Production of activated clostridial neurotoxins
Method of producing an activated clostridial neurotoxin. Composition comprising an activated clostridial neurotoxin. Method of treatment using a composition comprising an activated clostridial neurotoxin.
1. A method for producing a composition comprising an activated clostridial neurotoxin and less than 10% A single-chain or truncated clostridial neurotoxin, the method comprising:
contacting a single-chain clostridial neurotoxin comprising a BoNT/E activation loop with trypsin at a concentration of 0.5 to 3 μg per mg of clostridial neurotoxin and a pH between 6 and 7 for a duration of 15 to 25 hours to convert the single-chain clostridial neurotoxin into di-chain clostridial neurotoxin; and
contacting the di-chain clostridial neurotoxin with a mixed mode chromatography resin.
2. The method of claim 1 , wherein the trypsin comprises an amino acid sequence that has at least 90% identity with SEQ ID NO: 1.
3. The method of claim 2 , wherein the trypsin is obtained from bovine pancreas or a recombinant bovine trypsin.
4. The method of claim 1 , wherein the single-chain clostridial neurotoxin is obtained by expressing a gene encoding the single-chain clostridial neurotoxin in a heterologous host cell.
5. The method of claim 2 , wherein the step of contacting the single-chain clostridial neurotoxin with the trypsin is performed at a pH of approximately 6.5.
6. The method of claim 1 , wherein the single-chain clostridial neurotoxin is obtained by expressing a gene encoding the single-chain clostridial neurotoxin in E. coli.
7. The method of claim 1 , wherein the mixed mode chromatography resin is a ceramic hydroxyapatite type II resin.
8. The method of claim 1 , wherein the clostridial neurotoxin is a chimeric clostridial neurotoxin or a re-targeted clostridial neurotoxin.
9. The method of claim 1 , wherein the clostridial neurotoxin is a mutated clostridial neurotoxin, a chimeric clostridial neurotoxin, or a re-targeted clostridial neurotoxin.
10. The method of claim 1 , wherein the composition comprises less than 5% single-chain or truncated clostridial neurotoxin.
11. The method of claim 1 , wherein the composition comprises less than 1% single-chain or truncated clostridial neurotoxin.
12. The method of claim 1 , wherein the composition comprises less than 0.1% single-chain or truncated clostridial neurotoxin.