Sterile filtration is the final step in aseptic processing for removing microorganisms from drug products. The reliability of sterilizing-grade filters is therefore a critical quality attribute. Integrity testing of these filters has been a regulatory expectation for decades, with post-use integrity testing serving as confirmation that the filter performed as intended during the batch.
However, post-use testing cannot detect a filter that was already defective before filtration. Damage can occur during sterilization, handling, or assembly, and a compromised filter at the start of processing would result in an undetected sterility failure until the batch is already at risk.
To address this gap, the revised EU GMP Annex 1 requires Pre-Use Post-Sterilization Integrity Testing (PUPSIT), confirming filter integrity after sterilization but prior to product use.
Annex 1 makes PUPSIT the default expectation, allowing exceptions only when justified through a documented, science- and risk-based rationale. This has placed increased attention on filter integrity testing strategies within contamination control frameworks.
At the same time, PUPSIT remains one of the most debated requirements of Annex 1. Regulators emphasize its role in preventing product contamination, while manufacturers highlight concerns about test feasibility, potential introduction of contamination risk, and process complexity. Despite these challenges, regulators expect clear justification whenever PUPSIT is not implemented.
In this article, we will examine what PUPSIT is, the requirements outlined in Annex 1, the scientific and technical rationale, industry concerns, and practical considerations for implementation in aseptic manufacturing.
What is PUPSIT?
Pre-Use Post-Sterilization Integrity Testing (PUPSIT) is the verification of sterilizing-grade filters after they have undergone sterilization but before product filtration begins. The objective is to demonstrate that the filter remains intact and fully functional at the point of use.
The requirement originates from the concern that sterilization methods (commonly autoclaving or steam-in-place) or subsequent handling can cause damage to the filter. If this occurs, a filter may not retain microorganisms as intended, and relying solely on post-use integrity testing would detect the failure only after the batch has already been exposed.
PUPSIT therefore provides an additional assurance step, bridging the gap between sterilization and use. It is intended to:
- Confirm that sterilization has not compromised filter integrity.
- Prevent the use of a defective filter in critical aseptic processing.
- Strengthen the contamination control strategy (CCS) by ensuring filter reliability before batch contact.
In practice, PUPSIT is typically performed using validated physical integrity tests, such as bubble point, diffusive flow, or pressure hold methods, all of which are applied under aseptic conditions. These tests are non-destructive and confirm that the filter meets its specified integrity criteria.
Annex 1 Requirements for PUPSIT
EU GMP Annex 1, paragraph 8.87 defines the expectation for integrity testing of sterilizing-grade filters in aseptic processing. The clause establishes both mandatory requirements and conditional allowances, depending on process feasibility.
Integrity Testing Before and After Use
Annex 1 requires that the sterilized filter assembly be tested for integrity before use. This is PUPSIT. The regulator’s concern is that sterilization itself, usually steam-in-place or autoclaving, can introduce mechanical or thermal stress that damages the membrane. For example:
- Steam cycles can deform pore structures.
- Thermal expansion or contraction can stress filter seals.
- Handling during set-up can compromise filter housing connections.
If such damage occurs, a filter might already be non-functional before the product passes through it, making post-use testing irrelevant because the sterility breach would have already happened. PUPSIT closes this risk gap.
Annex 1 also requires a post-use integrity test. This is equally important: even if the filter is intact at the start, fouling or operational stress during the batch could damage it. Post-use testing proves that the filter maintained sterility assurance throughout the run. Inspectors will expect to see both test results in batch records as routine practice.
Validation and Correlation to Microbial Retention
The regulation explicitly states that the integrity test process must be validated and its results must correlate with the microbial retention capability of the filter. What does this mean in practice?
- The chosen physical test (bubble point, diffusive flow, water intrusion, or pressure hold) is not valuable on its own. Its acceptance criteria must be directly linked to microbial challenge data with Brevundimonas diminuta (0.22 µm retention).
- Manufacturers must hold validation reports, which are typically provided by filter suppliers, showing that when a filter passes integrity test limits, it can reliably remove microorganisms under process conditions.
- Inspectors often ask sites to present this validation package, not only the test results. Without this link, the test is not considered scientifically meaningful.
This requirement is often misunderstood. It is not enough to say, “we performed a bubble point test.” Companies must demonstrate that their bubble point limits are validated and tied to microbial retention.
