1. History of Sterilising Grade Filtration
Since the early 1900’s, when the first parenteral drugs were manufactured in large scale, Sterilising grade filters have been used as part of aseptic processes for sterilisation of products unsuited to terminal sterilisation such as autoclaving. These products include heat sensitive compounds, blood products etc. Until the early 1960’s, 0.45µm rated membrane filters were considered "Sterilising grade", and were successfully used to achieve sterilisation of parenteral products. Serratia marcescens (approximate dimensions: 0.6 x 1µm) continues to be used to validate the performance of 0.45µm filters. In the mid-60’s, an organism was identified by Frances Bowman of the FDA that would penetrate 0.45µm rated membranes. This organism was identified as Pseudomonas diminuta (approximate dimensions: 0.3 x 0.6-0.8µm) now reclassified as Brevundimonas diminuta ATCC 19146. The PDA identified advantages and disadvantages (see below) in the use of this organism, and in consideration of these, this strain was accepted by filter manufacturers and the pharmaceutical industry, as the new standard challenge organism for qualifying sterilising grade membrane filters.
Advantages:
- Originally a process stream isolate, and therefore representative of potential contaminating organisms in parenteral manufacture.
- Generally regarded as non-pathogenic to humans, thus minimising safety concerns for personnel involved conducting challenge tests.
- Organisms can be consistently cultured under controlled conditions to produce very small, monodispersed cells with a narrow size distribution which can penetrate 0.45µm filters reproducibly in small numbers at high challenge levels. Under these conditions the organisms, thus represents a potential worst-case challenge.
Disadvantages:
- Not viable in many pharmaceutical formulations.
- May not be the smallest bacterium potentially encountered in all formulations.
- In spite of being stable, batch to batch variation may exist.
During the 1990’s work has been reported identifying diminutive organisms that can penetrate 0.2µm sterilising grade products. Since 1985 domnick hunter has offered 0.1µm rated Sterilising grade filter products capable of removal of even these organisms. These 0.1µm filters are validated at domnick hunter using Acholeplasma laidlawii as the challenge organism. This organism belongs to the class mollicutes, which are the smallest living organisms capable of self-reproduction and are further characterised by having a flexible tri-laminar membrane rather than a cell wall. This gives the organism plasticity with a cellular morphology which is variable and pleomorphic, allowing potential deformation and passage through 0.2µm rated filter membranes. For these reasons, Alcholeplasma laidlawii is now selected by the majority of filter manufacturers in their validation protocols and is increasingly considered as the industry standard for 0.1µm rated sterilising grade filters. There is continuing debate regarding redefinition of "sterilising grade" products as only being 0.1µm rated product rather than the current 0.2µm rated filters. Until this situation is further resolved, pharmaceutical users must make a risk assessment regarding their selection of appropriately rated products. Regulators continue to specify 0.2µm rated filters, validated using Brevundimonas diminuta as the defining standard for sterilisation grade filtration.
2. Introduction
The Parenteral Drug Association guidance document "Sterilizing Filtration of Liquids" published as Technical Report No. 26 in 1998, defines sterilising filtration as "the process of removing all micro-organisms, excluding viruses, from a fluid stream". Bacterial Challenge testing then serves two major functions.
- Filter manufacturers, including domnick hunter, use bacterial challenge data to define the performance of "Sterilising grade" filters within an agreed industry performance window.
- Filter users, utilise this data as a benchmark for validation of the filter use within their specific application.
However, as this test is effectively destructive to the filter and could not be performed on-line, practical use of challenge test data means that it must be correlated to a non-destructive physical integrity test in the laboratory. This allows users to test filters to confirm their performance meets claims in terms of bacterial reduction efficiency. The process of performing a micro-organism challenge is defined in two documents. HIMA (Health Industry Manufacturers Association) now redefined as AdvaMed (Advanced Medical Technology Association) issued in April 1992 a guidance document No.3 Vol. 4 Microbiological Evaluation of Filters for Sterilizing Liquids. ASTM (American Society for Testing and Materials) subsequently issued a standard F883-83 Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration. Both documents are referenced by the industry, although ASTM F883-83 is the now adopted standard method discussed in the PDA Technical Report No. 26.
3. Brevundimonas diminuta Challenge - Overview
Both HIMA and ASTM documents provide a documented procedure to bacterial challenge a sterilising grade filter, but differ slightly in terms of rigour and protocol detail. The domnick hunter procedure is written in accordance with ASTM F838-83, which is becoming widely accepted as the most apprpriate test procedure. Some minor modifications have been incorporated into the domnick hunter procedure to allow specified key test parameters to be met. In summary the procedure is as follows:
After sterilisation of filter and test assembly, the test filter is challenged with a suspension of Brevundimonas diminuta (ATCC 19146), cultured under specified conditions at a minimum concentration of 107 organisms per cm2 of Effective Filtration Area (EFA). The maximum differential pressure across the filter is 30 psi (206 kPa) and the flowrate is 2–4 l/min per 1000 cm2 EFA. The entire filtrate is then filtered through an analytical membrane filter disc that is subsequently incubated on a solid growth medium. Organisms that have not been retained by the filter on test develop into visible colonies on the analytical membrane disc and can then be enumerated. A sterilising grade filter is thus defined as:
A filter which, when challenged with the micro-organism Brevundimonas diminuta, at a minimum concentration of 107 organisms per cm2 of filter surface, will produce a sterile effluent.* Therefore no organisms should be detected in the filtrate collected downstream of the test filter. Under these circumstances, the filter is said to produce a sterile filtrate at a Log Reduction Value (LRV) of greater than 7. This can be compared to the validation of thermal based sterilisation systems where a probability of sterilisation of greater than 106 is required i.e. LRV >= 6.
