PRODUCTION OF ACTIVATED CLOSTRIDIAL NEUROTOXINS

Abstract

The present invention relates to a method of producing activated clostridial neurotoxins that are essentially free of unactivated products, to compositions comprising such and to their use in therapy.

Claims

1. Method for producing an activated clostridial neurotoxin, comprising contacting a single chain clostridial neurotoxin with an activation enzyme until at least 90% of the single chain clostridial neurotoxin is converted into a di-chain clostridial neurotoxin.

2. Method according to claim 1, wherein said activation enzyme is a trypsin.

3. Method according to claim 2, wherein said trypsin is a bovine trypsin and wherein said bovine trypsin has an amino acid sequence which has at least 90% identity to SEQ ID NO: 1.

4. Method according to claim 3, wherein said bovine trypsin is selected from a native trypsin obtained from bovine pancreas and a recombinant bovine trypsin.

5. Method according to claim 3 or 4, wherein said step of contacting said single chain clostridial neurotoxin with a bovine trypsin is performed at a pH of between 5 and 7,5, preferably between 6 and 7, for example at a pH of approximatively 6,5.

6. Method according to claim 5, wherein the step of contacting the single chain clostridial neurotoxin with said bovine trypsin is carried out at room temperature at a pH between 6 and 7 for a duration of 15 to 25 hours, and wherein the concentration of said bovine trypsin is between 0.5 and 3 g per mg of clostridial neurotoxin.

7. Method according to any one of claims 1 to 6, further comprising a step of removing truncated clostridial neurotoxin.

8. Method according to claim 7, wherein said step of removing truncated clostridial neurotoxin comprises contacting said activated clostridial neurotoxin with a mixed mode chromatography resin.

9. Method according to any one of claims 1 to 8, wherein said clostridial neurotoxin is selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT.

10. Method according to any one of claims 1 to 9, wherein said clostridial neurotoxin is a modified botulinum neurotoxin as compared to a wild-type botulinum neurotoxin, eg a mutated clostridial neurotoxin, a chimeric clostridial neurotoxin or a retargeted clostridial neurotoxin.

11. Method according to any one of claims 1 to 10, wherein said clostridial neurotoxin comprises a BoNT/E activation loop.

12. Method according to anyone of claims 1 to 11, wherein said clostridial neurotoxin is a BoNT/E.

13. Method according to any one of claims 1 to 12, wherein said single chain clostridial neurotoxin is obtained by expressing a gene encoding said single chain clostridial neurotoxin in a heterologous host cell, preferably in E. coli.

14. An active di-chain clostridial neurotoxin obtainable by the method of any one of claims 1 to 13.

15. A pharmaceutical composition comprising an active di-chain clostridial neurotoxin according to claim 14, wherein said composition is essentially free of single chain clostridial neurotoxin.

16. A pharmaceutical composition according to claim 15 for use in therapy, eg for treating ophtalmological disorders, movement disorders, otorhinolaryngological disorders, gastrointestinal disorders, urogenital disorders, dermatological disorders, pain disorders, inflammatory disorders, secretory disorders, respiratory disorders, hypertrophic disorders, articular disorders, endocrine disorders, autoimmune diseases, proliferative diseases, traumatic injuries, veterinary uses.

17. Use of a pharmaceutical composition according to claim 15 in cosmetics or esthetics.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Embodiments of the invention will now be described, by way of example only, with reference to accompanying drawings, in which:

[0124] FIG. 1 shows the relative amounts of full length activated botulinum neurotoxin (endonegative) and truncated activated botulinum neurotoxin heavy chains following incubation of unactivated botulinum neurotoxin samples at pH 8 and a protein concentration of 0.55 mg/mL at 20 C. with recombinant porcine trypsin (Roche) at final concentrations of 0.3 and 0.4 g/mL respectively. Samples were removed for analysis by SDS-PAGE. Each SDS-PAGE sample was analysed by densitometry.

[0125] FIG. 2 shows analysis by SDS-PAGE under reducing conditions, after activation of single chain endonegative botulinum neurotoxin E (0.55 mg/mL) at pH 8.0 with bovine trypsin (Sigma-Aldrich) at various concentrations and incubation for 8 hours at 20 C. with, (Lane 1: Molecular weight marker; lane 2: 20 C. control; lane 3: +20 C. control; lane 4: 0.2 g/mL trypsin; lane 5: 0.4 g/mL trypsin; lane 6: 0.6 g/mL trypsin; lane 7: 0.8 g/mL trypsin; lane 8: 1.0 g/mL trypsin).

