin has a salt concentration of 0.1 mol / L or less and a pH in the range of 6.8 to 8.0, preferably a buffer solution such as phosphate or Tris buffer having a salt concentration of 0 mol / L and a pH of 7.5. Equilibrate with. The buffer solution to be used may be a salt concentration of 0.1 mol / L or less and a pH in the range of 6.8 to 8.0, and is not particularly limited to phosphate or Tris buffer. As long as NTX and NTNH can be adsorbed, the contaminating protein may be either adsorbed or non-adsorbed. Preferably, NTX and NTNH are adsorbed, and contaminating proteins are not adsorbed.

  Next, in step (f), the column on which NTX and NTNH are adsorbed is washed with the buffer solution, and then highly purified NTX is eluted with a linear gradient using the same buffer solution with a salt concentration of 0 to 0.3 mol / L. Fractionation. The anion exchange resin used is DEAE Sepharose Fast Flow (GE Healthcare), Q Sepharose Fast Flow (GE Healthcare), Capto-Q (GE Healthcare), DEAE Toyopearl (Tosoh), QAE Toyopearl (Tosoh), etc. Commercially available anion exchange resins can be used. The type of salt is not limited, and the salt may be used at a concentration corresponding to sodium chloride having the above-mentioned concentration.

  In the step (e), NTX and NTNH may be dissociated in advance before being developed into an anion exchange resin. For example, a botulinum toxin complex solution consisting of M toxin is subjected to ultrafiltration by a salt concentration of 0 to 0.1 mol / L and a pH of 6.8 to 8.0, preferably a salt concentration of 0 mol / L and a pH of 7. NTX and NTNH are dissociated by exchanging the buffer with 5 phosphate or Tris buffer. This ultrafiltration step can be performed using an industrial tangential flow ultrafiltration apparatus or the like. The ultrafiltration membrane used in this step is not particularly limited, and commercially available ultrafiltration membranes such as hydrozalto and hydrophilic polyethersulfone can be used. The membrane pore diameter may be 10 to 30 KDa.

  When the highly purified botulinum toxin obtained by the above process is used as a medicine, a pharmaceutically usable additive is used. For example, albumin, amino acids, sugars, surfactants and the like can be used as stabilizers, and a preferred amino acid is arginine monohydrochloride. Further, when the highly purified botulinum toxin is stored for a long time, it can be stored frozen.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the description of the examples.

The figure which shows the activity residual rate in the liquid preservation | save of highly purified A2 type NTX.

<< Test Example: Test and Method >>
(1) SDS-PAGE test In this method, the purity of highly purified NTX solution or botulinum toxin complex solution is examined. An SDS-PAGE test was performed by partially modifying the method of Laemmli et al. (Nature 227: 680-685). As a marker, Precision plus blue stained (Bio-Rad Laboratories, Inc.) was used. The toxin solution was reduced with DTT (Nacalai Tesque) and then electrophoresed on a 10% single concentration or 5-20% linear gradient gel (ATTO). After electrophoresis, it was stained with Coomassie Brilliant Blue R250 (Nacalai).
(2) Mouse intraperitoneal LD ₅₀ test (ip method)
In this method, the toxin activity of the botulinum toxin solution is examined. Botulinum toxin was administered intraperitoneally to mice, and 50% lethal dose (LD ₅₀ ) was estimated from the cumulative lethality over 4 days. The toxin solution was diluted and 0.5 mL of diluted sample was administered per mouse. LD ₅₀ was calculated by the Probit method.
(3) Mouse tail vein administration test This method replaces the mouse intraperitoneal LD ₅₀ test (ip method) or rat CMAP method (WO 2007/125604 A1), which is commonly used as a method for measuring toxin activity of botulinum toxin. It is a simple method. A high concentration of toxin was administered to the tail vein of the mouse, and the survival time was examined. The toxin activity concentration of the specimen was calculated from the regression line between the survival time (min) and the toxin activity concentration (LD ₅₀ / mL) prepared in advance.
(4) Protein concentration test (Raleigh method)
In this method, the protein concentration of the botulinum toxin solution is examined. A DC protein assay kit (BIO-RAD) was used. Bovine serum albumin (PIERCE) was used as the calibration curve solution.
(5) Size exclusion HPLC analysis (SEC-HPLC)
In this method, the purity of highly purified NTX solution is examined. TSK-GEL G3000SW _XL (7.8 mm x 300 mm, Tosoh) was equilibrated with 50 mmol / L sodium phosphate containing 0.2 mol / L sodium chloride and 0.05 w / v% sodium azide, pH 5.8, and a flow rate of 0.25 mL / Analyzed in min. The percentage (%) of the area area of NTX in the total area area was calculated as purity.
(6) Nucleic acid content test In this method, the concentration of nucleic acid contained in highly purified NTX solution is examined. CyQUANT Cell Proliferation Assay Kit (Molecular Probes) was used.
(7) Endotoxin test In this method, the concentration of endotoxin contained in the highly purified NTX solution is examined. Tested according to the Japanese Pharmacopoeia 15 revision endotoxin test.

