1 PARAMETERS OF BIOLOGICAL INDICATORS FOR HEAT STERILISATION
Sterilisation processes can be operated at temperatures lower than the standard 121 °C (for longer exposure times) or at higher temperatures (for shorter exposure times). The z-value (the temperature difference that leads to a 10-fold change in the D-value of the biological indicator) is used to compare the efficacy of 2 cycles operated at different temperatures. For a z-value determination, the D-value must be determined at 3 or more temperatures. The intended process temperature should be within the range of the 3 temperatures. The log10 of the D-value is plotted against the temperature in degrees Celsius. The z-value is equal to the negative reciprocal of the slope of the best-fit linear curve as determined by log10-linear regression analysis.
1-2 Establishment of validation cycle
The characteristics of the sterilisation process (e.g. time- temperature combination, level of sterility assurance or F0 required) are the basis for the choice of the biological indicator (type of biological indicator, test micro-organism, and initial viable count).
Inactivation of micro-organisms under sterilising conditions can be described by lethality kinetics and statistical probabilities. For a number of biological indicator units with an initial population of N0 micro-organisms per unit and a given D-value, the exposure time in minutes where all units are expected to carry survivors (average of 100 surviving spores per unit) is calculated by equation (1).
The exposure time in minutes where all units are expected to be inactivated (average of 10-4 surviving spores per unit) is calculated by equation (2).
The objective of a validation study is to demonstrate that the sterilisation effectiveness anticipated from the physical process parameters is equivalent to the biological sterilisation effectiveness. As part of that objective, the exposure time during validation tvl, shall not exceed tk. If a too high tvl is chosen, even a relatively large increase in the D-value would still result in biological indicator units with no surviving micro-organisms. In this case, the suboptimal sterilising conditions would not be detected. It is considered reasonable to choose a tvl not higher than required to expect 1 in 1000 biological indicator units having surviving micro-organisms. However, too short a tvl shall not be chosen. If a tvl is chosen such that 50 per cent of the biological indicator units have surviving micro-organisms, changes in the sterilising conditions (e.g. time, temperature) could still result in 100 per cent of the biological indicator units having surviving micro-organisms, and the test would be meaningless. For these reasons, a tvl is chosen such that a theoretical survival rate between 10-1 and 10-3 is expected, thus:
In general, biological indicators are subjected to the intended sterilisation process. However, for highly effective sterilisation processes, the calculated effectiveness of the cycle may be such that the tk is exceeded by a wide margin. In such instances, biological validation is carried out with reduced sterilisation cycles. Such reduced cycles may be shorter in time (e.g. half cycle) or be performed at a lower temperature. In the latter case the z-value for the test micro-organism under the actual sterilising conditions shall be known. A reduced cycle is chosen such that the temperature is not more than 1 z-value below the reference sterilisation process temperature. Biological indicators of an appropriate resistance for that cycle show an expected micro-organism survival rate within a window between the lower tvl and the tk (see equation (3)). A decision not to perform this test must be justified.
Depending on the D-value of the test micro-organism and the tvl chosen, biological indicators having surviving micro-organisms can be expected with a low frequency (not more than 1 in 10). If it can be demonstrated that the frequency of biological indicators having surviving micro-organisms is within the expected range and is not due to inappropriate sterilising conditions, the process can be accepted.
Following a full sterilisation cycle, all biological indicators in a validation study must be inactivated, thereby proving at least a 106 reduction in micro-organisms. It can then be concluded, from the resistance of the spore preparation used, that the process has delivered sufficient lethality to achieve the required sterility assurance level.
2 BIOLOGICAL INDICATORS FOR MOIST HEAT STERILISATION
Geobacillus stearothermophilus is the most widely accepted biological indicator micro-organism for moist heat sterilisation processes. Reported D121 °C-values for its spores are in the range of 1.5 min to about 4.5 min, depending on sporulation conditions, the carrier material on which the spores are inoculated, the primary package surrounding the inoculated carrier, and the environment during sterilisation. Strains ATCC 7953, NCTC 10007, CIP 52.81, NCIMB 8157 and ATCC 12980 (equivalent to NRRL B-4419) have been found to be suitable. Other strains may be used, provided equivalent performance has been demonstrated. It is recognised that a 105 or 106 population of Geobacillus stearothermophilus may not be suitable for sterilisation processes delivering an F0 between 8 and 15, therefore a lower spore number (i.e. 103 or 104) or a different test micro-organism may be used. Where a test micro-organism other than Geobacillus stearothermophilus (e.g. Bacillus subtilis ATCC 35021) is used, the resistance of the test micro-organism is evaluated to ensure its suitability for the process.
BIOLOGICAL INDICATORS FOR DRY HEAT STERILISATION
The reference conditions are stated in general chapter 5.1.1. Heat transfer is less effective with dry heat than with steam, and temperature distribution in dry heat sterilisers is less homogeneous compared to steam sterilisers.
For example, biological indicators available for dry heat sterilisation have D160 °C-values within a range of 1 to 5 min. When exposed to the reference cycle of 2 h at 160 °C, a biological indicator with a D160 °C-value of 2.5 min would be inactivated by 48 log10 scales. For dry-heat sterilisation processes, z-values of about 20 °C are typically assumed in calculations of equivalence of cycle effectiveness (FH-calculations). FH is the equivalent time in minutes at a temperature of 160 °C delivered by the sterilisation process to the product in its final container. For a biological indicator with a D160 °C-value of 5 min, the D150 °C-value would be about 16 min, and inactivation in the reference cycle would be 7.5 log10 scales. The use of a sterilisation process at a temperature reduced from the target temperature by 10 °C would give an expected 1 in 30 biological indicator units having surviving micro-organisms.
3-3-1 Test micro-organisms
Spores of Bacillus atrophaeus (e.g. ATCC 9372, NCIMB 8058, NRRL B-4418, or CIP 77.18) have been found to be suitable for use as biological indicators for dry heat sterilisation processes performed at temperatures between 160 °C and 180 °C. Where a test micro-organism other than Bacillus atrophaeus is used, to ensure its suitability, the resistance of the test micro-organism for the sterilisation process is evaluated as described in section 3-1-2
4 BIOLOGICAL INDICATORS FOR GAS STERILISATION
The use of biological indicators is necessary for the development, validation and monitoring of all gaseous sterilisation processes. Gas sterilisation is a multi-factorial process: gas concentration, humidity, temperature, time, surface characteristics interact in a complex manner. A number of gas sterilisation processes are currently used, including ethylene oxide, hydrogen peroxide and peracetic acid or combinations of the latter.
Gas surface disinfection is widely used for medical devices, isolators, chambers, etc. Use for such purposes is outside the scope of the European Pharmacopoeia but the use of biological indicators as described in this general chapter may assist in the validation of such disinfection processes.