[0126] FIG. 3 shows the respective percentages of endonegative botulinum neurotoxin E heavy chain (HC), light chain (LC) and truncated heavy chain (tHC) after activation with recombinant bovine trypsin at pH 6.5, 7.0, 7.5 and 7.8 and incubation for 16 hours at 20 C. (neurotoxinconcentration: 0.55 mg/mL, recombinant bovine trypsin (Sigma-Aldrich) concentration: 1,5 g/mL). Samples were analysed by SDS-PAGE under reducing conditions by densitometry.

[0127] FIG. 4 shows that the separation of full-length, di-chain botulinum neurotoxin E (endonegative) from truncated di-chain botulinum neurotoxin can be achieved following activation with bovine trypsin and separation using a ceramic hydroxyapatite type II chromatography column. The elution of full length di-chain botulinum neurotoxin and truncated di-chain botulinum neurotoxin from the column was monitored by online A.sub.280 nm readings, selected fractions containing only full length di-chain botulinum neurotoxin were pooled and analysed by SDS-PAGE under reducing and non-reducing conditions.

[0128] FIG. 5: protein sequence of BoNT/AUniProtKB Accession Number P10845 (SEQ ID NO: 2).

[0129] FIG. 6: protein sequence of BoNT/BUniProtKB Accession Number P10844 (SEQ ID NO: 3).

[0130] FIG. 7: protein sequence of BoNT/CUniProtKB Accession Number P18640 (SEQ ID NO: 4).

[0131] FIG. 8: protein sequence of BoNT/DUniProtKB Accession Number P19321 (SEQ ID NO: 5).

[0132] FIG. 9: protein sequence of BoNT/EUniParc I.D UPI00000010A3 (SEQ ID NO: 6).

[0133] FIG. 10: protein sequence of BoNT/FUniProtKB Accession Number YP_001390123 (SEQ ID NO: 7).

[0134] FIG. 11: protein sequence of BoNT/GUniProtKB Accession Number Q60393 (SEQ ID NO: 8).

[0135] FIG. 12: protein sequence of TeNTUniProtKB Accession Number P04958 (SEQ ID NO: 9).

[0136] FIG. 13: protein sequence of bovine trypsin (SEQ ID NO: 1).

EXAMPLES

[0137] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Example 1

[0138] Single chain botulinum neurotoxin E (endonegative) samples at pH 8 and a protein concentration of 0.55 mg/mL were incubated at 20 C. with recombinant porcine trypsin (Roche) at final concentrations of 0.3 and 0.4 pg/mL. Samples were removed for analysis by SDS-PAGE under reducing conditions every 30 minutes up to 6 hours and every 60 minutes afterwards up to 9 hours. Each SDS-PAGE samples was analysed by densitometry to determine the relative amounts of full length di-chain botulinum neurotoxin, truncated di-chain botulinum neurotoxin heavy chains and single chain botulinum neurotoxin. The values for the full length di-chain botulinum neurotoxin and truncated di-chain botulinum neurotoxin were then plotted on a chart (FIG. 1). Truncated di-chain botulinum neurotoxin occurs before full activation of the botulinum neurotoxin is achieved when contacted with porcine trypsin.

Example 2

Activation with Different Concentrations of Bovine Trypsin

[0139] Single chain botulinum neurotoxin E (endonegative) samples at pH 8.0 with a protein concentration of 0.55 mg/mL were incubated at 20 C. with bovine trypsin (Sigma-Aldrich) at final concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 g/mL respectively. Samples were removed for analysis by SDS-PAGE under reducing conditions after 8 hours. The results, presented in FIG. 2, show that heavy chain truncation was observed in each sample before complete activation had been achieved.

Example 3

Activation with Bovine Trypsin at Different pH

[0140] Single chain botulinum neurotoxin E (endonegative) samples at pH 6.5, 7.0, 7.5 and 7.8, with a protein concentration of 0.55 mg/mL were incubated at 20 C. with recombinant bovine trypsin (Sigma-Aldrich) at a final concentration of 1,5 g/mL. Samples were removed for analysis by SDS-PAGE under reducing conditions after 16 hours. Each SDS-PAGE sample was analysed by densitometry to determine the relative amounts of truncated di-chain botulinum neurotoxin heavy chain. The results are presented in FIG. 3 and table 1). Truncated di-chain botulinum neurotoxin formation occurs more readily at higher pH.

