In addition to characterization of the sterilizing effect at worst-case positions, the properties of the actual most-resistant bioburden isolates compared with those of the customized spore preparations must be considered in detail. Is the process bioburden well-enough characterized to ensure that the most resistant isolates are indeed known, and what are the cultivation and harvesting conditions needed to produce the most resistant spores from these isolates? What is the difference when resistant spores of these isolates are tested on paper or at a worst-case position? How do spores from process isolates react in a reference suspension in product or on the surface to be tested?

Using biological indicators

Use in product or process development. Ease of sterilization should be a criterion during the development of sterile products. It is the position of the European authorities that a heat-labile container-closure system alone is not a sufficient justification to choose a sterilization cycle other than the Standard Sterilization Cycle defined in the European Pharmacopoeia (10, 11). Although product reformulation may not be an option in many cases, primary packaging materials such as stoppers or delivery systems should at least be chosen in due consideration of their influence on sterilization effectiveness.

In addition, the primary packaging (container-closure) system should be evaluated with respect to possible problem points during sterilization. In the experience of the authors, spore inactivation is not the same on all types of stoppers. Specific effects may be attributed to the material, the surface finish, or both.

For vials with elastomeric stoppers, the space in between the stopper and the vial is always a critical position because this is a position where steam does not easily penetrate. For BI studies, it is necessary to ensure that the bacterial endospores are in contact with the elastomeric surface during sterilization.

During the development of processes for equipment sterilization (e.g., SIP), it is important to ensure steam penetration throughout all parts that may affect the sterility of any product processed with that equipment. Product-delivery systems also may contain positions that are very difficult to penetrate during steam sterilization. As a general rule, the more complicated the geometry of equipment or a system, the more difficult steam penetration will be. Any equipment or system to be sterilized should be analyzed to define worst-case positions, and, wherever possible, the effect of sterilizing conditions should be tested on model systems simulating worst-case conditions as closely as possible.

Studies conducted to investigate the specific effect of sterilization conditions must be quantitative and the results must be seen relative to the effect obtained under reference conditions. It is scientifically correct and easier to conduct these studies under scale-down conditions in a laboratory using a precision autoclave that delivers heat exposure with square-wave characteristics.

Process and cycle validation. Not all steam-sterilization processes are the same. Whether a prevacuum process (also called a porous-load process), a hot-water-shower process, or a steam-air mixture process is used will depend on the properties of the product, load, or equipment to be sterilized. Depending on the configuration, there will be various considerations as to where worst-case positions are to be expected and what cycle is needed to achieve the expected sterilizing conditions in the worst-case position.