PUPSIT In Cell & Gene Therapy: Risk Reduction Or Operational Complexity?

By Juergen Metzger, Pharma-Technology-Consulting LLC

Few topics introduced by Annex 1 have generated as much discussion as pre-use post-sterilization integrity testing (PUPSIT).

PUPSIT refers to the integrity testing of a sterilizing-grade filter after sterilization and before use for product filtration. The objective is to provide additional assurance that the filter remains intact and capable of performing its intended function during aseptic processing.

For some, PUPSIT represents a logical additional safeguard intended to strengthen contamination control. For others, it introduces another layer of operational complexity into already demanding aseptic manufacturing processes. The discussion itself is not new. What may be newer, however, is the environment in which the discussion is taking place.

Many traditional aseptic manufacturing concepts evolved around large-scale commercial production. Batch sizes were significant, manufacturing campaigns were repetitive, and process designs often remained relatively stable for years. The economics, operational assumptions, and risk profiles were fundamentally different from those encountered in many advanced therapy facilities today.

Cell and gene therapies rarely fit that model. Manufacturing campaigns may involve only a handful of containers. In some cases, a single patient effectively represents an entire batch. Product values can be extraordinary, available material may be extremely limited, and manufacturing timelines are often compressed. Flexibility frequently becomes just as important as efficiency.

This raises an interesting question: Does the operational value of PUPSIT remain identical across all manufacturing environments, or does the discussion become more nuanced when applied to advanced therapies?

From a regulatory perspective, Annex 1 clearly establishes the expectation that sterilizing-grade filters should be integrity tested before use unless an alternative approach can be scientifically justified. The discussion therefore is typically not whether filter integrity matters, but how PUPSIT can be implemented within a facility's overall contamination control strategy (CCS) while balancing product protection, operational practicality, and process robustness.

Nobody Is Questioning Filter Integrity

Before discussing complexity, it is important to clarify one point. The pharmaceutical industry broadly agrees on the importance of sterilizing-grade filtration. The sterilizing filter remains one of the most critical contamination control elements within aseptic manufacturing. Verifying filter integrity is not controversial. The discussion typically begins when evaluating how, when, and under what circumstances integrity testing should be performed.

The intent behind PUPSIT is understandable. If a sterilizing-grade filter represents a critical control point, verifying its integrity before product filtration provides additional assurance that the filter has not been compromised during installation, assembly, sterilization, transport, or preparation activities. Viewed from that perspective, the rationale is straightforward.

The challenge is not understanding the intent; it is understanding how the benefit integrates into increasingly specialized manufacturing environments.

Complexity Rarely Arrives Alone

One observation repeatedly encountered in pharmaceutical manufacturing is that risk reduction measures rarely arrive without introducing something else. Every additional process step brings its own operational consequences, including new procedures, training requirements, documentation activities, investigations, maintenance considerations, and additional opportunities for execution errors.

None of this automatically means the control strategy is inappropriate. It simply means that complexity itself becomes part of the risk equation. This is particularly relevant in advanced therapy manufacturing, where production processes are already highly controlled and often involve sophisticated contamination control strategies, single-use technologies, closed processing concepts, environmental monitoring programs, and extensive procedural controls.

Adding PUPSIT into these environments may be entirely appropriate. However, doing so is rarely as simple as inserting another box into a process flow diagram. Implementation frequently requires careful consideration of system design, operator interaction, process timing, equipment configuration, documentation requirements, and overall manufacturing practicality.

Automated Versus Manual PUPSIT

Another aspect that increasingly influences the PUPSIT discussion is the method of execution itself. Traditionally, many integrity tests have been performed using manually operated test equipment requiring operator interaction, system connections, documentation activities, and procedural execution steps.

Modern filling platforms increasingly offer integrated and automated PUPSIT solutions that minimize operator involvement and reduce the potential for human error. Automated execution may improve repeatability, simplify documentation, and support data integrity objectives while reducing the number of manual interventions required within the manufacturing process.

