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.