1What PUPSIT is.
PUPSIT stands for pre-use post-sterilisation integrity test. It is an integrity test of a sterilising grade filter after the filter assembly has been sterilised and before it is used, “to check for damage and loss of integrity caused by the filter preparation prior to use.” EU GMP Annex 1 sets the expectation in point 8.87 of its 2022 revision, in operation since 25 August 2023.¹
A sterilising grade filter is one that, when validated, “will remove a defined microbial challenge from a fluid or gas producing a sterile effluent”, usually with a pore size of 0.22 µm or less.¹ PUPSIT is one of two tests on it. The other comes after use, before the filter leaves its housing.
Annex 1 gives bubble point, diffusive flow, water intrusion and pressure hold as examples of the tests used. The test process should be validated, and its results “should correlate to the microbial retention capability of the filter established during validation.”¹ The filtration system should be designed to permit in-place integrity testing of the final 0.22 µm filter, “preferably as a closed system, both prior to, and following filtration as necessary” (8.82).¹
2What Annex 1 requires.
Point 8.87 says “the integrity of the sterilised filter assembly should be verified by integrity testing before use”, and that a filter used to sterilise a fluid should have a non-destructive integrity test after use, before it is removed from its housing.¹
There is one exception. Annex 1 recognises that PUPSIT “may not always be possible after sterilisation due to process constraints”, giving the example of filtering very small volumes. An alternative is then acceptable only after “a thorough risk assessment” and with controls that mitigate the risk of a non-integral filtration system. That assessment should consider at least:¹
- in-depth knowledge and control of the filter sterilisation process, so the potential for damage is minimised;
- in-depth control of the supply chain, including contract sterilisation, defined transport and packaging that protects the sterilised filter;
- the product itself, including its particle burden and whether it could “alter integrity-testing values and therefore prevent the detection of a non-integral filter during a post-use filter integrity test”;
- the pre-filtration steps that clarify the product before the final sterilising filter.
The joint PIC/S, EMA and WHO interpretation, PI 058-1, in force from 28 October 2026, explains how the exception is read.²
| Question | What PI 058-1 says |
|---|---|
| Which filters need PUPSIT? | Filters used for final product sterilisation, such as of solutions or liquids, “whenever feasible”. For other sterilising filters, such as upstream bioburden control, integrity testing is based on risk and the CCS. |
| When can PUPSIT be omitted? | Only when it is not feasible “due to process constraints that are not linked to design flaws”, justified in the contamination control strategy and supported by a product and patient safety focused risk evaluation and mitigation. Manufacturers should first consider process design changes to make PUPSIT possible. |
| Can a risk assessment replace it? | “Risk assessments should not be used inappropriately to justify the omission of PUPSIT.” |
| Redundant filters | PUPSIT should be performed on every filter declared as potentially used to support batch release. A redundant filter without PUPSIT can’t show a successful sterilising filtration if the primary filter fails its post-use test. |
PI 058-1 states that its purpose is not to introduce new GMP requirements. It also records that “verification of the integrity of sterilising filters before and after use is a requirement.”² EMA’s own GMP questions and answers on Annex 1 cover sterility test sampling, bioburden, water for injections, rapid methods, isolators and parametric release, not filter integrity.³ WHO’s 2022 sterile GMP, harmonised with the EU and PIC/S, has the same PUPSIT text.⁴
3Multiple, redundant and gas filters.
Annex 1 treats filter configurations differently.¹
- Validated as a system. Where several filters together achieve sterility, the system is one sterilising unit, and every filter in it should pass integrity testing after use (8.91).
- Redundant backup. Where a second filter is present as a backup but the process is validated with one, the primary filter is tested after use. If it passes, the backup needs no post-use test. If it fails, the backup is tested, with an investigation and risk assessment of the primary failure (8.92).
- Close to the point of fill. Because sterile filtration carries potential additional risks compared with other sterilisation processes, a further sterile filtration as close to the point of fill as possible should be considered as part of the CCS (8.80).
- Gas and vent filters. Critical sterile gas and air vent filters, directly linked to product sterility, are tested after use with the filter still in its housing. Non-critical ones are checked at appropriate intervals (8.88 and 8.89).
The batch record should include the critical filtration parameters, such as the minimum time to filter a known volume and the pressure difference across the filter, and significant differences should be investigated (8.86). All non-conformities should be investigated before the batch is certified or released, including whether other batches are affected (3.2).¹
4What FDA expects.
