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Single-Use Container Closure Integrity I: Using Microbial Ingress

Test Method to Determine the Maximum Allowable Leakage Limit
(MALL)
Saeedeh Aliaskarisohi, Marc Hogreve, Carole Langlois, et al.

PDA Journal of Pharmaceutical Science and Technology 2019,

Access the most recent version at doi:10.5731/pdajpst.2018.009688
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Single-Use Container Closure Integrity I: Using a

Microbial Ingress Test Method to Determine the
Maximum Allowable Leakage Limit (MALL)
Saeedeh Aliaskarisohi1*, Marc Hogreve1, Carole Langlois1, Magali Barbaroux1, Jean-Marc Cappia1 and Marie-Christine Menier1
1
R&D, Sartorius Stedim Biotech GmbH

* Corresponding Author

ABSTRACT objective, this paper also presents the microbial ingress

An aerosol microbial ingress test was specifically test data obtained so far for two extreme conditions.
designed and used to create a predictive model in order Potential constraints such as vibration, shock,
to determine the maximum allowable leakage limit acceleration, liquid movement and pressure
(MALL) of single-use systems (SUS). The MALL is differentials observed during normal usage were
defined as the greatest leak size that does not pose any simulated using two extreme differential pressures,
risk on the product. The procedure involved taking test 0 mbar and 300 mbar. The estimated MALL for typical
samples of film material of the single-use bags. As test use-case conditions are 10 µm to 20 µm for storage
samples, an EVA multilayer film (300 μm thick) and a applications and 2 µm – 10 µm for shipping conditions.
PE multilayer film (400 μm thick) was cut into 50 mm The microbial integrity test method used in this article
patches. Artificial defects of 1 µm to 100 µm were was able to detect bacterial ingress down to 3 µm.
laser-drilled in the middle of each film patch. The patch
was assembled on a holder and sealed properly. The
test units were filled aseptically with culture media and Index Terms: Container closure integrity (CCI),
placed inside an aerosol chamber. Certain pressures microbial container closure integrity (mCCI),
were applied to the test unit to simulate the constraints maximum allowable leakage limit (MALL), single-use
that single-use systems may overcome under real-life system (SUS), microbial ingress testing
conditions. After an aerosolization cycle with spores of
Bacillus atrophaeus, a minimum concentration of 106 LAY ABSTRACT
CFU/cm2 was formed on the film surface. The test units As single-use systems (SUS) are increasingly
were incubated for 14 days at 30°C–35°C and visually expanding into all process steps of commercial
inspected for bacterial ingress. Thirty samples per manufacturing, integrity failure can significantly
defect size were tested. Logistic regression was used to impact drug safety, availability and costs.
indicate the maximum allowable leakage limit (MALL) Consequently, growing industry scrutiny on single-use
for a single-use system according to the required risk container closure integrity (SU CCI) is raising the need
level. With this method, the probability of the to develop good science behind reliable determination
occurrence or absence of ingress for a specific defect of liquid leakage and microbial ingress as well as the
size was reported according to the experimental data. appropriate physical integrity testing technologies. In
Besides physical parameters, such as pressure applied the current study, microbial ingress testing by
and film material, the effect of the probabilistic nature aerosolization method is used to determine the
of the microorganisms in determining the MALL is maximum allowable leakage limit (MALL) for single-
considered. use systems. To define the MALL, it is generally
assumed that a system or product will not show any
microbial ingress or leakage under a certain defect size.
This study revealed, however, that statistical analysis
Although finding an experimental model to predict the of the experimental data indicated the probability of
MALL for real-life process conditions was the ultimate MALL encountered at a certain defect size for each
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system. As a result, the method studied provides a more transportation by airfreight (8), (12), (22,23). This test
accurate way of predicting ingress, increasing safety can reflect “intended use” or “worst case” conditions.
down the line for drug manufacturers and patients According to the severity of the case and ambient
alike. conditions, various types of microorganisms have been
used: Brevundimonas diminuta, Escherichia coli and
Serratia marcescens (6)– (8), (12), (24). In the present
study Bacillus atrophaeus was utilized as a common
INTRODUCTION reference microorganism employed for microbial
An important aspect of pharmaceutical product quality ingress testing by aerosolization. The rationale behind
assurance is to demonstrate the integrity of the this selection is explained in details in material and
container closure system throughout the product life method.
cycle, as it is critical to ensure product sterility and Various studies have already reported on microtubes,
safety (1,2,3). Sterility tests performed on final microwires, pinholes and orifices that were positioned
products have certain limitations. Therefore, in different parts of the intact container, such as on
alternative controls are recommended to complement welds and folds, to representatively simulate physical
this method in order to confirm the integrity of the defects (5)– (8) (12)- (13) , (21)- (22).
container closure system as a component of the stability
protocol for sterile products (4). These alternative tests However, the authors have selected the laser-drilled
include physical, chemical or microbiological holes to mimic the type of failure mode that most
container closure integrity tests. There are valuable needed to be considered (25). Laser-drilled holes were
studies in which physical and microbiological integrity also a more practical and controllable option among all
tests were used to check the integrity of glass vials of the artificial leak types that currently exist (26).
(5,6,7,8), food cans (9,10) and even flexible bags like Furthermore, evaluating the defects of the welds and
retort pouches (11,12,13). Although these non- folds was beyond the scope of this paper.
destructive, deterministic physical tests are capable of
identifying the existence of defects, they are not able to Traditionally, microbial ingress testing is performed
directly measure the threshold defect size (12). with a liquid immersion method as described in USP
<1207> (27) that entails exposing sterile packaging to
Since one of the major concerns associated with the test microorganism in worst-case conditions
package integrity is identifying the MALL, validation (bacteria concentration, exposure time and full
tests are required to demonstrate the microbial integrity immersion as a contact method with the challenge
of packages up to a certain defect size. solution). However, it is expected that no flexible bulk
Two main microbial container closure integrity (mCCI) container will be subjected to liquid immersion during
test methods have been used so far: A microbial liquid storage and shipping. Therefore, microbial ingress
immersion test in which sterile culture-media-filled testing by aerosolization is better suited to SUS, as it is
containers are challenged by immersion in a liquid more representative of the real conditions of use for
microbial suspension (8), (14,15,16,17) and a microbial flexible packaging (12). In addition, Table I
test by aerosolization, which is the focus of the present summarizes each MALL obtained for different
paper. This latter method uses an airborne microbial products using liquid immersion and aerosol methods
suspension (nebulization) to challenge containers filled performed in former studies (8,22,23,28). A MALL of
with sterile growth-support media (12,13) 2 µm resulting from the integrity test performed by
(18,19,20,21,22). In both methods, incubation is Keller is a good example confirming that a microbial
performed at a growth-promoting temperature, and challenge test using aerosolization can be as stringent
cultures are subsequently checked for evidence of as a liquid immersion test.
microbial growth.

