Sterile barrier packaging must meet the requirements outlined in ISO 11607-1 Packaging for terminally sterilized medical devices Part 1: Requirements for materials, sterile barrier systems and packaging systems. The package must demonstrate strength, integrity, and microbial barrier properties to maintain the sterile barrier through the intended shelf life. However, it is left to the individual medical device manufacturers to determine how to meet the requirement and create acceptance criteria for their sterile barrier packaging validations.
Defining an integrity specification is simple. By default, any sterile barrier integrity breach renders the package non-sterile. Therefore, the acceptance criteria for the packaging validation are zero integrity failures.
Strength specifications are based on the materials, coatings, and user requirements selected for the package. Guidance on setting specifications can be found in ISO 16775 Packaging for terminally sterilized medical devices – Guidance on the application of ISO 11607-1 and ISO 11607-2 and ASTM has a guidance document in process.
However, when it comes to acceptance criteria for the microbial barrier of porous packages, there is no guidance on what value or result is acceptable to prevent microbial ingress.
The most common test method used to evaluate microbial barrier performance is ASTM F1608 -Standard Test Method for Microbial Ranking of Porous Packaging Materials (Exposure Chamber Method). The ASTM F1608 method was designed to compare and rank materials. To detect differences in barrier performance, a high flow rate was included to create ingress through materials not seen under real world conditions. Any spores that penetrate the material are captured on a filter and enumerated. The log reduction value (LRV) is calculated by comparing the log of the positive control (no material) to the log of the test result. As the positive control is ~1 x 106, the maximum LRV is ~6.
But what is the minimum LRV that is acceptable to prevent microbial ingress? Knowing the minimum value would simplify setting acceptance criteria by allowing for a universal minimum based on ingress data.
One source of published data on LRV values shows that medical grade papers typically demonstrate 1-3 LRV, whereas high-density polyethylene options normally fall between 4-6 LRV. While this data is useful in setting an expectation range for these materials, it is less helpful in setting acceptance criteria for a validation. Different coatings will change the LRV for any given material and different sterilization modalities can also influence the LRV values. Therefore, published data is specific to the material tested, not to acceptance criteria. LRV values can range from 1 to 6, which is useful in selecting a better barrier, but still doesn’t answer the question: What is an acceptable minimum value?
To answer this question, a custom study was designed and conducted by Nelson Labs to determine if microbial ingress would occur in a material with a low LRV under typical storage conditions.
Here’s how the study to explore the minimum log-reduction value was done.
The first challenge was finding a material with a consistent LRV, near the lower end of the LRV range for packaging materials. A melt blown fabric, previously used as a reference control for the test method, was selected, as there were years of LRV data showing this material has a consistent LRV of ~2.
The next challenge was finding a method for testing the material that was more representative of real-world conditions rather than the high flow rate in ASTM F1608. An aerosol challenge exposure method was chosen. This is a Nelson Labs custom method that showers the samples with a high load/uniform spread of Bacillus atrophaeus spores.
Test samples were created by placing sterilized material over an open petri dish with growth agar and securing the material with a custom lid (Figure 1). The excess material was trimmed around the lid with scissors. The lid acts as a clamp to hold the material tight above the agar surface, this enables the largest surface area to be exposed to the microbial aerosol and provides a means to handle and move the test sample without touching the material.
Figure 1. Test sample
The agar is designed to support growth of a variety of microbes, including the indicator organism (Bacillus atrophaeus) used in the study. If any organisms penetrate the material, they will fall to the agar and form colonies, which will be visible after incubation.
Thirty-six of these test samples were prepared and placed in the Nelson Labs aerosol chamber. The test samples were evenly spaced to ensure coverage over the entire chamber floor (Figure 2). This sample size was selected as a statistically significant number that would fit within the chamber, while still allowing sufficient space for the fallout controls and adequate airflow around the samples to ensure an even dispersion of the aerosol and verify a valid test.
Figure 2. Samples during exposure
The aerosol chamber exposure runs a liquid suspension of Bacillus atrophaeus spores through a nebulizer, creating an aerosol that is allowed to fall via gravity to the bottom of the chamber. This exposure is designed to simulate real-world fallout conditions. The number of organisms that fall on the chamber floor represent the expected fallout of multiple years of actual on-the-shelf storage. After exposure, the test samples were removed from the chamber, placed on trays (single layer), and the trays were placed in plastic bags. Samples were incubated at 30-35°C, then enumerated.
The strain of Bacillus atrophaeus used in the exposure forms an orange colony with a distinct morphology that allows it to be distinguished from typical environmental organisms present in the air. The organism is also a spore former, and resistant to desiccation, which allows it to survive for longer periods in the environment and aids in keeping a stable titer (concentration) in the test suspension. These characteristics make Bacillus atrophaeus an ideal organism for this application.
The results of the study.
The aerosol suspension was characterized for both concentration (number of spores) and droplet size. The concentration was measured in the liquid suspension prior to nebulization, from the mid chamber air in the middle of the exposure (30 minutes) and from fallout gauze swatches placed in the middle and corners of the chamber floor. The initial concentration of the spore suspension delivered to the chamber nebulizer was 1.2 x 1010 cfu/mL. The mid chamber air was measured at a concentration of 2.9 x 104 cfu/ft3. The average fallout was measured at ~470 cfu/cm2. These values are all within the established acceptance criteria for the test method.
The droplet size was measured at both 15 minutes and 45 minutes during the 1-hour exposure and both timepoints were averaged. The aerosol mean particle size (mps) was measured at 2.8 mps for this study. This size is within the acceptance criteria for an aerosol exposure.
The LRV of the material was measured per ASTM F1608, where five replicates of the reference material were tested with an average LRV of 1.8. For the 36 test samples exposed to the high microbial load in the aerosol challenge exposure, there were no Bacillus atropheaus colonies on the growth media on any of the 36 test samples after incubation.
Conclusions and next steps.
Demonstration of no visible penetration of the indicator organism after exposure to a high level of aerosolized spores indicates that the LRV of this material is sufficient to prevent microbial ingress under static conditions. Therefore, a minimum LRV of 1.8, should reliably prevent microbial contamination in a sterile barrier system during shelf storage.
While this study begins to answer the minimum LRV question, there are still other variables to consider. For example, what happens when a lower LRV material is tested? Can an LRV of 1, or less, still provide the same level of protection? What effect does non-static conditions like those experienced during transportation have on the barrier performance? Does the influence of time on materials affect the results?
Further work will be required to answer the minimum LRV question fully, but this study is a promising start.