Navigating The Pros And Cons In Annex 1’s PUPSIT Requirements

By Maik W. Jornitz, principal consultant, BioProcess Resources LLC

Sterilizing grade filtration has been used for decades with reliability and assurance and has become the more prevalent sterilization method with biologics. The reliability of this aseptic processing step increased with filter and membrane stability improvements, advanced integrity tests of filters, and essential process validation activities, which evaluate the performance of a particular sterilizing grade filter under process conditions utilizing the actual fluid.1, 2, 3 The process validation is not just limited to the retentivity performance of the filter but also the stability during steam and/or gamma sterilization, pressure pulsations, packaging, transportation, etc. Oxygen Package Device

Navigating The Pros And Cons In Annex 1’s PUPSIT Requirements

Yet, to routinely confirm that the sterilizing grade filter is flawless, nondestructive integrity tests like bubble point, diffusive flow, or pressure decay have been used successfully since the first regulatory requests in the mid-'70s. The mandatory integrity test is typically performed post-use.4

However, the Pre-use Post Sterilization Integrity Test (PUPSIT) of final sterilizing grade filters has been a topic of discussion in recent years. It has been part of the EU Annex 1, Manufacture of Sterile Medicinal Products, guideline since inception, but sporadic enforcement only started happening around 2007 and solidified with the newest version of Annex 1.5. The necessity of this test is debatable because its implementation and accompanying increase in process design and handling complexities cause concerns.6, 7

This article reviews the potential benefits and downfalls of PUPSIT, as well as the recommendation to utilize risk assessment evaluation instead of the generic enforcement of the test.

The integrity test of sterilizing grade filters must be done and is most commonly performed after the filtration process, or post-use. Some filter users test the integrity before the filtration process and before the filter is sterilized. Exceptionally rare are integrity tests pre-use/post-sterilization, as such tests would require downstream sterile filtrate side manipulation and therefore can be considered precarious as they are accompanied with higher process complexity and increased risks. Consequently, most regulatory authorities mandate post-use testing of the integrity of a sterilizing grade filter. Pre-use testing, without specifying whether pre- or post-sterilization, is listed as a proposal and is not even mentioned in some of the guidance, as the complexity is understood by these organizations.

However, European regulators started enforcing pre-use testing, PUPSIT, in 2007 because of a debatable Q&A document8 that speculatively states: “The filter-sterilisation process may be physically stressful for the filter. For example, high temperatures during the process may cause the filter to distort, potentially leading to fluid pathways that allow the passage of particles greater than 0.2 µm in size. The performance of a filter can improve with use, as particles begin to block individual pathways and remove larger pathways that smaller particles could successfully navigate. For these reasons, filters should be tested both before use but after sterilisation and again after use.”

Reviewing the speculative statement, the filtration expert community submitted their opinions,9, 10, 11 and multiple publications, presentations, and position papers4, 6, 7 pointed out the elevation of risks when using the pre-use/post-sterilization tests.

Furthermore, masking trials performed by the joint Parenteral Drug Association (PDA) and BioPhorum task force showed that a minute filter flaw, blocking by high foulant fluids, happened in extreme and rare cases.12 The probability of such an event is extremely low and, if appropriately risk-assessed, immaterial. A data mining task group of the same collaboration showed equivalent results.13

Regulatory authorities, like the industry at large, focus on patient safety. It has to be the utmost focus and it must be the EU regulatory authority’s preamble to enforce PUPSIT. With that said, there must be benefits to enforcing such tests, which are listed and discussed in this section.

The benefits of PUPSIT include:

This list is likely incomplete, but, at the very least, it includes the main benefits. Are these benefits only achievable by PUPSIT or are there other means to gain equally beneficial certainty? For example, the potential damage listed in the first one is prevented by appropriate validation of the heat or gamma irradiation sterilization process, as it is typically done in the industry. The industry is not letting chance rule whether the filter outlives a sterilization process. Such processes are well-qualified, documented, and often automated. However, if a filter is damaged by the sterilization cycle, such damage is catastrophic and would be detected by the post-use integrity test (nowadays, a forgotten filter performance assurance).