The mCCI test can be performed without pressurization Table I: MALLs obtained with mCCI testing by liquid
as well as with pressure or vacuum to simulate the immersion and aerosol methods in different studies.
constraints observed in different conditions, e.g.,
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Though in the study conducted by Pethe et al. (29), the As test samples, an EVA multilayer film (300 μm
MALL achieved by the aerosol method is much larger thick) and a PE multilayer film (400 μm thick), the
than that obtained in the liquid immersion method, the materials used in Flexboy®, Celsius® and Flexsafe®
integrity test method proposed in the Technical Report bags (Sartorius Stedim Biotech), were each cut into
27 (30) does not necessarily consider worst-case testing 50 mm diameter patches.
parameters. Therefore, to design an appropriate
microbial ingress test using representative conditions,
the purpose for which the container or bag will be used To simulate a pinhole defect, the patches were laser-
must be considered. drilled in the center with different micro-hole sizes
ranging from a nominal size of 1 μm to 100 µm. The
The objective of the present study is to describe an orifice leak size of laser–drilled holes was calibrated by
appropriate microbial testing method and its Lenox Laser using flow measurement (27). Laser
validation with the final goal of determining the drilling was done by Lenox Laser.
maximum allowable leakage limit (MALL) for single-
use systems, during their product life cycles, including A polypropylene patch holder secured the film patch on
storage, mixing and shipping after filling. each screw cap with two silicon gaskets on both sides
of the patch. To fill the test unit aseptically, a needleless
The single-use systems under study are made of connector was glued to the holder body. To permit air
ethylene vinyl acetate film (EVA) and polyethylene to circulate in the media, a silicon tube with a 0.2 µm
(PE) film, respectively. The microbial integrity test filter was connected to the bottom of the holder. Using
method used by the authors is the aerosol test. This a 0.2 µm filter also kept the test unit sterile during
method, the equipment and its qualification are integrity testing. The test unit is shown in Figure1.
presented in the following. In addition, this paper This test unit with a laser-drilled defect is
considers the different parameters that can affect representative of actual full-size bags with an artificial
microbial ingress by aerosol exposure, such as leak defect. Different aspects of this similarity and
size, material thickness, pressure inside and outside representativeness have been previously tested.
the packaging, process conditions, type of
microorganisms and concentration of the inoculum.
In addition, assembling the laser-drilled film patch on
a small holder instead of attaching it to the actual
package enables the bacteria ingress test to be
MATERIALS AND METHODS performed by aerosolization for more than 30 samples
in each run. This improves the statistical results and is
a key benefit of the method presented by authors.
Test Microorganisms

