Unmasking endotoxins in solution

Abstract

The invention relates to unmasking endotoxins in compositions so that previously present, but undetectable endotoxins are rendered detectable.

1 . A method of unmasking an endotoxin in a composition, the composition comprising an endotoxin masker and known or suspected of comprising such an endotoxin, said method comprising unmasking the endotoxin by adding to said composition a modulator that unmasks said endotoxin, said modulator comprising a first heteroatom-substituted aliphatic, said first heteroatom-substituted aliphatic being a branched aliphatic comprising 8 to 16 carbon atoms. 
     
     
         2 . The method of  claim 1 , further comprising detecting said endotoxin. 
     
     
         3 . The method according to  claim 1 , further comprising the step of adding to said composition an agent which influences hydrogen bonding stability in solution. 
     
     
         4 . The method according to  claim 3 , wherein said agent which influences hydrogen bonding stability in solution is added to said solution prior to the addition of said modulator. 
     
     
         5 . The method of  claim 3 , wherein the agent which influences hydrogen bonding stability in solution is a chaotropic agent, a cation or a combination thereof. 
     
     
         6 . The method of  claim 5 , wherein said chaotropic agent is chosen from the group consisting of: urea; guanidinium chloride; butanol; ethanol; lithium perchlorate; lithium acetate;
 magnesium chloride; phenol; propanol; and thiourea.   
     
     
         7 . The method of  claim 5 , wherein the cation is a divalent cation. 
     
     
         8 . The method of  claim 7 , wherein the divalent cation is chosen from Ca 2+ , Mg 2+ , Sr 2+  and Zn 2+ . 
     
     
         9 . The method of  claim 1 , wherein said first heteroatom-substituted aliphatic is an oxygen-substituted aliphatic. 
     
     
         10 . The method of  claim 9 , wherein said oxygen-substituted aliphatic is an aliphatic alcohol. 
     
     
         11 . The method of  claim 1  wherein said modulator further comprises a second heteroatom-substituted aliphatic, wherein the main chain of said second heteroatom-substituted aliphatic comprises 8 to 16 carbon atoms. 
     
     
         12 . The method of  claim 11 , wherein said second heteroatom-substituted aliphatic is an oxygen-substituted aliphatic. 
     
     
         13 . The method of  claim 12 , wherein said oxygen-substituted aliphatic is an aliphatic sulfate. 
     
     
         14 . The method of  claim 13 , wherein said aliphatic sulfate is sodium dodecyl sulfate (SDS). 
     
     
         15 . The method of  claim 1 , wherein said modulator further comprises a protein capable of binding a detergent so as to break up micelles formed by said detergent. 
     
     
         16 . The method of  claim 15 , wherein said protein is an albumin. 
     
     
         17 . The method of  claim 16 , wherein said albumin is human serum albumin, bovine serum albumin and/or ovalbumin. 
     
     
         18 . The method of  claim 1 , wherein said endotoxin masker is a detergent and/or an active pharmaceutical ingredient (API). 
     
     
         19 . The method of  claim 18 , wherein the detergent is selected from the group consisting of an anionic detergent; a cationic detergent; a nonionic detergent; an amphoteric detergent; and
 any combination thereof.   
     
     
         20 . The method of  claim 19 , wherein said detergent is an anionic detergent selected from the group consisting of: alkyl sulfates; alkyl-ether sulfates; cholesterol sulfate; sulfonates; alkyl sulfo succinates; sulfoxides, preferably dodecyl methyl sulfoxide; phosphates and carboxylates. 
     
     
         21 . The method of  claim 19 , wherein said detergent is a cationic detergent selected from the group consisting of: primary amines; secondary amines; tertiary amines; and quaternary ammonium cations; and quaternary ammonium detergents. 
     
     
         22 . The method of  claim 19 , wherein said detergent is a nonionic detergent selected from the group consisting of: polyoxyethylene glycol sorbitan alkyl esters (polysorbates); polyoxyethylene glycol alkyl ethers; polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers; polyoxyethylene glycol octylphenol ethers; polyoxyethylene glycol alkylphenol ethers; glycerol alkyl esters; sorbitan alkyl esters; block copolymers of polyethylene glycol and polypropylene glycol; sterols; cyclodextrins; and poloxamers. 
     
     
         23 . The method of  claim 19 , wherein said detergent is an amphoteric detergent selected from the group consisting of: CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate); sultaines; betaines; amino oxides and amine oxides of the general formula R 3 N + O − , wherein R 3  is C 8 -C 18  alkyl, C 8 -C 18  alkenyl, or C 8 -C 18  alkynyl. 
     
     
         24 . The method of  claim 18 , wherein said API is a protein API. 
     
     
         25 . The method of  claim 24 , wherein the protein is selected from the group consisting of an antibody; an antibody fragment; a hormone; an enzyme; a fusion protein; a protein conjugate; and any combination thereof. 
     
     
         26 . The method of  claim 25 , wherein the protein is an antibody fragment selected from the group consisting of a Fab; a Fab′; a F(ab′)2; an Fv; a single chain antibody; and any combination thereof. 
     
     
         27 . The method of  claim 25 , wherein the protein is an antibody selected from the group consisting of a fully human antibody; an anti-idiotype antibody; a humanized antibody; a bispecific antibody; a chimeric antibody; a CDR-grafted antibody; a monoclonal antibody; a polyclonal antibody; and any combination thereof. 
     
     
         28 . The method of  claim 1 , wherein the composition is further defined as a pharmaceutical composition. 
     
     
         29 . The method of  claim 20 , wherein the detergent is sodium stearate, sodium lauroyl sarcosinate, dodecylbenzensulfonate, sodiumlauryl sulfoacetate, xylene sulfonate, ammonium lauryl sulfate, sodium lauryl sulfate (SDS), sodium laureth sulfate, sodium myreth sulfate, disodium lauryl sulfosuccinate or trilaureth-4 phosphate. 
     
     
         30 . The method of  claim 21 , wherein the cationic detergent is a alkyltrimethylammonium salts selected from the group consisting of cetyl trimethylammonium bromide (CTAB); cetyl trimethylammonium chloride (CTAC); and cetylpyridinium chloride (CPC). 
     
     
         31 . The method of  claim 21 , wherein the cationic detergent is a quaternary ammonium detergents selected from the group consisting of tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate; and polyquaternium-10. 
     
     
         32 . The method of  claim 22 , wherein the nonionic detergent is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60; polysorbate 80; cocamide MEA; cholesterol; and cocamide DEA. 
     
     
         33 . The method of  claim 23 , wherein the amphoteric detergent is selected from the group consisting of cocamidopropyl hydroxysultaine; cocamidopropyl betaine; palmitamine oxide; laurylamine oxide and lecithin. 
     
     
         34 . The method of  claim 1 , wherein the branched aliphatic is a branched alkanol. 
     
     
         35 . A method of detecting an endotoxin in a composition, the composition comprising an endotoxin masker and being known or suspected of comprising said endotoxin, said method comprising the steps of:
 a) adding to said composition a modulator capable of unmasking said endotoxin; and   (b) detecting said endotoxin,   
       wherein said modulator comprises a first heteroatom-substituted aliphatic, said first heteroatom-substituted aliphatic being a branched aliphatic comprising 8 to 16 carbon atoms. 
     
     
         36 . The method of  claim 35 , wherein the branched aliphatic is a branched alkanol.