When PUPSIT Is Not Possible
Annex 1 acknowledges that PUPSIT may not always be technically feasible. Examples include:
- Very small-volume products (e.g., orphan drugs, clinical batches) where pre-use testing would consume too much product or compromise sterility.
- Closed single-use systems that cannot be accessed for testing without breaching aseptic conditions.
- Special filtration set-ups where performing PUPSIT would introduce more risk than benefit (e.g., unnecessary aseptic manipulations).
In these cases, companies are not automatically excused. Instead, Annex 1 requires them to perform a thorough risk assessment and implement appropriate risk controls.
Risk Assessment Considerations
The guideline provides examples of what such a risk assessment should address:
- Filter sterilization process: Companies must show a deep understanding and control of sterilization cycles. This includes validated autoclave/SIP cycles, monitoring parameters (time, temperature, pressure), and evidence that the process does not routinely damage filters.
- Supply chain controls: If filters are sterilized externally or transported, risks of physical damage must be assessed. This covers packaging design, transport validation, and oversight of contract sterilization facilities.
- Process knowledge: The product itself can affect filter performance. For example:
- High particle burden can clog the filter, affecting test results.
- Certain product properties can interfere with integrity testing (e.g., viscosity, surface tension), making failures harder to interpret.
- Upstream pre-filtration or clarification steps reduce particle load, decreasing the likelihood of false results.
These elements demonstrate to inspectors that the company has not simply declared PUPSIT “not possible,” but has critically analyzed risks and designed controls to compensate.
SEE ALSO: Bioburden Testing in the Pharmaceutical Industry
Regulatory Intent
The underlying intent of paragraph 8.87 is to shift sterility assurance from being reactive (detecting failures after product exposure) to being proactive (verifying integrity before exposure).
Regulators expect PUPSIT to be the default practice, and any deviation must be justified with scientific evidence, validated data, and documented risk management.
Inspectors will expect to see:
- PUPSIT performed as routine unless a process-specific justification exists.
- Validation packages linking test values to microbial retention data.
- Risk assessments detailing why PUPSIT cannot be done, and what alternative controls are in place.
- Documentation of both pre-use (if feasible) and post-use test results in batch records.
In simple terms, Annex 1 paragraph 8.87 means: do PUPSIT unless you can prove with complex data and documented risk management that it is impossible, and even then, regulators will challenge your rationale.
Scientific and Technical Basis for PUPSIT
The scientific rationale for PUPSIT lies in the recognition that sterilizing-grade filters may lose integrity during sterilization or subsequent handling, before product use. Without a pre-use test, such failures would only be detected after the batch has been exposed, compromising sterility assurance.
Why Post-Use Testing Alone Is Not Sufficient
Post-use integrity testing has long been an established control step, but it has inherent limitations. The test confirms that the filter was intact at the end of processing, yet it cannot prevent the use of a filter that was already defective at the start.
- Detection after exposure: Post-use integrity testing confirms that the filter was intact at the end of processing but does not prevent the use of a defective filter at the start.
- Batch at risk: If the filter was already compromised before filtration, the entire batch may be contaminated before the failure is discovered.
- Regulatory concern: This gap in sterility assurance is why Annex 1 requires pre-use verification.
Potential Causes of Filter Damage Before Use
Annex 1 emphasizes PUPSIT because several factors can compromise filter integrity between sterilization and use. These risks include both process-related stresses and handling issues that may not be visible during set-up.
- Thermal stress during steam-in-place or autoclaving, leading to pore enlargement or rupture of the membrane.
- Mechanical stress from installation, tightening, or handling of filter housings.
- Chemical exposure to cleaning agents or sterilants that may degrade filter material.
- Improper wetting or air entrapment, which can create false readings or mask existing damage.
Integrity Test Methods Applied in PUPSIT
PUPSIT relies on non-destructive, validated physical integrity tests that are sensitive enough to detect damage or changes in pore structure:
- Bubble Point Test – measures the minimum pressure required to displace liquid from wetted pores.
- Diffusive Flow Test – quantifies the diffusion of gas through a wetted membrane under pressure.
- Pressure Hold Test – monitors pressure decay over time to detect leaks or defects.
Each method is linked to the manufacturer’s filter validation data, ensuring that passing results correlate with the ability to retain microorganisms (e.g., Brevundimonas diminuta challenge).
