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

Clayton McNeff, Qianhua Zhao, Elisabeth Almlöf, Michael Flickinger, Peter W. Carr

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

The synthesis and use of a zirconia-based, alkalistable strong anion- exchange stationary phase are described for the removal of pyrogenic lipopolysaccharides (LPS) from insulin. The strong anion-exchange material is produced by deposition of polyethyleneimine (PEI) onto porous zirconia particles, followed by cross-linking with a novel reagent, 1,2-bis-(2-iodo- ethoxy) ethane, and quaternization with iodomethane. Physical characterization of the chromatographic support shows that it has an ion- exchange capacity of 0.6 mmol/g, and 82% of the amine sites on the surface are in quaternized form. Isocratic elution of small benzoic acid derivatives shows good column efficiency. The two primary virtues of this material are its chemical stability under alkali conditions (up to pH 13) and its lower hydrophobicity compared to previously described alkali-stable PEI-coated zirconia supports cross-linked with 1,10-diiododecane. Using this new zirconia-based phase, a purification protocol is developed for the efficient removal of Escherichia coli 0111:B4 LPS from bovine insulin samples. An endotoxin clearance rate of up to 1.3 x 108 was attained. Endotoxin levels were reduced to less than 5 endotoxin units/ml even at initial contamination levels as high as 5.0 x 106 endotoxin units/ml. Furthermore, endotoxin adsorbed to the porous zirconia column may be easily removed (depyrogenated) using alkali for repeated purification cycles.

Original languageEnglish (US)
Pages (from-to)181-187
Number of pages7
JournalAnalytical Biochemistry
Volume274
Issue number2
DOIs
StatePublished - Oct 15 1999

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