| LRV = | Log10 | number of organisms in the challenge |
| | |
|
| | | number of organisms in the filtrate |
*FDA Guideline on Sterile Drug Products Produced by Aseptic Processing (June 1987) Reprinted June 1991
4. domnick hunter Bacterial Challenge Methodology
Preparation of the Bacterial Challenge Stock Suspensions 0.2µm filter challenges For the bacterial challenge of 0.2µm rated membrane cartridges, a single colony of Brevundimonas diminuta ATCC 19146 is inoculated aseptically into 20ml of Tryptone Soya Broth (TSB). This is then statically incubated at 30°C ± 2°C (86°F) for 24 hours ± 4 hours. 2ml of this starter culture is then aseptically inoculated into 3 litres of sterile Saline Lactose Broth (SLB) and statically incubated for 24 hours ± 2 hours at 30°C ± 2°C (86°F) to provide a challenge culture. The challenge culture is then used to make up 4 litres of challenge suspension by aseptically transferring the culture into a flask containing 1 litres of sterile 0.9% saline. Following gentle mixing the suspension is then used to challenge the cartridge on test. A sample of the challenge suspension is also plated on to TSA and incubated at 30°C ± 2°C (86°F) and read at 2 and 5 days to determine the actual microbial concentration in the challenge suspension (colony forming units per ml).
0.45µm filter challenges
Whilst challenges of 0.45µm rated membrane cartridges fall outside the remit of this document on sterilising grade filters, they are included for completeness. In this case, a single colony of Serratia marcescens ATCC 14756 is sub cultured as described above for Brevundimonas diminuta ATCC 19146, with the exception that only 2 litres of sterile SLB is used in the final transfer.
0.1µm filter challenges
For 0.1µm rated cartridges, challenge testing is essentially similar to that described above although the culture conditions required to generate a challenge suspension of Acholeplasma laidlawii NCTC10116 are somewhat more complicated. Acholeplasma laidlawii is obtained as a lyophilised culture, to which is aseptically added 1ml of sterile Acholeplasma laidlawii broth (ALB). The ampoule is shaken to mix the solution and then aseptically transferred in equal portions into 15-20ml sterile universal bottles. A small portion of penicillin G is transferred to each universal bottle. This is shaken thoroughly and incubated for 4 days at 30°C ± 2°C (86°F). After 4 days the culture is semi opaque, and yellow in colour. This seed is transferred aseptically into 500ml of sterile ALB and inclubated again for 4 days at 30°C ± 2°C (86°F). For challenges of 10" cartridge filters, it is necessary to perform a further transfer of the above challenge culture into a 1 litre bottle containing 500ml of sterile ALB, followed by incubation at 30°C ± 2°C (86°F) for 4 days. The challenge culture is then used to make up 4 litres of challenge suspension by aseptically transferring the culture into a flask containing an appropriate volume of sterile ALB. Following gentle mixing the suspension is then used to challenge the cartridge on test.
5. The Challenge Process
ASTM and HIMA specify 2–4 l/min and 3.86 l/min per 1000cm2 EFA respectively as the challenge flowrate. The bacterial challenge facility at domnick hunter allows a range of challenge flux rates to be used dependant on the challenge requirements. For standard testing the ASTM recommendations apply. 
Liquid Bacterial Challenge Rig Schematic
i) Testing Procedure Overview
- Fit pre-wetted filter cartridge under test to the housing in the rig.
- Integrity test the filter and housing.
- Fit sterile pre-validated 142mm-assay filter discs to the rig.
- Steam sterilise rig and cartridge.
- Cool with sterile water flush.
- Integrity test the filter and housing.
- Challenge cartridge and rig with sterile SLB control to verify sterility.
- Challenge cartridge with bacterial challenge solution, using sterile compressed air to feed the challenge solution at the required flowrate.
- Collect challenge through assay filter discs and plate each disc onto TSA (Tryptone Soya Agar)
- Integrity test the filter and housing.
- Incubate test discs for 5 days.