Comparative Example 1: Separation and purification of A2 type M toxin by conventional technology
Type A2 Clostridium botulinum strain Chiba-H was cultured by the method of Sakaguchi et al. (Biochemical aspects of botulism: Purification and oral toxicities of Clostridium botulinum progenitor toxins., 21-34, Lewis GE., 1981, Academic Press, New York). 3N sulfuric acid was added to 30 L of the culture solution after 96 hours until pH 3.5, and the mixture was allowed to stand overnight at room temperature. The precipitate was dissolved in 0.2 mol / L phosphate buffer, pH 6.0, and extracted at 37 ° C. for 1 hour. Ammonium sulfate was added to the extract and left at 4 ° C. overnight. The precipitate recovered by centrifugation was dissolved in 50 mmol / L acetate buffer, pH 4.2. The lysate was placed in a dialysis tube and dialyzed against the buffer at 4 ° C. for 2 days. The precipitate collected by centrifugation was dissolved in 50 mmol / L acetate buffer containing 0.5 mol / L sodium chloride, pH 4.2. A 50 mmol / L citrate buffer solution, pH 4.2, containing an equal amount of 0.5 mol / L sodium chloride was added to the lysate, and then treated with protamine. The supernatant from which the precipitate (nucleic acid) was removed by centrifugation was developed and adsorbed on SP Sepharose Fast Flow equilibrated in advance with 50 mmol / L acetate buffer containing 0.2 mol / L, pH 4.2. After washing with the buffer, elution was performed with a linear gradient using the same acetate buffer containing 0.7 mol / L sodium chloride. The first peak of the eluted fraction was fractionated, and the collected solution was concentrated with a stirring type ultrafiltration device (Amicon, YM-30). This concentrated solution was developed on Sephadex G-200 previously equilibrated with 50 mmol / L acetic acid buffer containing 0.2 mol / L sodium chloride, pH 6.0. The eluted second peak was fractionated to recover A2 type M toxin. The collected A2-type M toxin solution was concentrated with a stirring type ultrafiltration device (Amicon, YM-30).

<< Comparative Example 2: Separation and purification of A2 type NTX by conventional technology >>
The A2 type M toxin solution concentrated in Comparative Example 1 was placed in a dialysis tube and dialyzed overnight at 4 ° C. with 10 mmol / L phosphate buffer, pH 7.5. The dialyzed solution was developed and adsorbed on DEAE Sepharose Fast Flow equilibrated in advance with 10 mmol / L phosphate buffer, pH 7.5. After washing with the buffer solution, elution was performed with a linear gradient using the same acetate buffer solution containing 0.3 mol / L sodium chloride. The first peak of the eluted fraction was fractionated, and the collected solution was concentrated with a stirring ultrafiltration device (Amicon, YM-10), and then buffer exchanged with 10 mmol / L phosphate buffer, pH 7.5. . This was designated as highly purified A2 type NTX solution. The production period from the culture solution to the purification of the highly purified A2-type NTX solution was 7 days. The results obtained from this test are shown in Table 2.

Example 1: Bacterial Clostridium Clostridium Botulinum Filtration with 0.2 μm Filter
30 L of the A2-type Clostridium botulinum culture solution was centrifuged at 6,700 × g and 4 ° C. for 20 minutes, and the centrifuged supernatant was recovered. The culture supernatant was sterilized and filtered with hydrosalt (Sartorius) having a membrane pore size of 0.2 μm using a tangential flow filtration device (Sartorius). The filtration throughput was 25 L / m ² / hr.

