Recombinant elastase proteins and methods of manufacturing and use thereof
View Patent ↗The present invention relates to methods for the manufacture, purification, formulation, and use of biologically active recombinant elastase proteins. Described are recombinant methods for producing therapeutically useful elastase proteins, as are pharmaceutical compositions comprising said elastase proteins. Novel recombinant elastase proteins and protein preparations are also disclosed. Methods are described for treating and preventing diseases of biological conduits using pharmaceutical compositions containing the elastase proteins of the invention.
1. A pharmaceutical composition comprising a mature, enzymatically active human type I elastase protein comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 wherein the amount of trypsin corresponds to less than 5 ng of trypsin per 1 mg of mature elastase protein and wherein said mature elastase protein has an activity of 20 to 40 U/mg of protein as quantified using a colorimetric N-succinyl-Ala-Ala-Ala-pNitroanilide (SLAP) hydrolysis assay.
2. The pharmaceutical composition of claim 1 , wherein trypsin is undetectable by an ELISA assay.
3. The pharmaceutical composition of claim 1 which is produced by a process that does not utilize trypsin.
4. The pharmaceutical composition of claim 1 , which is free of any protein consisting of SEQ ID NOS:70 and 71.
5. The pharmaceutical composition of claim 1 , which contains less than 0.5% by weight of a protein consisting of SEQ ID NO:2 and less than 0.5% by weight of a protein consisting of SEQ ID NO:3.
6. The pharmaceutical composition of any one of claims 1 , 2 , and 3 , which is a liquid pharmaceutical composition.
7. The pharmaceutical composition of claim 6 , wherein the concentration of mature elastase protein is 0.001 mg/ml to 50 mg/ml.
8. The pharmaceutical composition of claim 6 , which is in the form of a unit dosage of 0.0033 mg to 200 mg of said mature elastase protein.
9. The pharmaceutical composition of claim 6 , which is free of bacterial proteins.
10. The pharmaceutical composition of claim 6 which is free of mammalian proteins other than said mature elastase protein.
11. The pharmaceutical composition of claim 6 , which contains pharmaceutically acceptable levels of endotoxins.
12. The pharmaceutical composition of claim 6 , which comprises phosphate ions.
13. The pharmaceutical composition of claim 6 , which comprises potassium ions.
14. The pharmaceutical composition of claim 6 , which comprises sodium and chloride ions.
15. The pharmaceutical composition of claim 6 , which comprises polysorbate-80.
16. The pharmaceutical composition of claim 6 , which comprises phosphate, sodium, potassium and chloride ions.
17. The pharmaceutical composition of claim 16 , which comprises polysorbate-80.
18. The pharmaceutical composition of claim any one of claims 1 , 2 , and 3 , which is a lyophilized pharmaceutical composition.
19. The pharmaceutical composition of claim 18 , which is in the form of a unit dosage of 0.0033 mg to 200 mg of said mature elastase protein.
20. The pharmaceutical composition of claim 18 , which is free of bacterial proteins.
21. The pharmaceutical composition of claim 18 , which is free of mammalian proteins other than said mature elastase protein.
22. The pharmaceutical composition of claim 18 , which contains pharmaceutically acceptable levels of endotoxins.
23. The pharmaceutical composition of claim 18 , which comprises phosphate ions.
24. The pharmaceutical composition of claim 18 , which comprises potassium ions.
25. The pharmaceutical composition of claim 18 , which comprises sodium and chloride ions.
26. The pharmaceutical composition of claim 18 , which comprises polysorbate-80.
27. The pharmaceutical composition of claim 18 , which comprises phosphate, sodium, potassium and chloride ions.
28. The pharmaceutical composition of claim 27 , which comprises polysorbate-80.
29. The pharmaceutical composition of claim 18 , wherein the mature elastase protein in said composition maintains 60% to 100% of its specific activity after one month of storage at 4° C.
30. The pharmaceutical composition of claim 18 , wherein the mature elastase protein in said composition maintains 60% to 100% of its specific activity after three months of storage at 4° C.
31. The pharmaceutical composition of claim 18 , wherein the mature elastase protein in said composition maintains 60% to 100% of its specific activity after six months of storage at 4° C.