Sterile Filtration and PUPSIT – Questions and Answers at a Glance (Part 1)
Sterile filtration is a key step in the aseptic manufacture of pharmaceutical products. It serves to remove microbiological contamination and ensure the sterility of the end product. A closely related topic is PUPSIT (Pre-Use Post-Sterilization Integrity Testing), which checks the integrity of the filter after sterilization but before the actual filtration. PUPSIT is not an easy system to implement. Many companies face technical, spatial, and organizational challenges. Existing systems in particular often cannot be easily adapted for PUPSIT implementation. While some manufacturers have already successfully implemented PUPSIT, others are still experiencing difficulties or need to conduct a thorough risk analysis to justify an alternative approach or the complete avoidance of PUPSIT.
To understand the relevance and complexity of this topic, it is essential to consider the regulatory background. The revised Annex 1, which came into force in 2023, introduced significantly more de tailed and stringent requirements for the manufacture of sterile medicinal products.
Despite its importance from a regulatory perspective and in terms of patient safety, implementing PUPSIT in practice can be complex. Many systems in aseptic production environments were not developed in accordance with the new Annex 1 requirements. Significant redesigns, investments, and operational adjustments are often necessary to adapt these systems to enable reliable and validated PUPSIT procedures. In addition, space constraints in cleanrooms, the need for additional equipment, and changes to SOPs and training programs add to the burden.
We therefore collected questions about sterile filtration from seminar participants, discussed them with an expert, and compiled them. Read the first of three sets of questions below.
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Cologne, Germany13-15 October 2026
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1. What are the expectations regarding filter validation for a clinical Drug Product?
For clinical drug products you don't need to submit validation data in the dossier according to FDA Guidance "INDs for Phase 2 and Phase 3 Studies" However, that doesn't mean you don't have to do anything as other guidelines for manufacturing of sterile products (such as EU - Annex 1) do not distinguish between clinical and commercial production. If you do not have a full validation package you need to have data in the back of your hand which justify the sterilization process (sterile filtration) and drug product quality attributes are not compromised by filtration. This includes at mini mum potential adsorption or compatibility issues, leachables/ extractables and successful sterilization (removal of bacteria). So, as soon as you manufacture a sterile product on the line which shall be released for patient it also must include a contamination control strategy which includes convincing information about robust sterile filtration process. We were also challenged that an aseptic process is considered as a non-standard process. Therefore, validation data were requested according to EMA sterilization guideline (Sterilisation of the medicinal product, active substance, excipient and primary container - Scientific guideline) but a data based risk assessment was accepted at the end.
In summary, you should validate sterile filtration. However, I think it is acceptable when you have arguments to not perform a full validation but use other data (batch monitoring, lab data, historical data etc.) to justify drug product quality attributes and sterility.
2. How is the risk for PUPSIT assessed, that there must be 4 additional filters to test a redundant filtration setup? There is threefold risk to have a non-integral filter.
There is no requirement to perform a risk assessment if you per form PUPSIT as it fulfills the Annex 1 requirement. Nevertheless, if your line design requires a complex system to fulfill the PUPSIT requirement (e.g. additional manual manipulations etc) which might put the sterility at risk you could bring arguments why you believe that the contamination risk increases due to the design versus applying PUPSIT. Nevertheless, you also need to bring arguments why you can't simplify the process design (e.g. automisation, closed systems etc) to reduce additional risks. In any case, the failure of additional gas filters in the PUPSIT process is quite low if designed properly. I would be more worried about integrity of additional piping/tubing rather than integrity of additional filters, which by the way, you could also implement redundant if you are worried.
3. PUPSIT: "very small volume" what does that mean, is there any threshold? To specify: Is 1 liter a very small volume?
There is no threshold given. You would need to define it and hope that it will be accepted. To give you a number, what was running through my mind: If you would lose 20% of your full batch size due to PUPSIT this could be a starting point to argue. Or, if I don't have enough clinical material to support he project because I lose it for PUPSIT this could be an argument. However, in this case you could be challenged why not using "water" or any other wetting agent for PUPSIT, if the dilution effect to rinse out the wetting agent before start filtration is quite low due to the line design.
4. PUPSIT: what is the added value of a pre-use filter test if the filter integrity is tested after filtration of the product?
