Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by non flammable solvents.

Abstract

During the production of recombinant proteins from gram negative bacteria, lipopolysaccharides (LPS, endotoxin) are released along with the protein of interest. In many instances, LPS will copurify with the target protein due to specific or non-specific protein-ILPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on anion or cation exchange chromatographic media. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile due to their lower toxicity and decreased flammability. In addition, they are less costly than many of the detergents that have been used for such purposes. LPS removal efficiency increased with increasing alkane chain length. 1,2-alkanediols were more effective than terminal alkanediols in the separation of LPS from protein LPS complexes.

1 . A process for removing an endotoxin from recombinantly produced proteins comprising a Lipopolysaccharide (LPS)-protein complex comprising the steps of: 
 immobilizing the complex to an chromatographic resin;    washing the resin with an alkanediol whereby at least a portion of the LPS is separated from the complex; and,    eluting at least a portion of the protein from the resin.    
   
   
       2 . The process of  claim 1  wherein the alkandiol is selected from the group consisting of 1,5-pentanediol, 1,6-hexanediol, 1,2-hexanediol, 1,2-butanediol, 1,4-butanediol, and 1,7-heptanediol.  
   
   
       3 . The process of  claim 1  wherein the resin is selected from the group consisting of a cation exchange resin and an anion exchange resin.  
   
   
       4 . The process of  claim 1  wherein the protein is selected from the group consisting of bovine albumin (BSA), bovine holo-transferrin, lactoferrin, lysozyme, and heat shock proteins.  
   
   
       5 . The process of  claim 1  wherein the protein is affixed to the resin.  
   
   
       6 . The process of  claim 1  wherein the LPS is affixed to the resin.  
   
   
       7 . The process of  claim 1  wherein greater than about 95% of the protein eluted from the resin is free of LPS or other endotoxin.  
   
   
       8 . The process of  claim 7  wherein a change in pH or conductivity is used to elute the protein from the resin.  
   
   
       9 . The process of  claim 1  wherein the protein is produced from a bacterial expression system.  
   
   
       10 . The process of  claim 9  wherein the bacterial expression system is selected from the group consisting of an  E. coli  expression system, a  Caulobacter crescent  expression system, and  Proteus mirabilis  expression system.  
   
   
       11 . The process of  claim 1  wherein the resin is in a high salt environment.  
   
   
       12 . The process of  claim 11  wherein the alkanediol is 1,2-hexanediol.  
   
   
       13 . The process of  claim 12  wherein the protein is eluted by changing the pH.  
   
   
       14 . A process for separating an endotoxin from a protein comprising the steps of affixing the protein to a resin and washing the resin with an alkanediol whereby at least a portion of the endotoxin is separated.  
   
   
       15 . The process of  claim 14  wherein the endotoxin is an impurity created from the production of the protein.  
   
   
       16 . The process of  claim 14  wherein greater than 99.9% of the enndotoxin is separated from the protein.  
   
   
       17 . A process for removing an endotoxin from recombinantly produced proteins comprising a Lipopolysaccharide (LPS)-protein complex comprising the steps of: 
 affixing the protein of the complex to a cation exchange chromatographic resin;    washing the resin with an alkanediol whereby at least a portion of the LPS is separated from the complex; and,    eluting at least a portion of the protein from the resin by either changing the pH or the conductivity.    
   
   
       18 . The process of  claim 17  wherein the LPS of the complex is affixed to an anion exchange chromatographic resin.