Endotoxin destruction

Endotoxin destruction should be ascertained at the coolest location of the load. Load configurations should be identical to those used in the microbial validation studies. 

F values required for endotoxin destruction at various temperatures and/or cycle time-temperature variations can be determined using a Z value of 54°C and the following equation ... 

There is basically no endotoxin destruction at temperatures below 80°C, and the D values can be as high as 20 min for dry-heat temperatures of around 170°C. The second-order models give a better estimate of endotoxin destruction kinetics at temperatures above 250°C than in the 170-250°C range. Generally, the following conditions are required for endotoxin destruction ... 

Temperatures required for dry heat sterilization (over I60 C> are in a far higher range than those necessary for saturated steam sterilization (110 to 140 C). Depyrogenation requires even higher temperatures (2(X) to 400 C) if very long periods of exposure are to be avoided. Temperature/time combinations that are sufficient to meet requirements for endotoxin destruction are more than sufficient to sterilize the most highly contamina items. Temperature/ time combinations that are only sufficient to meet requirements of sterility are not sufficient to meet pharmacopoeial and regulatory requirements for endotoxin destruction. 

For most practical purposes the term endotoxin can be regarded as synonymous with pyrogen, depyrogenation with endotoxin destruction, and pyrogen-free with endoioxin-free. 

Dry heat destruction of bacterial endotoxins is complex and poorly understood much of the research data is contradictory [2]. Most experimental evidence has shown that destruction follows second order chemical kinetics with a high initial rate of decrease of endotoxin followed by a much slower terminal rate. These second-order models give a better estimate of the kinetics of endotoxin destruction at temperatures above 250 C than in the 170 to 250 C temperature band. Impure endotoxin may account for these anomalies. There is practically no endotoxin destruction at temperatures below 80 0, and D-values for dry heat sterilization temperatures of around I70°C are as high as 20 min [2], These observations are in agreement with published c-values of around 40 C for endotoxin destruction. Evidence of endotoxin destruction in practical situations is usually taken from empirical observation because of the uncertainty over its theoretical basis. 

For endotoxin destruction, items must be spiked with bacterial endotoxin and passed through preferably shortened (for tunnels this means fa.ster belt speeds than specified) or cooler cycles than the proposed process specification. The spiked items should be placed in previously identified cold spots. They are then retrieved and assayed by the LAL method for remaining traces of endotoxin. The key to doing this successfully is to be sure that each item is spiked with at least 1000 times as much endotoxin as the minimal quantity detectable by the available recovery and assay techniques. 

All ancillary reagents used in the LAL assay system (e.g. WFI, test tubes, pipette tips for liquid transfer, etc.) must obviously be endotoxin free. Such items can be rendered endotoxin free by heat. Its heat-stable nature, however, renders very vigorous heating necessary in order to destroy contaminant endotoxin. A single autoclave cycle is insufficient, with total destruction requiring three consecutive autoclave cycles. Dry heat may also be used (180 °C for 3 h or 240 °C for 1 h). 

Akers, M. J., Ketron, K., Thompson, B. F value requirements for the destruction of endotoxin in the validation of dry heat sterilization/depyrogenation cycles. J Parenter Drug Assoc 36 23-27 (1982). 

Avis, K. E., Jewell, R. C., Ludwig, J. D. Studies on the thermal destruction of Escherichia coli endotoxin. J Parenter Drug Assoc 41 49 (1987). 

Dry-heat processes have two main targets microorganisms and their by-products, pyrogens or endotoxins. Depyrogenation is the process that destroys the chemical activity of these by-products. Destruction of microorganisms and endotoxins by dry heat is considered an oxidative process, which is almost a combustion. Depyrogenation requires a higher temperature than sterilization and can be summarized as follows ... 

The dry-heat destruction of endotoxins is complex and poorly understood. Most evidence has shown that destruction follows second-order kinetics with an initial high rate of destruction followed by a slower terminal rate. The kinetics of dry-heat destruction for lipopolysaccharide are expressed in terms of and D, for the initial and secondary first-order reaction rates, respectively. Previous research found that there was greater reduction in the dry-heat resistance of lipopolysaccharide in whole cells than that in the semipurified state. The value for the whole cells was found to be 1.6, and the value was 12. In the semipurified state, was 3.7 and was 29.4. Thus the rate of the destruction was two times faster when whole cells were used. ... 

Low endotoxin level (required for WFI only). The term endotoxin applies to organic compounds that cause harmful effects when injected in the bloodstream of laboratory animals. Such com-poimds are lipopolysaccharides produced as a result of microbial growth or microbial destruction. 

Dry beat should be the method of choice for sterilization of heat-stable items that are damaged by moisture or are impervious to steam. It can in fact serve one or both of two functions it may serve as a method of sterilization or as a method of steriliziUion and depyrogenation (destruction of bacterial endotoxins). Absence of bacterial endotoxins is a biological quality of equal or greater importance to sterility for pharmaceutical products and medical devices intended for parenteral... 

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