In the pharmaceutical, biotechnology and food industries, production, maintenance and laboratory operators frequently refer to sterilising filtration, and more specifically to 0.22 µm (0.22 micron) filters, also known as sterilising filters.
Key questions on sterilising filtration:
- What does sterilising filtration mean?
- How is a 0.22 µm filter qualified?
- When can a filter be considered absolute?
- What does an integrity test demonstrate?
These questions often arise when discussing sterilising filtration. The questions are simple, but the answers are far more complex, especially as the terminology has become increasingly generic. Many industries use 0.2 µm filters, but only the pharmaceutical and biotech sectors define the specifications that 0.2 µm filters must meet. It is not uncommon for suppliers to provide 0.2 µm filters with certificates, yet these elements do not comply with the standards required by the pharmaceutical and biotechnology industries.
Let’s clarify this broad topic.
Background: origin of the concept of sterilising filtration
In the 1970s, a group made up of major pharmaceutical companies and filter manufacturers created an organisation called HIMA (Health Industry Manufacturer’s Association). One of the first actions undertaken by this organisation was to define the criteria a sterilising filter had to meet. It is important to understand that in the 1970s, a 0.45 µm filter was considered a sterilising filter. However, as analytical methods improved, relying solely on the retention threshold of a filter to define sterilising filtration—or sterility—was somewhat empirical and not necessarily representative or sufficient.
HIMA therefore developed a highly detailed challenge protocol, which became the reference standard that sterilising filters had to comply with to be recognised as sterilising in the pharmaceutical industry.
Bacterial challenge required for a sterilising filter
The challenge was developed using a micro-organism called Pseudomonas diminuta (ATCC 19415), now replaced by Brevundimonas diminuta (ATCC 19146). This reference organism measures approximately 0.3 µm in width and 0.8 µm in length. The protocol also specifies the growth medium to be used, as well as the temperature and duration of cultivation. The HIMA challenge defines both the concentration and the quantity of this bacterium used to test the filter, with a precise microbial load per cm².
0.2 µm or 0.22 µm then became a label, but it did not necessarily mean that filters marketed as sterilising had passed the HIMA tests. Manufacturers with HIMA-validated products found themselves in competition with non-HIMA-certified filters, and for non-critical markets (solvents, demineralised water, acids, etc.), they were forced to develop lower-cost product ranges.
To maintain clarity and credibility, filter manufacturers therefore started referring to filters validated according to the HIMA challenge as 0.2 µm absolute filters or 0.22 µm filters. Today, the HIMA standard has been replaced by ASTM Standard F838-83. The testing procedures remain the same—the only changes are the name and the governing body.
Example of sterilising cartridges
Bacterial challenge: HIMA and ASTM 838-83 standards
The ASTM F838-83 standard, titled “Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration,” is the new reference replacing the HIMA test (“Microbiological Evaluation of Filters for Sterilising Liquids”).
The reference micro-organism for ASTM is Brevundimonas diminuta (ATCC 19146) (diameter 0.3 µm – length 0.6 to 0.8 µm), and for HIMA, Pseudomonas diminuta (ATCC 19415) (diameter 0.3 µm – length 1 µm), grown in a saline lactose solution according to the parameters described in the procedure. This micro-organism was selected due to its extremely small size achieved when cultured under stress or nutrient deficiency. The filter membrane must withstand a minimum challenge of 10⁷ organisms per cm² of effective filtration surface.
After preparing the challenge solution, the membrane filter is installed in the filter housing and autoclaved. Once cooled, system sterility is checked and the integrity test can take place. The solution is filtered through both the test membrane and the control filters. Once the entire test solution has passed through, the control filters are placed in 150 mm Petri dishes containing Soybean Casein Digest Agar. All developing colonies are then counted and identified.
The Log Reduction Value (LRV) is calculated as follows:
LRV = log₁₀ (Number of challenge micro-organisms / Number of micro-organisms in the filtrate)
Brevundimonas (Pseudomonas) diminuta zoom 3000x
In summary: key takeaways on sterilising filtration
| ASTM | HIMA | |
|---|---|---|
| Flow rate | 20 lpm/m² | 30 lpm/m² |
| Pressure drop | 2 bar max | 2 bar min |
| Bacterial challenge (Micro-organisms/cm²) | 107 | 107 |
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