Water for Pyrogen-Free

Application Overview

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Endotoxin-Free Water, Pyrogen-Free Water

Endotoxin-free water is required for mammalian cell cultures, as well as for the rinsing or the preparation of a solution or a device that will further be in contact with human and other mammals.

The major and most significant components of endotoxins are lipopolysaccharides (LPS) from Gram negative bacteria walls. The LPS can be formally divided into three substructures: a hydrophobic and highly conservative structure formed by Lipid A (non-polar), a core oligosaccharide conservative part, and a variable heteropolysaccharide surface structure, the O-antigen (Figure 1). The two glycosidic parts can vary largely in structure and size, depending on the bacteria strain and species. This leads to a wide range of molecular weights, 3,000 to 25,000 Da (average 10-12 kDa), and a variety of biological activities. Introduction of LPS into the blood or spinal fluid causes toxic responses and induces the development of fever (i.e. a “pyrogenic” effect). LPS are known to generate early toxic effects and slow toxic actions.

Early Toxic Effects

• Effect on ion channels (Reduction of Ca²⁺ influx)
• Activation of a metabolic cascade leading to superoxide anion radical (O₂^-.), in turn generating other toxic radicals and compounds OH·, OH^-, H₂O₂ 

Slow Toxic Actions

• Cellular loss-of-adherence
• Disruption of basic vital functions
• Necrosis/apoptosis

The words endotoxin and pyrogen have become used in a wider sense to designate the active LPS molecules. In high purity water, potentially pyrogenic substances are predominantly, meaning here quasi-exclusively, endotoxins. Both terms endotoxin-free- and pyrogen-free-water are used therefore, to designate water free of LPS.

Figure 1. Endotoxin Structure

Resources

• R&D Notebook – Qualification study of a point-of-use ultrafiltration device for the production of pyrogen-free ultrapure water. S Mabic, D Darbouret, and I Kano.
• Application Note - Endotoxin-Free Water for Tissue Culture. E Riché, J Bôle and S Mabic.  

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