<< Example 2: Study on coarse filtration and sterilization filtration >>
For the purpose of increasing the throughput of sterilization filtration, the introduction of a coarse filtration step in which filtration was performed with a membrane having a relatively large membrane pore diameter was studied in advance. Zeta plus (CUN0) and zeta carbon R53SLP (CUNO) were used as membranes for rough filtration. Zeta Plus used four types of membranes with different filtration accuracy. Filtration accuracy is high in the order of 90LA, 60LA, 30LA and 05SP. Further, a filter in which 0.65 μm Sarto Clean CA (Sartorius) and 0.2 μm Sartopore 2 (Sartorius) were connected was used as a membrane for sterilization filtration. 30 L of a culture solution of A2-type Clostridium botulinum Chiba-H strain was subjected to constant pressure filtration (0.03 MPa) with each membrane for coarse filtration and membrane for sterilization filtration, and the amount of filtration was examined. The results obtained in this test are shown in Table 1. By introducing the coarse filter, the amount of sterilization filtration by the 0.2 μm filter was 40 times that of Example 1 (25 L / m ² / hr).

Example 3: Separation and purification of A2 type M toxin according to the present invention
A2-type Clostridium botulinum Chiba-H strain was cultured by partially modifying the method of Siegel, LS et al. (Appl. Microbiol. Vol. 38, No. 4: 606-611., 1979). After 48 hours, 30 L of the culture solution was coarsely filtered at a pressure of 0.03 MPa with a filter in which a zeta plus filter (05SP) and a zeta plus filter maximizer (30LA + 60LA) were connected. This filtrate was subjected to sterilization filtration at a pressure of 0.03 MPa through a filter in which 0.65 μm Zaltclean CA and 0.2 μm Zaltpore 2 were connected. After filtration, the 0.2 μm filter was confirmed to meet the integrity test. The filtrate from which botulinum cells have been removed is concentrated using a tangential flow ultrafiltration device (Sartorius, 30 KDa), and then 20 mmol / L citric acid / phosphate mixed buffer containing 0.1 mol / L sodium chloride, pH 6 The buffer was exchanged at 0.0. This concentrated solution was developed and used further in Q Sepharose Fast Flow (GE Healthcare) equilibrated with 20 mmol / L citrate / phosphate mixed buffer containing 0.1 mol / L sodium chloride in advance, pH 6.0. The non-adsorbed fraction was collected by washing with about 7 times (7 CV) of the gel amount (CV) of column chromatography. In this step, the nucleic acid was effectively removed, and at the same time, crude purification could be achieved. The collected non-adsorbed fractions were concentrated with a tangential flow ultrafiltration apparatus (Sartorius, 30 KDa), and then buffer exchanged with 50 mmol / L acetate buffer containing 0.2 mol / L sodium chloride, pH 4.2. This concentrated solution was developed and adsorbed on SP Sepharose Fast Flow (GE Healthcare) previously equilibrated with 50 mmol / L acetate buffer containing 0.2 mol / L sodium chloride, pH 4.2. Next, after washing with about 10 CV of the buffer solution, elution was performed with a linear gradient using the same acetate buffer solution containing 0.7 mol / L sodium chloride. The first peak of the eluted fraction was fractionated to recover A2 type M toxin. The collected A2-type M toxin solution was concentrated with a tangential flow ultrafiltration device (Sartorius, 30 KDa), and then buffer exchanged with 50 mmol / L acetate buffer containing 0.2 mol / L sodium chloride, pH 6.0. . As a result of SDS-PAGE test of the obtained A2-type M toxin solution, no band other than M toxin was observed.

Example 4: Separation and purification of A2 type NTX according to the present invention
Furthermore, after concentrating the A2 type M toxin solution collect | recovered in Example 3 with a tangential flow type | formula ultrafiltration apparatus (Sartorius, 30 KDa), buffer exchange was carried out by 10 mmol / L phosphate buffer and pH7.5. . This concentrated solution was developed and adsorbed on DEAE Sepharose Fast Flow (GE Healthcare) previously equilibrated with 10 mmol / L phosphate buffer, pH 7.5. Next, after washing with about 10 CV of the buffer solution, elution was performed with a linear gradient using the same phosphate buffer solution containing 0.3 mol / L sodium chloride. The first peak of the eluate was fractionated and collected. This was designated as highly purified A2 type NTX solution. The production period from the culture solution to purification of the highly purified A2 type NTX solution was 3 days. The results obtained in this test are shown in Table 2. As a result, it was shown that NTX with high purity and high specific activity can be prepared in large quantities with a very small number of steps as compared with the prior art. It was shown that it was possible to provide fully automated equipment and in-line equipment for the production line because the operator did not collect sediment. By eliminating / reducing the opportunity for workers to be exposed to high concentrations of Clostridium botulinum or botulinum toxin, it was expected that the worker's occupational safety could be significantly improved and that GMP compliance and sterility assurance could be improved. Furthermore, it was shown that the production period can be significantly shortened compared to the prior art, and highly purified NTX can be efficiently produced on an industrial scale.