As challenge microorganisms, spores of Bacillus Figure 1: The test unit used to simulate a defect in a
atrophaeus (ATCC®9372TM) were used. This bacteria single-use system.
species was selected for two main reasons: First, it is
spore-forming, with a small spore size of 0.1 to 0.4 µm.
Second, B. atrophaeus spores are resistant to dry The fully assembled test unit, including the patch, was
conditions and not destroyed by aerosolization. In air-leak-tested using a Sartorius Sartocheck® 4 plus
addition, under its former name of B. subtilis, it is also Bag Tester at 300 mbar pressure, with a pressure
recommended to be used for bacteria ingress testing by stabilization time of 240 s and a test time of 240 s.
liquid immersion in the ISO15747 (31).
This test is required to check the integrity of the entire
assembly besides the defect itself and to confirm that
Test Unit Assembly the micro-hole is not blocked in the process.
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The test units were gamma-irradiated between 25 kGy representing a compromised sample that had a large
and 50 kGy. To check whether irradiation had an defect (3 mm). The compromised sample was exposed
impact on the integrity of the patches and the micro- to aerosol to confirm that the aerosolization process
hole behavior, 10% of the gamma-irradiated test units was able to cause bacterial growth inside the holder.
were air-leak-tested again using the Sartocheck® One non-exposed negative control was used to
device, applying the same testing parameters. challenge aseptic conditions and to visually compare
growth after incubation to evaluate the results. In
addition, one exposed negative control was used to
Aerosol Chamber and Aerosolization Cycle check the integrity of the test unit during the entire
process, from preparation to incubation.
The exposure chamber with a volume of approximately
1 m3 is made of stainless steel. It has two air inlet ports Test units, the compromised sample and exposed
and four stainless steel ducts for aerosol circulation. A negative controls aseptically filled with tryptic soy
support plate with 36 slots and plate holders is located broth, TSB (Merck), pressurized and were exposed to
inside the chamber for positioning the samples. aerosol, removed from the aerosolization chamber for
microbial ingress testing and after individual
A volume of spore suspension corresponding to a protection, incubated for 14 days at 30–35°C. The non-
minimum of 4×1011 spores was placed in the reservoir exposed negative control was left under a laminar flow
of the liquid nebulizer. This quantity used in equipment hood during aerosolization and incubated later with the
performance qualification is the minimum quantity other test units.
needed to attain 106 CFU/cm2 on the surface of the film
patch in each test unit. Finally, the presence or absence of growth was
determined by visual inspection and compared with the
To achieve a microbial concentration of a minimum of non-exposed negative control. The growth promotion
106 CFU/cm2 on the surface of each test unit, the test was conducted by spiking B. atrophaeus (10 to 100
following parameters were applied and controlled CFU) in the exposed negative control after the
during the aerosolization cycle. The pressure was incubation period to verify that the media had retained
3.5±0.5 bar inside the liquid nebulizer; the temperature their growth-promoting characteristics throughout the
was 20±5°C and the humidity was 50±10% inside the entire process.
aerosol chamber. To measure and control the
temperature and humidity, two probes were used to During its life cycle, each single-use system may be
connect the thermometer and hygrometer to the aerosol exposed to different pressures. Therefore, to qualify
chamber. and assess the effect of different pressure levels applied
to SUS, apart from atmospheric pressure, the film
The above-mentioned spore challenge of 106 CFU/cm2 patches were tested under different use-case pressure
on the film surface is selected to represent worst-case conditions representing storage, shipping and mixing.
conditions with regard to usual applications for single- This can be used later to establish a predictive model to
use systems. Even though these systems are typically determine the MALL under use-case conditions.
used in ISO 7 conditions, the ISO 8 surface However, testing has only been performed so far at
contamination specification was used and augmented atmospheric pressure and 300 mbar as the extreme
by additional 6 logs (32). Concentration of spores was points used to build the predictive model.
verified on two film patches, which were placed on an The sequence of the routine runs for these testing
empty holder and exposed to an aerosol cycle based on pressures are presented in figure 2.
the qualified method.
Figure 2: The sequences done for each run of
Microbial Ingress Procedure microbial ingress testing by aerosolization

