PUPSIT Risk Assessment

EU GMP Annex 1 contains a requirements for Pre-Use Post Sterilization Integrity Test (PUPSIT) of sterilizing-grade filters. The performing of PUPSIT significantly influences pharmaceutical production operations. When designing PUPSIT, the primary objectives are mitigating safety risks, minimizing product loss, and reducing operational disruptions. In this article we will explain and summarize the key attention points when performing PUSIT.  

1.Selection of Integrity Testing Methods for PUPSIT Systems

Bubble point and diffusion flow are the two most commonly used integrity testing methods for hydrophilic filters. The selection between these two methods must consider the Effective Filtration Area (EFA) of the filter and limitations of the test system.

Selection Criteria Based on EFA:

1) Small EFA Filters (e.g., <0.03m²): Only bubble point testing is feasible. Because Diffusion/forward flow values are too low for accurate detection by standard integrity testers.

2) Moderate EFA Filters (Typical range: 0.03–2.2m²): Both bubble point and diffusion flow methods are applicable. Diffusion flow testing is preferred because its test pressure is more stable and controllable. Upstream test pressure is at least 20% lower than bubble point pressure (typically 80% of minimum bubble point).

3) Large EFA Filters (e.g., >2.2m²): Only diffusion flow testing is viable. 

Given the pressure resistance limitations of single-use tubing and connectors in PUPSIT systems, diffusion flow testing is recommended for moderate EFA filters to enhances system safety and reliability. While for small EFA Filters which can only be integrity tested by bubble point, it is crucial to ensure that the system's pressure resistance is always higher than the maximum measured bubble point, or the upper limit of the upstream pressure set by the test program, when designing single-use PUPSIT system.

2. Selection of Wetting Liquid for PUPSIT

The choice of liquid for wetting the filter membrane significantly impacts both process design and operational efficiency before performing PUPSIT. Water, filtered process solution, or compatible buffers may all be considered as potential wetting liquid. This decision requires careful evaluation of multiple factors. In the following scenarios water or buffer solution can be clearly used as the wetting solution:

1) Pre-rinsing with water/buffer is required to minimize extractables (e.g., for sensitive biologics). Pre-rinse liquid will be disposed.

2) When process fluid contains surfactant properties. The surfactant will cause variability in integrity test values across different processing volumes, resulting in complicated validation of integrity testing methods using process fluid.

3) For undefined or evolving process formulations. Formula changes may alter surface tension characteristics of wetting fluid and wetting contact angle with membrane, leading to misjudgment of integrity test.

4) When process fluid is incompatible with test conditions or test gases (typically air or nitrogen). Water/buffer substitution is recommended.

3. Implementation of PUPSIT Inside Isolator/RABS Systems

One important point to consider is the location of the final sterilizing filtration unit with respect to barrier systems, particularly when employing single-use filter capsules and tubing assemblies. Process design requires a careful balancing of risks, and no single solution is right for all applications. A number of aspects can inform the decision. Annex 1: Manufacture of Sterile Medicinal Products of the European Union good manufacturing practices guidelines, states that the “final sterile filtration should be carried out as close as possible to the filling point” (8). This philosophy encourages minimization of the sterile boundary and, thus, minimization of risk of contamination. Care must be taken when considering placing the final sterilizing filtration inside an isolator or RABS enclosure, however, especially if executing PUPISIT, for two primary reasons:

• Execution of PUPISIT requires extensive manipulation of the filtration assembly, that could include movement of clamps, rotation of filter capsules, actuation of filter bleed valves, high pressurization, flush bag handling, and/or filter blow-down with compressed air. In general, addition of such operations in a Grade A environment is undesirable. Attempting to execute these manual processes through glove ports can present significant challenges to reliable performance.

• High pressures experienced during filter integrity testing and filter capsule venting (of the non-sterile side of a filter) present nonnegligible risks of causing non-sterile air or fluid to leak into the interior of the enclosure system which, in turn, could contribute particulates and bioburden into the environment immediately surrounding sterile filling steps. Everything exposed to the pressure of the integrity test, which could exceed 60 psi, must be designed to handle this force.

