Extraction
Solvent extraction is used to separate endotoxins from target
therapeutics based on their relative solubilities in two immiscible
liquids. Endotoxins form partition in the organic phase, while
hydrophilic target molecules remain in the aqueous phase. Endotoxins
have been effectively removed from the bacteriophages T4, HAP1, and F8
using 1-octanol with endotoxin removal efficiencies varying between 64 -
99.9%. 36. Additional processing is required to
remove any trace quantities of 1-octanol present in the aqueous phase as
the presence of 1-octanol interferes with the LAL test for endotoxin
detection 36. Even though solvent extraction technique
gives high endotoxin removal from various therapeutics solutions, the
product yield is significantly low and varies between 30-60% impacting
the profits associated with the method where it may not be a practical
choice for this application 36.
Two-phase extraction using detergent Triton X-114, a non-ionic
surfactant 112, has been explored to remove endotoxins
from target therapeutics. Endotoxin was successfully removed from the
green fluorescent protein using Triton X-114 and temperature
transitions. Triton X-114 is miscible with water at a temperature of
0°C, but a phase separation occurs at temperatures above 23°C
113. Endotoxins are partitioned in the detergent phase
while the target therapeutics are partitioned in the aqueous phase.
Endotoxin removal efficiencies using Triton X-114 ranged between 45-99%
113. In addition to high endotoxin removal, Triton
X-114 results in high product recovery of over 80%
16.Triton X-114 isothermal extraction using sodium
dodecyl sulfate (SDS) has also been very effective in removing
endotoxins from pDNA with residual endotoxin concentration of around 16
EU/mg. Moreover, using this extraction technique, a pDNA recovery of
over 80% was reported. While isothermal extraction was proven effective
for plasmid-endotoxin removal, this method is not applicable for the
removal of endotoxins from protein solutions because SDS is reported to
have strong interactions with proteins causing significant changes to
protein conformation 114. One major disadvantage of
temperature transition extraction using Triton X-114 is that the
repeated heating and cooling degrades therapeutic products
114. Extraction processes provide a rapid separation
that is easily scalable and can achieve high removal efficiencies,
especially with high initial concentrations
36,113,115. However, final endotoxin concentrations in
the aqueous phase for both solvent extraction and Triton X-114
extraction remained above desired specifications, meaning additional
processing is required.