Of the different test methods described, the assessment of the container-closure is arguably the most important because it
indicates whether the device is at risk from extraneous microbial contamination. Pharmaceutical containers constructed of
materials such as plastic and glass must be qualified and meet USP <661> Containers and <671> Containers-Permeation standards. The user will therefore need to undertake additional tests that
examine the physical seal of the closure in the vial, i.e., when the stopper is fully inserted and crimped, usually by of
an aluminium band. The choice to conduct a physical test or a microbial ingress test for this purpose is a matter of debate.
Some practitioners argue that the physical methods of measuring the system's integrity are preferred because they are more
reproducible, faster, less expensive, more reliable, and quantitative. Others argue that, as the objective is to ensure that
the product is safe from microbial contamination, a microbial test is the only true test. Some opt to undertake both physical
and microbial tests.
A review of industry practices suggests that failures occur with container-closure seals for a variety of reasons (8). These
failures include poor quality starting materials, an improper fit of the container-closure combination, the lack of sufficient
inspection as part of batch release, insufficient process monitoring or process control, the use of unreliable manual or visual
inspection techniques, the use of methods that produce subjective results, and the lack of proper process validation. The
latter point is addressed through the tests described below.
Physical tests include the dye test, vacuum testing, gas leakage determined using a bubble test, liquid leakage detected by
atomic absorption of a copper ion tracer solution, or a helium leak rate test (9). Of these, the helium leak test is one of
the most widely conducted; the objective is to detect leaks by monitoring changes in headspace gas composition or changes
in total headspace pressure. This test measures the rate of helium leak from the vial as well as the actual percentage of
helium that is filled within the vial. Mass spectrometry can be used to measure the rate of leakage. Mass spectrometry-based
leak detection is accomplished by measuring the amount of a tracer gas that escapes from the container-closure system. Tracer
egress is facilitated by a pressure difference across the container-closure barrier.
Alternative and novel test methods to assess container-closure integrity include the use of hygroscopic powder and near-infrared
(NIR) spectroscopy as a means of visualization. A second example is with airborne ultrasonic technology where a sound wave
is directed towards the container-closure and visualized through the creation of a high-resolution image. An alternative to
ultrasound is the use of a laser diode or the utilization of high- voltage technology. These new techniques have the advantage
of being non-destructive and they allow for a larger proportion of the batch to be tested, which increases the level of confidence
in the integrity of the seal. These techniques are also more accurate in allowing identification of small pinholes, micro
cracks and seal imperfections that cannot visually be seen.
With microbiological testing, a sterility test of the end product or a microbial ingress test can be considered. The sterility
test is unsuitable because the test will only detect viable microorganisms present at the time of the test and those that
are capable of growth within the culture media used. The microbial ingress test involves direct microbial challenge and is,
therefore, a more robust test. The objective is to detect microbial ingress based on 1) the probability that the challenge
microorganisms can find a container-closure leak, 2) the ability of the microorganisms to traverse the leak, and 3) the capability
of the microorganisms to grow in the internal container environment.
The microbial ingress test can be performed in different ways. One of the key criteria is the selection of the microorganisms.
It is more common to use two different microorganisms of different sizes and with different methods of motility. For example,
Brevundimonas diminuta, a very small bacterium, and Escherichia coli, a bacterium with a relatively powerful motility, are often used in combination (10). The complexity with the test relates
to achieving a sufficiently high microbial population.
To conduct a microbial challenge test, vials are filled with a microbiological growth medium before stoppering and crimping,
and are immersed in a 35 °C bath containing magnesium ion as well as 8 to 10 logs of viable bacterial cells for 24 hours.
The test units are then incubated at 35 °C for 7 or 14 days. Microbial ingress is detected by turbidity and plating on blood
The described tests, or a selection thereof, should ensure that the integrity is verified over the product's shelf-life, simulating
the stresses the product will be subjected to, including sterilization, handling, and storage conditions. The tests, therefore,
need to be made more rigorous in order to simulate "real life" events, for example by exposing test vials to stresses of temperature
and pressure conditions, which the vials are subjected to when being transported for distribution and sales. The level of
confidence is increased if three different batches are assessed. Another option is to assess vials as part of a stability
trial program, which includes a time point at the end of the shelf-life.