Removal of endotoxins from recombinant antithrombin

Endotoxins (also known as lipopolysaccharides or ETs) are a pyrogenic byproduct of the breakdown of gram-negative bacterial cell-walls, such as E. coli. They cause fever and septic shock in humans, and are therefore highly regulated by the FDA and USP. ETs are a common contaminant in recombinant protein solutions, derived from cell cultures which also have ETs. ETs are angiogenic and stimulate the inflammatory response, making them extremely important to remove for research on these cellular functions. The high stability of ETs makes them very difficult to remove and methods must be designed specifically for this purpose. The protein of interest in this study is recombinant antithrombin (AT), which is potentially antiangiogenic and therefore could be used for reatment of diseases caused by excess angiogenesis, like diabetic retinopathy and cancer. This study used two methods of endotoxin removal shown to be effective in cleaning protein solutions: phase extraction with Triton X-114 and affinity column chromatography. Heparin affinity columns were used, due to the strong interaction between heparin and AT. Triton X-114 is also used to wash the affinity column. Results show that both a combination protocol of the phase extraction and affinity chromatography and an affinity column only protocol are effective in removing endotoxins.