Each routine test examined a maximum of 34 test units

with defined defect sizes and one positive control
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QUALIFICATION regression to calculate and predict the probability of

Qualification of the chamber and aerosolization having growth or no growth for a certain defect size
process, was performed according to the classic 3-step based on the experimental data.
plan of installation qualification (IQ), operational
qualification (OQ) and performance qualification (PQ).
Table II: Number of tested samples which have
RESULTS AND DISCUSSION shown bacterial ingress at certain defect size
compared to the total number of tested samples for
A total number of 30 patches per defect size were
PE and EVA films at atmospheric pressure
evaluated in different experimental runs using the
aerosol method of the microbial ingress test. However,
based on the prediction of having microbial ingress at
The results of microbial ingress testing by
a defect size of around 20 µm for atmospheric pressure,
aerosolization for PE and EVA films with a defect size
a fewer number of samples were tested for 2 µm. The
rationale behind the selection of number of test samples between 2 m to 100 m at atmospheric pressure are
is explained in the Appendix. For all runs, the negative reported in Table II.
controls (exposed and non-exposed) demonstrated that
no contamination occurred during preparation of the For EVA film at an applied pressure of 0 mbar, one
test units. The results obtained from compromised single incident of bacterial growth was reported at 40
samples show that test units with a defect allowed m, and for sample with defect size below 40 m, no
microbial growth. The growth promotion test growth was reported.
demonstrated that the growth-inducing characteristics For PE film at an applied pressure of 0 mbar, one single
of the media were maintained up to the end of the incident of bacterial growth was reported at 20 m, and
incubation period. The spore concentration on the film for sample with defect size below 20 m, no growth
patch used for verification of the concentration (film was reported.
patch without a defect and on an empty holder) was a
minimum of 106 CFU/cm2 for each run. The Figure 3 shows the probability of bacterial ingress as a
aerosolization parameters were compliant with the function of defect size based on statistical analysis of
cycle validated in performance qualification. Since all the experimental data at an applied pressure of 0 mbar
acceptance criteria were met, the results were for PE and EVA film.
conclusive. Logistic regression indicates that the probability of
bacterial ingress for PE film at a defect size of 20 µm
In most studies conducted in the field of integrity is 1.3%. For a defect size of 15 µm, even though no
testing, a specific defect size is commonly reported as bacterial ingress reported experimentally, this
the MALL for individual systems (6,13,22,24,33). Yet probability is around 0.89%.
in the case of microbial integrity testing, it is not
realistic to sharply define the transition between the Similar results for EVA film indicates that the
actual absence or occurrence of microbial ingress due probability of bacterial ingress at a defect size of 40 µm
to the variability of microorganisms in terms of their is 2.5%. For a defect size of 30 µm, even though no
size, shape, spore-forming capability, motility and bacterial ingress reported experimentally, this
environmental growth conditions. probability is around 1.3%.

