Validation of dry heat inactivation of bacterial endotoxins.

Bacterial endotoxins (ETs) are lipopolysaccharides from the cell wall of Gram negative bacteria. ETs get into the environment as a result of autolytic desintegration of the bacterial cells. There exist a number of depyrogenation methods, either serving to remove or to inactivate ET. The most common means of ET inactivation is dry heat. Unfortunately no uniform regulation exists describing the conditions for sufficient ET inactivation. While the USP and FDA require an ET reduction of 3 log steps, no European regulation exists regarding depyrogenation of final containers for parenterals. However, the Ph. Eur. specifies the temperature and time conditions for depyrogenation of glassware in the pyrogen test monograph, allowing to choose between the two variants 250 degrees C/30 minutes or 200 degrees C/60 minutes which are not equivalent. In the present study those conditions for depyrogenation of glass containers in production of parenterals were investigated which, on the one hand, are technically feasible and, on the other hand, comply with the requirements of the main Pharmacopeias; furthermore, an ET preparation suitable for validation studies was selected. The preparation of ET indicators, the dry-heat inactivation and the recovery of ET are described in detail. Based on the results obtained, it is recommended to follow a defined treating temperature and period for safe depyrogenation of glass containers for parenterals, which results in a 4 log step reduction in ET without fillers. Thereby the USP/FDA requirement for a 3 log step reduction as well as the 200 degrees C/60 minutes requirement variant given in the Ph. Eur. can be fulfilled.