Process for the reduction of endotoxins in a plasmid preparation using a carbohydrate non-ionic detergent with silica chromatography

Abstract

The present invention provides methods for the reduction of endotoxins in a plasmid preparation using a carbohydrate non-ionic detergent with silica chromatography.

1 . A method for reducing endotoxin levels in a nucleic acid solution, the method comprising:
 a) contacting the nucleic acid solution with a carbohydrate non-ionic detergent selected from the group consisting of an alkyl thiomaltoside, a sucrose monoalkyl ester, and combinations thereof;   b) contacting the solution resulting from step a) with an inorganic binding matrix; and   c) washing the binding matrix resulting from step b) to obtain a nucleic acid composition having an endotoxin content of less than 100 EU/mg.   
   
   
       2 . The method of  claim 1 , further comprising eluting the nucleic acid composition from the inorganic binding matrix of step c). 
   
   
       3 . The method of  claim 2 , wherein the eluted nucleic acid composition comprises an average plasmid DNA yield of about 15 μg to about 50 μg and an average endotoxin level from about 20 EU/mg to about 100 EU/mg. 
   
   
       4 . The method of  claim 1 , wherein the alkyl thiomaltoside is selected from the group consisting of n-decyl-β-D-thiomaltopyranoside, n-nonyl-β-D-thiomaltoside, and n-octyl-β-D-thiomaltopyranoside. 
   
   
       5 . The method of  claim 1 , wherein the sucrose monoalkyl ester is selected from the group consisting of sucrose monodecanoate, n-monododecanoate-β-D-glucopyranoside, sucrose monolaurate, and n-octanoylsucrose. 
   
   
       6 . The method of  claim 1 , wherein the inorganic binding matrix is selected from the group consisting of silica, diatomaceous earth, aluminum oxides, glass, titanium oxides, zirconium oxides, and hydroxyapatite. 
   
   
       7 . The method of  claim 1 , wherein the inorganic binding matrix is silica. 
   
   
       8 . The method of  claim 1 , wherein the nucleic acid solution is a plasmid DNA solution. 
   
   
       9 . The method of  claim 1 , wherein the nucleic acid solution comprises double-stranded RNA or DNA, RNA/DNA hybrids, DNA fragments, oligonucleotides, amplified DNA or RNA, BACs, plasmid DNA, or a combination thereof. 
   
   
       10 . The method of  claim 1 , wherein the nucleic acid solution from step a) comprises a binding solution. 
   
   
       11 . The method of  claim 10 , wherein the binding solution comprises a chaotropic substance selected from the group consisting of a guanidinium salt, urea, an alkali thiocyanate, an alkali halide, an alkali perchlorate, and combinations thereof. 
   
   
       12 . The method of  claim 11 , wherein the guanidinium salt is guanidine hydrochloride. 
   
   
       13 . The method of  claim 1 , wherein step c) comprises washing with a binding solution, an alcohol solution, a carbohydrate non-ionic detergent, or a combination thereof. 
   
   
       14 . The method of  claim 1 , further comprising obtaining the nucleic acid solution to be used in step a) from a biological source by a digestion method. 
   
   
       15 . The method of  claim 14 , wherein the digestion method comprises alkaline lysis, centrifugation, filtration, or precipitation. 
   
   
       16 . The method of  claim 1 , wherein the nucleic acid solution is a cleared lysate. 
   
   
       17 . A kit for reducing endotoxin levels in a nucleic acid solution, the kit comprising:
 a) a binding solution;   b) a carbohydrate non-ionic detergent; and   c) an inorganic binding matrix.   
   
   
       18 . The kit of  claim 17 , wherein the binding solution comprises a chaotropic substance selected from the group consisting of a guanidinium salt, urea, an alkali thiocyanate, an alkali halide, an alkali perchlorate, and combinations thereof. 
   
   
       19 . The kit of  claim 18 , wherein the guanidinium salt is guanidine hydrochloride. 
   
   
       20 . The kit of  claim 17 , wherein the carbohydrate non-ionic detergent is selected from the group consisting of an alkyl thioglucoside, an alkyl glucoside, an alkyl thiomaltoside, an alkyl maltosides, a sucrose monoalkyl esters, and combinations thereof. 
   
   
       21 . The kit of  claim 17 , wherein the inorganic binding matrix is selected from the group consisting of silica, diatomaceous earth, aluminum oxides, glass, titanium oxides, zirconium oxides, and hydroxyapatite. 
   
   
       22 . The kit of  claim 17 , wherein the inorganic binding matrix is silica. 
   
   
       23 . The kit of  claim 18 , further comprising concentrated solutions for the preparation of wash solutions. 
   
   
       24 . The kit of  claim 18 , further comprising an elution buffer.