GB2272697A

GB2272697A - Process for producing crystalline type A botulinum toxin

Info

Publication number: GB2272697A
Application number: GB9321794A
Authority: GB
Inventors: Genji Sakaguchi; Tomoko Shimizu
Original assignee: Chiba Prefectural Government
Current assignee: Chiba Prefectural Government
Priority date: 1992-10-28
Filing date: 1993-10-22
Publication date: 1994-05-25
Also published as: SE9303323D0; FR2697163B1; SE9303323L; DE4335366A1; JPH06192296A; DK112793A; GB9321794D0; FR2697163A1; DK112793D0

Description

2272697 PROCESS FOR PRODUCING CRYSTALLINE TYPE A BOTULINUM TOXIN This

invention relates to a process for producing crystalline type A botulinum toxin which is suitable as a therapeutic medicine and, more specifically. it relates to a process for producing crystalline type A botulinum toxin utilising a nucleic acid removing agent, e.g. a natural substance such as, for example, chitoxan (trade name of products manufactured by Dainippon Seiyaku Co.).

Botulinum toxin, as a typical example of bacteriaderived neurotoxin is a proteotoxin produced from Clostridium botulinum. Botulinum toxin is classified into eight sero types of A, B, Cl, C2, D, E, F, G depending on the antigenicity. Among them the type A toxin was the first one which was purified and crystallized (Lamnna, C.O.E.McElroy and H.W. Eklund: Science, 103: 613-614, 1946).

Heretofore, the production process for crystalline botulinum type A toxin has required complicated procedures conducted in the following order: acid precipitation (1), water washing, extraction with calcium chloride and acid precipitation (2), extraction with phosphate buffer, precipitation with ethanol and extraction and deposition in ammonium sulfate solution. In addition, since the production operation is complicated, it requires the skill and perception of experienced engineers. Further, the process gives only a low toxin recovery rate and one cannot expect to obtain a required and effective amount thereof. Moreover, although a method comprising combination of, for example, a protamine treatment, ion exchange chromatography and gel filtration has also been adopted, nevertheless crystallization efficiency of such methods is not optimal.

One of the problems involved in the crystallization of botulinum type A toxin is how to obtain a toxin of an easily crystallizable. macro molecule in good yield. Another of the problems is how to enable elimination of crystallization inhibiting substances mainly comprising nucleic acid contained in the culture solution without denaturing the toxin. The first problem can be solved by selecting a strain yielding botulinum type A toxin having a molecular weight of 500,000 to 900,000 and the solution to the second problem depends on how the nucleic acid can be removed under mild conditions.

The present invention is directed to the foregoing disadvantages in the prior art and an object of the invention is to provide a process for producing crystalline type A botulinum toxin having improved stability and effectiveness as a therapeutic medicine such as a medicine for the treatment of strabismus and blepharospasm, by obtaining a pure toxin.

Thus, in one aspect the present invention provides a process for producing a crystalline Type A botulinum toxin, which process comprises the step of removing nucleic acid using a nucleic acid removing agent, whereby to produce crystalline type A botulinum toxin substantially without loss of the toxicity thereof. With such a process, the botulinum type A toxin is crystallized substantially without loss of its toxicity. The toxin is obtained substantially without denaturation and deactivation, in a substantially pure form, in an improved yield for toxic activity. The product is suitable as a therapeutic medicine, e.g. to treat strabismus or blepharospasm.

9 Botulinum type A toxin is crystallized without loss of toxicity by removing, during the process, nucleic acid with a nucleic acid removing agent. The nucleic acid removing agent preferably comprises a natural substance such as chitoxan.

First, among strains yielding the botulinum type A toxin, strains producing L toxin of molecular weight 500.000 and LL toxin of molecular weight 900,000 with high hemoagglutination titer and showing high toxicity are conveniently selected. The strains are conveniently cultured in an appropriate culture medium, for example, NZ-amine culture medium, an acid is added to a culturefiltrate to cause precipitation, and a phosphate buffer solution with the molar concentration being changed is used instead of calcium chloride to extract the toxic ingredient effectively from the precipitate. Further, the liquid extract is repeatedly subjected to pH control, overnight standing, centrifugation, dissolution in an acetate buffer solution, pH modification and centrifugation to obtain a clear supernate. A nucleic acid removing agent is added to the supernate under most effective conditions.

