Allantoin as a solid phase adsorbent for removing endotoxins

• A new adsorbent for removing endotoxins from proteins solutions is described.
• Solid allantoin removes >99.9% endotoxins in <5 min.
• Endotoxin binding on allantoin crystals appears to be mediated by hydrogen-bonding.
• The method is more effective and faster than other methods for endotoxin clearance.

In this study we present a simple and robust method for removing endotoxins from protein solutions by using crystals of the small-molecule compound 2,5-dioxo-4-imidazolidinyl urea (allantoin) as a solid phase adsorbent. Allantoin crystalline powder is added to a protein solution at supersaturated concentrations, endotoxins bind and undissolved allantoin crystals with bound endotoxins are removed by filtration or centrifugation. This method removes an average of 99.98% endotoxin for 20 test proteins. The average protein recovery is ∼80%. Endotoxin binding is largely independent of pH, conductivity, reducing agent and various organic solvents. This is consistent with a hydrogen-bond based binding mechanism. Allantoin does not affect protein activity and stability, and the use of allantoin as a solid phase adsorbent provides better endotoxin removal than anion exchange, polymixin affinity and biological affinity methods for endotoxin clearance.