These challenges become more significant if attempting to perform PUPSIT on both filters in a redundant filtration train inside of an isolator. Therefore, locating the filters inside of the isolator probably directs the user to use a single filter (see “Need for Redundant Filtration of the Process Stream” under this section) and makes doing product-wet PUPSIT more practical, avoiding the additional connections and source for water wetting, blow-down or flushing. This may also be preferred when filtering potent compounds or products with higher biosafety levels, where locating the filter inside the isolator provides greater protection for operators.

Source: Parenteral Drug Association (PDA), Points to Consider for Implementation of Pre-Use Post-Sterilization Integrity Testing (PUPSIT), 2020, Section V, Figure 2, p. 9.

These risks must be weighed against the risks of moving the filter train outside the isolator and slightly upstream. Moving the train potentially incorporates a new aseptic connection into the sterile boundary or the use of a transfer port but eliminates the risks of performing PUPSIT inside the isolator. In a way, the isolator now protects the critical aseptic filling operation from the inherently risky process of pressurizing the nonsterile side of a filter to 60 psi or more, or bleeding the air out of the upstream side of the filter.  

An additional operational benefit of locating the filters outside the enclosure is the ability to change out the filter assembly in the event of a PUPSIT failure without needing to set up the isolator again.

4. Design Points for PUPSIT Flushing

To maintain sterility, wetting liquid and test gas will be discharged from the downstream (sterile side) of the system. For single-use systems, integrated waste bags can be connected downstream of the sterilizing-grade filter.

Downstream container must accommodate full wetting volume (calculated based on filter EFA and pore size). Additional safety margin (typically 20–30% extra volume) to account for potential re-wetting if initial wetting fails.

5. Backpressure for PUPSIT

To ensure reliable and reproducible integrity testing results, the downstream pressure of the filter must be maintained at atmospheric pressure or a constant known value. The integrity tester compensates by increasing the test pressure associated with the higher downstream pressure. If the pressure downstream of the valve under test differs significantly from the atmospheric pressure and is not compensated (or varies greatly in different tests), the test is invalid.

6. Temperature for PUPSIT

1) The temperature of the wetting liquid on the filter membrane directly affects integrity test values. Bubble point value typically decreases with rising temperature due to reduced surface tension. While diffusion/forward flow test value usually increases with higher temperatures. Integrity test criteria are valid only within a predefined temperature range. If the liquid temperature exceeds this range during testing, the results are invalid.

2) The temperature of the test gas in the tubing between the integrity tester and the filter under test must remain stable during the test.

Operation Guidance of PUPSIT

Operation guidance 1: Complete wetting of sterilizing-grade filter to be tested

Operation guidance 2: Integrity test of the first-level filter

Operation guidance 3: Integrity test of the second-level filter

Operation guidance 4: Sampling/Waste Discharge

Operation guidance 5: Sterile filtration of product

Once PUPSIT is implemented in production and system changes are involved, the potential impact of the integrity testing process, test results or inspection standards must be evaluated by professionals. The impact of items on the PUPSIT including but not limited to the following:

• Changes in formulation of the wetting liquid
• Variations in the temperature of the wetting liquid
• Changes in the test gas or temperature of the gas
• Changes in the downstream tubing of the filter undergoing integrity test (potential back pressure)
• Changes in the materials of single-use system supplier
• Changes in the configuration of the test program of the integrity tester
• Changes in the manufacturer of the sterilizing-grade filters
• Changes in the moisture or temperature of the air used for drying
• Changes in the volume of the downstream collection container

For over twenty years, Cobetter has been deeply engaged in the filtration industry. Backed by the rich experience of its single-use technology experts and filtration experts, we have summarized the highly applicable PUPSIT practical experience. From the perspective of risk control, we provide users with high-quality solutions for single-use PUPSIT systems and full-process training and verification services.

References：Points to Consider for Implementation of Pre-Use Post-Sterilization Integrity Testing (PUPSIT), Parenteral Drug Association, Inc. 2020

PDA's guidance provides a valuable framework for PUPSIT implementation. Building on these industry recommendations, Cobetter is well positioned to develop and deliver PUPSIT solutions tailored to customers' manufacturing needs.