With the increasing utilization of ultracold frozen storage to maintain product potency, the question arises: how do we verify container closure integrity at these extremely low temperatures? The container closure integrity tests presented in USP <1207> are typically conducted at ambient conditions, as condensation can contribute to skewed results, limiting the integrity evaluation methods that are available for this application. This blog will aim to provide insight into a possible solution for assessing integrity of parenteral package systems at ultra low temperatures.
In a blog written by my colleague describing preventative vs. reactive CCI programs, capping studies to optimize sealing parameters to yield integrally assembled parenteral vial packages are offered as a potential preventative approach to ensuring CCI. However, even packages well-sealed at ambient conditions may experience leakage at temperatures below the glass transition temperature of the elastomeric stopper (Tg of many elastomeric formulations is around -65°C). When the elastomer is stored at frozen conditions, whether a result of packing on dry ice (-80°C) or even exposure to cryogenic temperatures (-180°C), the material loses its viscoelastic properties. These properties are responsible for the material’s ability to flow into slight variances in the vial land seal surface while resisting deformation under compressive forces to create an integral seal. At these ultra low temperatures, the elastomer becomes brittle and exhibits behaviors more similar to glass than rubber. This glass-like behavior, coupled with slight dimensional changes of the vial and aluminum crimp seal, can result in the formation of gaps and integrity breaches between the vial and land seal surface at these very low temperatures.
When the packages are removed from cold storage, the viscoelastic properties are regained once the elastomer is warmed above its Tg. Once the sealing properties are regained, the breaches and gaps present at cold storage are essentially resealed – the package is no longer leaking. Evaluation of these packages by an ambient CCI method would deem these packages integral, as the breaches are not present at the time of test. For this reason, integrity breaches at ultracold conditions may be considered “temporary”. How then are we able to detect these temporary leaks that only occur at ultra low temperatures?
Let’s consider the kinetics by taking, for example, a vial that has been capped at ambient conditions with atmospheric headspace. When the package is placed into cryogenic storage, the gas within the vial shrinks while the external environment remains at ambient pressure. This pressure differential drives the ingress of dense, cold gaseous environment into the package through the formed breaches. When the vial is taken out of storage, however, and the elastomer is thawed above its Tg, the material will regain its viscoelastic properties – causing any formed gaps to reseal and therefore trapping cold gas within the vial. As the package conditions to ambient, the trapped dense headspace warms and expands, resulting in an over pressure within the package.
Another example would be a vial processed with a nitrogen overlay headspace and stored in an ultracold air environment or on dry ice. Environmental ingress through the temporary breaches between the elastomer and vial land seal will displace the nitrogen within the package, resulting in increased headspace oxygen or CO2 levels upon package thaw.
By utilizing a headspace analysis method in these two scenarios, these packages can be evaluated for internal pressure or headspace oxygen/CO2 concentration post-removal from ultra cold storage. Any rise in pressure or increase in oxygen/CO2 levels, respectively, would be indicative of leakage. A non-destructive laser-based headspace analysis method is optimal as packages are spared and no disruption of gases occurs.
While the mechanism is simple to understand, it is important to note that the extent of the gas exchange is dependent on several variables, such as package headspace volume, the specific gases involved, temperature severity, pressure differentials, and the nature of the temporary breaches. As a result, the kinetics may be difficult to calculate. While theoretical calculations are useful, the importance of quality by design and the employment of positive controls is prevalent.
The question that remains unanswered is when will technology allow for integrity evaluation at temperature, allowing for real-time leakage assessment. While anecdotal indications suggest that these technologies are currently being explored, at temperature integrity evaluation is not yet mature nor reliable enough for employment. The headspace analysis studies proposed above are currently the best available option for this application. To aid completion of such studies, Whitehouse Labs houses Lighthouse Instruments laser-based headspace analyzers and an MVE cryogenic freezer capable of reaching -196°C.
Associate Director, Container Qualification & CCIT