TABLE-US-00001 TABLE 1 Percentage of truncated heavy chain at different pH (<LOD: below limit of detection) pH Truncated Heavy Chain (%) 6.5 <LOD 7.0 <LOD 7.5 8.9 7.8 12.1

Example 4

Purification of Activated Neurotoxin After Activation with Bovine Trypsin

[0141] 26 mg of total protein containing endonegative BoNT/E that had been activated by incubation with 78.57 g Trypzean (bovine trypsin) for 18 hours at 20 C. was applied to a 5 mL ceramic hydroxyapatite type II column. The column was washed with binding buffer (25 mM sodium phosphate, pH 6.5) and then eluted over 35 column volumes increasing the sodium phosphate concentration with a linear gradient using binding buffer and elution buffer (500 mM sodium phosphate pH 6.5), collecting 2.5 mL fractions. The elution of full length, di-chain botulinum neurotoxin and truncated di-chain botulinum neurotoxin from the column was monitored by online A280 nm readings, selected fractions containing only full length di-chain botulinum neurotoxin were pooled and analysed by SDS-PAGE under reducing and non-reducing conditions (FIG. 4).

Example 5

Full Length Activated Botulinum Neurotoxin E Preparation

[0142] A botulinum neurotoxin E inoculum E. coli culture was prepared by thawing a seed bank vial and inoculating a shake flask containing 100 mL modified Terrific Broth (mTB). The flasks were then incubated at 25 C. for 17 hours in a shaking incubator. The inoculum culture was used to inoculate five shake flasks, each containing 1 L of mTB. The cells were cultivated at 37 C. in a shaking incubator into exponential growth phase; the temperature of the cultures reduced to 16 C.; and the cultures were induced by adding isopropyl -D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. The cells were harvested 20 hours post-induction by tangential flow filtration (TFF) using a hollow fibre membrane. The culture was first concentrated five-fold and then diafiltered with five volumes of lysis buffer (100 mM sodium phosphate, 100 mM NaCl, 1.3 M (NH4)2SO4, pH 7.8).

[0143] The resulting cell paste slurry was homogenised by two passes through a mechanical cell disrupter. The insoluble cell debris was sedimented by centrifugation and the supernatant was recovered and applied to a column packed with Butyl Sepharose 4 FF (GE Lifesciences), which was washed with binding buffer (100 mM sodium phosphate, 100 mM NaCl, 1.25 M (NH.sub.4).sub.2SO.sub.4, pH 7.8). The unactivated botulinum neurotoxin E was eluted from the column using three step gradients with the following mixtures of binding and elution (100 mM sodium phosphate, 100 mM NaCl, pH 7.8) buffers, with the product eluted in step 2.

TABLE-US-00002 Step 1 88% loading buffer; 12% elution buffer Step 2 58% loading buffer; 42% elution buffer Step 3 100% elution buffer

[0144] The material from step 2 was then concentrated approximately two-fold by TFF using a hollow fibre membrane and then diafiltered with 10 volumes of 25 mM sodium phosphate pH 6.5 buffer. After diafiltration any insoluble material in the retentate was sedimented by centrifugation and the supernatant applied to a column packed with Q Sepharose HP (GE Lifesciences) in a negative chromatography step. The flowthrough containing the unactivated botulinum neurotoxin was collected and the column was washed with 25 mM sodium phosphate pH 6.5 to maximize product recovery.

[0145] The flowthrough was then diluted to a total protein concentration of 0.5 mg/ml with 25 mM sodium phosphate pH 6.5 and incubated with 7.27 USP units/mL recombinant bovine trypsin (TrypZean) for 21 hours at room temperature. After incubation the activated botulinum neurotoxin E was then applied to a ceramic hydroxyapatite type II column, which was washed with binding buffer (25 mM sodium phosphate pH 6.5). The activated botulinum neurotoxin was eluted from the column with a linear gradient using binding buffer and elution buffer (500 mM sodium phosphate pH 6.5).

[0146] Fractions containing full-length, activated botulinum neurotoxin were pooled and applied to a column packed with Benzamidine Sepharose FF (high-sub) (GE Lifesciences) in a negative chromatography step. The flowthrough containing the full-length, activated botulinum neurotoxin was collected and the column was washed with loading buffer (110 mM sodium phosphate, pH 6.5) to maximize product recovery. The flowthrough was diafiltered by TFF using a hollow fibre cartridge into the final storage buffer with 5 volumes of 25 mM sodium phosphate, 100 mM NaCl, pH 6.5.