US regulation sets the requirement in general terms. 21 CFR 211.113(b) requires written procedures to prevent microbiological contamination of sterile drugs, including validation of all aseptic and sterilisation processes.⁵
FDA’s 2004 aseptic processing guidance, which contains nonbinding recommendations, says integrity testing of the filter “can be performed prior to processing, and should be routinely performed post-use.” Testing after filtration matters “to detect any filter leaks or perforations that might have occurred during the filtration”. It names forward flow and bubble point tests, and says a production filter’s test specification should be consistent with the bacterial retention validation data.⁶
| Point | EU GMP Annex 1 (2022) | FDA guidance (2004) |
|---|---|---|
| Before use, after sterilisation | “Should be verified”, unless process constraints prevent it and a thorough risk assessment supports an alternative | “Can be performed prior to processing” |
| After use | Non-destructive test before the filter is removed from its housing | “Should be routinely performed post-use” |
| Tests named | Bubble point, diffusive flow, water intrusion, pressure hold | Forward flow, bubble point |
| Redundant filters | Defined post-use testing of primary and backup filters | “Use of redundant sterilizing filters should be considered in many cases” |
5How it runs in Seal.
In Seal, sterile filtration binds the approved filtration design, product-specific validation, the assembly and its sterilisation state, installation, PUPSIT, process conditions and post-use integrity into one evidence chain for each filter position. Where PUPSIT is not performed under an approved exceptional rationale, the risk assessment, alternative controls, scope and approval are recorded as such rather than shown as a passing test. Interventions and filter replacements divide the run into distinct filtration intervals, so a failed post-use test is bounded to the material and filled units it can affect, and the original result is kept while the investigation separates test failure from membrane failure.
For sterile injectables, the batch record is linked to environmental conditions, interventions, qualification, equipment state, materials, microbiology and release on one timeline, so a filter lot concern traces forward to each filling batch.
References
- 1EudraLex Volume 4, Annex 1: Manufacture of Sterile Medicinal Products, C(2022) 5938 final, sections 3.2, 8.79 to 8.93 and the glossary, European Commission, 22 August 2022. https:/
/ health. ec. europa. eu/ system/ files/ 2022- 08/ 20220825_ gmp- an1_ en_ 0. pdf (read 6 November 2026) - 2PI 058-1, Interpretation of the most important changes in the Annex 1 on the manufacture of sterile products (2022), sections 2.1 and 4.8, PIC/S, 28 October 2026. https:/
/ picscheme. org/ docview/ 11495 (read 6 November 2026) - 3Guidance on good manufacturing practice and good distribution practice: questions and answers, EU GMP guide annexes, Annex 1, European Medicines Agency. https:/
/ www. ema. europa. eu/ en/ human- regulatory- overview/ research- development/ compliance- research- development/ good- manufacturing- practice/ guidance- good- manufacturing- practice- good- distribution- practice- questions- answers (read 6 November 2026) - 4WHO good manufacturing practices for sterile pharmaceutical products, WHO Technical Report Series No. 1044, Annex 2, World Health Organization, 2022. https:/
/ cdn. who. int/ media/ docs/ default- source/ medicines/ norms- and- standards/ guidelines/ production/ trs1044- annex- 2- gmp- for- sterile- pharmaceutical- products. pdf (read 6 November 2026) - 521 CFR 211.113, Control of microbiological contamination, eCFR. https:/
/ www. ecfr. gov/ current/ title- 21/ chapter- I/ subchapter- C/ part- 211/ subpart- F/ section- 211. 113 (read 6 November 2026) - 6Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing, Current Good Manufacturing Practice, section IX.B, FDA, September 2004. https:/
/ www. fda. gov/ media/ 71026/ download (read 6 November 2026)
AQuestions and answers
Does FDA require PUPSIT?
No. The US regulation requires validation of all aseptic and sterilisation processes without naming filter tests, and FDA’s 2004 aseptic processing guidance says integrity testing can be performed before processing and should routinely be performed after use. Annex 1 goes further, expecting the test before use as well unless process constraints prevent it.
Is PUPSIT needed for every sterilising filter in the process?
The PIC/S, EMA and WHO interpretation says PUPSIT is required for filters used for final product sterilisation whenever feasible. For other sterilising filters, such as those controlling bioburden upstream, integrity testing is decided by risk and the contamination control strategy.
Which integrity tests can be used?
Annex 1 gives bubble point, diffusive flow, water intrusion and pressure hold as examples. The method should be validated and its results should correlate with the filter’s microbial retention established in validation, using a wetting fluid chosen from the filter manufacturer’s recommendation or the fluid being filtered.
Can a risk assessment justify skipping PUPSIT?
Only where process constraints that aren’t design flaws make it infeasible, such as very small volumes. The interpretation says manufacturers should first consider changing the process design, and that risk assessments should not be used inappropriately to justify leaving PUPSIT out.
What happens if the post-use integrity test fails?
It should be investigated before the batch is certified, as Annex 1 expects for any non-conformity. With a redundant filter, the backup is tested after use along with an investigation of the primary failure, and the backup can support the filtration step only if it had its own PUPSIT.