What is pre-use, post-sterilization integrity testing (PUPSIT)?

When manufacturing medicinal products, aseptic processing is an essential step to ensuring the safety, purity, and quality of a medicine. While aseptic processing includes both filtration and filling, this article will focus on the filtration step. In this critical step, sterilizing-grade filters are used, and these filters need to be integrity tested to confirm that they have performed as expected. Sterile filter integrity testing, whether it’s done pre-use, post-sterilization, and/or post-use, helps confirm the structural integrity of a filter and ultimately reduces the risk of contamination of the drug product. Regulatory bodies across the world continue to amend their guidelines to include higher aseptic processing standards. In the context of filtration, the latest guidance highlights a requirement to perform pre-use, post-sterilization integrity testing (PUPSIT) if possible.

What is PUPSIT and why is it important?

A critical step in aseptic processing is developing an effective contamination control strategy (CCS), which seeks to identify, evaluate, and control potential risks to the quality of the drug product. Sterile filtration and filter integrity testing are parts of the CCS. Manufacturers are encouraged, and sometimes required, to perform filter integrity tests at various points in the process, informed by the results of a risk assessment and the criticality of the filter in question.

As part of a CCS, PUPSIT is one way by which manufacturers help ensure the sterility of their aseptically-filled medicinal drug products. PUPSIT determines the integrity of the sterilizing-grade filter and assembly, including filter housing, support structure, and connections, after the assembly has been installed and sterilized but before it is used to filter product.

Why is PUPSIT performed?

PUPSIT is carried out to ensure that the filter has not been damaged during shipping, handling, installation, and sterilization prior to use. A damaged filter could result in a non-sterile product, which can ultimately affect drug safety.

How to perform PUPSIT filter testing

PUPSIT tests are filter integrity tests that are used to verify a sterilizing-grade filter has remained intact after sterilization but before product filtration. These tests include bubble point, forward flow, pressure hold, and water intrusion (for air/gas filters) tests.

What tests are performed during PUPSIT?

A bubble point test measures the minimum pressure required to force gas through a wetted membrane. A forward flow test measures gas flow across a wetted membrane at a set pressure. A pressure hold test checks if a system can maintain a set pressure over time, indicating absence of leaks. Water intrusion tests are used on hydrophobic air/gas filters, and this type of test checks resistance to water entering the pores under pressure. These methods are specifically referenced as acceptable integrity tests under Annex 1 guidelines.

Which filters require PUPSIT testing?

PUPSIT is required for all sterilizing-grade filters used in aseptic processing, especially the final sterilizing-grade filters used before filling or direct product contact. These filters typically have a pore size of 0.2/0.22 µm.

Manual and automated solutions for PUPSIT

Cytiva offers both a manual PUPSIT system as well as a variety of automated solutions. These are designed to control the risk of improper installation and reduce the time of implementation without adding significant risk. Our standard, manual single-use system allows manufacturers to confidently meet Annex 1 guidelines in critical downstream applications such as formulation and filling. On the other hand, our automated solutions can be used for a variety of molecules and concentrations, to deliver simple operation and process consistency. Both systems use the Palltronic Flowstar integrity test instruments, trusted for their accuracy and reliability.

Standard single-use manual PUPSIT system designs are available with single and redundant filtration options incorporating a range of sterilizing-grade filter sizes from 1 in. to 10 in. and a variety of filtration materials. Auxiliary manifolds such as inlet manifolds, flush bags, and sampling manifolds are also available as ready-to-order standard solutions. The flexibility of the manual solution enables one shadowboard to be suitable for all filter sizes and manifold configurations (Fig 1).

Fig 1. Cytiva manual drug product filtration system.

Our Cytiva drug product filtration system is an automated solution for performing PUPSIT and post-use integrity testing that is suitable for a wide range of batch sizes and optimized for low and high-concentration biologics (Fig 2). This further reduces the chance of operator error and minimizes the requirement for training on how to implement PUPSIT.

Automated solutions offer:

• Simple operation – Shadowboard design for easy, step-by-step installation, guided on the human-machine interface (HMI)
• Process consistency – Recipe driven with fully editable recipes to help ensure maximum consistency for batch runs
• Minimized risk of errors – Highly decreased human intervention to dramatically reduce processes most prone to error
• Automated filter integrity testing – Forward flow and bubble point procedures for PUPSIT and post-use integrity testing
• Batch recording and process trend analysis – Automatic recording of batch process elements that reduce risk on data integrity
• Enhanced product recovery – Filters oriented in the vertical position and air bag included to facilitate recovery of fluid in the downstream flow path
• Flexible filter design – Manufacturer agnostic

Fig 2. Cytiva drug product filtration system, an automated solution for performing PUPSIT.