Before start sterile filtration, the filter may be damaged due to sterilization in place, transport, handling etc. In the theoretical case that there is a damage, the product solution could be blocking the damage during sterile filtration. In that case this blockage could mask the damage which would lead in the post-use integrity test to a false passed result. By performing PUPSIT such damages shall be detected before a masking effect happens.
5. PUPSIT: what is the risk that the PUPSIT test damages/has a negative impact on the filter?
PUPSIT has no risk of damaging the filter as long as non-destructive test methods such as bubble point test, diffusion flow test, water intrusion test or pressure decay tests are applied.
Recommendation
Vienna, Austria3-6 November 2026
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6. Does the acceptance of a risk assessment (RA) for filter tests finally depend on the mood of the Inspector/Auditor? What to do if the inspecting health authority does not accept the RA?
In general Annex 1 accepts also performing not PUPSIT if justified according to the points listed in the Annex 1 (chapter 8.87). The justifications you may provide may not always be accepted by HA. I wouldn't say it depends on the "mood" but on the perspective. In any case - not only for PUPSIT - if you are working with risk assessments this might not be acceptable for everybody. Hence, the goal should be to strive for compliance. Then there is no room for discussion/opinions.
7. Under what special conditions can a filter be used more than once or for longer than one working day?
According to Annex 1, chapter 8.95 allows longer usage of the filter "where campaign manufacture of a product has been appropriately justified in the CCS and validated, …". There are clear points followed in this chapter which must be fulfilled. The longer usage of the filter must be assessed for sterile filtration which includes performance of filtration and quality of the filtrate. Also, the validated duration must be controlled and not exceeded. Controls must also be implemented that filters are removed from usage if contaminated or defective. I would assume that a strong argumentation for such a process should be in place since chapter 8.94 clearly advises to discard the filter after a single batch and the same filter should not be used continuously for more than 1 working day. I also would strongly recommend to align with the filter supplier on usage time of the filter.
8. Annex 1, Chapter 8.87: What other cases ("process cons traints") in which the PUPSIT can be dispensed "very small volume of solution", are known in the industry and accepted by the authority?
Other process constraints than "very small volume of solution" could be from my point of view higher risks in potential microbiological contamination due to additional manipulation. Especially in non-closed systems, like cleanrooms where SIP of the filter cannot be/is not performed, additional interventions in grade A due to the PUPSIT process might increase the risk of contamination which from my point of view could be reason enough to justify pre-use pre-sterilization integrity test.
9. Annex 1, chap. 8.91 & 8.92: Which approach "redundant" (8.92) or "filtration system" (8.91) is preferred in the industry? Do authorities specifically ask about the validation approach when inspecting the validation?
So far, we have not yet been to strongly challenged with regard to filtration system. Nevertheless, I'm still convinced of a single sterile filtration system with business redundancy. Since I validate sterility with 1 filter, the redundant filter is considered as minimising business risk in case one of both fails integrity. Since both filters are of the same type, filter validation (bacterial retention test) is applicable also for both since the same process conditions apply for each filter in the line (redundant (downstream from primary filter) even less stress). Of course, adsorption and L&E must be considered cumulative. If I decide to use a redundant filter in the process this filter is also part of the process validation but it should be validated in that way that even one filter would be sufficient.
10. Differentiation / definition of sterile filtration, prefiltration, particle filtration, sterilization filtration
I think there should be a clear definition for each process what function the respective filter has. We have defined "sterile filtration" as the filter which assures sterility of the solution (sterilization step of the product), prefiltration is considered as low bioburden filtration (prior to sterile filtration or even further up in the process during compounding) and particle filtration is any filter which assures reducing particles as main function but has no microbiological reduction "responsibility". "Sterilization filtration" is for me the same as "Sterile filtration". Finally, there are different synonyms for that. It is important that everybody has the same understanding of what main function the respective filter has in the process.
In summary
Sterile filtration and PUPSIT are integral components of a robust aseptic production strategy. While the new EU GMP Annex 1 defines clear requirements, there is still room for scientifically sound, risk-based decisions - especially for clinical batches or small volumes. It is important that every decision is documented in a comprehensible manner, justified in relation to the process, and embedded in the contamination control strategy (CCS). This is the only way to ensure long-term regulatory acceptance and product safety. Read the next sets of questions in part 2 and 3.
About the Author
Matthias Schaar has been working for Novartis in Switzerland since 2007. He began building up his knowledge in microbiological quality assurance and quality control. Currently, his main focus is on supporting the validation team and routine manufacturing in the context of sterile filter validation and its application.