For the second on the list — identifying filter damage due to packaging, transportation, handling, and installation — it is not just the responsibility of the filter end user to make sure damage does not happen but also the filter supplier. Consequently, the packaging of filters and transportation of such undergo rigorous designs and testing to avoid any damage. Once received, the end user understands the handling of these filters as well as the installation. Frequent training sessions and appropriate SOPs are in place to make sure that damage does not occur. In addition, any filter damage due to the above-mentioned activities is discovered by visual inspections and the post-use integrity test.

The third benefit addresses the potential of minute flaw masking or blocking due to the particulate or foulant load within the fluid to be filtered, so the post-use test would not detect it. This possibility was debated over many years and required scientific evidence, which the industry brought forward and verified that, in rare and extreme conditions, a minute filter flaw can be masked.12 What was anecdotal became a good scientific basis to work from to determine whether masking of a minute flaw may be a possibility within the sterile filtration application at hand. Data are available and can be utilized as a solid foundation to understand the risks, very much as one can understand the risks of sterilization and/or handling. 

There are inherent risks when one installs and performs PUPSIT. All of us, including regulators, act under the preface: "You shall not test quality into a process, but design it into the process." PUPSIT is a test, and it creates complexity within a process. It was designed as a safeguard, but in many cases, it may do more harm than good.        

To perform PUPSIT, the sterilized downstream side of the filter will need to be redesigned to allow wetting fluids to be captured and keep the filtrate side under atmospheric pressure. This redesign effort will inevitably make the filtration downstream setup more complex, and examples of such have been presented.6, 7 Higher complexity within processes typically elevates risks, including risk of mishandling, mistakes in setup, more connections on the sterilized filtrate side, vent filters that require testing, no more over pressure line holds, which previously enabled the determination of potential filtrate side breach, and single-use closed systems being more convoluted with increasing risk of flaws — to name a few. Highly complex processes are normally avoided to eliminate the risk of errors. The complexity of a PUPSIT filtrate side opposes the desire of ease of use and therefore undeniably elevates risks. Additionally, any change in the design of an existing critical sterilizing grade filter downstream side must be classified as a post-approval change with all consequences attached.

PUPSIT requires sterilized filtrate side manipulations, a fact that is highly objectionable and not common practice. Since the filter membrane must be wetted to perform the integrity test, the downstream fluid path must change with additional valves, vent filters, connections, and other process needs. Once done, the filter undergoes the integrity test, which means the filtrate side must be drained and under atmospheric pressure. After the test, the filter needs to be dried if the wetting fluid is other than the product. That would mean the filter must reach bubble point to expel the wetting fluid from the membrane matrix, a rather stressful and potentially damaging process. If the wetting fluid is product, what will be done with the product and what does the prolonged residence time of the product within the membrane matrix during the test mean to the membrane or fluid?

The combination of the two – high filtrate side complexity and filtrate side manipulation – also must be reviewed for the elevated possibility of missing a flaw, a mistake, or a breach, resulting in a potential for microbial ingress. Such ingress is not detectable, which raises the risk. In sterilizing grade filtration, it has always been the commandment: “Do not touch the sterile filtrate side,” as this has to be the highest criticality of sterility. PUPSIT necessitates touching the filtrate side, which is logically a risk elevation.  

As of this moment, the EU has not addressed these risks.

The new Annex 15 states in paragraph 8.87 “The integrity of the sterilised filter assembly should be verified by integrity testing before use (pre-use post sterilisation integrity test or PUPSIT), to check for damage and loss of integrity caused by the filter preparation prior to use.” However, it also admits that PUPSIT “may not always be possible” and in such instances “an alternative approach may be taken providing that a thorough risk assessment has been performed.”

The suggestion of the use of risk assessments is encouraging.

The decades of sterilizing grade filtration for the sole purpose of post-use integrity testing show us that high-quality and patient-safe injectables can be supplied reliably. The hope is that the tripartite of regulators, industry, and suppliers come together to truly determine when PUPSIT is needed within a process and, conversely, when it does not create any patient safety benefit.

Maik W. Jornitz is a technical expert with over 35 years of experience in bioprocesses, especially sterilizing grade filtration and single-use technologies, including regulatory requirements, integrity testing, systems design, and optimization. Jornitz has published 11 books, 18 book chapters, and more than 100 scientific papers. He is a member of multiple PDA Task Forces, a former chair of the PDA Board of Directors, and the PDA’s Science Advisory Board.

Navigating The Pros And Cons In Annex 1’s PUPSIT Requirements

Air Separation Equipment Get the latest articles from Bioprocess Online delivered to your inbox.