PACKAGING SYSTEMS - Container Closure Integrity in Cryogenic Storage Environments

INTRODUCTION

Just a few decades ago, cryogenics – the study of the production and behavior of materials at very low temperatures – was often regarded as being in the realm of sci-fi. In recent years, however, cryogenics has become firmly enmeshed in a number of applications across industries, including fuel production, power transmission, and food transportation. Cryogenics has also proven to be especially useful in the pharmaceutical industry, where cryogenic storage is being utilized to ensure the efficacy of advanced therapeutics, such as biologics and stem cell therapies.

Injectable biologics and cell-based therapies often require low-temperature storage in order to maintain the stability and integrity of the drug product. This presents a significant challenge for drug packaging systems and components: many packaging containers are made of glass, which can’t handle the challenges of cryogenic storage, nor can glass systems maintain seal integrity at such low temperatures. Issues like breaking and cracking when the vials are dropped or brought to room temperature can lead to cell death. In addition, many cryovials are not transparent, making visual inspection of the drug product contained within the vials difficult.

This is a good point to comment on some of the risks associated with the loss of container closure integrity (CCI) during cold and cryogenic shipment and storage. Clients initially reported pH change in their aqueous drug product caused by the ingress of carbon dioxide gas (CO₂) during cold chain shipment. Loss of CCI while using cryopreservation techniques may lead to the possibility of microbial ingress due to cold gas rushing into the vial under septic conditions of storage. Cryopreservation techniques could also lead to an overpressurization condition in the vial. Cold gas trapped in the vial when the elastomer regains its elasticity could become dangerously high after the vial returns to room temperature (remember the Ideal Gas Law? PV=nRT), which could lead to healthcare worker injury. Just picture the scenario whereby a nurse introduces a disposable syringe into a pressurized vial, and before he knows it, the plunger flies out, hitting his face due to the high pressure in the vial.

STUDY OVERVIEW

Driven by advances in biotechnology, pharmaceutical products, such as stem cells and biologics, are now more frequently stored and shipped in rubber-stoppered vials at cryogenic conditions (eg, -165°C/vapor phase liquid nitrogen). Commonly used butyl stoppers lose their elastic properties and sealability below their glass transition temperature (T_g), raising concerns about CCI failure.

A study was performed to better understand how rubber-stoppered glass and plastic drug vials function in cryogenic storage conditions and to provide guidance on the use of packaging components and the testing regimen used to qualify cold and cryogenic storage as part of a cold-chain strategy.

The study compared the CCI of two types of parenteral vials (glass and plastic), as well as two elastomeric formulations for the closure. Changes in headspace pressure and oxygen content were used to determine whether the empty vial samples maintained CCI. Headspace pressure and oxygen content measurements were made using a Lighthouse non-destructive headspace oxygen analyzer. Vials were sealed with FluroTec^®-laminated elastomeric closures under ambient conditions and tested for seal integrity to ensure the crimping process was successful prior to placement in a cryogenic storage chamber for 8 days.

METHODS

The study utilized sterilized Type 1A borosilicate glass and Daikyo Crystal Zenith^® cyclic olefin polymer vials sealed and capped with stoppers containing two chlorobutyl elastomer formulations (West 4432/50 Gray and D21-7S with fluoropolymer lamination on the closures’ plugs). A total of 170 sample vials and stoppers were used to determine CCI when stored at cryogenic temperatures. Four different vial/closure combinations were prepared using a Crimptronic CR-5000 USB device. All components were ready to use, and no further sterilization step was performed. Glass and elastomeric components were internally sourced, and vials were made to US GPI (Glass Producers Institute) standard. Materials used included the following:

– Daikyo Crystal Zenith vial 2 mL x 13 mm #19550057
– Afton Ready-to-Fill^® sterile vial 2 mL x 13 mm # 68000367
– 13mm Flip-Off^® seal #54131329
– 13-mm Serum NovaPure^® RP S2-F451 4432/50 Gray #19700302
– S2-F451 D21-7S R B2-40 WESTAR^® RS #19560253

All vials were empty with an initial air headspace. A total of 10 positive control vials of two container types were created by puncturing the stopper with a single 10-μm capillary tube. Two different vials and two different stoppers were used, resulting in four combinations. All containers were empty with an initial air headspace, and all vials were capped with Afton Ready-to-Fill sterilized seals 3767 (Table 1).

