US2003031684A1PendingUtilityA1

Methods for the production of 3-O-deactivated-4'-monophosphoryl lipid a (3D-MLA)

48
Assignee: CORIXA CORPPriority: Mar 30, 2001Filed: Mar 14, 2002Published: Feb 13, 2003
Est. expiryMar 30, 2021(expired)· nominal 20-yr term from priority
Inventors:Kent Raymond MyersD. Snyder
A61K 2039/55572C12P 19/04C07H 13/06C12N 1/20A61P 37/04
48
PatentIndex Score
0
Cited by
0
References
0
Claims

Abstract

Herein is disclosed a method for producing lipopolysaccharide (LPS), comprising: (a) growing a culture of a deep rough mutant bacterial strain in a medium; (b) maintaining the culture in stationary phase for at least about 5 hr; (c) harvesting cells from the culture; and (d) extracting LPS from the cells. The method allows for the production of an LPS which can be used to produce a 3-O-deacylated monophosphoryl lipid A (3D-MLA) having at least about 20 mol % of the hexaacyl congener group. Also herein is disclosed a method of extracting lipopolysaccharide (LPS) from a culture of deep rough mutant bacterial strain cells, comprising: (a) extracting the cells with a solution consisting essentially of at least about 75 wt % of an aliphatic alcohol having from 1 to 4 carbon atoms and the balance water, thereby producing cells with reduced phospholipid content; and (b) extracting the cells with reduced phospholipid content with a solution comprising chloroform and methanol, thereby yielding a solution of LPS in chloroform and methanol (CM). This method provides LPS solutions in CM that have reduced phospholipid content and are produced by relatively simple and inexpensive process steps.

Claims

exact text as granted — not AI-modified
What is claimed is:  
     
         1 . A method for producing a lipopolysaccharide composition (LPS), comprising: 
 (a) growing a culture of a deep rough mutant bacterial strain in a medium;    (b) maintaining the culture in stationary phase for at least about 5 hr;    (c) harvesting cells from the culture; and    (d) extracting LPS from the cells.    
     
     
         2 . The method of  claim 1 , wherein the deep rough mutant bacterial strain is of the genus Salmonella.  
     
     
         3 . The method of  claim 2 , wherein the deep rough mutant bacterial strain of genus Salmonella is of species  Salmonella minnesota.    
     
     
         4 . The method of  claim 3 , wherein the deep rough mutant bacterial strain of species  Salmonella minnesota  is strain  Salmonella minnesota  R595.  
     
     
         5 . The method of  claim 1 , wherein the medium is M9.  
     
     
         6 . The method of  claim 1 , wherein the maintaining is performed for between about 5 hr and about 6 hr.  
     
     
         7 . The method of  claim 1 , wherein the LPS can be used to produce a 3D-MLA having at least about 20 mol % of the hexaacyl congener group.  
     
     
         8 . The method of  claim 1 , further comprising subjecting the LPS to sequential acid hydrolysis and base hydrolysis, to form 3D-MLA.  
     
     
         9 . An LPS produced by the method of  claim 1 .  
     
     
         10 . A 3D-MLA produced by the method of  claim 8 .  
     
     
         11 . A method of extracting lipopolysaccharide (LPS) from a culture of deep rough mutant bacterial strain cells, comprising: 
 (a) extracting the cells with a solution consisting essentially of at least about 75 wt % of an aliphatic alcohol having from 1 to 4 carbon atoms and the balance water, thereby producing cells with reduced phospholipid content;    (b) extracting the cells with reduced phospholipid content with a solution comprising chloroform and methanol, thereby yielding a solution of LPS in chloroform and methanol.    
     
     
         12 . The method of  claim 11 , wherein the deep rough mutant bacterial strain is of the genera Salmonella or Escherichia.  
     
     
         13 . The method of  claim 12 , wherein the deep rough mutant bacterial strain of genus Salmonella is of species  Salmonella minnesota.    
     
     
         14 . The method of  claim 13 , wherein the deep rough mutant bacterial strain of species  Salmonella minnesota  is strain  Salmonella minnesota  R595.  
     
     
         15 . The method of  claim 12 , wherein the deep rough mutant bacterial strain of genus Escherichia is of species  Escherichia coli.    
     
     
         16 . The method of  claim 15 , wherein the deep rough mutant bacterial strain of species  Escherichia coli  is of strain  Escherichia coli  D31 m4.  
     
     
         17 . The method of  claim 11 , wherein the aliphatic alcohol has from 2 to 4 carbon atoms.  
     
     
         18 . The method of  claim 17 , wherein the aliphatic alcohol is ethanol.  
     
     
         19 . The method of  claim 11 , wherein the solution comprising aliphatic alcohol comprises between about 85 wt % and about 95 wt % aliphatic alcohol.  
     
     
         20 . The method of  claim 11 , wherein the extracting with aliphatic alcohol is performed at a temperature between about 35° C. and about 65° C.  
     
     
         21 . The method of  claim 20 , wherein the temperature is between about 45° C. and about 55° C.  
     
     
         22 . The method of  claim 11 , further comprising evaporating the chloroform and methanol from the LPS solution, thereby yielding a dry LPS residue.  
     
     
         23 . The method of  claim 22 , further comprising subjecting the dry LPS residue to sequential acid hydrolysis and base hydrolysis, to form 3D-MLA.  
     
     
         24 . An LPS formed according to the method of  claim 22 .  
     
     
         25 . A 3D-MLA formed according to the method of  claim 23.

Join the waitlist — get patent alerts

Track US2003031684A1 — get alerts on status changes and closely related new filings.

We store only your email — no account needed. See our privacy policy.