1. Summary
A filter within a sterile system can be aerosol integrity tested using the VALAIRDATA without compromising the sterility on the downstream side of the filter. This has been demonstrated in three separate tests where air contaminated with approx 107 cfu/litre of Brevundimonas diminuta is passed across the downstream integrity test point at 10 l/min, prior to, and during an integrity test. The maintenance of sterility is guaranteed due to the VALAIRDATA always providing a positive pressure to the system under test.
2. Introduction
In some instances users may want to integrity test air filters using the VALAIRDATA aerosol integrity test following steam sterilisation. As it is necessary to connect the VALAIRDATA into the sterile side of the filtration system it is important to ensure that this connection does not compromise the sterility of this downstream section. To overcome this potential problem, sterility is maintained with the VALAIRDATA by ensuring there is always a positive pressure within the filtration system during a test. Under these circumstances where a positive pressure is maintained, ingress into the downstream section is impossible. The studies reported here were conducted to show that the sterility of the downstream sector is maintained even if high levels of contamination exist around the downstream VALAIRDATA connection.
3. Methods
To simulate a worst case situation, a bacterial aerosol of Brevundimonas diminuta containing approximately 107 CFU per litre of air was passed over the VALAIRDATA male and female Rectus couplings both prior to connecting the tubing and during a 2 minute aerosol integrity test See Figure 1). The bacterial challenge aerosol was contained around the couplings with the use of a sealed polythene bag. The aerosol was flowed over the couplings at 10 l /min.
3.1 Test procedureThe test rig used for these studies is shown in Figure 2. Prior to initiating the test procedure all equipment was sterilised using the following procedures; Nebuliser : The nebuliser bowl was filled with approximately 100 ml of sodium hypochlorite solution (5% v/v), and pressurised to 30 psi to generate aerosol for a period of 5 minutes. Following sterilisation, the nebuliser was rinsed with water for injection, repressurised prior to a final rinse and emptying of the bowl. Disc housings : The disc housings were autoclaved complete with ASYPOR 0.2 micron membrane at 121°C (250°F) for 30 minutes. Impingers : The impingers were autoclaved complete with glycerol at 121°C (250°F) for 30 minutes Test Rig : The complete test rig, including the sterile air filter and all valves, was steam sterilised at 121°C (250°F) for 30 minutes. The VALAIRDATA was disconnected during this process.
3.1.1 Following steam sterilisation of the test rig close all valves.
3.1.2 Pressurise the rig with air at 1.5 to 2 barg and open all valves to dry the system.
3.1.3 When condensate free air exits the Rectus couplings and the rig has cooled to ambient temperature, isolate the Nebuliser from the test rig by closing V4.
3.1.4 Fill the nebuliser with 100ml of the Brevundimonas diminuta suspension and connect the glass impingers to the outlet of V8. Two impingers were used in series to ensure efficient sampling of all the micro-organisms.
3.1.5 Pressurise the nebuliser to 2 bar and open valve V11 . Air will flow from the nebuliser through the vent filter to act as a carrier gas for the bacterial aerosol. Crack open the diaphragm valve V8 until a flow of 10 l / min is shown on the rotometer downstream of the impingers. Allow aerosol to pass through the two impingers for 10 minutes . The challenge concentration was calculated by plating out a proportion of the glycerol from the impingers.
3.1.6 Isolate the air supply to the nebuliser.
3.1.7 Power up the VALAIRDATA and initiate an integrity test
3.1.8 Connect the VALAIRDATA to the upstream and downstream connections V1 and V2 .
3.1.9 Open V1 to allow the VALAIRDATA to pressurise the system to 3 barg as indicated by the pressure gauge on the housing.
3.1.10 Connect the flexible tube from valve 8 to the side inlet on the polythene bag.
3.1.11 Pressurise the nebuliser and adjust the flow to 10 l/min via V8. Challenge aerosol was then allowed to pass over the downstream connection for 10 minutes.
3.1.12 Connect the two halves of the downstream coupling and open V2 to allow air to pass through the VALAIRDATA.
3.1.13 A 10 minute VALAIRDATA integrity test was then performed whilst bacterial aerosol challenge was still being passed across the downstream connection.
3.1.14 On completion of the aerosol integrity test, isolate the VALAIRDATA by closing V2 then V1 and disconnect the VALAIRDATA.
3.1.15 Isolate the air supply to the nebuliser and close V8.
3.1.16 Remove the containment bag from the base of the valve V2 and shut the diaphragm valves V5 and V6.
3.1.17 Remove the "t-piece" from the test rig and transfer to a laminar flow cabinet. The possible ingress of bacteria was then determined as follows.
3.1.18 Flame the exposed ends of the pipework either side of the valves V5 and V6.
3.1.19 Open V5 and fill with WFI. Close V5 and agitate the pipework to ensure any bacterial contamination is removed from the internal walls.
3.1.20 Open V5 and empty the water into the pre-sterilised disc housing ; V6 may have to be cracked open to ensure all the water is drained.
3.1.21 Pull the sample through the 0.2-micron disc using a vacuum.
3.1.22 Remove the disc and place in a petri dish containing soya Tryptone agar. Incubate the petri dish at 30°C for five days prior to counting the colonies.
4. Results and Discussions
The data from three separate studies are shown in Table 1.
Total challenge level from the nebuliser = 2.94 x 107 cfu
Table 1: Recovery of Brevundimonas diminuta from downstream pipework
The total challenge of Brevundimonas diminuta passed across the VALAIRDATA connections prior to and during an integrity test sequence ranged from 4500 CFU to 6200 CFU. In all cases it can be seen that no organisms were recovered from the downstream pipework indicating no ingress of bacteria into the sterile section of the pipework. These data show that the sterility of a filtration system is maintained during an aerosol integrity test with the VALAIRDATA even under worst case conditions where extremely high levels of bacteria are present immediately adjacent to the VALAIRDATA connections. The bacterial count were conducted over a 5 minute period at 10 l/m therefore the average number of cfu per litre is:
This is the number of cfu passing across the sterile downstream connection before and during the aerosol integrity test.
6. Technical Support Group activities
domnick hunter have a trained team of scientists and engineers available to answer questions regarding the technical capabilities of our products, to assist in the selection and design of appropriate filtration systems and to provide user training programs. The following services can be delivered both on site and in-house;
- filterability testing to optimise filter system design
- advice on the development of integrity testing, steam sterilisation and clean in place procedures
- development of validation procedures
- troubleshooting
- facility audits to ensure continued optimisation of filter use
- operator training including filtration theory, filter system design and management, validation, etc.
For more information on any of the above support services please contact your local domnick hunter representative. email: tsg@domnickhunter.com
