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 - 18 . (canceled) 
     
     
         19 . A method of removing a lipopolysaccharide from a recombinantly produced protein comprising a lipopolysaccharide-protein complex of a lipopolysaccharide and a protein comprising the steps of:
 (a) loading the recombinantly produced protein comprising the complex on to a substrate; and either   (b) washing the substrate with an alkanediol whereby lipopolysaccharide is separated from the complex and the protein is affixed to the resin; and,   (c) eluting the protein from the resin to form an eluent, such that the ratio of the protein to the lipopolysaccharide in the eluent is increased relative to the ratio of the protein to the lipopolysaccharide in the complex present in the recombinantly-produced protein loaded on to the substrate in step (a), or   (d) washing the substrate with an alkanediol whereby protein is separated from the complex and the lipopolysaccharide is affixed to the substrate, such that the ratio of the protein to the lipopolysaccharide in the eluent is increased relative to the ratio of the protein to the lipopolysaccharide in the complex present in the recombinantly-produced protein loaded on to the chromatographic resin in step (a).   
     
     
         20 . The method of  claim 19 , wherein the alkanediol 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. 
     
     
         21 . The method of  claim 19 , wherein the substrate is a chromatographic resin 
     
     
         22 . The method of  claim 21 , wherein the chromatographic resin is an affinity resin. 
     
     
         23 . The method of  claim 19 , wherein the protein is selected from the group consisting of bovine albumin (BSA), bovine holo-transferrin, lactoferrin, lysozyme, and heat shock proteins. 
     
     
         24 . The method of  claim 19 , wherein greater than about 95% of the protein eluted from the substrate is free of the lipopolysaccharide. 
     
     
         25 . The method of  claim 24 , wherein a change in pH or conductivity conditions applied to the substrate compared to the pH or conductivity conditions employed to load the recombinantly-produced protein comprising the complex on to the substrate is used to elute the protein from the substrate. 
     
     
         26 . The method of  claim 19 , wherein the recombinantly-produced protein is produced from a bacterial expression system. 
     
     
         27 . The method of  claim 26 , wherein the bacterial expression system is selected from the group consisting of an  E. coli  expression system, a  Caulobacter crescent  expression system, and a  Proteus mirabilis  expression system. 
     
     
         28 . The method of  claim 20 , wherein the alkanediol is 1,2-hexanediol. 
     
     
         29 . The method of  claim 28 , wherein the protein is eluted by a change of pH conditions applied to the substrate compared to the pH conditions employed to load the recombinantly-produced protein comprising the complex on to the substrate. 
     
     
         30 . A process for separating an endotoxin from a protein-endotoxin complex comprising protein and endotoxin comprising the steps of:
 (a) affixing the protein-endotoxin complex to a substrate; and   (b) washing the substrate with an alkanediol whereby the endotoxin is separated from the protein-endotoxin complex such that the ratio of the protein to the endotoxin in the protein-endotoxin complex is increased.   
     
     
         31 . The process of  claim 30 , wherein the substrate is a chromatographic resin 
     
     
         32 . The process of  claim 31 , wherein the chromatographic resin is an affinity resin. 
     
     
         33 . The process of  claim 30 , wherein the endotoxin is an impurity created from the production of the protein. 
     
     
         34 . The process of  claim 30 , wherein greater than 99.9% of the endotoxin is separated from the protein. 
     
     
         35 . A method for removing a lipopolysaccharide from a recombinantly produced protein comprising a lipopolysaccharide-protein complex of lipopolysaccharide and protein comprising the steps of:
 (a) affixing the lipopolysaccharide-protein complex to a substrate under a first pH and/or conductivity condition;   (b) washing the chromatographic resin with an alkanediol whereby the lipopolysaccharide is separated from the lipopolysaccharide complex such that the ratio of the protein to the lipopolysaccharide in the lipopolysaccharide-protein complex is increased; and,   (c) eluting the protein from the chromatographic resin by either changing the pH and/or the conductivity relative to the first pH and/or conductivity condition.   
     
     
         36 . The method of  claim 24 , wherein the substrate is a chromatographic resin. 
     
     
         37 . A method of removing a lipopolysaccharide from a recombinantly-produced protein comprising a lipopolysaccharide-protein complex of lipopolysaccharide and protein comprising the steps of:
 (a) loading the recombinantly-produced protein comprising the complex to a substrate;   (b) affixing the lipopolysaccharide to the substrate;   (c) washing the substrate with an alkanediol; and   (d) eluting the lipopolysaccharide from the substrate such that the ratio of the protein to the lipopolysaccharide in the lipopolysaccharide-protein complex is increased.   
     
     
         38 . A method of removing a lipopolysaccharide from a recombinantly-produced protein comprising a lipopolysaccharide-protein complex of lipopolysaccharide and protein comprising the steps of:
 (a) loading the recombinantly-produced protein comprising the complex to a substrate;   (b) affixing the protein to the substrate;   (c) washing the substrate with an alkanediol; and   (d) eluting the protein from the substrate such that the ratio of the protein to the lipopolysaccharide in the lipopolysaccharide-protein complex is increased.