Endotoxin

Bacterial Endotoxin (Pyrogen)

Endotoxin is lipopolysaccharide shed from the cell wall of Gram-negative bacteria. The critical property for manufacturing is that it survives conditions that kill the organism: routine moist-heat sterilisation destroys the bacterium but leaves the endotoxin intact. A product can therefore be perfectly sterile and still be pyrogenic — which is why endotoxin is controlled as its own attribute rather than assumed away by sterilisation.

Because it cannot be sterilised out, endotoxin must be prevented and removed. Prevention means controlling the water system (WFI), component washing, and hold times so Gram-negative populations never establish. Removal means depyrogenation — typically dry-heat at 250 °C for glass and equipment, validated to a 3-log reduction of a known endotoxin challenge.

Limits are calculated, not guessed. The endotoxin limit is K/M, where K is the threshold pyrogenic dose per kilogram and M is the maximum human dose per kilogram per hour. For most parenteral products K is 5 EU/kg; for intrathecal products it drops to 0.2 EU/kg, reflecting the far greater hazard of direct CNS exposure.

• —Heat-stable: sterilisation kills the organism but does not destroy the endotoxin — sterile does not mean non-pyrogenic.
• —Endotoxin limit = K/M (K = 5 EU/kg for most parenterals; 0.2 EU/kg intrathecal).
• —Tested by the bacterial endotoxin test — gel-clot, turbidimetric, or chromogenic LAL; recombinant Factor C is a recognised animal-free alternative.
• —Depyrogenation (typically dry heat, validated to ≥3-log reduction) is the removal step for glass and equipment.
• —Water systems are the usual root cause — control WFI, component washing, and hold times to prevent Gram-negative growth.

USP <85> (Bacterial Endotoxins Test) and USP <86> (Bacterial Endotoxins Test Using Recombinant Reagents); Ph. Eur. 2.6.14; USP <151> (Pyrogen Test); EU GMP Annex 1 (2022) for contamination control and depyrogenation.