Role in Contamination Control
By providing assurance that the filter is intact at the moment of product use, PUPSIT adds a critical layer of control to the overall Contamination Control Strategy (CCS). It complements:
- Filter validation studies (microbial retention, compatibility, and robustness).
- Post-use integrity testing, which confirms ongoing performance.
- Environmental and process monitoring programs.
The technical justification for PUPSIT is therefore not only regulatory compliance but also alignment with the risk-based approach underpinning modern GMP.
Risk-Based Approaches for PUPSIT
Annex 1 establishes PUPSIT as the default regulatory expectation. However, it also recognizes that there may be limited cases where performing the test is either not technically possible or could introduce a higher risk of contamination.
This concession does not mean that companies are excused from compliance. Instead, manufacturers must provide a documented, science-based, and risk-driven justification explaining why PUPSIT cannot be implemented and how sterility assurance will still be guaranteed.
The quality of this justification determines whether it will withstand regulatory scrutiny. Inspectors look for three main elements: the thoroughness of the risk assessment, the strength of the supporting data, and the effectiveness of alternative controls integrated into the facility’s contamination control framework.
| Area | Examples to Cover | Why Important |
|---|---|---|
| Sterilization Method | SIP vs autoclave, cycle robustness | Prevents filter damage from heat/pressure |
| Equipment Design | Accessibility for PUPSIT | Determines feasibility |
| Historical Data | Filter failure trends | Supports likelihood analysis |
| Process Risks | Product viscosity, volume | Affects feasibility of testing |
Comprehensive Risk Assessment
A structured risk assessment is the starting point. This evaluation should identify the possible ways in which a filter might fail before use and assess the likelihood and impact of such failures. Considerations typically include:
- The sterilization method used (e.g., steam-in-place vs. autoclave) and its potential to stress or damage the filter
- Equipment design and whether it allows or restricts PUPSIT
- Historical filter performance data, including any known failure modes
- Process-specific risks, such as product volume or viscosity that may affect testing feasibility
Formal tools such as FMEA (Failure Mode and Effects Analysis) or fault tree analysis are strongly recommended. These provide structured, transparent, and traceable decision-making, which is exactly what inspectors expect to see.
Scientific Justification
Risk assessment alone is not enough. Regulators expect scientific evidence to support the conclusion that omitting PUPSIT does not compromise sterility assurance. This may include:
- Data from microbial retention validation studies
- Filter robustness studies showing tolerance to sterilization cycles
- Supplier-provided data on material stability and long-term performance
- Historical process records showing a very low rate of pre-use filter failures
Importantly, justifications must be specific to the process and product. Generic arguments such as “we have never observed failures in the past” are insufficient and are frequently challenged during inspections.
| Evidence Type | What It Proves |
|---|---|
| Microbial retention studies | Filter passes B. diminuta challenge |
| Robustness data | Tolerance to sterilization cycles |
| Supplier stability data | Material compatibility & long-term use |
| Process history | Very low pre-use failure rate |
Alternative Controls
Where PUPSIT cannot be performed, compensatory measures must be implemented to maintain sterility assurance. Examples include:
- Using redundant sterilizing filters in series to provide backup capacity
- Tightening control of sterilization cycles and monitoring parameters
- Performing enhanced in-process monitoring to detect potential failures earlier
- Implementing stricter acceptance criteria for post-use integrity testing
- Strengthening operator training and procedural safeguards for filter handling and assembly
The objective is to demonstrate that, even without PUPSIT, the risk of a non-integral filter being used is reduced to an acceptably low level.
Documentation and Traceability
Every decision must be fully documented. This includes:
- The rationale for not performing PUPSIT
- The supporting data used in the justification
- The risk controls applied in its place
- Evidence in batch records that these measures are consistently followed
Lack of documentation or vague justifications are treated as critical findings during inspections.
Regulatory Scrutiny
Authorities review these justifications closely. Weak or generic rationales are consistently rejected.
In contrast, well-documented, process-specific arguments, supported by robust validation data, are more likely to be accepted. Inspectors will expect companies to show not only why PUPSIT is not possible, but also how sterility assurance is otherwise guaranteed.