Heretofore, calcium and alcohol have been used customarily for removing nucleic acid. However, no remarkable result can be obtained even when conditions such as concentration and pH of the nucleic acid removing agent are changed. Further, protamine has been used as a nucleic acid removing agent but it causes precipitation of not only nucleic acid but also necessary proteins though it has an excellent effect as a removing agent. In addition, it brings about the problem of differences in the homogeneity between batches, which is a fatal defect of natural products. Further, it is necessary to use an additional procedure for eliminating excess protamine from the resultant precipitate. On the other hand. chitoxan (trade name products manufactured by Dainippon Seiyaku Co.) is a natural high molecular weight substance, which is a natural substance obtained by deacetylating chitin (poly-N-acyl-P-D-glucosamine) contained in outer shells, for example, of crabs and shrimps and C-7, C-9 obtained by reducing the molecular weight thereof can effectively remove nucleic acid without loss of the toxin. When ammonium sulfate salting out is conducted after removal of nucleic acid with chitoxan. excessive chitoxan does not precipitate and, after dissolving the salted out toxin into a phosphate buffer solution, ammonium sulfate is conveniently gradually added and stood still to easily crystallize the toxin.

In the production process using Duff's method, the yield of the crystalline type A botulinum toxin was as low as 1.4 - 3.1 assuming the culture solution as 100. Although it could be increased up to about 5% by improving the extraction method, the yield of the toxic activity can be increased to 10% by the method according to the present invention, particularly using chitoxan.

According to the present invention, nucleic acid can be removed efficiently when producing the crystalline type A botulinum toxin. The yield of the toxic activity can be improved and, as a result, a remarkable effect can be attained as a therapeutic medicine such as for treating strabismus and blepharospasm.

Other features and advantages of the present invention will become apparent from the following Example:

Examl:)le 1 (Culturing) Clostridium botulinum, strain Hall. NZ-amine culture medium, standing culture at 350C for 4 days.

(Acid precipitation 1) sulfuric acid added: pH adjusted to 3.5 with 3N H2SO4 stood still overnight (40C) Supernate removed (siphon) Centrifugation Precipitate: Dissolved in distilled water, pH adjusted to 5.0 with 0.1N NaCl Centrifugation (Extraction) Precipitate: 0.2M phosphate buffer solution (pH 6.0) added Centrifugation Supernate Precipitate: 0.5M NaCl incorporated 0.1M tris- HCl buffer solution (pH 7.5) added Centrifugation (Acid precipitation 2) HCl added: pH adjusted to 3.7 with 2N HCl, stood still overnight (4C) Supernate removed (siphon) Centrifugation Precipitate: Dissolved in 0.05M acetate buffer solution (pH 5.0) Centrifugation (C-9 treatment) Supernate: 5% C-9 solution added by 0.1% for one min, stirred for 10 min in an ice box Centrifugation Supernate Ammonium sulfate added: Solid ammonium sulfate added to make 50% saturation solution. Stood still overnight at 4C. Centrifugation Precipitate: Precipitate dissolved in 0.03M phosphate buffer solution at pH 6.8 Dialysis: Dialyzed with 0.03M phosphate buffer solution with pH 6.8 (Crystallization) Ammonium sulfate added: Ammonium sulfate added until the solution exhibits pearlescent color, providing that the final concentration does not exceed 0.9M concentration Crystal deposition: Left at 40C [(PreDaration of 5% C-9 solution) Distilled water was added to C-9 (Kurimover, manufactured by Dainippon Seiyaku, Lot No. K143) at 10 to 1 ratio. Further, 1N HCl was added at a ratio of 2 and stirred by a stirrer for 2 hours. pH was adjusted with 1N sodium hydroxide to 4-6. The solution was diluted with distilled water to a 5% solution.] 1

Claims

1. A process for producing a crystalline type A botulinum toxin, which process comprises the step of removing nucleic acid using a nucleic acid removing agent, whereby to produce crystalline type A botulinum toxin substantially without loss of the toxicity thereof.

2. A process as claimed in claim 1 wherein said nucleic acid removing agent is a natural substance.

3. A process as claimed in claim 1 or claim 2 wherein said nucleic acid removing agent is chitoxan.

4. A process as claimed in any one of the preceding claims wherein said type A botulinum toxin is produced from a strain of bacteria yielding botulinum type A toxin having a molecular weight of 500,000 to 900,000.

5. A process as claimed in any one of the preceding claims, which process comprises the steps of culturing a strain of bacteria yielding botulinum type A toxin, precipitating a culture filtrate with an acid, extracting the precipitate with a phosphate buffer solution, adjusting the pH of the liquid extract, standing the adjusted solution overnight, centrifugally separating the adjusted solution, dissolving the separated precipitate with an acetate buffer solution, modifying the pH value of the dissolved solution, centrifugally separating the modified solution and adding said nucleic acid removing agent to the supernate.

6. A process for producing a crystalline type A botulinum toxin substantially as herein described with reference to the Example.

7. Crystalline type A botulinum toxin when made by a process as claimed in any one of claims 1 to 6.

8. A pharmaceutical composition comprising a crystalline type A botulinum toxin as claimed in claim 7 together with at least one pharmaceutical excipient.

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