The Complete Guide to Pre-Use, Post-Sterilization Integrity Testing (PUPSIT)

PUPSIT (Pre-Use Post Sterilization Integrity Testing) is a European regulatory requirement for sterile medicinal products. The rule first came out in the 1996 version of Annex 1 (26 years ago), but PUPSIT came into sharper focus when the European Medicines Agency (EMA) released the latest official version of Annex 1 in 2008. PUPSIT is a mandatory work step in final fill filtration (final filling) for biopharmaceutical manufacturers, except when proven unfeasible.

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The U.S. Food and Drug Administration (FDA) and Pharmaceutical Inspection Co-operation Scheme (PIC/S) are collaborating with the EMA on another Annex 1 revision. The upcoming revision maintains the requirement for PUPSIT with some minor modifications. The working group revising Annex 1 wants to create a standard that is as close as possible to a worldwide consensus, applicable to virtually every manufacturer in any country.

This overview will:

• Outline what PUPSIT is, where it came from, and why it matters
• Provide detailed information on how to perform PUPSIT
• Describe some of the risks and benefits of using PUPSIT
• Review current developments related to PUPSIT
• Provide resources to implement and improve PUPSIT

What is PUPSIT, and Why Does it Matter?

PUPSIT measures the integrity of the sterilizing filter and all the components—filter housing, connections, and support structures—after installation and sterilization, but before the drug manufacturers use it.

Since 1971, Annex 1 of the European Union’s Good Manufacturing Practice (GMP) offers guidance to help the pharmaceutical industry produce safer medicinal drugs. The latest revision, started in 2017, is all about Quality Risk Management (QRM). As with prior versions, it maintains the requirement for PUPSIT.

Regulatory bodies first introduced PUPSIT to address concerns that manufacturers would not detect microscopic flaws in the post-use test. Flaws may arise during the in-line steam sterilization process allowing contaminants to pass through during the filtration process. The clogging during filtration would result in a passed integrity post-use. This theoretical “filter flaw masking” could have serious consequences for downstream processing, significantly affecting the quality of the final product, and potentially putting patients at risk.

This theoretical 'filter flaw masking' could have serious consequences for downstream processing, significantly affecting the quality of the final product.

How to Perform PUPSIT

During PUPSIT, manufacturers must maintain sterility on the downstream side and avoid uncontrolled bioburden on the upstream side of the tested filter. That means installing additional components for a filtration setup:

• Sterilizing hydrophobic filters — Install these filters between the integrity tester and the filter to be tested to avoid increased bioburden. Install another on the downstream side of the filter to be tested to evacuate the test gas and avoid pressure increase.
• Sterile container, single-use bag, or hydrophilic filter — Implement these downstream to evacuate the wetting liquid.

The integrity of the filter can be determined in two different ways:

• Bubble point test — This test determines the differential pressure required to expel the wetting liquid from the largest pores.
• Diffusion | forward flow test — This test measures the gas flow rate through the porous structure at a defined differential pressure. It is the preferred test for single-use setups because it requires lower pressure.

Because each test has strengths and weaknesses, some drug manufacturers perform both types, although there is no regulatory requirement to do so.

Evaluating the Risks and Benefits of PUPSIT

The revised EU Annex 1 is focused on QRM, with increased emphasis on the patient. One important question is whether “flaw masking” is an actual risk that justifies the increased complexity of PUPSIT. Some experts believe the increased complexity and additional steps in the process increase the risk for breaking sterility, with little to no patient safety benefit.

The Risks

PUPSIT tests are complex, leaving more room to introduce errors that otherwise would not exist. Undetected errors could affect the efficacy and safety of the final product. The biggest risks are:

• Operator errors (opening or closing the wrong valves at the wrong time)
• Leaks (from additional components like T-pieces and connectors)

While rare, some additional risks that should be taken into consideration include:

• Non-integral vent and drain filters on the downstream side
• Non-integral draining bag

As opposed to pre-use, pre-sterilization integrity testing, and post-use integrity testing, the pre-use post-sterilization integrity testing requires:

• Wetting the filter to be tested under sterile conditions
• Evacuating the wetting liquid under sterile conditions
• Adding complex filtration systems with additional components and connections
• Intervening and aseptic handling from the operator

Any errors in these steps could introduce additional contaminants, negating any benefit of the PUPSIT process.