Example 5 Separation and Purification of A1 Type M Toxin According to the Present Invention
A1 type Clostridium botulinum Hall strain was cultured by partially modifying the method of Siegel, LS et al. (Appl. Microbiol. Vol. 38, No. 4: 606-611., 1979). After 48 hours, 30 L of the culture broth was removed in the same manner as in Example 3 in the order of cell removal, concentration, buffer exchange, Q Sepharose Fast Flow chromatography, and SP Sepharose Fast Flow chromatography. In SP Sepharose Fast Flow chromatography, the third peak of the eluted fraction was fractionated to recover A1 type M toxin. The collected A1 type M toxin solution was concentrated with a tangential flow ultrafiltration device (Sartorius, 30 KDa), and then buffer exchanged with 50 mmol / L acetate buffer solution, pH 6.0 containing 0.2 mol / L sodium chloride. . As a result of SDS-PAGE test of the obtained A1 type M toxin, no band other than M toxin was observed.

Example 6 Separation and Purification of A1 Type NTX According to the Present Invention
Furthermore, after concentrating the A1 type M toxin solution collected in Example 5 with a tangential flow ultrafiltration device (Sartorius, 30 KDa), a 50 mmol / L acetate buffer containing 0.2 mol / L sodium chloride, pH 6 The buffer was exchanged at 0.0. This concentrated solution was developed on Sephacryl S-300 High Resolution (GE Healthcare) previously equilibrated with 50 mmol / L acetate buffer containing 0.2 mol / L sodium chloride, pH 6.0, and subjected to gel filtration. The eluted first peak was fractionated and collected. Subsequent operations were carried out in the same manner as in Example 4 in the order of concentration, buffer exchange with pH 7.5 buffer, and DEAE Sepharose Fast Flow chromatography to obtain a highly purified A1-type NTX solution. The production period from purification of the highly purified A1-type NTX solution from the culture was 4 days. The results obtained in this test are shown in Table 2. As a result, it was shown by the present invention that A1 type NTX having high purity and high specific activity can be purified in the same manner as highly purified A2 type NTX. Therefore, according to the present invention, NTX can be purified with high purity and high specific activity even from strains (types B to G) having different botulinum toxin complex components (LL toxin / L toxin / M toxin). Can be expected.

<< Example 7: Separation and purification of A1 type LL and L toxins according to the present invention >>
In SP Sepharose Fast Flow chromatography in Example 5, the second peak of the eluted fraction was fractionated to recover LL toxin and L toxin. Concentrate the collected LL toxin and L toxin mixed solution with a tangential flow ultrafiltration device (Sartorius, 30 KDa), then buffer with 50 mmol / L acetate buffer containing 0.2 mol / L sodium chloride, pH 6.0 Exchanged. As a result of SDS-PAGE test of the obtained LL toxin and L toxin mixed solution, bands other than LL toxin and L toxin were not recognized.

<< Example 8: Comparison of stability of highly purified A2 type NTX >>
The stability at 23 ° C. of highly purified A2 type NTX solutions prepared in the prior art (Comparative Example 2) and the present invention (Example 4) was compared. The result of the activity remaining rate is shown in FIG. As a result, the highly purified A2 type NTX prepared by the present invention did not have a decrease in toxin activity compared to the toxin prepared by the prior art. In the SDS-PAGE test, no degradation product was observed in the highly purified A2-type NTX prepared in the present invention. This result indicates that the highly purified A2 type NTX prepared in the present invention is more stable than the toxin prepared by the prior art.

  According to the present invention, compared with the prior art, the membrane filtration method and the chromatography method are combined without performing the precipitation method / centrifugation, so that the botulinum culture solution is extremely easily and stably stabilized in a short time. It became possible to prepare a large amount of highly specific botulinum toxin. In addition, by eliminating the sediment collection work in the precipitation method and centrifugation, it has become possible to provide fully automated equipment and in-line equipment for the production line. In addition, there is no operator intervention and the toxin solution is not exposed to the environment, which makes it possible to improve workers' occupational safety and health environment, GMP compliance and sterility assurance.

