James Rickloff, Advanced Barrier Concepts, provides an overview of how to validate sterility test isolator systems and, using a case study, how to streamline the process when equivalent isolators are installed in a sterility test suite.
In general, the validation work for a sterility test isolator project involves all of the major steps that are summarised in the table below. The goal is first to qualify the room and equipment contained within it for mechanical and electrical operation, including HVAC characteristics. Following equipment qualification, the decontamination process for bioburden reduction of test materials (including the exterior of sample containers) needs to be qualified to provide evidence that sufficient kill is achieved in order to provide a germ-free environment for sterility testing.
Additionally, aeration times and conditions are established, verifying the removal of the residual sterilant upon completion of the decontamination process.
The final phase of testing involves verification that the decontamination process does not interfere with the results of the sterility test on the target product sample. This is achieved by subjecting the testing materials (including the sample containers and sterility testing supplies) to the sterilant (back-to-back decontamination cycles), then demonstrating that low levels of microbial contamination can still be detected with the sterility test.
This procedure is termed ‘false-negative’ testing and it is a form of a process simulation that provides evidence that the decontamination process does not interfere with the sterility test or environmental monitoring results.
| Table 1: A summary of the major steps required in validating a sterility test isolator | |
| 1. Facility Qualification | Key facility attributes include: |
| a. HVAC/temperature control | |
| b. safety monitoring | |
| c. controlled access | |
| 2. Equipment Qualification | Standard IQ/OQ of isolator and sterilant generator installation and functional testing, including alarms testing. Also, the exhaust system is verified as an extension of the isolator |
| 3. BI D-value Determination | Provides a baseline resistance under specific conditions for the BIs used for testing. This baseline value can be compared with subsequent BI lots (used in revalidation studies) to verify that a reasonably equivalent challenge is being utilised |
| 4. Decontamination Cycle | Cycle parameters for the VHP1000 are established, initially based upon computer models using the temperature ranges established. Parameters are then verified in PQ. Consistent temperature and gas distribution provides optimal performance |
| 5. Sporicidal Efficacy Qualification | A minimal (fractional) cycle is validated in triplicate with the intention of providing a total kill of all 10-6 BIs (Geobacillus stearothermophilus spores) |
| 6. Aeration Qualification | The normal cycle (typically, the validated exposure time is increased by 20% to account for potential system variability) is challenged in triplicate to verify removal of the sterilant residues that may interfere with the sterility test |
| 7. Sterilant Ingress Testing | Product containers filled with distilled water are exposed to back-to-back decontamination cycles and then chemically assayed to verify no sterilant penetration |
| 8. Residue Effects Testing (Process Simulation to rule out false negatives) | Typically select 3–6 organisms (10–100 CFU) based upon USP growth promotion organisms and lab history (key environmental isolates) to provide evidence that the sterilant does not interfere with the sterility test and environmental monitoring techniques |
Case study
A vaccine manufacturer provided Walker Barrier Systems (New Lisbon, WI, US) with a set of requirements for testing a single lot of final product containers (vials) and bulk samples (Hyclone bags) on a daily basis in a fully automated isolator system that would both require the use of Millipore Steritest kits for sample recovery. At maximum loading, the total decontamination cycle time including aeration would need to be <5 hours to permit time for the testing within a single 8-hour shift.
The end-user decided to purchase two identical turbulent flow isolator systems (pictured above) to handle current and anticipated test requirements for the facility and to provide for redundancy in the event that one system was down for maintenance, etc. Two STERIS VHP 1000ED-AB Generators were purchased for the decontamination of the enclosures using hydrogen peroxide (H2O2) gas.
Initially, a robust decontamination cycle was developed for the maximum final product load configuration in Sterility Test Isolator No 1 using VHP 1000ED-AB Generator No 1. The sterilant gas concentration was based on the minimum surface temperature within the enclosure at the end of the Dehumidify Phase (20°C). It was determined that a 30-minute exposure was sufficient to inactivate all of the biological indictors (BIs) placed throughout the enclosure and load. The developed ‘fractional’ cycle was then validated in triplicate for the maximum final product and bulk sample load configurations, which was successful.
Aeration studies using a ‘normal’ exposure time of 36 minutes (20% longer than the validated time listed above) were performed in triplicate on both maximum load configurations. Three hours of isolator exhaust (pressure set at 1.0” of water column) were required to consistently reduce the H2O2 gas concentration to at or below 0.5ppm for each load configuration. The isolator exhaust used a ‘thimble’ connection that allowed isolator air to be diluted into a room exhaust air line, which eliminated the concern of pressure drop on reducing the airflow rate if a hard connection was made to the isolator’s exhaust pipe.
Sporicidal efficacy
The next goal of the validation effort was to verify that temperature distribution and sporicidal efficacy of H2O2 gas during the decontamination of Sterility Test Isolator No 1 was equivalent to the decontamination of Sterility Test Isolator No 2 for each validated load configuration. Temperature distribution and sporicidal efficacy tests were also performed using VHP 1000 ED-AB Generator No 2 attached to Sterility Test Isolator No 1 to demonstrate generator equivalency for each validated load configuration. A single total coliforms (TC)/ BI study was performed on each load configuration to demonstrate isolator and generator equivalency and each of the tests were successful.
The aeration of Sterility Test Isolator No 2 to H2O2 gas levels considered acceptable for sterility testing and for ensuring operator safety (≤0.5ppm) was then shown to be equivalent to the aeration time validated for Sterility Test Isolator No 1 for each validated load configuration.
Finally, a single test verified that VHP 1000ED-AB Generator No 2 was equivalent to VHP 1000ED-AB No 1 in terms of aerating Sterility Test Isolator No 1 to ≤21ppm of H2O2 gas for each validated load configuration after exposure to the ‘normal’ time of 36 minutes.
In conclusion, a 4 hour, 14 minute “robust” decontamination cycle was validated for the sterility test isolator system in this facility, which met the user requirement of <5 hours. The validation data supported the use of either isolator connected to either generator for the decontamination and sterility testing of either final product or bulk samples using the same cycle settings, thus providing the end-user with sufficient operational flexibility in the event that some of the equipment was out of service.
Process simulation studies were also successful in verifying that false negative sterility test or environmental monitoring results were not occurring even following back-to-back decontamination cycles. This data supported the use of product samples or supplies that were exposed to more than one decontamination cycle in the event that the previous cycle was aborted for some reason.