For sealed containers, it is important to verify during the development of the sterilization cycle that sterilizing conditions
are achieved in all parts of the containers when they reach the sterilization temperature.
In cases in which porous goods are sterilized with the direct access of steam, it is important to verify full penetration
of the steam through the pores of the product. Because sterilization conditions in this case are achieved by the direct action
of the steam, steam saturation is a critical parameter.
Penetration of steam also can be notoriously difficult to achieve, for example, with filling equipment that has pipes or tubing
that is sterilized in place (SIP). Even in processes where air is removed by evacuation, complete air removal may be difficult.
There also may be positions in a load to be steam sterilized that are occluded from the access of steam; for example, the
space between the barrel and piston of a filling pump, or the interface between elastomeric stoppers and the necks of glass
vials.
Effect of the microenvironment on sterilization efficiency.
In addition, the effect of sterilizing conditions can be strongly modulated by the microenvironment encountered by bacterial
endospores during sterilization. The decimal reduction time (D-value) of a spore preparation is notoriously different when the spores are presented on a paper strip, suspended in water,
or attached to a polymeric surface (1). D-values of spores in solutions can depend on the composition of the solution (2–4). For example, the presence of divalent
cations has significant influence on endospore resistance, and D-values are lower in solutions containing high concentrations of glucose (5). Thus, spore inactivation is not dependent solely
on the conditions in the autoclave. There are additional chemical and possibly other surface effects that may strongly influence
the D-values of suspended or attached endospores.
Such influences of the microenvironment cannot be measured by any physical probes. Using BIs is the only method to directly
measure the sterilizing effect, and, therefore, an ideal BI should indicate any effect of product and microenvironment.
Definition of the worst-case position in a sterilizer load.
The sterilization effect is achieved as a result of the combined influence of temperature, heat transfer, surface hydration,
and all other protecting or inactivating factors that influence endospores during the sterilization process. Therefore, the
most difficult-to-sterilize position (the worst-case position) in a sterilizer load should be defined as the position where
the sum of all influences on microorganisms results in minimal inactivation.
The relevance of the worst-case position to product safety also should be considered. Although there may be occluded positions
in a piece of SIP equipment that are never reached by steam during the sterilization process, such positions also may never
come in contact with product. As long as there is no potential to jeopardize the sterility of any product manufactured with
that equipment, there would be no good reason why the position should be sterilized. This, however, can be correctly judged
only with a thorough understanding of the equipment and the process.
The effect of steam sterilization on microorganisms trapped between the polymeric stopper and the vial in terminally sterilized
pharmaceuticals has been discussed (6). The relevance of that position to the sterility of the contents of the vial is critical
for the decision of whether that is, in fact, the worst-case position of the load. As long as deformation of elastomeric stoppers
during the cooling phase of autoclaves cannot be excluded, such a position certainly would have to be considered as relevant
for the sterilization effect.
Overkill processes.
The necessary sterilization assurance is described in United States Pharmacopeia: "A sterilization process must result in a biologically verified lethality sufficient to achieve a probability of obtaining
a nonsterile unit that is less than one in a million" (7).
In cases in which the product to be sterilized is very heat resistant, sterilization processes are usually designed to achieve
inactivation of microorganisms by a wide margin of safety. Such overkill processes are frequently defined on the basis of
their ability to inactivate a given number of microorganisms. Overkill processes are defined in USP 30 as follows:
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