- Report number of colony forming units on sterile control and challenge.
ii) Correlation of Bacterial Challenge to Integrity Test Methods
Integrity testing (IT) of filters, before and after use is a requirement for sterilising filtration applications in accordance with current Good Manufacturing Practice (cGMP). A non-destructive physical test correlated to bacterial challenge is the first step in sterility assurance monitoring in the production of sterile products, confirming that acceptable criteria will be met when the system is put into operation. The FDA, in 1976, established the requirements for the integrity testing of filters in its proposed GMP for large volume parenterals (LVP). Although never implemented and withdrawn in 1994, the following forms the basis for inspection of both LVP and SVP operations: a) The integrity of all air filters shall be verified upon installation and maintained throughout use. A written testing program adequate to monitor integrity of filters shall be established and followed. Results shall be recorded and maintained as specified in 212.83. b) Solution filters shall be sterilised and installed aseptically. The integrity of solution filters shall be verified by an appropriate test, both prior to any large-volume parenteral solution filtering operation and at the conclusion of such operation before the filters are discarded. If the filter assembly fails the test at the conclusion of the filtering operation, all materials filtered through it during that filtering operation shall be rejected. Rejected materials may be refiltered using filters whose integrity has been verified provided that the additional time required for refiltration does not result in a total process time that exceeds the limitations specified in 212.111. Results of each test shall be recorded and maintained as required in 212.188(a). In effect there is a guideline requirement to integrity test sterilising grade air and liquid filters, both before and after the filtration operation. PDA Report 26 also states that "It is generally regarded as cGMP requirement that filters or filter systems routinely be integrity tested both prior to and after use." The PDA document goes on to define IT requirements for sterilising grade filters when redundant or serial filtration is a requirement or has been validated. Additional information is available from domnick hunter. A variety of IT methods exist for hydrophilic and hydrophobic based liquid filtration products. For filters in small-scale device and disc format, the most commonly used method is "Bubble Point", while for larger scale cartridge and capsule based products "Diffusional Flow" or "Pressure Decay" based methods are considered more appropriate. domnick hunter uses the "Bubble Point" IT method to verify flatstock and disc, and "Diffusional Flow" to verify cartridge and capsule based product. When a differential pressure is applied to a wet filter, gas initially dissolves into the surface layer of liquid then diffuse through its depth and come out of solution on the low-pressure side. This is referred to as diffusional flow. As the differential pressure is increased, the wetting liquid will be forced from the largest filter pores, as their bubble point is exceeded, and the gas flow will start to increase dramatically. This increase in flow is related to the change from relatively low diffusional flows to much higher, levels of mass air flow through the pores where the wetting liquid has been displaced.
The diffusional flow IT method depends on the transfer of gas from the upstream side of a wet filter to the downstream side when a test pressure below the bubble point of the media is applied. With readily wetted media, purified water is used as the wetting fluid. To ensure that less hydrophilic media are fully wetted, low surface tension fluids such as Isopropyl Alcohol (IPA) or mixtures of IPA and Water may be used. It should be noted that the use of IPA, or other wetting agents, used to allow successful integrity testing of hydrophobic filter products (e.g. air and vent filters) can in some cases be judged inappropriate where it is preferred not to add a chemical agent into the processing system. To overcome this problem, a new methodology has been introduced for the integrity testing of hydrophobic sterilising grade filters. The Water Intrusion Test (WIT) method relies on measurement of the change in volume when the water filled upstream face of a hydrophobic filter media is pressurised. As the pressure is increased, so water ‘intrudes’ into the pore structure of the media, the more open the media, the higher the level of instrusion. However, regardless of the test method specified, the filter manufacturer must specify an integrity test value which can be directly correlated to a bacterial challenge meeting HIMA/ASTM guidelines. The integrity test values for any particular validated filter can then be used to verify that the cartridge will perform within defined limits in terms of its ability to remove bacterial contaminants from the process stream. The process by which correlation between the maximum allowable integrity test result (e.g. ml per minute for a diffusional flow test) and the bacterial challenge is achieved is relatively straight forward. A number of filter devices are sourced from the manufacturing process and submitted to the bacterial challenge process. In each case the integrity test value associated with individual filters is determined before conducting the challenge test. domnick hunter uses standard format 10" cartridges as the base product for correlation of integrity testing values to bacterial challenge, and specifies Diffusional Flow as the standard integrity test methodology. Cartridges are used for these studies as this enables not only the membrane performance but also the processing methods and other build components of the cartridge, that can modify the performance of the finished product, to be assessed. In the following chart, the bacterial challenge result for each product is either a ‘PASS’ , the effluent was sterile, or ‘FAIL’, the effluent was non-sterile.
It can be seen that all cartridge exhibiting a diffusional flow of less than X ml/min passed the bacterial challenge. However, at higher diffusional flow values some of the cartridges failed challenge test. In this example, the maximum allowable diffusional flow would be set at Y ml/min to provide a degree of safety from the first identified fail point (i.e. X ml/min). Integrity test values for filter formats (of a given filter type) are determined using a ratio of the effective filter area for the alternative filter format compared to the standard 10" product used in the challenge test work. In cases where integrity testing will not be performed in water, or will use product as the wetting solution, it is necessary to contact domnick hunter for advice on applying appropriate integrity test values. Water wet integrity test values will not be applicable in the majority of these instances.
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
