Application Note

Removal Of Endotoxin From Monoclonal Antibodies

Source: Sartorius Stedim Biotech

It is desirable to minimise endotoxins in purified protein preparations prior to their use in cell-based assays. Vivapure® centrifugal anion exchange membrane devices can remove endotoxin from research grade monoclonal antibody solutions simply with high protein recovery.

Endotoxins are lipopolysaccharides present in the cell wall of most Gram-negative bacteria, and are frequently present as contaminants in protein solutions purified in research environments. They have profound biological effects and thus must be minimised prior to use of such preparations in cell-based assays. The term EU is used to describe the activity of endotoxins, and typically the limit for endotoxin is set at 50 EU/mg for bioactive proteins destined for cell-based assays.

Achieving this low level is often a challenge in research as endotoxins are robust molecules surviving extremes of temperature and pH. Endotoxins are negatively charged under conditions commonly encountered during protein purification. This negative charge facilitates the use of anion exchange chromatography for their removal. If the binding of endotoxin can be achieved under conditions at which the protein of interest carries a net positive charge (i.e. at a pH below it’s isoelectric point) then the protein will be repelled from the positively charged matrix and flow through with the mobile phase, in what is often termed negative chromatography mode. However, this will often result in dilution of the protein, which may call for an additional concentration step.

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