The Sterilization Process
Different biological indicators are used for different types of sterilization processes. For moist- heat or steam sterilization, one might use Bacillus stearothermophilus, also known as Geobacillus stearothermophilus, Bacillus subtilis variety 5230, Bacillus coagulans ATCC 51232, or Clostridium sporogenes. However, these same indicators may not be appropriate for another type of sterilization process. One should select a system that is appropriate for the desired biological indicator and the type of sterilization process used.
The Sterilization Model Utilized
The sterilization model or approach utilized refers to the methodology used to determine the appropriate lethality required in the cycle to achieve the desired sterility assurance level. The most commonly used model is the "overkill" cycle. In this type of cycle, one would expect all of the biological indicators to be inactivated, i.e., killed.
Another type of model is based upon the actual bioburden (environmental isolates) present in the facility. Testing is performed to assess the resistance of this bioburden to the desired sterilization process and then a sterilization cycle is selected to achieve the desired sterility assurance level. In this type of cycle, the most resistant bioburden organism is cultured and maintained in the facility to be used as the biological indicator. This type of model is an "absolute bioburden" cycle. All of the challenge microorganisms should be inactivated in this type of cycle. These challenge organisms may be different for each facility.
The last type of sterilization model is a combination of these two models. It uses a biological indicator with a known resistance to the sterilization process and it also utilizes the resistance of the actual bioburden to the sterilization process. By knowing the bioburden resistance and the relationship of the bioburden resistance to the biological indicator resistance, one can use a biological indicator in the cycle to predict the sterility assurance level that will be achieved for the bioburden. For example, if the biological indicator has a resistance of 2 minutes to the sterilization process, and the bioburden worst case resistance in the facility is 0.5 minute, for every one log of biological indicator inactivated, one would expect a four log inactivation of environmental bioburden. This type of model is a "combined biological indicator – bioburden based model." This type of cycle may or may not inactivate all of the biological indicator challenge units. Following the cycle, the biological indicators are typically enumerated to determine the actual number of survivors of the cycle. The log reduction achieved for the biological indicator can be used to calculate the sterility assurance level achieved for the bioburden with the most resistance to the sterilization process.
The D-value is the measurement used to assess resistance to the sterilization process. It is defined as the time in minutes that is required at the sterilization process conditions specified, e.g., 121oC, to reduce the biological indicator population by one log. The comparison of D-values between the biological indicator and the worst-case bioburden establishes the relationship in heat resistance between the microorganisms, for the combined biological indicator-bioburden based model.
Any of these types of sterilization processes may be approved in a product application to FDA however "overkill" cycles are the preferred methodology for Europe . No one model is the only choice for sterilization. It is important to assess the sterility assurance needs and the business needs to choose a model that is appropriate for the company.
Depending upon the sterilization model used, the requirement for a rapid microbiology system may be different, e.g., is it necessary to enumerate the living cells or only evaluate if all cells are killed?
Stressed Microorganisms
When evaluating a system for rapid detection of biological indicators and / or sterilization process efficacy it is critical to understand that stressed microorganisms do not always have the same responses to methods as laboratory stock cultures. This is true even with traditional recovery methods. It is not unusual for stressed microorganisms (post sterilization process counts) to show different growth characteristics on lots of microbiological media than cultures that have not been stressed (control counts). There are also concerns regarding the potential for dying and injured biological indicators at the end of a sterilization process, e.g., will they be recovered by the selected method?
A simple method to evaluate the effects of stressed biological indicators on a potential rapid microbiology system is to test the system with stressed organisms. For example, if one were to evaluate a moist-heat sterilization biological indicator with a rapid microbiology method, stressed organisms could be prepared by exposing containers of biological indicators to short sterilization processes and then testing them with the system. When determining time intervals for the "short" cycles, one might use sterilization cycles that differ by the D-value time, e.g., if the D- value is 2 minutes, one might want to try a sterilization cycle of 2, 4, 6, and 8 minutes at the same conditions for the D-value determination, e.g., 121oC. The premise behind this is that one would expect the recovery of organisms to show approximately a one-log difference for each 2 minutes. By performing a series of time intervals, it is easier to make a critical assessment of the results obtained. This same type of testing scheme can be applied to other sterilization processes.
This type of evaluation is useful even when one intends to use an overkill cycle, to provide assurance that one will have confidence in the results obtained in validation studies.
Ease of Use
A concern with biological indicators is the potential to contaminate the manufacturing environment with an organism that is extremely difficult to kill in the sterilization process. It is important that the rapid system does not increase the risk of operator error/failure and potentially contaminate the environment
Financial Benefits
There are several different systems currently available for testing of biological indicators. They range in price from a few hundred dollars to many thousands of dollars. A more expensive method does not mean that it is the best method for your application. There are rapid systems sold by several biological indicator manufacturers. Some of these systems use actual biological indicators and other systems utilize enzymes from the biological indicators. Degradation of the enzymes in these test systems has been shown to have a relationship to the kill of biological indicators. All of these technologies are based upon the premise that all sterilization validation studies should result in total kill of the biological indicators (overkill cycles). Some of these technologies state that they are for Bacillus stearothermophilus only. They may also state specific sterilization cycle parameters. These inexpensive, quick test methods are useful for the manufacturer who performs overkill sterilization cycles under the conditions specified in the product literature.
For those companies who do not utilize Bacillus stearothermophilus or do not perform overkill cycles, these systems are not appropriate. Recently, presentations were given at conferences indicating that the Chemunex and AATI systems could be used to enumerate a variety of biological indicators. Although these systems may be more expensive, the cost has to be evaluated along with the benefits of having qualification data more quickly, e.g., reduced number of batches processed at risk, inventory hold time, time to perform biological indicator testing, etc.
Method Development
For many of the rapid systems it may be necessary to refine or optimize a method for biological indicators. This may be due to the stressed microorganisms, the number of organisms to be recovered, or the effects of cellular debris (biological indicators that have died, are dying or are injured) on the method. Don't assume that because you don't get the expected results on the first try that the method won't work. Work with your vendors and colleagues in rapid microbiology!
The Celsis Advance System
A special thank you to Ken Shore of Celsis for the following:
The Celsis Advance system is the most widely used rapid micro testing solution in the world today. First developed in the 1980s, it is the only solution in the market that has been purchased and validated by such pharmaceutical and personal care companies as Wyeth, Pfizer, P&G, Colgate Palmolive, Unilever and Johnson and Johnson. It has been reviewed by the FDA and implemented at more than 300 locations worldwide.
Using ATP bioluminescence, Celsis' customers are able to determine the presence of bacteria, yeast or mold in 24-48 hours, which is many days faster than their previous method of testing (agar plates). Instead of having extremely valuable capital in the form of finished goods tied up in the warehouse waiting for micro results, Celsis' customers are able to get their products to market 3-5 days faster, saving their company millions of dollars.
They are also able to identify a contamination much quicker. In as few as 1-2 days, they now know if their finished goods are contaminated, saving them upwards of three full production days of wasted product that must be destroyed. Depending on the product, this benefit alone could be worth millions of dollars to the company.
As the leader in the industry, Celsis learned early on the importance of technical and validation support. Each customer is assigned a dedicated technical manager with years of microbiology experience and training. They also receive complete installation qualification (IQ), operational qualification (OQ), and performance qualification (PQ) documentation to make the validation process as simple and as quick as possible.
Each instrument also comes with Advance.im (Information Management) software that is fully compliant with 21 CFR Part 11. Clients can easily store, analyze and share data with others at the plant and throughout their organization.
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