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
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.
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 (Tg),
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.
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
- Afton Ready-to-Fill® sterile
vial 2 mL x 13 mm # 68000367
- 13mm Flip-Off® seal
- 13-mm Serum NovaPure® RP
S2-F451 4432/50 Gray #19700302
- S2-F451 D21-7S R B2-40 WESTAR®
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.
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.
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 Tg 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 Tg 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