In addition, there are experimental limitations in Therefore, determining the MALL for an SUS needs to
considering all of the physical parameters that can be statistically evaluated.
affect the microbial integrity of a SUS. Therefore, to
report a MALL for an SUS, it is more appropriate to
talk about the probability of the occurrence or absence
of ingress for a specific defect size. The binary
character of the results makes it easier to use logistic
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Figure 3: The bacterial growth or absence of growth is Figure 4: The bacterial growth or absence of growth is
shown with gray circles as a function of defect size for shown with gray circles as a function of defect size for
0 mbar of applied pressure. The probability of bacterial 300 mbar of applied pressure. The probability of
ingress expressed as a function of different defect sizes bacterial ingress expressed as a function of different
(solid curve) with 95% confidence interval of (0.5%, defect sizes (solid curve) with 95% confidence interval
3.4%) and (0.97%, 6.4%) for (a) PE and (b) EVA of (3.3%, 12.4%) and (11%, 24.2%) for (a) PE and (b)
respectively (dashed curves). EVA respectively (dashed curves).

Figure 5 shows the threshold defect size above which

According to prior studies, it is necessary to have liquid microbial ingress can occur as a function of applied
in the defect pathway in order for microbial growth to pressure. Threshold pressure required to initiate liquid
occur (33,34,30). The threshold pressure required to flow through a known defect is an inverse function of
initiate liquid flow through a microchannel is an the defect size. Therefore, the existing experimental
inverse function of the microchannel diameter size. data for PE film are fitted to the predictive model using
Performing a microbial integrity test at higher pressure non-linear fit. Based on the application-specific
provides the opportunity to search for a smaller MALL pressure conditions, MALL can be predicted with a
at the same time. Therefore, for 300mbar, the defect certain probability using this model.
sizes are chosen in a smaller range.
Correspondingly, the MALL given for low-pressure
Table III shows the results of the bacteria ingress test storage conditions is significantly higher, likely in the
for PE and EVA films with a defect size between 1 µm range of 10 µm to 20 µm, compared with the one
and 10 µm at an applied pressure of 300 mbar. associated with high-pressure shipping conditions, in
Statistical analysis of the above results is shown in the range of 2 µm to 10 µm. To improve the validity of
Figure 4. the model, testing with additional pressure data points
will be performed in a future study.
Table III: Number of tested samples showing bacterial
ingress at a certain defect size compared to the total
number of tested samples for PE and EVA films at 300
mbar. Figure 5: Threshold defect size as a function of
pressure applied. The dashed line is the fitted line to
the PE film experimental data according to the
Two incidents of positive growth out of the thirty predictive model. Two ovals show the estimated
samples with 2µm and nine incidents of positive position of defect size for certain applications.
growth out of the thirty samples with 3 µm defects are
reported for PE and EVA films respectively. Similar to
the evaluation of the test results in atmospheric SUMMARY AND CONCLUSION
pressure, statistical analysis shows that the probability
of bacterial ingress for a defect size of 2 µm of PE film
is 6.5%. The same statistical analysis of EVA film
This paper examines the effect that defect size, as
shows that the probability of bacterial ingress for a
represented by laser-drilled holes, and pressure applied
defect size of 3 µm of EVA film is 16.7%.
have on microbial ingress. A novel integrity test system
Although the probability of bacterial ingress for 1 µm
designed to be representative of a single-use systems
of PE film and 2µm of EVA film is around 4.2% and
made from PE and EVA multilayer films.
12.7% respectively.
These test systems were challenged by microbial
aerosols to determine their maximum allowable
leakage limit (MALL).
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The experimental results obtained for PE and EVA interests.