Claims (23)

A method for purifying botulinum toxin, comprising combining membrane filtration, anion exchange chromatography, and cation exchange chromatography. The purification method according to claim 1, comprising the following steps.
(a) a step of removing botulinum cells from the Clostridium botulinum culture solution by a membrane filtration method,
(b) developing the botulinum toxin solution from which the botulinum cells have been removed in the step (a) on an anion exchange chromatography column,
(c) developing the recovered fraction of the anion exchange chromatography column obtained in the step (b) on a cation exchange chromatography column under conditions such that the botulinum toxin component is adsorbed on the column; and
(d) washing away impurities from the cation exchange chromatography and then eluting the botulinum toxin. The purification method according to claim 1 or 2, wherein the membrane filtration method is a precision membrane filtration method. The purification method according to claim 3, wherein the pore size of the membrane used in the precision membrane filtration method is 0.22 µm or less. The purification method according to claim 2, further comprising a step of performing coarse membrane filtration with a membrane having a larger pore diameter than that of the membrane used for the precision membrane filtration before performing the precision membrane filtration in the membrane filtration method. The purification method according to claim 1 or 2, wherein the pH of the buffer in the anion exchange chromatography column is 4.5 to 6.8. The purification method according to claim 1 or 2, wherein the salt concentration of the buffer in the anion exchange chromatography column is 0 to 0.3 mol / L. In the anion exchange chromatography, when the botulinum toxin component is adsorbed on a column, the botulinum toxin adsorbed on the column is eluted with a gradient having a salt concentration of 0 to 2.0 mol / L. Method. Before the step (b), further comprising the step of adjusting the botulinum toxin solution from which the botulinum cells have been removed in the step (a) to the conditions of the salt concentration and pH of the anion exchange chromatography. 2. The purification method according to 2. The purification method according to claim 1 or 2, wherein the pH of the buffer in the cation exchange chromatography column is from 4.0 to 4.5. The purification method according to claim 1 or 2, wherein the salt concentration of the buffer in the cation exchange chromatography column is 0 to 0.2 mol / L. Before performing the step (c), the method further includes a step of adjusting the recovered fraction of the anion exchange chromatography column obtained in the step (b) to the salt concentration and pH conditions of the cation exchange chromatography. The purification method according to claim 2. The purification method according to claim 1 or 2, wherein in the step (d), the botulinum toxin adsorbed on the cation exchange chromatography is eluted with a gradient having a salt concentration of 0.2 to 0.7 mol / L. The purification method according to claim 2, wherein the botulinum toxin eluted in step (d) is LL toxin, L toxin or M toxin. The method for purifying botulinum toxin according to claim 1 or 2, further comprising the following steps after the steps (a) to (d).
(e) The cation exchange chromatography obtained in the step (d) under the condition that the botulinum toxin component is adsorbed on the column and the botulinum neurotoxin (NTX) and non-toxic non-HA protein (NTNH) are dissociated. Developing the elution fraction of the chromatography on a second anion exchange chromatography column; and
(f) washing away impurities from the second anion exchange chromatography and then eluting botulinum neurotoxin (NTX); The purification method according to claim 1, wherein the buffer solution in the second anion exchange chromatography column has a pH of 6.8 to 8.0. The purification method according to claim 1, wherein the salt concentration of the buffer in the second anion exchange chromatography column is 0 to 0.1 mol / L. 16. The purification method according to claim 1, wherein in the step (f), NTX adsorbed on the second anion exchange chromatography is eluted with a gradient having a salt concentration of 0 to 0.3 mol / L. Before performing the step (e), the step of adjusting the elution fraction of the cation exchange chromatography obtained in the step (d) to the salt concentration and pH conditions of the second anion exchange chromatography is further performed. The purification method according to claim 15, comprising: The purification method according to any one of claims 1 to 19, wherein the botulinum toxin is a botulinum toxin derived from A, B, C, D, E, F, and G botulinum. A botulinum toxin purified by the purification method according to any one of claims 1 to 20. The botulinum toxin according to claim 21, wherein the botulinum toxin is any one of LL toxin, L toxin, M toxin, and NTX. A drug containing the botulinum toxin according to claim 21 or 22 as an active ingredient.

Images