Simulating PUPSIT or gas purges in bacterial challenge tests

For aseptic processing, devising an effective contamination control strategy (CCS) for final sterile filtration is a critical step for ensuring drugs are safe and effective. The CCS for final sterile filtration must identify, evaluate, and control any potential risks to product quality (1,2). One aspect of the risk assessment for final sterile filtration must consider the impact of performing pre-use, post-sterilization integrity testing (PUPSIT), which aims to identify sterile filter integrity test failures after the filter is sterilized and before the drug product is passed through the sterilizing-grade filter. Examples of some factors to consider in a PUPSIT risk assessment are shown in Table 1.

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

The bacterial retention (challenge) study is part of process‑specific sterile filter validation, showing that the filter can retain microorganisms under worst‑case conditions. This validation step may also include a simulated PUPSIT phase if relevant to the process.

In this article, we provide recommendations for manufacturers who wish to evaluate the risk of PUPSIT for bacterial penetration by simulating integrity testing in their process-specific bacterial challenge test. Our recommendations are based on end users who use either water or the product (when product-wet integrity test [PWIT] values are generated) as the wetting fluid. In addition, after final sterile filtration of the product, some end users apply a gas purge to maximize product recovery through the filter. This, too, should be covered by a risk assessment to mitigate the risk of bacterial penetration through the final filter.

How to perform and evaluate a PUPSIT risk assessment

PUPSIT with water as the wetting fluid

To perform a risk assessment for a potential breach to sterility when running PUPSIT, the fluid used to perform the integrity testing must be considered. For cases where water is used as the wetting fluid, Cytiva considers the risk of bacteria penetrating through the filter negligible, due to the very low, if any, bioburden that the filter would be exposed to. For example, consider the following scenario:

WFI: Water for Injection; LPM: liters per minute; EFA: Effective filtration area

If the following assumptions are made:

• Water for Injection (WFI) is used with a maximum bioburden level = 10 colony-forming units (CFU)/100 mL
• Total water flush volume = 80 L
• Maximum bioburden level in water flush = 8000 CFU

The sterilizing-grade filter would therefore be exposed to a maximum of < 1 CFU/cm².

Based on core validation data in which sterilizing-grade filters are demonstrated to retain ≥ 1.0 x 10⁷ CFU/cm² of Brevundimonas diminuta (B. diminuta) ATCC 19146 in a reference fluid (e.g., water), the risk of a sterilizing-grade filter being unable to retain < 1 CFU/cm² is negligible. In addition, the sterilizing-grade filters are qualified to withstand the elevated pressures the filters are subjected to during integrity testing.

The risk of bacterial penetration was evaluated using the wetting conditions described above with three PUPSIT simulations performed on five different sterilizing-grade types that had been previously exposed to a minimum B. diminuta load of 3.0 x 10³ CFU/cm². Even under such extreme conditions, no bacterial penetration was observed through any of the sterilizing-grade filters (3).

Consequently, Cytiva considers it unnecessary to simulate PUPSIT using water as the reference fluid as part of the process-specific bacterial retention testing.

However, if the WFI is known to contain any of the following waterborne bacteria, even at levels < 10 CFU/100 mL, then it is recommended that appropriate actions are implemented immediately to eliminate these bacteria from the WFI. Using WFI contaminated with these bacteria increases the risk of penetration through 0.2 µm sterilizing-grade filters (4-6), with potential contamination of the batch:

• Ralstonia pickettii
• Stenotrophomonas maltophila
• Sphingomonas paucimobilis
• Burkholderia cepacia

Therefore, a risk assessment should be performed to determine the potential for bacterial penetration through the filter following PUPSIT.

PUPSIT with product as the wetting fluid

For applications where the end user performs PUPSIT using the process fluid as the wetting agent, the sterilizing-grade filter would again only be exposed to a very small bioburden load, similar to the example given for WFI. Cytiva also does not consider this a significant risk for bacterial penetration through the sterilizing-grade filter. However, as part of the risk assessment, consideration should be given to:

• Properties of the process fluid (bactericidal vs non-bactericidal)
• Maximum allowable bioburden (presumably ≤ 10 CFU/100 mL)
• Qualitative assessment of prefiltration bioburden
• Volume of process fluid for filter wetting/integrity testing

For end users who want to evaluate the potential risk of bacterial penetration if using process fluid to perform PUPSIT, this can be incorporated into the design of the process-specific bacterial challenge study. For such a request, Cytiva recommends simulating PUPSIT at the start of the bacterial challenge study, which more closely simulates the full-scale processing conditions (including a more representative bioburden load on the sterilizing-grade filter). For such a request, generation PWIT values is a prerequisite to the bacterial challenge study to determine product-wet forward flow test pressures and/or minimum expected bubble point values.

Post-use sterile filter integrity testing using product as the wetting fluid

For post-use sterile filter integrity testing using the process fluid as the reference fluid, the product used to perform the post-use integrity test would be diverted to waste, and therefore, does not present a sterility risk to the final batch. The risk to batch sterility is only a concern if the process fluid used during integrity testing is directed to the final fill. While not a common occurrence, it could be simulated as part of the process-specific bacterial retention study to assess any risk of bacterial penetration. For such a request, generation of PWIT values is a prerequisite to the bacterial challenge study to determine product-wet forward flow test pressures and/or minimum expected bubble point values.

If required, the PWIT would be simulated at the end of the process-specific bacterial challenge test. However, careful consideration is needed to assess if simulating a post-use PWIT on a filter that has been challenged at ≥1.0 x 10⁷ CFU/cm² may add an excessive safety factor compared to what a process filter would be exposed to in the actual manufacturing process.

Application of a post-filtration gas purge to maximize product recovery

If a gas purge is applied at the end of the sterile filtration step to maximize product recovery, Cytiva recommends that this is simulated in the process-specific bacterial challenge study. Gas pressure would be applied at the end of the bacterial challenge to achieve the requested upstream pressure to the test filters. This approach results in a very high bacterial load on the filters (≥1.0 x 10⁷ CFU/cm² as required to validate the filter for bacterial retention). However, this bacterial load would not be representative of what the filter would be exposed to during processing. Therefore, it is also possible to perform this simulation as a separate test that uses a lower bacterial load on the filters (based on prefiltration bioburden load, batch size, and a safety factor [e.g., 2 logs]).

The approach taken to incorporate a gas purge in a process-specific bacterial challenge is dependent on the fluid properties towards the challenge organism (B. diminuta ATCC 19146).

If requested, a separate set of recovery membranes can be attached prior to the gas purge to specifically evaluate any impact of the gas purge on bacterial retention.

Assessing PUPSIT risk is crucial in final sterile filtration integrity testing

Demonstration of a robust CCS for final sterile filtration is a regulatory expectation. While there are many factors to consider for final sterile filtration, inclusion of a risk assessment for pre- and post-use integrity testing is a critical consideration. Assuming cGMP conditions are maintained, Cytiva considers the risk of bacterial penetration due to the increased pressures associated with PUPSIT to be very low. Similarly, if the process fluid is used to perform post-use integrity testing of the filter, the risk for contamination to the batch is also deemed negligible assuming the fluid used to perform post-use integrity testing is diverted to waste. For end users who apply a gas purge post-filtration to maximize product recovery, Cytiva recommends that they perform testing to evaluate the risk of bacterial penetration under the specific conditions used.

CY58561-23MAR26-AR