Claims for Patent: 4,450,103

Summary for Patent: 4,450,103

Title:	Process for recovering human IFN-.beta. from a transformed microorganism
Abstract:	A process for recovering IFN-.beta. from transformed bacteria comprising: disrupting the cell membranes of the bacteria; solubilizing the IFN-.beta. from the disruptate into an aqueous medium with a solubilizing agent such as sodium dodecyl sulfate; extracting the IFN-.beta. from the aqueous medium with 2-butanol, 2-methyl-butanol, or mixtures thereof under conditions that maintain phase separation between the aqueous meduim and the extractant; and isolating the IFN-.beta. from the extractant such as by precipitating the IFN-.beta. from an aqeous buffer mixture of the extractant by lowering the pH thereof.
Inventor(s):	Konrad; Michael W. (Alameda, CA), Lin; Leo S. (Fremont, CA)
Assignee:	Cetus Corporation (Emeryville, CA)
Application Number:	06/353,360
Patent Claims:	1. A process for extracting unglycosylated human IFN-.beta. solubilized with an appropriate solubilizing agent from an aqueous medium comprising contacting the aqueous medium containing the unglycosylated human IFN-.beta. with an organic extractant of the formula ##STR2## where R is hydrogen or methyl under conditions wherein an aqueous and an organic phase are formed. 2. The process of claim 1 wherein the solubilizing agent is an anionic surface active agent. 3. The process of claim 1 wherein the solubilizing agent is sodium dodecyl sulfate or sodium laurate. 4. The process of claim 1 wherein the contacting is carried out under conditions that facilitate phase separation between the aqueous phase and the extractant phase. 5. The process of claim 1 wherein the ionic strength of the aqueous medium is about 0.05 to about 0.15. 6. The process of claim 1 wherein the ionic strength is provided by sodium chloride. 7. The process of claim 1, 2, 3, or 4 wherein the extractant is 2-butanol. 8. The process of claim 1, 2, 3, or 4 including the step of isolating the unglycosylated human IFN-.beta. from the extractant. 9. A process for recovering unglycosylated human IFN-.beta. from a transformed microorganism containing unglycosylated human IFN-.beta. comprising: (a) disrupting the cell membrane of the microorganism; (b) solubilizing the unglycosylated human IFN-.beta. in the disruptate into an aqueous medium with a solubilizing agent that forms an extractable complex with the unglycosylated human IFN-.beta.; (c) extracting the complex from the aqueous medium with an organic extractant of the formula ##STR3## where R is hydrogen or methyl; and (d) isolating the unglycosylated IFN-.beta. from the extract. 10. The process of claim 9 wherein the microorganism is a bacterium. 11. The process of claim 10 wherein the microorganism is E. coli. 12. The process of claim 9 or 11 wherein the microorganism is contained within a fermentation medium and is concentrated before step (a). 13. The process of claim 9 or 11 wherein the disruption is effected by subjecting the microorganism to pressure cycling to rupture its cell membrane. 14. The process of claim 9 wherein the solubilization of step (b) dissolves substantially all the particulate matter in the disruptate. 15. The process of claim 9 or 14 wherein the pH of the aqueous medium is one that maintains the solubilizing agent in solution. 16. The process of claim 9 wherein the solubilizing agent is an anionic surface active agent. 17. The process of claim 9 wherein the solubilizing agent is an alkali metal alkyl sulfate or an alkali metal salt of a fatty acid wherein said sulfate or said salt contains 10 to 14 carbon atoms. 18. The process of claim 15 wherein the anionic surface active agent is sodium dodecyl sulfate or sodium laurate. 19. The process of claim 15 wherein the pH is about 7 to about 8. 20. The process of claim 16 wherein the surface active agent is sodium dodecyl sulfate and the weight ratio of sodium dodecyl sulfate to protein in the aqueous medium is in the range of about 1:1 to about 5:1. 21. The process of claim 9 wherein the weight ratio is about 3:1. 22. The process of claim 9 wherein the extraction is carried out under conditions that facilitate phase separation between the aqueous phase and the extractant phase. 23. The process of claim 22 wherein the ionic strength of the aqueous medium is about 0.05 to about 0.15. 24. The process of claim 23 wherein said ionic strength is provided by sodium chloride in the aqueous medium. 25. The process of claim 9, 22, 23, or 24 wherein the organic extractant is 2-butanol. 26. The process of claim 9 wherein the isolation includes: (i) mixing the extract with an aqueous buffer; (ii) precipitating the unglycosylated human IFN-.beta. from the mixture of (i) by lowering the pH of the mixture; and (iii) isolating the precipitated unglycosylated human IFN-.beta. from the supernatant. 27. The process of claim 26 wherein the pH is lowered to below about 5. 28. The process of claim 26 or 27 wherein the solubilizing agent is sodium dodecyl sulfate. 29. A process for recovering unglycosylated human IFN-.beta. from transformed E. coli bacteria containing unglycosylated human IFN-.beta. said bacteria being contained in a fermentation medium comprising: (a) concentrating said bacteria in the fermentation medium; (b) disrupting the cell membrane of the bacteria by subjecting the bacteria to pressure cycling; (c) separating the solid cellular material from the remainder of the disruptate; (d) suspending the cellular material in an aqueous medium at a protein concentration in the range of 2 to 15 mg/ml and a pH in the range of about 7 and 8; (e) solubilizing the cellular material with sodim dodecyl sulfate at a sodium dodecyl sulfate:protein weight ratio of about 1:1 to 1:5; (f) extracting the unglycosylated human IFN-.beta. from the aqueous medium with 2-butanol wherein the ionic strength of the aqueous medium is sufficient to maintain the aqueous medium and 2-butanol substantially immiscible; (g) separating the aqueous phase from the unglycosylated, human IFN-.beta.-containing 2-butanol phase; and (h) precipitating the human IFN-.beta. from the 2-butanol phase by mixing the 2-butanol phase with an aqueous buffer and lowering the pH of the mixture to below about 5.

Details for Patent 4,450,103

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Bayer Healthcare Pharmaceuticals Inc.	BETASERON	interferon beta-1b	For Injection	103471	July 23, 1993		2039-03-29

International Patent Family for US Patent 4,450,103

Country	Patent Number	Estimated Expiration
Austria	20090
Australia	1331783
Australia	549721
Canada	1187013
Germany	3363647
European Patent Office	0102989
Italy	1164626