A scientifically ideal procedure would be to place bacterial endospores during cycle development at worst-case positions.
The inactivation characteristics of the spores at that position could then be correlated to the inactivation of the same spore
preparation achieved at a reference position. For such studies in theory, it is vital that the worst-case positions are well
defined and bacterial endospores are correctly positioned without alteration of worst-case conditions. Most sterilization
processes, however, are not easily amenable to such an analysis. Worst-case positions tend not to be freely accessible or
easily inoculated with endospores, and it can be difficult to recover endospores from worst-case positions. In addition, such
studies must be performed in production autoclaves or production equipment because various large-scale sterilization processes
cannot be simulated with a biological indicator evaluator resistometer (BIER) vessel. Production autoclaves do not deliver
heat with square-wave characteristics and, therefore, precise quantitative studies of D-values are not feasible therein.
To evaluate the biological effect of large-scale sterilization processes, test pieces with a defined number of microorganisms
and defined resistance to saturated-steam exposure (D-value) should be exposed to actual or simulated worst-case conditions. Test pieces can be paper strips inoculated with resistant
spores, units of inoculated product, vials with inoculated stoppers inserted, or pieces of tubing of a length simulating a
worst-case exposure. The inactivation characteristics of the test pieces under reference conditions should be determined in
a laboratory using a BIER-vessel. The requirements for manufacturing quality control of such test pieces (confectioned BI)
are standardized in ISO 11138 (12).
Unfortunately, the definition of true worst-case positions at which BIs are to be exposed is not well understood by many users.
The necessary availability of saturated steam or any possible influence of the microenvironment of spores are neglected. Standard
paper strips or self-contained BIs are frequently used to simulate various worst-case conditions, and the coldest position
measured in a temperature-mapping study of a load is often assumed to be the worst-case position, although this is not necessarily
a valid assumption.
To correctly validate a sterilization cycle, it is necessary to use data gathered during product or process development to
identify the conditions or positions where inactivation of spores is most difficult to achieve. These conditions should be
simulated as closely as possible by suitable BIs. Whether this can be achieved by using a spore preparation on paper strips
or a self-contained BI must be decided in each case. In many cases, a better simulation will be achieved with a customized
BI that uses units or assembled parts of the product to be sterilized.
Although using BIs as an additional means of monitoring autoclave cycles is recommended in hospitals, this practice is not
common in the manufacture of pharmaceuticals or medical devices. Once a sterilization cycle has been validated for standardized
defined loads, manufacturers typically rely on physical measurements for cycle control.
Again, the approach taken should be governed by what is intended with the process. In a hospital setting it is impossible
to define reproducible loads and, thus, by convention a defined pack of tissue is considered the standard worst-case position.
Defined loads are common practice in the manufacture of medicinal products. A pack of tissue would not be a good representation
of a worst-case for typical pharmaceutical sterilization processes for the reasons discussed previously.
The situation may again be different in pharmaceutical laboratories engaged in development or quality work, where standardized
sterilizer loads also are difficult to define. The less defined a sterilization process is with regard to worst-case positions,
the higher the advantage of arriving at a conventional definition of a standard worst-case model. For production processes
governed by the rules of good manufacturing practices (GMP), validation of load-specific cycles is generally required, and
reliance on standard worst-case models is not accepted in most cases.
Properties and quality of bacterial endospore preparations and biological indicators
D-value of biological indicators.
An important prerequisite for the suitability of endospore preparations is their D-value in correlation with the theoretical effectiveness of the process. When BIs are used to validate a sterilization cycle,
the normal expectation is that all BIs exposed during the cycle are completely inactivated at the end of the exposure time.
For quantitative determination of the sterilizing effect, it is necessary to apply reduced-exposure conditions that leave
a fraction of viable endospores that can be quantified. The resistance of the endospore preparations used must be such that
meaningful exposure times can be applied to obtain fraction-negative results. Graded fraction-negative conditions typically
are used to evaluate the resistance of BIs.