The efficient removal of endotoxins from insulin using quaternized polyethyleneimine-coated porous zirconia

The synthesis and use of a zirconia-based, alkali-stable strong anion-excha nge stationary phase are described for the removal of pyrogenic lipopolysac charides (LPS) from insulin, The strong anion-exchange material is produced by deposition of polyethyleneimine (PEI) onto porous zirconia particles, f ollowed by cross-linking with a novel reagent, 1,2-bis-(2-iodoethoxy) ethan e, and quaternization with iodomethane. Physical characterization of the ch romatographic support shows that it has an ion-exchange capacity of 0.6 mmo l/g, and 82% of the amine sites on the surface are in quaternized form. Iso cratic elution of small benzoic acid derivatives shows good column efficien cy. The two primary virtues of this material are its chemical stability und er alkali conditions (up to pH 13) and its lower hydrophobicity compared to previously described alkali-stable PEI-coated zirconia supports cross-link ed with 1.10-diiododecane. Using this new zirconia-based phase, a purificat ion protocol is developed for the efficient removal of Escherichia coli 011 1:B4 LPS from bovine insulin samples. An endotoxin clearance rate of up to 1.3 x 10(8) was attained. Endotoxin levels were reduced to less than 5 endo toxin units/ml even at initial contamination levels as high as 5.0 x 10(6) endotoxin units/ml. Furthermore, endotoxin adsorbed to the porous zirconia column may be easily removed (depyrogenated) using alkali for repeated puri fication cycles. (C) 1999 Academic Press.