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. Risks and Considerations of Performing PUPSIT Inside vs. Outside RABS

EU GMP Annex 1 "Manufacture of Sterile Products" mandates that "the final sterilizing filtration should be as close as possible to the filling point." This principle aims to minimize the aseptic boundary, thereby reducing contamination risks. When positioning the final sterilizing-grade filter within RABS, careful evaluation is required especially for PUPSIT implement - due to two primary risks:

1) Performing PUPSIT requires a large number of operations on the filter assemblies including clamp adjustments, capsule filter rotation, vent valve switch-on, high-pressure purging (compressed air/fluid) waste bag drainage. Generally it is not advisable to have these operations in an A-level environments. Attempting them through glove ports compromises reliability and increases particulate generation.

2) The high pressure generated during the filter integrity test and venting process is a risk that cannot be ignored. It may cause non-sterile air or liquid to leak into the RABS system, thereby bringing particles and bioburdens to the surrounding environment of the sterile filling. The pressure for integrity testing may exceed 60psi, and all equipment exposed to this pressure must have the corresponding load-bearing capacity.

If attempt to perform PUPSIT in the redundant filtering system within the isolator, these challenges will become even more severe. Therefore, it is recommended to install a single filter in the isolator,  and to use PUPSIT in the form of product wetting, which can avoid the additional connection and introduction of water wetting, purging or rinsing.

Moving the filtration system upstream outside the isolator may incorporate new aseptic connections or aseptic transfers into the aseptic boundary, but it eliminates the risk of performing PUPSIT within the isolator. The isolator can avoid the inherent risks such as the pressure on the non-aseptic side of the internal filter rising to 60 psi or higher, or the exhaust from the non-aseptic side of the filter affecting the isolator. Another advantage of installing the filter outside the isolator is that when the PUPSIT test fails, only the filter assemblies need to be replaced without operating the isolator.

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.