VALIDATION OF STERILIZATION PROCESSES

The goal of a sterilization process is the complete destruction of all micro- organisms present in a test article. To perform the process in a reproducible,

consistent, and reliable way, the sterilization process must be validated. Validation of a sterility process comprises the demonstration of the absence of microbial growth and the different parameters to achieve microbial death [24]. To determine the efficacy of the sterilization process, BIs are used [25]. BIs provide direct evidence that sterilization lethal conditions have been achieved during the treatment. Other process indicators such as temperature, gas concentration, pressure, humidity, etc. can be recorded by instruments and are critical parameters during the validation studies [26].

BIs are used during the validation process to determine the lowest probability to detect a nonsterile unit in a sterile load. BIs are standard preparations of bacterial spores specific to different types of sterilization processes. Table 2 shows the different types of BIs used for validating different sterilization treatments. For instance, if a sample is sterilized using irradiation processes, B. pumilus is the BI used, whereas for ethylene oxide treatments, Bacillus subtilisvar. niger is the choice. Different types of BIs are used for wet (steam) sterilization validation studies. A chapter in this book describes the use and validation of BIs.

BIs are used to show a reproducible logarithmic inactivation of microorganisms due to their resistance to some of the sterilization processes. Bacterial spores are most resistant to these processes than vegetative bacteria. Therefore, if spores are inactivated, so are other types of vegetative bacteria. In sterilization science, the D value is used to measure the rate of microbial death. The D value is the time in minutes required at the specified conditions to reduce the numbers of viable microorganisms by 90%. The D values are obtained when the numbers of colony-forming units (CFU) (on a logarithmic scale) is plotted against the exposure sterilization time. A slope of the line will be the D value. The D value is used to predict the lethal effect of the sterili- zation process on the microorganism. If the conditions where the D values are

TABLE 2 Bacterial Spores Used as Biological Indicators for Different Sterilization Treatments Wet heat Bacillus stearothermophilus

Bacillus subtilis Bacillus coagulans Clostridium sporogenes Dry heat Bacillus subtilis

Bacillus subtilis var. niger Bacillus stearothermophilus Ethylene oxide Bacillus subtilis var. niger Radiation Bacillus pumilus

changed (e.g., temperature change from 121jC to 105jC), then the D values will also change. For instance, the D value for Bacillus stearothermophilus is approximately 2 min at 121jC whereas at 105jC, it will be closer to 35 min. When other sterilization processes such as gas sterilization are used, then other factors (e.g., relative humidity and gas concentration) affect the D values. For irradiation processes, the D value is sensitive to time of exposure and radiation dosage.

The Z value is the numbers of degrees of temperature required to pro- duce a 10-fold change in the D value. The Z value is only important for thermal sterilization processes. The reason is that temperature is the main factor for the sterilization process to be effective. Using the Z value, we can predict the lethality of the treatment at different temperatures from which the Dvalue was determined. Another indicator in the evaluation of moist and dry heat sterilization processes is the Fo value, which can be used to estimate process lethality. The Fovalue indicates the integration of the instantaneous lethality over the duration of the sterilization process. More detailed infor- mation on D, Z, and Fovalues and their importance to sterilization processes is discussed elsewhere [16,24].

An example of a sterilization cycle is the overkill method. The overkill method provides a cycle with a minimum of a 12-log reduction of a resistant BI with a known D value of not less than 1 min. However, overkill ensures a greater log reduction than that. The assumption is that the natural bioburden in the product has less resistance to the sterilization process than the BI, and that the destruction of large numbers of resistant indicator organisms results in an even greater destruction of the biological bioburden. Cycle times are established by considering the time required to inactivate the indicators to achieve the 12-log reduction. Validation of an overkill cycle is based upon the use of BIs in a load adjacent to items at different locations inside the chamber. The BB approach is a process commonly used for medical devices sterilization. It provides a probability of survival of less than one in a million for the most resistant microorganisms (BB) expected in the load. It requires information on the number and heat resistance of the BB and ongoing monitoring and control over the BB. BB sterilization requires knowledge of the quantity and resistance of any BB present in or on the items to be steri- lized. Initial screening of the BB is performed to identify the most resistant microorganisms. The process involves the suspension and washing of the medical devices in a buffer. The buffer removes the bioburden from the devices. The buffer is then pooled and filtered through a 0.45-Am membrane. The membrane is then placed on growth media plates such as soybean–casein digest agar (SCDA) and Sabouraud dextrose agar (SDA). Incubation times range from 2 to 5 days. Colony-forming units on the plates are recorded and the final CFU per device is averaged. Once enumerated and identified, then

these microorganisms are used as the BB. The BB approach is mostly used for medical devices sterilization. Continuous monitoring of the BB of medical devices prior to sterilization provides valuable information to determine the sterilization parameters that will deliver a reproducible and reliable steri- lization process.