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色谱 2012年第11期30卷 1194-1202页

作者： André Moreni LOPES Jorge Sánchez ROMEU Rolando Páez MEIRELES gabriel marquez perera Rolando Perdomo MORALES Adalberto PESSOA Lourdes Zumalacárregui CáRDENAS Department of Biochemical and Pharmaceutical Technology School of Pharmaceutical SciencesUniversity of So Paulo(FCF/USP) Purification Development Department Center for Genetic Engineering and Biotechnology(CIGB) Center for Pharmaceuticals Research and Development(CIDEM) Higher Polytechnic Institute José Antonio Echeverría(CUJAE)

Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their removal one of the most difficult tasks in downstream processes during protein purification.The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration.Immobilized metal affinity chromatography(IMAC) enables the affinity interactions between the metal ions(immobilized on the support through the chelating compound) and the target molecules,thus enabling high-efficiency separation of the target molecules from other components present in a mixture.Affinity chromatography is applied with Ca2+-iminodiacetic acid(IDA) to remove most of the LPS contaminants from the end product(more than90%).In this study,the adsorption of LPS on an IDA-Ca2+ was investigated.The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal.It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads.The factors such as pH(4.0 or 5.5) and ionic strength(1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than100 EU/mL and 100 000 EU/mL.This new protocol represents a substantial advantage in time,effort,and production costs.

关键词： immobilized metal affinity chromatography(IMAC) lipopolysaccharides(LPS) endotoxin removal recombinant proteins isotherms protein purification

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