PUPSIT risk assessment

Controlling risks associated with performing PUPSIT is critical. While PUPSIT is designed to detect damage that could occur to the filter during transportation, handling, or the sterilization process, if not performed properly, it may introduce a new risk of sterility breach. This includes but is not limited to:

1. Additional operator steps to perform PUPSIT (operator errors)
2. Increased number of components and connections (leaks)
3. Manual operation of PUPSIT

These risks may be further increased where complex filtration systems, such as a system including redundant filtration, are used.

Actions that may mitigate this risk include:

• carefully optimized system design
• control of the bioburden in the wetting fluid used to perform PUPSIT
• operator training to help ensure accurate execution of critical tasks
• the adoption of automated solutions to further control human risk factors

The addition of PUPSIT without robust process design and usability controls may increase the risk of adulteration of the drug product. This risk-based argument to support the absence of PUPSIT is common. This may even be accepted by some inspectors when it can be shown that there are no practical ways to overcome these challenges.

Benefits vs risks of PUPSIT testing

Table 1. Examples of factors to consider for PUPSIT risk assessment

Benefit	Risk
Detects filter damage caused during sterilization or handling.	Potentially introduces contamination risk during setup or handling of PUPSIT.
Prevents “filter flaw masking," when filter clogging masks, or hides, a pre existing defect.	Increases process complexity, especially in multi-filter or single-use systems.
Offers sterility assurance by verifying microbial retention capability right before product contact.	Presents potential feasibility issues for certain processes (e.g., very small-volume batches) due to design constraints.
Strengthens contamination control strategy.	Adds risk of operational disruptions and product loss if PUPSIT fails.
Meets regulatory compliance requirements of Annex 1.	Increases the burden of validation.

 

PUPSIT and EU GMP Annex 1 guidelines

Regulatory bodies across the world continue to update their aseptic processing guidelines to include stipulations for when to perform filter integrity testing. Testing can be done pre-use, post-use, and/or pre-use post-sterilization. Currently, United States (US) Food and Drug Administration (FDA) guidelines require post-use integrity testing to release a batch. However, European Union (EU) regulatory requirements mandate that manufacturers in the EU perform PUPSIT unless the end user can justify not doing it based on a thorough risk assessment.

PUPSIT updates to Annex 1

While not absolutely required, performing PUPSIT is expected in many countries. Recently, European regulators have amended the Guidelines for Good Manufacturing Practice (GMP) for Medicinal Products for Human and Veterinary Use, Annex 1: Manufacture of Sterile Medicinal Products (1) to include the option to perform a risk assessment if a manufacturer determines that PUPSIT is not possible. This risk assessment should consider the impact and/or risks of performing integrity testing, which could include damage to the filter setup and a potential breach to the sterile pathway through the process of performing PUPSIT.

In other words, Annex 1 requires manufacturers to perform integrity tests both pre-use post-sterilization, then again post-use, for filtration of liquid drug product. Different approaches to PUPSIT can be taken, including bubble point, diffusive flow (also known as forward flow), or pressure hold tests. In cases where integrity testing can’t be performed, Annex 1 requires the manufacturer perform a risk assessment.

The recent update to Annex 1 provides greater clarification on PUPSIT implementation and puts quality risk management and periodic review of the CCS at the heart of this guidance. You can view our e-book, "Manufacturing sterile drug product", to learn more about PUPSIT and its role in a CCS.

What is the global regulatory position on PUPSIT?

Even though regulatory bodies in both Europe and around the world are now stipulating PUPSIT should be performed, these regional guidelines and their adoption globally have not been consistent. The US FDA’s Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice (2) only says that pre-use testing can be performed, but it does not mandate it. Pharmaceutical Inspection Co-operation Scheme (PIC/S) adopted EU Annex 1 into its own guidelines (3). Because PIC/S is a global organization whose aim is to develop common GMP standards, this effectively means that standards around PUPSIT can impact manufacturers not just in Europe but globally. That said, regional regulatory bodies around the world have instated their own wording around when and how integrity testing should be performed (1-8) (Fig 3). Read more about our perspective on global regulatory positions on PUPSIT.

Fig 3. Regulatory positions on filter integrity testing and PUPSIT.

A holistic approach to risk during process validation

A risk assessment to investigate the potential for microbial penetration through the sterilizing-grade filter as a result of PUPSIT can be done in-house or it can be performed as part of a validation services package. While every biomanufacturer understands the importance of validating their filters, single-use system (SUS) components, and aseptic filtration process as a whole, not every organization can perform these tests on their own.

