Container closure integrity in the deep cold

Advanced modalities such as cell and gene therapies (CGT) represent a novel approach to treating diseases by addressing their root causes rather than just symptoms. These therapies often involve gene or cell manipulation to replace defective components, restore normal function, potentially provide cures for incurable conditions, or bolster the immune system to detect and destroy cancer cells.

Despite their potency, these therapies are highly sensitive and susceptible to loss of efficacy if not stored properly. To maintain their efficacy post-manufacturing, the therapies must be stored at ultra-low (around -80°C) for gene therapies to cryogenic temperatures (below -150°C) for cell therapies. Preserving biological materials at extremely low temperatures halts biochemical reactions, thereby maintaining their integrity, functionality and efficacy over extended periods, ensuring the reliability of advanced therapies.

However, storing these advanced therapies at extremely low temperatures poses significant challenges. Container closure integrity (CCI) may be at risk if the appropriate packaging components are not utilized, putting these costly therapies in jeopardy of degradation and efficacy loss. Typically, when maintained at temperatures between -20°C and -196°C, packaging materials can become brittle, potentially compromising the seal and allowing contaminants to breach.

To ensure CCI, it is crucial to select materials and engineer closures that can endure these conditions without losing integrity. Moreover, it is imperative to develop test methods that can detect leaks that are smaller than the product-specific maximum allowable leak limit (MALL). This article discusses the considerations necessary for conducting CCI studies for ultra-low and cryogenic applications.

Sample preparation considerations

CCS configuration

The CCI of a container closure system (CCS) and the experimental outcome of CCI testing are heavily dependent on the configuration of the CCS. Achieving optimal CCI relies on several factors: (1) the materials constituting the CCS must be break-resistant at low temperatures, (2) components of the CCS assembly should have similar coefficients of thermal expansion (CTE) to ensure synchronized thermal contraction/expansion during freezing/thawing, (3) the closure process (e.g., vial crimping parameters, plunger placement) should establish an effective seal against temperature-dependent pressure differentials and any asynchronous thermal contraction, and (4) secondary packaging and load patterns in the low-temperature equipment for freezing, storage, shipping and thawing must provide a temperature profile within the tolerance of the CCS design. It is crucial to optimize these parameters during product development and assemble the CCS samples in a manner representative of the product during CCI testing.

Fill content

Fill volume is another important factor to consider. Using a liquid fill of representative formulation and volume can mimic the heat transfer, volumetric expansion and headspace pressure changes of the CCS under use case conditions. However, it also introduces the risk of liquid or frozen droplets partially or completely blocking leak paths, leading to false negative leak data and false confidence in CCI performance. This trade-off must be considered when designing CCI studies, evaluating both scenarios and/or the worst case.

Freezing and thawing steps

To effectively evaluate leak risk in a CCS, it is recommended that testing replicates, as closely as possible to real-life scenarios, the product’s manufacturing process, which includes the freezing method, storage duration/condition, and thawing method.

For instance, in cell therapy, controlled rate freezing (CRF) is typically used since cells are more sensitive than traditional therapies. Depending on the heat transfer profile across the CCS configuration, processing samples using a CRF prior to storage may present a better, worse, or similar CCI case compared to direct storage at the terminal temperature. Additionally, the thawing process, whether passive under ambient conditions or assisted by a water bath or thawing device, must be considered. Slower freezing/thawing rate may prolong the exposure of a transient leak path but, conversely, allow more consistent and gentle thermal contraction, preventing leak paths. Regardless of the exact impact of a freezing/thawing method on the CCI, it is best to use a freezing and thawing process representative of the use case, to avoid over- or under-estimation of integrity risks inherent in the CCS.

Storage duration

The low-temperature storage duration of CCI samples is relevant to both the detectability of a static leak path and the progression or degradation of CCS integrity over time. When designing a CCI study, it is essential to select a storage duration long enough to detect leak sizes at and above the MALL but also representative of the failure mode(s) expected throughout the product’s shelf life.

Test method considerations

The selection of an appropriate method depends on the packaging type (e.g., flexible vs. rigid), study timeline (e.g., fast fail vs. long-term stability), failure mode of interest (e.g., leak during the freeze/thaw process vs. at final cold chain temperature vs. aging), and available resources (e.g., equipment, expertise, budget). USP <1207>,1 Annex 1,2 and USP <382>3 provide detailed regulatory references useful for method construction and comparison in integrity testing. This section discusses considerations regarding selecting and constructing a test method for integrity testing in low-temperature applications.

Headspace analysis vs. helium leak detection

Among the various methods used for evaluating CCI under ultra-low and cryogenic conditions, headspace analysis and tracer gas leak detection (helium leak detection) are notably two of the most common deterministic techniques. Their compatibility with extreme temperatures and general sensitivity to leak detection make them ideal choices.

Procedurally, headspace analysis measures the cumulative change in headspace gas composition after the sample undergoes the entire process of freezing, storage, shipping/transport and thawing. In contrast, helium leak detection measures the leak rate of helium through defects present at the terminal cold chain temperature. Consequently, headspace analysis is often more suitable for capturing transient leaks that materialize during dynamic freeze/thaw steps, whereas helium leak detection is more precise in determining the defect size present within the CCS assembly, especially the seal integrity throughout the product’s shelf life.