However, automation does not eliminate complexity entirely. Automated systems require validation, maintenance, software lifecycle management, and appropriate integration into the overall manufacturing platform. In highly automated isolator-based filling systems, an automated PUPSIT sequence may reduce operator interventions and improve execution consistency compared with manually performed testing activities. The resulting impact on overall process risk should be evaluated within the context of the overall manufacturing platform and CCS.

The discussion therefore is not simply manual versus automated execution, but rather which approach best supports the overall CCS while maintaining process robustness and operational practicality.

Small Batches Change The Discussion

One aspect that deserves particular attention is the relationship between process complexity and batch size. A commercial biologics process producing millions of doses annually distributes operational complexity across a very large manufacturing output. The economics are different when a manufacturing campaign produces only a handful of units. It’s not necessarily whether PUPSIT can be performed; rather, the focus should be on determining the most appropriate way to implement it within a manufacturing environment that must balance sterility assurance with operational flexibility and product supply demands.

The discussion becomes even more interesting when considering the consequences of an unexpected filter integrity test result. In a traditional commercial manufacturing environment, the impact of a failed integrity test may be substantial, but organizations often have access to additional inventory, established production schedules, and future manufacturing opportunities. Cell and gene therapy manufacturing frequently operates under conditions where material is limited, batch sizes are small, and manufacturing windows are tightly defined. Under these circumstances, even modest increases in process complexity can have meaningful operational consequences. The product itself may represent weeks or months of upstream processing before it ever reaches the fill/finish stage. Under those circumstances, any event that interrupts production carries implications extending well beyond operational efficiency alone. In a patient-specific manufacturing campaign, a failed integrity test may have implications extending far beyond batch disposition, potentially affecting manufacturing schedules, patient treatment timelines, and product availability.

Equally important is the distinction between identifying a filter integrity issue before product filtration and discovering it only after product processing has been completed. Particularly in patient-specific manufacturing, early identification of a compromised filter may prevent the loss of highly valuable material, manufacturing time, and potentially critical treatment opportunities.

This does not diminish the importance of filtration integrity. Rather, it highlights the reality that advanced therapy manufacturing often requires decisions to be evaluated through a different operational lens than traditional large-scale production.

Single-Use Technologies And Process Integration

Another factor worth considering is the extensive use of single-use technologies throughout modern cell and gene therapy manufacturing. Many advanced therapy facilities rely heavily on disposable assemblies, pre-sterilized fluid paths, sterile connectors, closed transfer systems, and highly integrated single-use processing platforms. These technologies have delivered significant benefits, including improved flexibility, reduced cleaning requirements, shorter turnaround times, and lower facility complexity. At the same time, they have fundamentally changed how many manufacturing processes are designed. PUPSIT must ultimately integrate into these increasingly sophisticated processing architectures.

The practical hurdle is often not the integrity test itself. The objective is designing a process that allows integrity testing to be performed efficiently while maintaining overall system simplicity, minimizing unnecessary manipulations, and preserving operational robustness.

As manufacturing systems become more advanced, the industry must continuously evaluate whether additional control measures integrate naturally into existing process designs or whether they introduce new layers of complexity that require separate management.

Where Should PUPSIT Be Performed?

Annex 1 establishes expectations regarding PUPSIT but provides limited direction regarding where the test should physically be performed. As a result, manufacturers are left to determine the most appropriate implementation strategy based on product characteristics, containment requirements, facility design, and the overall contamination control strategy.

The industry discussion often centers on whether PUPSIT should be performed. Yet an equally important practical question receives far less attention: where and how should it be executed?

Some organizations perform PUPSIT directly within the aseptic processing environment, while others use dedicated testing locations separated from the primary manufacturing operation. The chosen approach may vary significantly between facilities and applications. This may be particularly relevant in cell and gene therapy manufacturing, where closed processing technologies and single-use systems are frequently employed throughout the manufacturing process.

The decision is rarely driven by contamination control considerations alone. For highly potent compounds, ADCs, radiopharmaceuticals, viral vector products, and other products requiring elevated operator protection, containment requirements may become equally important as sterility assurance considerations. In such cases, performing PUPSIT within a contained environment may offer advantages by reducing operator exposure while maintaining the required level of process control. Depending on facility design and process architecture, some manufacturers may choose to perform PUPSIT within a dedicated process isolator upstream of the filling operation rather than within the filling isolator itself. This approach may help maintain containment while reducing additional interventions and activities within the primary filling environment.

