Unmasking endotoxins in solution
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 a protein active pharmaceutical ingredient (a protein API) and said endotoxin, said method comprising unmasking the endotoxin by adding to said composition a saturated unbranched 1-alkanol comprising 8 to 16 carbon atoms, a divalent cation that is Ca2+, sodium dodecyl sulfate (SDS) and an albumin,
said protein API being selected from the group consisting of an antibody, an antibody fragment, a hormone, an enzyme, a fusion protein, and a protein conjugate.
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 saturated unbranched 1-alkanol comprising 8 to 16 carbon atoms.
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 1 , wherein said saturated unbranched 1-alkanol is 1-dodecanol.
8. The method of claim 1 , wherein said albumin is human serum albumin, bovine serum albumin and/or ovalbumin.
9. The method of claim 1 , 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.
10. The method of claim 1 , 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.
11. The method of claim 1 , wherein the composition is a pharmaceutical composition.
12. A method of detecting an endotoxin in a composition, the composition comprising a protein active pharmaceutical ingredient (a protein API) and said endotoxin,
said method comprising the steps of:
a) adding to said composition a saturated unbranched 1-alkanol comprising 8 to 16 carbon atoms, a divalent cation that is Ca2+, sodium dodecyl sulfate (SDS) and an albumin; and
(b) detecting said endotoxin by means of a detection method,
said protein API being selected from the group consisting of an antibody, an antibody fragment, a hormone, an enzyme, a fusion protein, and a protein conjugate.