Claims for Patent: 7,763,451

Summary for Patent: 7,763,451

Title:	Methods for preparing Bacillus anthracis protective antigen for use in vaccines
Abstract:	The invention relates to improved methods of producing and recovering B. anthracis protective antigen (PA), especially modified PA which is protease resistant, and to methods of using of these PAs or nucleic acids encoding these PAs for eliciting an immunogenic response in humans, including responses which provide protection against, or reduce the severity of, B. anthracis bacterial infections and which are useful to prevent and/or treat illnesses caused by B. anthracis, such as inhalation anthrax, cutaneous anthrax and gastrointestinal anthrax.
Inventor(s):	Shiloach; Joseph (Rockville, MD), Leppla; Stephen H. (Bethesda, MD), Ramirez; Delia M. (Bethesda, MD), Schneerson; Rachel (Bethesda, MD), Robbins; John B. (Chevy Chase, MD), Hsu; S. Dana (Bethesda, MD), Rosovitz; Mary Jo (Germantown, MD)
Assignee:	The United States of America as represented by the Department of Health and Human Services (Washington, DC) N/A (N/A)
Application Number:	10/290,712
Patent Claims:	1. A method of producing a Bacillus anthracis protective antigen protein, comprising: fermenting in a fermentation medium a cell or microorganism comprising a nucleotide sequence encoding said B. anthracis protective antigen protein in a manner to cause expression of said protective antigen, wherein the fermentation medium comprises dissolved oxygen at 30% saturation and is maintained at about pH 7 to about pH 8 throughout the fermenting. 2. The method of claim 1 further comprising recovering said protective antigen. 3. The method of claim 2 wherein said recovering step further comprises using hydrophobic interaction chromatography, ion exchange chromatography and gel filtration. 4. The method of claim 1 or 2 wherein the cell or microorganism is a protease-deficient nonsporogenic avirulent strain of B. anthracis. 5. The method of claim 1 wherein the B. anthracis protective antigen is a modified B. anthracis protective antigen wherein the receptor-binding domain has been altered. 6. The method of claim 5 wherein the B. anthracis protective antigen comprises an N657A substitution (SEQ ID NO: 5). 7. The method of claim 1 wherein the B. anthracis protective antigen is a modified B. anthracis protective antigen which cannot be cleaved at the chymotrypsin cleavage site. 8. The method of claim 7, wherein the modified protective antigen comprises an D315A substitution in SEQ ID NO: 3. 9. The method of claim 7, wherein the modified protective antigen comprises an F313A and an F314A substitution in SEQ ID NO: 3. 10. The method of claim 7, wherein the modified protective antigen comprises an F313C substitution in SEQ ID NO: 3. 11. The method of claim 1 wherein the B. anthracis protective antigen is a modified B. anthracis protective antigen which cannot be cleaved at the furin cleavage site. 12. The method of claim 11 wherein the B. anthracis protective antigen amino acid sequence RKKR.sup.167 (SEQ ID NO: 14) in SEQ ID NO: 3 has been changed to SNKE.sup.167 (SEQ ID NO: 15). 13. The method of claim 1 wherein the B. anthracis protective antigen is a modified B. anthracis protective antigen which cannot be cleaved at either the chymotrypsin or furin cleavage site. 14. The method of claim 13 wherein the amino acid sequence RKKR.sup.167 (SEQ ID NO: 14) has been changed to SNKE.sup.167 (SEQ ID NO: 15), the two phenylalanines at positions 313-314 are deleted and the glutamic acid at position 308 is substituted with aspartic acid. 15. The method of claim 2 wherein EDTA is added to the culture medium prior to the recovery step. 16. The method of claim 1, wherein the pH is maintained with HCl and NH.sub.4OH. 17. The method of claim 1, wherein the pH is maintained at about pH 7.5 throughout the fermentation. 18. The method of claim 3, wherein the hydrophobic interaction chromatography precedes the ion exchange chromatography. 19. The method of claim 1, wherein the B. anthracis protective antigen sequence comprises SEQ ID NO: 3. 20. The method of claim 1, wherein the B. anthracis protective antigen sequence is a fragment of a B. anthracis protective antigen sequence comprising SEQ ID NO: 13, and wherein the fragment B. anthracis protective antigen sequence can produce an immune response in a mammal. 21. The method of claim 1, wherein the B. anthracis protective antigen sequence comprises any of SEQ ID NOS: 3-13. 22. The method of claim 14, wherein the B. anthracis protective antigen amino acid sequence comprises SEQ ID NO: 4. 23. The method of claim 5, wherein the B. anthracis protective antigen amino acid sequence comprises SEQ ID NO: 5. 24. The method of claim 4, wherein the protease-deficient nonsporogenic avirulent strain of B. anthracis is BH445 (pXO 1-, pXO2-). 25. A method of producing a Bacillus anthracis exotoxin protein comprising fermenting in a fermentation medium a cell or microorganism comprising a nucleotide sequence encoding said B. anthracis exotoxin protein in a manner to cause expression of exotoxin protein wherein the-fermentation medium comprises dissolved oxygen at 30% saturation and is maintained at about pH 7 to about pH 8 throughout the fermenting. 26. The method of claim 25, wherein the exotoxin protein is a Bacillus anthracis lethal factor or protective antigen. 27. The method of claim 26, wherein the exotoxin protein is a Bacillus anthracis lethal factor. 28. The method of claim 27, further comprising recovering said lethal factor. 29. The method of claim 27, wherein the B. anthracis lethal factor is a modified B. anthracis lethal factor wherein a catalytic site of the lethal factor has been altered. 30. The method of claim 25, wherein the pH is maintained with HCl and NH.sub.4OH.

Details for Patent 7,763,451

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Emergent Biodefense Operations Lansing Llc	BIOTHRAX	anthrax vaccine adsorbed	Injection	103821	11/12/1998		2021-11-09