Quantitative Determination of Ca²⁺ Effects on Endotoxin Removal and Protein Yield in a Two-Stage Ultrafiltration Process

Abstract

Endotoxin subunits normally aggregate into vesicles with a molecular mass around 1,000,000. Some proteins can bind and disaggregate endotoxin to form protein–endotoxin complexes, which makes endotoxin removal from protein solution more complicated. It is also known that divalent cations such as Ca²⁺ can act as “bridges” between lipopolysaccharide subunits, causing large vesicles to form. Such a property has potential utility in endotoxin removal from biological solutions. A two-stage ultrafiltration was used in this study during which Ca²⁺ reaggregated lipopolysaccharide subunits in protein solutions into large vesicles that hence were retained by a 300,000 nominal molecular weight cutoff ultrafiltration membrane. After the reaggregation of lipopolysaccharide subunits, the endotoxin removal efficiency was in the range of 54.78 to 73.10%, meanwhile protein yield was in the range of 71.57 to 89.54% at various protein/endotoxin concentration ratios.

Keywords:

• Endotoxin removal
• Protein purification
• Membrane
• Ultrafiltration
• Lipopolysaccharide
• Protein–LPS complex

ACKNOWLEDGMENT

This work was supported, in part, by the New Jersey Institute of Technology under Grant 421280, and by the Colgate-Palmolive Company.