The Benefits

Those in favor of PUPSIT counter that it is necessary because current controls during filter manufacturing, transport, and installation are not sufficient to protect against minor flaws. Proponents also argue that those who say the system is too complex have failed to measure the risks properly, and there is bias in the current studies. They also note that we need more studies and more data on whether masking is occurring, and at what rate, before eliminating PUPSIT.  

Weighing Risks and Benefits

Manufacturers that do not need to comply with PUPSIT for regulatory reasons must decide whether to conduct PUPSIT by weighing the risks and benefits in an unbiased risk assessment. What is the risk of masking a minor flaw during filtration versus the risks generated by a more complex filtration setup?

The BPOG/PDA consortium Sterile Filtration Quality Risk Management Interest Group (SFQRM IG) has several publications and studies on the likelihood of the masking effect. These publications also outline failure modes and risks around PUPSIT. Even when PUPSIT is a regulatory requirement (e.g., for selling to European markets), the SFQRM IG only advocates for the implementation of PUPSIT if it reduces the risk of contamination and improves patient safety.

PUPSIT Updates and Changes

The EMA approved the current official version of Annex 1 in 1998 and updated it in 2008, but a lot has changed since then. A collaboration between the Parenteral Drug Association (PDA) and BioPhorum is evaluating what control measures—including but not limited to PUPSIT—are necessary to protect the patient and ensure sterility in the manufacturing process. PDA provides scientific guidance, laboratory support, and experts, while BioPhorum encourages its members to participate and contribute as thought leaders.

Many experts agree on the need for more objective, risk-based data to identify the benefits and risks of PUPSIT. In 2020, a draft version of the updated Annex 1 language came out, and if adopted, is likely to impact manufacturers.

The 2008 wording (adopted as the current official version) requires PUPSIT testing:
“113. The integrity of the sterilised filter should be verified before use and should be confirmed immediately after use by an appropriate method such as a bubble point, diffusive flow or pressure hold test.”

 The 2017 draft included additional wording following the statement above that said:
“It is recognised that for small batch sizes, this may not be possible; in these cases, an alternative approach may be taken as long as a formal risk assessment has been performed and compliance is achieved. There should be written integrity test methods, including acceptance criteria, failure investigation procedures, and justified conditions under which the filter integrity test can be repeated. Results of the integrity tests (including failed and repeated tests) should be included in the batch record.”

The opponents of PUPSIT have suggested that this addition could be a way for manufacturers to avoid more stringent requirements.

In the recently revised 2020 draft, the language changed again to include more detail about the materials used in PUPSIT, how to report results, and examples of compliant tests:
“The integrity of the sterilised filter assembly should be verified by integrity testing before use, to check for damage and loss of integrity caused by the filter preparation prior to use. A sterilising grade filter that is used to sterilise a fluid should be subject to a non-destructive integrity test post-use prior to removal of the filter from its housing. Test results should correlate to the microbial retention capability of the filter established during validation. Examples of tests that are used include bubble point, diffusive flow, water intrusion or pressure hold test. It is recognised that pre-use post sterilisation integrity testing (PUPSIT) may not always be possible after sterilisation due to process constraints (e.g. the filtration of very small volumes of solution). In these cases, an alternative approach may be taken providing that a thorough risk assessment has been performed and compliance is achieved by the implementation of appropriate controls to mitigate any risk of non-sterility.”

This language also acknowledges that smaller batches may not require PUPSIT, but drug manufacturers must still conduct a thorough risk assessment and report results.

Challengers to PUPSIT have also tried to outline the meaning of “should be” as opposed to “must be.” “Should” is understood to be a recommendation, a desirable goal, whereas the word “must” designates an obligation.

Regional translations of Annex 1 add to the confusion. For example, the French translation uses the equivalent of “must be” (doit être), which also reflects the uncompromising position of the French ANSM with regards to PUPSIT implementation.

Resources to Start or Improve PUPSIT

One of the main reasons manufacturers opt-out of PUPSIT, if it’s not required for the markets in which they sell, is the extensive resources and expertise required to do it right. Companies interested in starting PUPSIT to sell to European markets or mitigate contamination risks should partner with someone who understands the process and can provide expertise and guidance on what testing is appropriate based on your needs.