For aseptic products where operator containment is not a primary concern, the assessment may look different. In such cases, performing PUPSIT within an isolator may not necessarily provide additional benefit and may introduce additional operational complexity. Depending on facility design and process architecture, manufacturers may choose to perform PUPSIT for conventional aseptic products within Grade B or Grade C environments, often supported by localized Grade A protection or RABS technologies while maintaining appropriate contamination control measures.

Since Annex 1 provides limited direction regarding the specific execution environment, manufacturers may arrive at different solutions depending on facility design, CCS, containment requirements, and overall process requirements. The selected approach should ultimately support both sterility assurance and operational practicality.

Ultimately, the discussion extends beyond simply performing PUPSIT. It also involves determining how the testing strategy integrates into the overall manufacturing process while balancing sterility assurance, operator safety, operational practicality, and process complexity. In that sense, the industry discussion may gradually shift from "Should PUPSIT be performed?" toward a more practical question: "What represents the most appropriate PUPSIT strategy for a specific manufacturing platform?"

The Human Factor Never Completely Disappears

One assumption occasionally encountered in discussions around advanced manufacturing technologies is that automation and closed systems gradually eliminate human influence. Reality is usually less absolute.

Operators still prepare systems, assemble process paths, execute testing procedures, investigate deviations, and respond to unexpected events. The human factor may be reduced, but it is rarely eliminated entirely. PUPSIT is no exception. Regardless of the technology platform being used, successful execution ultimately depends upon process understanding, procedural discipline, training, and operational consistency. Technology can reduce certain risks. It rarely eliminates the need for human judgment.

PUPSIT Within The CCS

Ultimately, PUPSIT should not be viewed as an isolated activity. Like any contamination control measure, its effectiveness should be evaluated through a quality risk management (QRM) approach and within the broader context of the facility's CCS. Factors such as process closure, single-use technology, automation level, barrier systems, operator interventions, product characteristics, and containment requirements all influence how PUPSIT contributes to overall sterility assurance.

The objective should not be compliance for its own sake, but a scientifically justified and operationally sustainable CCS.

The Bigger Question

Perhaps the most valuable discussion surrounding PUPSIT is not whether it should exist. The more interesting question may be how contamination control measures should evolve alongside the manufacturing technologies they are intended to support.

Cell and gene therapies continue to reshape many assumptions inherited from traditional pharmaceutical manufacturing. Batch sizes are smaller. Manufacturing platforms are more flexible. Product values are higher. Patient expectations are immediate.

As these therapies continue to mature, manufacturers will likely continue searching for the appropriate balance between contamination control, operational practicality, process complexity, and product availability. PUPSIT is simply one example of a broader industry discussion. How do we introduce additional safeguards without unintentionally introducing additional complexity?

The answer may not be identical for every manufacturing platform, every product, or every patient population. And perhaps that is precisely why the discussion remains so relevant.

The objective remains unchanged: protecting the patient.

For most applications, PUPSIT will likely remain an important element of sterility assurance. The more relevant industry discussion may therefore be less about whether PUPSIT should be performed and more about how it can be integrated into modern manufacturing platforms in a manner that remains scientifically justified, operationally practical, and aligned with the overall CCS.

About The Author:

Juergen M. Metzger is founder and principal of Pharma-Technology-Consulting, LLC, specializing in aseptic fill/finish, containment systems, and Annex 1 compliance. He has nearly 30 years of experience in the pharmaceutical manufacturing industry, with a focus on isolator technology, aseptic processing, and contamination control strategies. He began his career with Bosch Packaging Technology in 1999 and has held technical and leadership roles across engineering, product management, business development, and global project coordination. He has worked extensively on aseptic fill/finish systems worldwide, supporting the design, implementation, and optimization of complex manufacturing environments. Over the course of his career, he has also served in director-level and strategic advisory roles focused on new technologies, market development, turnkey projects, and aseptic manufacturing strategy across the Americas and international markets.