In the early 1980s, Dr. Jerry Nelson, founder of Nelson Laboratories, began developing a whole-package aerosol-challenge test. The original intent of the test method was to simulate real-time shelf life using a high-challenge microbial-spore load. In essence, this involved bombarding the packages with the equivalent of years of natural microbial fallout using a hardy microorganism which had a high survival rate and unique growth pattern. Now, almost four decades later, the landscape of package testing and the types of sterile barrier systems has changed considerably, and the time has come to re-examine this test method and review its place in today’s package testing strategy.
Aerosol challenge testing continues to succeed at its original intent to rapidly deliver a high microbial load to packages, representing real-time shelf life. The equivalent of more than five years of microbial fallout is delivered during the exposure phase of the test. In the process the aerosol is evenly dispersed throughout the chamber and allowed to fall naturally to the bottom of the chamber. Empty chamber loads demonstrate that there is an even dispersion pattern across the chamber floor, though this can change during actual use if the chamber configuration is overloaded. Overloading can disrupt the dispersion pattern and can create shielding or dead zones, leaving areas with little or no challenge exposure.
The aerosol exposure method utilizes ambient fallout, which most closely represents real world exposure conditions. However, as there is no mechanism to force spores through defects or to move organisms around, the test method relies solely on distribution probability that the spore will happen to fall on the defect. Fortunately, this probability increases as the concentration of spores in the chamber increases, which is why there is a minimum fallout concentration specification that each run must meet.
The aerosol is characterized in multiple ways. The initial concentration of the spore suspension is measured before the exposure begins. All-glass impingers (AGIs) are used to sample the aerosol concentration in the middle of the chamber during exposure. Anderson samplers are used to determine the mean particle size of the aerosol droplets to ensure droplet size is small. The product level of the chamber is sampled with fallout gauze swatches to monitor the concentration of spores reaching the samples. Exposure acceptance is based on maximum mean particle size and minimum fallout concentration.
Real-world contamination relies on the probability of an organism opportunistically falling and entering a defect in the packaging, landing on the product and surviving. If a defect is shielded by the packaging, or the defect is in a part of the packaging which does not have direct contact with the product, or the organism doesn’t happen to land in the right place, then microbial contamination will not occur. Microbial tests are not designed to find defects and breaches. Therefore, the aerosol challenge exposure was not designed to find defects or integrity breaches in packaging.
The current preferred methods for microbial barrier performance are physical material tests. If the package is non-porous, a Gurley non-porous verification per ASTM F2981 is used to confirm the material meets the standard definition of non-porous. If the material is porous, a microbial ranking test per ASTM F1608 is used to calculate a log reduction value (LRV) based on the number of spores that penetrate the material at a high flow rate vs. the control. However, there are some sterile barrier systems that cannot be tested with material tests. In such cases a whole-package test, such as microbial aerosol exposure, may be the only alternative.
Another good use of the aerosol challenge test is to create a highly contaminated environment to challenge a drug-delivery system during simulated use. For example, a repeated-use dispenser could be actuated during the exposure phase, creating a worse case microbial challenge to determine whether the closure will prevent contamination over the use-life of the product.
Devices that do not have an accessory package pose a unique challenge for microbial testing. Closures that rely on a tortuous path to prevent microbial ingress often cannot be tested with vacuum/pressure-leak methods if the leak rate is too high. The aerosol challenge method could be a viable option for these types of sterile barrier systems, but there are some points to consider. First, the preferred test option is to pre-fill the system with a liquid growth media, pre-incubate to ensure the filling didn’t contaminate the system, and then run the exposure. Many vented closures will leak if growth media contacts the closure, causing contamination during incubation. Second, in order for the challenge organism to grow and propagate, an adequate headspace is required. Often the headspace is at the closure, meaning the area with the highest probability of penetration has the lowest chance for detection.
The other alternative to pre-fill is flushing the interior after exposure. However, flushing is typically not an effective extraction method; therefore, organisms (if they are present) may not be detected using this method, and extraction efficiency studies should be included to understand the confidence in the data. Flushing can also be prone to contamination if there is significant manipulation involved and flushing through a closure may extract organisms that were near the actual closure but had not penetrated the barrier.
Additionally, the fluid path often contains features that restrict flow to one direction, further complicating the filling or extraction process. It may be necessary to subdivide complex fluid-path systems into smaller components and test them separately to adequately challenge each closure in a complex device.
To answer the original question, Is aerosol challenge testing still relevant in today’s package testing strategy? I would say yes—in certain circumstances. The aerosol challenge exposure continues to perform as designed and achieves its goal of delivering a microbial load exceeding years of real-time storage in a short duration of time. Though the current industry approach for shelf-life determination is focused on physical testing after real-time aging, there are situations where the microbial aerosol challenge may be the only viable alternative for microbial-barrier determination. When the preferred options prove unsuitable, aerosol challenge can fill the gap.