Integration with the Contamination Control Strategy (CCS)
Any deviation from PUPSIT must be addressed within the site’s Contamination Control Strategy. The CCS should demonstrate how sterility is ensured holistically, with filter integrity risks effectively controlled alongside other preventive measures, such as aseptic practices, environmental monitoring, and equipment design.
Practical Implications
In practice, the threshold for excluding PUPSIT is extremely high. Regulators expect companies to implement it wherever technically possible. As a result, many manufacturers are:
- Redesigning filtration systems to enable pre-use testing
- Investing in automated integrity testing equipment
- Modifying aseptic processes to reduce contamination risks during PUPSIT
These investments are often less burdensome than defending a justification for not performing PUPSIT, particularly under the scrutiny of EU inspectors.
Best Practices for Implementing Pre-Use Post Sterilization Integrity Testing
Implementing PUPSIT is not simply a matter of placing an integrity tester into the process. It requires a structured program that integrates procedures, validation, training, and documentation into the broader framework for contamination control.
Companies that approach PUPSIT effectively treat it as a lifecycle requirement of sterile filtration, rather than a last-minute compliance exercise.
1. Develop Clear SOPs
Written procedures should define exactly how PUPSIT is to be performed. SOPs must describe the test method (bubble point, diffusive flow, pressure hold, or water intrusion), the acceptance criteria, and the sequence of activities.
They should also include defined steps for handling failures, for example, how to replace the filter, how to assess batch impact, and how to document and investigate deviations. A well-written SOP minimizes operator variability and ensures consistent execution.
2. Validate Integrity Test Methods
Regulators require that integrity tests are scientifically meaningful. This means that the method must be validated against microbial challenge data, showing a direct correlation between test results and microbial retention.
For example, a passing bubble point must correspond to successful retention of Brevundimonas diminuta. In addition to initial validation, companies are expected to requalify test methods periodically to confirm continued reliability. Without this link, integrity testing is considered incomplete.
3. Integrate PUPSIT with Equipment Design
Many challenges with PUPSIT stem from equipment that was not originally designed to accommodate it. Wherever possible, filtration systems should be engineered or adapted so that PUPSIT can be carried out without breaching sterility.
Automated integrity testers with closed, sterile connections reduce the need for manual interventions and minimize the risk of contamination. In practice, this often means retrofitting existing skids or specifying PUPSIT-ready features in new equipment procurement.
4. Train and Qualify Personnel
Operators must be more than mechanically competent. Training should cover aseptic handling, test principles, result interpretation, and troubleshooting.
For example, distinguishing between a genuine filter failure and an invalid result caused by inadequate wetting is critical. Competence should be assessed regularly through observed practical tests or refresher training, and training records should be readily available for inspectors.
5. Manage Test Variability
False failures are a well-known risk in PUPSIT. To prevent unnecessary filter changes or batch delays, companies should define a structured troubleshooting approach. This could include repeating the test once under controlled conditions to confirm the result, checking wetting procedures, or verifying test parameters.
Clear decision trees within SOPs ensure that responses to test anomalies are consistent and scientifically justified.
6. Ensure Data Integrity
Integrity test data are GMP-critical and must comply with ALCOA++ principles. For automated systems, this means validated software with audit trails, secure data storage, and access controls.
For manual systems, contemporaneous recording, verification, and review are essential. Inspectors routinely request raw integrity test data, not just summarized reports, so data governance must be watertight.
SEE ALSO: Data Integrity and Data Governance in GMP
7. Link PUPSIT to the Contamination Control Strategy (CCS)
PUPSIT cannot be treated in isolation. Annex 1 requires that it is explicitly addressed within the site’s CCS, alongside filter validation, post-use testing, environmental monitoring, and aseptic practices.
The CCS should explain how PUPSIT strengthens sterility assurance and how risks are managed when it cannot be performed. Inspectors expect to see this link clearly documented and defendable.
Tips for Effective Implementation of Pre-Use Post Sterilization Integrity Testing
Practical experience from industry highlights several measures that can support the reliable execution of PUPSIT:
- Perform dry runs during validation: Testing system set-ups without product exposure helps identify practical difficulties before routine implementation.
- Standardize filter wetting procedures: Consistent wetting (e.g., type of wetting fluid, time allowed, method of application) reduces variability and minimizes false failures.
- Use automated integrity testers when possible: Automated systems reduce operator error, ensure consistent parameters, and provide secure electronic data.