Fast Trak™ validation services cover a wide range of Cytiva sterilizing-grade filters and SUS components. Testing includes:

• Microbial retention testing
• Compatibility testing
• Adsorption testing
• Product-wet integrity testing (PWIT)
• Extractables and leachables (E&L) testing and consulting
• PUPSIT risk assessments
• Regulatory support
• Both standard and low-volume validation services

Conclusion: PUPSIT testing is integral to aseptic processing

PUPSIT provides assurance that a sterilizing-grade filter is intact and serves as a safeguard in the final filtration and filling stage of aseptic drug manufacturing. Regulatory standards continue to evolve, requiring more, not less, integrity testing; as such, it becomes increasingly important that manufacturers understand the benefits of PUPSIT, as well as how product and process can both mitigate and contribute to contamination risk. Integrity testing remains crucial to delivering safe and effective drugs to patients, and biomanufacturers must balance evolving regulatory expectations and practical implementation challenges of PUPSIT.

Frequently asked questions

What is PUPSIT?

Pre-use, post-sterilization integrity testing (PUPSIT) is used to confirm that the sterilizing-grade filter and assembly is intact, after the filtration setup has been installed and sterilized but before it is used to filter product.

Why should PUPSIT be performed?

PUPSIT confirms that the filter has not been damaged during sterilization, shipment, installation, or other pre-use steps, reducing filter failure and contamination risk.

What PUPSIT tests are typically performed?

Bubble point, forward flow/diffusion, and pressure hold tests are the main PUPSIT tests.

Is PUPSIT required by regulatory bodies?

Regulatory bodies across the world now require documented evidence of filter integrity for sterile filtration processes. Each has its own standards, including that the European Union (EU) now requires PUPSIT whereas the United States (US) Food and Drug Administration (FDA) stipulates post-use testing only.

What does Annex 1 say about PUPSIT?

In 2022 the EU issued updated Guidelines for Good Manufacturing Practice (GMP) for Medicinal Products for Human and Veterinary Use, Annex 1: Manufacture of Sterile Medicinal Products (1), requiring pre-use, post-sterilization integrity testing (PUPSIT). Specifically, PUPSIT is now mandatory for drugs marketed within the EU unless the manufacturer can justify not doing it based on a thorough risk assessment. Currently, US guidelines require only post-use integrity testing (2).

References

1. EudraLex - The Rules Governing Medicinal Products in the European Union, Volume 4, EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use, Annex 1 – Manufacture of Sterile Medicinal Products. European Commission (EC). August 2022. Accessed March 18, 2026. https://health.ec.europa.eu/document/download/e05af55b-38e9-42bf-8495-194bbf0b9262_en?filename=20220825_gmp-an1_en_0.pdf
2. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice. US Food and Drug Administration (FDA). September 2004. Accessed March 18, 2026 https://www.fda.gov/media/71026/download
3. Guide to Good Manufacturing Practice for Medicinal Products Annexes. Pharmaceutical Inspection Co-operation Scheme (PIC/S). August 25, 2023. Accessed March 18, 2026. https://picscheme.org/docview/8881
4. Regulation on Good Manufacturing Practices (GMP) for Medicinal Products. Ministry of Food and Drug Safety (MFDS). November 4, 2021. Accessed March 18, 2026. https://www.mfds.go.kr/eng/brd/m_18/view.do?seq=71530
5. Guidance on the Manufacture of Sterile Pharmaceutical Products by Aseptic Processing. Pharmaceuticals and Medical Devices Agency (PMDA). April 2011. Accessed March 18, 2026. https://www.pmda.go.jp/files/000153543.pdf
6. Good Manufacturing Practice for Drugs (2010 Revision). National Medical Products Administration (NMPA). March 1, 2011. Accessed March 18, 2026. https://english.nmpa.gov.cn/2019-07/25/c_390613.htm
7. Guidelines on Good Manufacturing Practice for Cell, Tissue and Gene Therapy Products. Health Sciences Authority (HSA). March 1, 2021. Accessed April 29, 2026. https://www.hsa.gov.sg/docs/default-source/hprg-ald/hsa_gmp_guidelines_for_ctgtp.pdf
8. Guidance Document for Risk Based Inspection of Drug Manufacturing Sites. Central Drugs Standard Control Organisation (CDSCO). March 27, 2024. Accessed April 29, 2026. https://cdsco.gov.in/opencms/resources/UploadCDSCOWeb/2018/UploadCircularFile/Guidance%20document%20for%20risk%20based%20inspection.pdf