The packaging components were shipped to Lighthouse laboratory in Amsterdam and prepared at this location. Stopper compression and residual seal force (RSF) were optimized for the different vial/closure combinations (Table 2.)

After preparation, the samples were stored for 48 hours in a nitrogen-rich environment to confirm there was no loss in CCI of the samples before storage. If a leak was present, the nitrogen was exchanged with oxygen in the vial, causing oxygen levels to decrease.

All vials were measured with the Lighthouse FMS-Oxygen Headspace Analyzer, and passed this pre-leak test. In addition, RSF testing was performed to check the seal quality of all test containers. Packages with low RSF (< 6 lbf) were eliminated from the study. At this point, every group (CA, CC, GA, and GC) contained 40 samples.

Samples were stored for 8 days in a Linde Gas K24 storage container and were placed in the gas phase of the liquid nitrogen at a temperature of -165°C. Prior to final analysis all samples were allowed to return to room temperature.

RESULTS

Prior to analysis of the test samples, a set of known oxygen standards were measured to determine the performance of the oxygen analyzer. For each measurement time point, the standards of known oxygen levels were each measured five consecutive times. In addition, one sample of each vial type was measured 10 times to give more insight on the standard deviation of the sample vials. The mean measured headspace oxygen levels and the corresponding standard deviations are listed in Table 3.

The headspace oxygen levels of all package combinations were measured before and after storage at cryogenic conditions. Lower headspace oxygen levels would be indicative of a loss of CCI. These measurements are detailed in Figures 1 and 2.

All glass vials that showed loss of closure during the cold storage period were analyzed again for headspace oxygen 1 day after the vials were removed from the cryogenic environment. The measured headspace oxygen levels were similar to the first analysis after removal from the cold storage. This analysis was performed to confirm that the glass vials only leaked temporarily during the cold storage period and that the leak was re-sealed once the samples were brought back to room temperature.

CONCLUSION

The ability to rapidly and non-destructively measure headspace conditions in product samples enabled quantitative insight into the CCI of glass and plastic containers stored at cryogenic temperatures. In this study, headspace inspection of a total of 170 samples from four different vial/stopper configurations was performed following storage at cryogenic temperatures for 8 days. A loss of CCI would be indicated by decreased levels of headspace oxygen. It was found that none of the Crystal Zenith vials lost container closure integrity. However, the measurements performed on the glass vials showed strikingly different results.

Only one glass vial from vial/stopper combination GA did not lose container closure integrity during the cold storage period. All of the vials from the GC vial/stopper combination, as well as the positive controls vials, showed lowered headspace oxygen levels measured after the storage period. Clues to the remarkable difference in behavior between vials made of Crystal Zenith and ones made of glass may be found in observations made after the headspace measurements had been completed. When the glass vials were analyzed after the study, it was observed that the stoppers were easily removed and there was a popping sound due to the release of pressure from within the container.

This is likely because the rubber had been exposed to temperatures below its T_g and could no longer form a seal with the crown finish of the vial; the nitrogen gas was able to ingress into the vial, effectively displacing the oxygen. When the vial was returned to room temperature, the stopper went above the glass transition point enabling it to form a seal against the glass and effectively trapping the cold dense gas inside the vial.

When the Crystal Zenith vials were examined after the study, the stoppers were difficult to remove and appeared to be bound to the top of the Crystal Zenith vial. It is believed that this is an example of polymer entanglement, a phenomenon not unfamiliar to manufacturers of elastomeric components, in which polymers will cold flow into each other effectively gluing them together.

Because of this process, the rubber should remain sealed against the Crystal Zenith polymer even while the rubber has gone through its T_g twice. Consequently, nitrogen will not have an opportunity to ingress the Crystal Zenith vials during storage at cryogenic temperature, resulting in a maintenance of CCI.

As the demand for biologics and stem-based therapies expands, the need for innovative components that preserve the integrity of drugs in cryogenic environments will also continue to grow. By partnering with drug packaging and delivery systems providers that offer innovative component solutions for maintaining the efficacy and integrity of injectable drug products in cryogenic storage environments, pharmaceutical manufacturers can focus on manufacturing a safe, effective drug and meeting increased market demand. The end result is a strengthened relationship between both parties.

To view this issue and all back issues online, please visit www.drug-dev.com.

Eugene Polini, formerly of West Pharmaceutical Services, has a background in analytical chemistry, biology, and international business. Mr. Polini earned his BSc in Biology from Villanova University and his MBA in Health Care Administration from St. Joseph’s University.