- Define clear troubleshooting pathways: Establish in advance how to respond to test failures, including re-tests, investigation triggers, and criteria for filter replacement.
- Engage filter suppliers: Work with filter manufacturers for validation support, robustness data, and training. Supplier expertise can often resolve technical challenges.
- Document everything during set-up and execution: Detailed records not only support compliance but also make troubleshooting and investigations faster and more transparent.
- Regularly review failure trends: Monitoring the frequency and causes of PUPSIT failures helps identify training needs, equipment issues, or procedural gaps.
When applied consistently, these tips help companies reduce operational burden, strengthen inspection readiness, and maintain the focus on sterility assurance that Annex 1 requires.
Regulatory Guidelines for PUPSIT Implementation
EU GMP Annex 1
- PUPSIT is explicitly required in Annex 1: filters must be tested for integrity after sterilization and before use, and again after filtration to ensure sterility assurance at both time points.
- Any exceptions must be justified with a science- and risk-based rationale, fully documented and integrated into the site’s Contamination Control Strategy (CCS).
- All test results (pass or fail) must be recorded in batch documentation and adhere to ALCOA++ data integrity principles.
U.S. (FDA)
- U.S. cGMP (21 CFR 210 & 211) does not mandate PUPSIT, either before or after sterilization.
- However, the FDA’s Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing (2004) recommends integrity testing post-filtration and allows (but does not require) pre-use testing when appropriate.
- Common integrity tests include bubble point and forward flow; test specifications must align with bacterial retention validation data.
USP <1207> Container Closure Integrity (CCI)
- USP <1207> provides guidance (non-binding standards) for package/container integrity evaluation in sterile products.
- While not directly about filter testing, the principles align: integrity assurance requires lifecycle-integrated, validated, and deterministic test methods (e.g., pressure decay, laser headspace, high-voltage leak detection) over probabilistic methods.
- The focus is on selecting methods correlated to microbial integrity and integrating them throughout product development and production.
FAQ
What Products Require PUPSIT Most Critically?
PUPSIT is most relevant in aseptically manufactured parenteral products where sterile filtration is the final step before filling. This includes injectables, biologics, vaccines, and other sterile drug products that cannot undergo terminal sterilization.
Does PUPSIT Apply to Gas Filtration or Only Liquids?
Annex 1 focuses primarily on liquid sterile filtration, but the same principle of integrity verification may be applied to critical gas filters when used in aseptic environments, especially where gases contact sterile product or sterile pathways.
How Should Failed PUPSIT Results Be Investigated?
Failed pre-use integrity tests require immediate investigation. The filter must not be used, and the failure should be treated as a deviation. Root cause analysis should consider handling, sterilization parameters, filter wetting procedures, and test equipment performance.
Is There a Regulatory Requirement to Retest After a Failed PUPSIT?
There is no regulatory allowance for requalifying a failed filter. If the initial test result is invalid, a repeat test may be justified to confirm proper execution. However, if failure is confirmed, the filter must be rejected, and the event must be documented and investigated.
Can Single-Use Systems Comply With PUPSIT?
Single-use filtration systems can comply if they are designed with test ports and connections that allow integrity testing under aseptic conditions. If system design prevents this, a science- and risk-based justification must be documented in line with Annex 1.
Final Thoughts
PUPSIT has evolved from an industry debate into a regulatory requirement under EU GMP Annex 1. By mandating pre-use filter integrity testing, regulators aim to close the gap left by relying solely on post-use confirmation and ensure sterility assurance at the point of product contact. While its implementation can present operational and technical challenges, these do not diminish its regulatory significance.
For manufacturers, compliance requires more than installing integrity testers. It involves validated methods, robust SOPs, operator training, integration into the Contamination Control Strategy, and clear documentation in batch records.
Where PUPSIT is not technically feasible, a strong risk-based justification supported by scientific data must be in place, with regulators closely scrutinizing its adequacy.
Global guidelines show varying degrees of alignment, but the trend is moving toward harmonization, with Europe setting the benchmark. Companies operating internationally must therefore anticipate that regulators outside the EU may also begin to expect PUPSIT or equivalent measures.
PUPSIT is not just about meeting Annex 1 expectations, but a critical step in protecting patients, maintaining sterility assurance, and demonstrating that aseptic processes are controlled with the highest level of rigor.