Headspace analysis, using techniques such as laser absorption spectroscopy, is a non-destructive, high-throughput method for testing intact vials, making it potentially suitable for in-process CCI testing of frozen products with headspace in advanced therapy manufacturing. On the other hand, helium leak detection can be performed in either a non-destructive or destructive manner, depending on the helium filling method and the measurement of headspace helium content. By weighing their respective advantages and limitations, both methods can be effectively utilized in tandem to ensure the robustness of the packaging system.

Controls

No matter the general method of choice, it is crucial to test, interpret and communicate inherent integrity relative to a point of reference (i.e., a control). This provides a checkpoint to determine whether a leak of interest can be detected and whether the measured value is the result of a leak.

Positive controls can be containers known to exhibit a consistent leak under experimental conditions, or ones modified with an intentional defect of a specific size anticipated to yield a detectable leak. Negative controls can be containers known to consistently show the absence of detectable leaks within the given conditions or ones with all possible leak paths intentionally sealed. Without appropriate positive control, a negative leak measurement might be due to instrument or process errors that prevent leak detection, or from the instrument’s insensitivity to detecting leaks of the size present in the sample. This could lead to incorrect conclusions about sample integrity. Similarly, without proper negative control, a positive measurement might be due to confounding factors like gas permeation through materials, rather than an actual leak path in the CCS. Establishing controls within a CCI testing method enhances data reliability and enables comparison of integrity data across different studies.

Gas permeation

Especially within CCS comprised of polymeric containers and/or closure components, gas permeation through construction materials is a common confounding factor potentially leading to elevated leak measurements in the absence of an actual defect or leak path. Although gas permeation rates are typically low at reduced temperatures, permeation concerns remain relevant in low-temperature applications. These extreme temperatures and freeze-thaw processes can potentially increase the intermolecular rearrangement of the packaging materials, and high-temperature steps are present on either side of the cold chain, such as fill-finish, cooling, warming and post-thaw holds.

To isolate the effect of actual leaks in CCI experiments, the impact of permeation can be accounted for using negative controls. It may also be mitigated by substituting the concerning component with a form-factor-equivalent counterpart made from low-permeability materials, or by modifying the component to incorporate an added permeation barrier while maintaining equivalent fit among components.

Headspace gas exchange scenario

According to the Ideal Gas Law, the total pressure inside a constant volume changes linearly with temperature. In ultra-low and cryogenic applications, this can result in pressure drops of up to 1.5-fold and 3-fold, respectively, upon freezing from ambient conditions or pressure rises during thawing to ambient conditions. This creates a pressure differential that challenges the integrity of the container closure by forcing gases through leak paths until pressure equilibrium is achieved.

Therefore, a test method’s capability to detect the leak of interest is influenced by factors such as the gases used to fill the sample headspace, the gas condition of the storage environment, whether the method monitors the egress of headspace gas or the ingress of environmental gas, the size of the potential defect, and the duration the sample is subjected to the low-temperature environment. Specifically, for headspace analysis and helium leak detection, a leak’s detectability in a particular experiment may depend on which parameter (e.g., volume fraction, partial pressure, or leak rate) is being measured by the instrument, as well as when the measurement occurs — whether when headspace pressure is above 1 atm or equilibrated to 1 atm.

Temperature-appropriate definition of acceptance criterion

In clinical applications, a primary function of integrity testing is assessing microbial contamination risk and inferring product sterility. Hence, defining a MALL relevant to microbial ingress is crucial, incorporating adjustments of standard MALLs (e.g., Kirsch limit) to low-temperature contexts. It is critical to note that the Kirsch limit, defined as a leak rate of 6E-6 mbar · L/s or an orifice size of 0.2 µm, was established by the Kirsch study4 at room temperature and is associated with a less than 10% probability of microbial ingress at 35°C.

On one hand, the leak rate through a given defect decreases at lower temperatures, making it essential to adjust the Kirsch limit to its defect-equivalent value for the low temperature of interest and to validate the detection limit of a given CCI test method using positive controls with Kirsch-equivalent defect sizes if the Kirsch limit is applied to define the MALL. On the other hand, a MALL originally established for higher temperatures corresponds to a lower probability of microbial contamination in ultra-low and cryogenic applications. This is due to the reduced microbial mobility and the solidification of the product formulation at such low temperatures, which necessitates careful consideration of the relevance and limitations of the Kirsch limit when assessing sterility risks for these low-temperature use cases.

CCI testing remains indispensable

CCI testing is a vital safeguard in pharmaceutical packaging, ensuring primary containers consistently provide complete barriers against environmental contamination, microbial ingress, and product loss throughout a drug product’s shelf life. By employing validated test methods with appropriate controls as described in this article, manufacturers can verify CCS performance under low-temperature conditions necessary for advanced therapies.

Robust CCI verification is essential not only for regulatory compliance but also for protecting product stability and patient safety. As advanced therapies become more complex, the rigor and precision of CCI testing will remain indispensable for delivering safe and effective medicines to patients. 

References

1. USP General Chapter, Package Integrity Evaluation—Sterile Products. (2017). United StatesPharmacopeia.
2. EU GMP Annex 1: Manufacture of Sterile Medicinal Products. (2022). European Commission.
3. USP General Chapter, Elastomeric Component Functional Suitability in Parenteral Product Packaging/Delivery Systems. (2020). United States Pharmacopeia.
4. Kirsch, L., et. al. (1997). Pharmaceutical container/closure integrity. II: The relationship between microbial ingress and helium leak rates in rubber-stoppered glass vials. PDA J Pharm Sci Technol. 51(5):195-202.