films with various defect sizes at different pressures
applied are provided and discussed. The number of test
samples showing bacterial growth, i.e., ingress, was
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Conflict of Interest Declaration

The authors declare that they have no competing
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AUTHORS
27. USP. 1207 Packaging Integrity Evaluation-Sterile
First Author – Dr. Saeedeh Aliaskarisohi, Scientist, Sartorius
Product.; United States Pharmacopia (USP), 2019. Stedim Biotech GmbH, Goettingen, Germany.
saeedeh.aliaskarisohi@sartorius.
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Package Sterility Maintenance, Dissertation; Food
Second Author – Marc Hogreve, Senior Engineer Integrity
Science and Technology: Blacksburg, Virginia, 1998. Testing Solutions, Sartorius Stedim Biotech, Goettingen,
Germany, [email protected]
29. Pethe, V., et al. Integrity Testing of Flexible Container
Third Author Carole Langlois, Senior Product Manager of Fluid
Using the Helium Integrity Testing (HIT) Platform (2-D Management Technology (FMT), Sartorius Stedim Biotech
and 3-D Bags and Manifolds). 2013. Aubagne, France. [email protected]

Fourth Author- Dr. Magali Barbaroux, Head of Bag

30. Technical Report No.27; PDA Journal of
Technologies Platform – Product Development, Sartorius Stedim
Pharmaceutical Science and Technology, 1998. Biotech, Aubagne, France [email protected]

31. International Organization for Standardization. ISO Fifth Author - Jean-Marc Cappia, Head of Vaccine Segment,
Sartorius Stedim Biotech GmbH, Aubagne, France. jean-
15747: Plastic Containers for Intravenous Injection;, [email protected]
2003.
Sixth Author- Marie-Christine Menier, Compliance and
32. International Organization for Standardization. Regulatory Affairs Manager, Sartorius Stedim Biotech GmbH,
Aubagne, France.
ISO14644: Cleanrooms and associated controlled marie-christine [email protected]
environments;, 2003.
Correspondence Author – Dr. Saeedeh Aliaskarisohi, Scientist,
Sartorius Stedim Biotech GmbH, Goettingen, Germany.
33. Keller, S., Marcy, J., Blakistone, B., Hackney, C., saeedeh.aliaskarisohi@sartorius.
Carter, W.H. and G., Lacy. Effect of Microorganism
Characteristics on Leak Size Critical to Predicting
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APPENDIX

There are several studies in the field of integrity testing with different number of samples tested for each defect
size. We used 30 samples per defect size; in the study done by Keller (12), he used seven samples per defect
size, and Moghimi (13), for example, used 250 per defect size. However, the rationale behind these numbers is
not clear enough or relevant, so we would like to explain how we proceeded to answer the following questions:

1. How many samples are appropriate for this kind of integrity test study?
2. How will these numbers affect the results and how we can explain these effects?

There are planning and performing tools like DoE or 3Pod to design the optimal number of experiments
required to achieve the appropriate levels of statistical power and sensitivity.
 In our study, due to the binary nature of the data we had, we were not able to use DoE. For 3Pod we
needed some input to design the rest of the experiments. We started with 30 patches per defect size,
however, this number of samples per existing defect size, is far above what 3Pod predicts for the rest of
the experiment.
 In this case, the probability approach, which is based on experimental data, can be used to explain the
results and predict the probability of growth occurring for any specific defect size, which has not been
examined before. This approach also shows that the growth for a certain defect size detected in a limited
quantity of tests cannot exclude the possibility that growth would never occur. In other words, this
transition from the occurrence of growth to its absence for a certain defect size is not an absolute
phenomenon, rather it is based on probability.
Furthermore, we did the following step by step:
 We performed 30 tests per each defect size; we examined how many of them exhibited bacterial ingress
and growth. We reported these results as is.
 We fitted our data with logistic regression, which is appropriate for binary data.
 From there, we calculated the probability of growth occurring for a certain defect size.
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