Claims for Patent: 10,030,238

Summary for Patent: 10,030,238

Title:	Recombinant clostridium botulinum neurotoxins
Abstract:	The invention provides a nucleic acid sequence comprising a sequence of contiguous nucleotides, wherein said sequence of contiguous nucleotides has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, and wherein said sequence of contiguous nucleotides encodes a single-chain BoNT/E1 protein. The present invention also provides methods for producing soluble single-chain BoNT/E1 protein in an E. coli host cell, together with methods for producing soluble di-chain BoNT/E1 protein.
Inventor(s):	Cossins; Aimee (Abingdon, GB), Beard; Matthew (Abingdon, GB), Marks; Philip (Wrexham, GB)
Assignee:	IPSEN BIOINNOVATION LIMITED (Abingdon, Oxfordshire, GB) IPSEN BIOPHARM LIMITED (Slough, Berkshire, GB)
Application Number:	14/427,234
Patent Claims:	1. A nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the sequence encodes a single-chain BoNT/E1 polypeptide. 2. The sequence of claim 1, wherein the sequence has a maximum of 160 slow codons. 3. A nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the sequence encodes a single-chain BoNT/E1 polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. 4. The sequence of claim 3, wherein the single-chain BoNT/E1 polypeptide comprises one or more of the following amino acids, wherein the amino acid position numbering starts with the N-terminal amino acid residue of the polypeptide and ends with the C-terminal amino acid residue thereof: glycine at position 177; serine at position 198; alanine at position 340; leucine at position 773; leucine at position 963; glutamine at position 964; alanine at position 967; and asparagine at position 1195. 5. A nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the sequence encodes a single-chain BoNT/E1 polypeptide and comprises at least 785 synonymous codons when compared to SEQ ID NO: 3. 6. A method for producing a soluble single-chain BoNT/E1 polypeptide, the method comprising expressing the nucleic acid sequence of claim 1 in an E. coli expression system. 7. The method of claim 6, wherein the soluble single-chain BoNT/E1 polypeptide is expressed in the cytoplasm of the E. coli host cell. 8. The method of claim 6, wherein the soluble single-chain BoNT/E1 polypeptide is expressed at a level of at least 5 mg/L. 9. The method of claim 6, further comprising lysis of the E. coli host cell to provide an E. coli host cell homogenate containing the soluble single-chain BoNT/E1 polypeptide. 10. A method for producing a soluble di-chain BoNT/E1 protein, the method comprising: providing a soluble single-chain BoNT/E1 polypeptide having an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 2; contacting the polypeptide with trypsin in solution, allowing for the trypsin to cleave the single-chain polypeptide, resulting in a di-chain BoNT/E1 protein; and separating the soluble BoNT/E1 protein from trypsin by contacting the solution containing soluble BoNT/E1 protein and trypsin with a hydrophobic surface, wherein the soluble BoNT/E1 protein preferentially binds to the hydrophobic surface. 11. The method of claim 10, wherein the polypeptide comprises one or more of the following amino acids, wherein the amino acid position numbering starts with the N-terminal amino acid residue of the polypeptide and ends with the C-terminal amino acid residue thereof: glycine at position 177; serine at position 198; alanine at position 340; leucine at position 773; leucine at position 963; glutamine at position 964; alanine at position 967; and asparagine at position 1195. 12. The method of claim 10, wherein the soluble single-chain BoNT/E1 polypeptide is provided by expressing a nucleic acid sequence in an E. coli expression system, the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1 and encoding a single-chain BoNT/E1 polypeptide. 13. The method of claim 10, wherein the hydrophobic surface is an inert matrix to which a ligand consisting of aryl or alkyl groups is attached. 14. The method of claim 13, wherein the hydrophobic surface comprises butyl ligands, phenyl ligands, and/or octyl ligands. 15. An active di-chain BoNT/E1 protein, wherein the first chain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of positions 1-419 of SEQ ID NO: 2; wherein the second chain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of positions 423-1252 of SEQ ID NO: 2; and wherein the first and second chains are joined together by a disulphide bond between cysteine 412 on the first chain and cysteine 426 on the second chain; wherein the sequences include comprises one or more of the following amino acids, wherein the amino acid position numbering starts with the N-terminal amino acid residue of the polypeptide and ends with the C-terminal amino acid residue thereof: glycine at position 177; serine at position 198; alanine at position 340; leucine at position 773; leucine at position 963; glutamine at position 964; alanine at position 967; and asparagine at position 1195. 16. An active di-chain BoNT/E1 protein produced using the method of claim 10. 17. A composition comprising the active di-chain BoNT/E1 protein of claim 15, wherein said composition is substantially free of trypsin. 18. The composition of claim 17, wherein the composition contains less than 10 pg trypsin per 100 ng BoNT/E1 protein. 19. A pharmaceutical composition comprising: the active di-chain BoNT/E1 protein of claim 15; a surfactant; and water; wherein the composition does not comprise a protein stabilizing agent and is substantially free of trypsin. 20. The pharmaceutical composition of claim 19, further comprising: sodium chloride; a buffer to maintain pH between 5.5 and 7.5; and a disaccharide; and wherein the water is sterile water. 21. An active di-chain BoNT/E1 protein produced by proteolytic cleavage of the single-chain BoNT/E1 polypeptide produced by the method of claim 6.

Details for Patent 10,030,238

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2032-10-31