色谱

色谱 ›› 2012, Vol. 30 ›› Issue (11): 1194-1202.DOI: 10.3724/SP.J.1123.2012.06007

• 研究论文 • 上一篇    下一篇

ADSORPTION OF ENDOTOXINS ON IDA-Ca+2 BY ION METAL AFFINITY CHROMATOGRAPHY

André Moreni LOPES1*, Jorge Sánchez ROMEU2#, Rolando Páez MEIRELES2, Gabriel Marquez PERERA2, Rolando Perdomo MORALES3, Adalberto PESSOA Jr1, Lourdes Zumalacárregui CáRDENAS4   

  1. 1. Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of So Paulo (FCF/USP), 05508000 So Paulo, Brazil; 2. Purification Development Department, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba; 3. Center for Pharmaceuticals Research and Development (CIDEM), Havana, Cuba; 4. Higher Polytechnic Institute José Antonio Echeverría (CUJAE), Havana, Cuba
  • 收稿日期:2012-06-06 修回日期:2012-08-25 出版日期:2012-11-28 发布日期:2012-11-21
  • 通讯作者: André Moreni LOPES. Tel: +551130913862, Fax: +551138156386, Email: andreml@usp.br.
  • 基金资助:

    This research was supported by grants from the Brazilian Agency Coordination of Graduate Level Training (CAPES, project 0366/09-9) and State of Sao Paulo Research Support Foundation (FAPESP-Brazil, project 2005/60159-7).

Adsorption of endotoxins on Ca2+ iminodiacetic acid by metal ion affinity chromatography

André Moreni LOPES1*, Jorge Sánchez ROMEU2#, Rolando Páez MEIRELES2, Gabriel Marquez PERERA2, Rolando Perdomo MORALES3, Adalberto PESSOA Jr1, Lourdes Zumalacárregui CáRDENAS4   

  1. 1. Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of So Paulo (FCF/USP), 05508000 So Paulo, Brazil; 2. Purification Development Department, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba; 3. Center for Pharmaceuticals Research and Development (CIDEM), Havana, Cuba; 4. Higher Polytechnic Institute José Antonio Echeverría (CUJAE), Havana, Cuba
  • Received:2012-06-06 Revised:2012-08-25 Online:2012-11-28 Published:2012-11-21

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摘要： 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 than 90%). 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 than 100 EU/mL and 100000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

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

Abstract: 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 than 90%). 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 than 100 EU/mL and 100000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.