Endotoxin removal from protein solutions by immobilized metal ion affinity chromatography
Department of Chemical Engineering, Chemistry and Environmental Science
Doctor of Philosophy
Luo, Robert G.
Kebbekus, Barbara B.
Knox, Dana E.
Immobilized Metal (ion) Affinity Chromatography (IMAC)
Limulus Amebocyte Lysate (LAL)
Sterogene Acticlean Etox
Protein Elution Process
In the biotechnology/pharmaceutical industry, the removal of endotoxins from final protein product has always been a challenge. A novel method for endotoxin removal from protein solutions, immobilized metal (ion) affinity chromatography (IMAC) has been developed in this study. Fe3+ was charged to a chelating gel that contains iminodiacetic acid (IDA). The IDA- Fe3+ IMAC column was exploited to remove endotoxin from protein solutions. This method involves the adsorption of the endotoxin on IMAC column. More than 99% endotoxin is removed and protein yield is higher than 90%. IDA-Fe3+ IMAC has proved to be an economical and efficient method to remove endotoxin from protein solutions.
The study consists four segments: endotoxin assay method development, the evaluation of current endotoxin removal methods, adsorption isotherm and capacity study regarding IMAC column, and process development including a leaching study.
First, to properly detect endotoxin content in experimental solutions, the effect of protein on Limulus Amebocyte Lysate (LAL) test was discussed. Subtraction of protein absorbance from total LAL reaction mixture is a very efficient way. The inhibition of phosphate ion on LAL test was observed. This was circumvented by a proper sample dilution before the test.
Second, the currently available technology, Sterogene Acticlean Etox was used in a comparison study. The column capacity for endotoxin is very low (less than 100 EU/ml gel). The efficiency to remove endotoxin from protein solution with IMAC column has proved to be better.
Third, adsorption isotherms for both endotoxin and bovine serum albumin on IDA- Fe3+ IMAC were investigated. This IMAC column has a high capacity for endotoxins: more than 10,000 EU/ml gel. It was found that endotoxin adsorption mechanism was due to the interaction between Fe3+ and phosphate group in lipid A, one of the important parts of endotoxins. The IMAC resin also has high capacity for proteins. The column adsorbs endotoxins more strongly than proteins at certain condition. The buffer conditions for selectively removing endotoxins from protein solutions were determined.
Fourth, the endotoxin or protein elution process was developed. Different elution schemes were investigated including buffer pH, ionic strength, and elution competing agent. The change of solution ionic strength and introduction of NH4Cl in elution buffer are not efficient to elute protein from the IMAC column. N-octyl-b-D-glucopyranoside (OBDG) at a low concentration could not elution protein while at a high concentration ferric ion leaching was observed. It was found that phosphate buffer was a very efficient elution agent. To prevent the leaching of metal ion from the column, elution buffer pH should be lower than 5.5.
njit-etd2000-023 (233 pages ~ 8,924 KB pdf)
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Created December 5, 2001