Novel process

Abstract

A single-phase LPS extraction composition comprising water, an alcohol and a further organic solvent, where the amount of water is between about 0.8 to 1.2% (v/v).

1 . An LPS extraction composition comprising water, an alcohol and a further organic solvent. 
     
     
         2 . The composition of  claim 1  wherein the LPS extraction composition is single-phase. 
     
     
         3 . The composition of  claims 1  or  2  wherein the amount of water in the LPS extraction composition is between about 0.1 and about 1.5% (v/v). 
     
     
         4 . The composition of any of  claims 1  to  3  wherein the amount of water is about 1% (v/v). 
     
     
         5 . The composition of any of  claims 1  to  3  wherein the amount of water is about 0.5% (v/v). 
     
     
         6 . The composition of any of  claims 1  to  5  wherein the alcohol is selected from the list: methanol, ethanol, isopropanol or butanol. 
     
     
         7 . The composition of any of  claims 1  to  6  wherein the percentage of alcohol in the LPS extraction composition is between about 5% and about 40% (v/v). 
     
     
         8 . The composition of  claim 7  wherein the percentage of alcohol is between about 10% (v/v) and about 30% (v/v). 
     
     
         9 . The composition of any preceding claim wherein the further organic solvent is selected from the group: chloroform, alkanes, toluene and petroleum ether. 
     
     
         10 . The composition of  claim 9  wherein the alkane is selected from the group: isooctane, ethane, heptane and hexane. 
     
     
         11 . The composition of any preceding claim wherein the percentage of the further organic solvent in the LPS extraction composition is between about 60% (v/v) and about 95% (v/v). 
     
     
         12 . The composition of  claim 11  wherein the percentage of the further organic solvent is between about 75% (v/v) and about 90% (v/v). 
     
     
         13 . The composition of any of  claims 1  to  12  wherein the LPS extraction solution comprises chloroform, methanol and water. 
     
     
         14 . The composition of any of  claims 1  to  12  wherein the LPS extraction composition comprises an alkane, ethanol and water. 
     
     
         15 . A composition according to any of  claims 1  to  14  for use in the extraction of LPS from gram negative bacterial cells. 
     
     
         16 . Use of an LPS extraction composition according to any of  claims 1  to  14  in a method for extracting LPS from gram negative bacterial cells. 
     
     
         17 . A method of lipopolysaccharide (LPS) extraction from gram negative bacterial cells comprising the step, extracting LPS from the cells in an LPS extraction composition according to any one of  claims 1  to  14 . 
     
     
         18 . The method of  claim 17  wherein the extraction of LPS is performed at a temperature of between about 35° C. and about 65° C. 
     
     
         19 . The method of  claim 18  wherein the temperature is between about 45° C. and about 55° C. 
     
     
         20 . The method of  claim 18  or  19  wherein the temperature is about 50° C. 
     
     
         21 . The method of any of  claims 16  to  20  wherein LPS extraction is performed at a pH of between 7.8 and 9. 
     
     
         22 . The method of  claim 21  wherein the pH is about 8.6. 
     
     
         23 . The method of any of  claims 16  to  22  wherein the LPS extraction is performed in about 1 to about 30 hours. 
     
     
         24 . The method of  claim 23  wherein the LPS extraction is performed in about 0.5 to about 20 hours. 
     
     
         25 . The method of  claim 23  or  24  wherein the LPS extraction is performed for about 1 hour. 
     
     
         26 . The method of any of  claims 16  to  25  further comprising the steps:
 i. Washing cells with a solution of ethanol or ethanol; and optionally 
 ii. Washing cells a second time with ethanol or methanol. 
 
     
     
         27 . The method of  claim 26  wherein cell are washed in i) and/or ii) with a solution of between about 75 and about 95% ethanol or methanol (v/v). 
     
     
         28 . The method of  claim 26  or  27  wherein the cells in step i) are washed in a solution of about 85% (v/v) ethanol or methanol. 
     
     
         29 . The method of  claim 26  or  27  wherein cells are washed in step ii) with a solution of about 90% (v/v) ethanol or methanol. 
     
     
         30 . The method of any of  claims 16  to  29  further comprising the step: washing cells with a solution of methanol. 
     
     
         31 . The method of any of  claims 16  to  30  further comprising the step: evaporating the water, alcohol and further organic solvent from the LPS solution, thereby yielding a dry LPS residue. 
     
     
         32 . The method of any of  claims 16  to  31  wherein the bacterial cell is that of a deep rough mutant bacterial strain of  Salmonella  or  Escherichia.    
     
     
         33 . The method of  claim 32  wherein the bacterial cells are that of  Escherichia coli.    
     
     
         34 . The method of  claim 32  wherein the bacterial cells are that of  Salmonella minnesota.    
     
     
         35 . The method of  claim 34  wherein the bacterial cells are that of  Salmonella minnesota  R595. 
     
     
         36 . An LPS composition produced by the methods of any of  claims 16  to  35 . 
     
     
         37 . The method of any of  claims 16  to  35  further comprising the step: subjecting the LPS to sequential acid hydrolysis and base hydrolysis, to form 3D-MPL. 
     
     
         38 . A 3D-MPL composition produced by the method of  claim 37 .