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org on February 2, 2015

Vacuum Decay Container Closure Integrity Leak Test Method

Development and Validation for a Lyophilized
Product-Package System
Jayshree Patel, Brian Mulhall, Heinz Wolf, et al.

PDA J Pharm Sci and Tech 2011, 65 486-505

Access the most recent version at doi:10.5731/pdajpst.2011.00780
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Vacuum Decay Container Closure Integrity Leak Test

Method Development and Validation for a Lyophilized
Product-Package System
JAYSHREE PATEL1, BRIAN MULHALL2, HEINZ WOLF3, STEVEN KLOHR4, and DANA MORTON GUAZZO5,*
1
Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, New Brunswick, NJ; 2Whitehouse Analytical
Laboratories, LLC, Whitehouse, NJ; 3Packaging Technologies & Inspection, LLC, Tuckahoe, NY; 4Analytical &
Bioanalytical Development, Bristol-Myers Squibb Company, New Brunswick, NJ, and 5RxPax, LLC, Bridgewater, NJ
©PDA, Inc. 2011

ABSTRACT: A leak test performed according to ASTM F2338-09 Standard Test Method for Nondestructive Detection
of Leaks in Packages by Vacuum Decay Method was developed and validated for container-closure integrity
verification of a lyophilized product in a parenteral vial package system. This nondestructive leak test method is
intended for use in manufacturing as an in-process package integrity check, and for testing product stored on stability
in lieu of sterility tests. Method development and optimization challenge studies incorporated artificially defective
packages representing a range of glass vial wall and sealing surface defects, as well as various elastomeric stopper
defects. Method validation required 3 days of random-order replicate testing of a test sample population of
negative-control, no-defect packages and positive-control, with-defect packages. Positive-control packages were
prepared using vials each with a single hole laser-drilled through the glass vial wall. Hole creation and hole size
certification was performed by Lenox Laser. Validation study results successfully demonstrated the vacuum decay
leak test method’s ability to accurately and reliably detect those packages with laser-drilled holes greater than or equal
to approximately 5 ␮m in nominal diameter. All development and validation studies were performed at Whitehouse
Analytical Laboratories in Whitehouse, NJ, under the direction of consultant Dana Guazzo of RxPax, LLC, using a
VeriPac 455 Micro Leak Test System by Packaging Technologies & Inspection (Tuckahoe, NY). Bristol Myers
Squibb (New Brunswick, NJ) fully subsidized all work.

KEYWORDS: ASTM F2338, Container closure, Container-closure integrity, Defects, Glass vial, Glass vial defects,
Leak, Leakage, Leak detection, Leak test method, Lyophilized product, Package, Package defects, Package integrity,
Package integrity method, Vacuum decay, Vacuum decay leak detection, VeriPac

LAY ABSTRACT: A leak test performed according to ASTM F2338-09 Standard Test Method for Nondestructive
Detection of Leaks in Packages by Vacuum Decay Method was developed and validated to detect defects in stoppered
vial packages containing lyophilized product for injection. This nondestructive leak test method is intended for use
in manufacturing as an in-process package integrity check, and for testing product stored on stability in lieu of sterility
tests. Test method validation study results proved the method capable of detecting holes laser-drilled through the glass
vial wall greater than or equal to 5 ␮m in nominal diameter. Total test time is less than 1 min per package. All method
development and validation studies were performed at Whitehouse Analytical Laboratories in Whitehouse, NJ, under
the direction of consultant Dana Guazzo of RxPax, LLC, using a VeriPac 455 Micro Leak Test System by Packaging
Technologies & Inspection (Tuckahoe, NY). Bristol Myers Squibb (New Brunswick, NJ) fully subsidized all work.

Introduction environmental contaminants such as microorganisms,

Parenteral product packaging must be able to prevent
product loss due to leakage and to avert ingress of
* Corresponding author: RxPax, LLC, 920 Ardsley
Lane, Bridgewater, NJ 08807; Phone: 908.507.3905;
Email: dguazzo@rxpax.com
debris, or reactive gases. Stability studies to support
new product market approval applications or to assure
marketed product quality are required to include tests
proving container closure integrity (1). The U.S. Food
and Drug Administration has encouraged the use of
validated physicochemical leak test methods in lieu of
product sterility tests for verifying container closure
integrity as a component of product stability studies

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(2). Integrity tests are also an important component of
packaging line qualification studies and as in-process
checks of routine manufacturing operations.
The current research describes the development, opti-
mization, and validation of a container-closure integ-
rity test intended for a lyophilized dosage form con-
tained in a parenteral vial package system. The method
chosen follows ASTM F2338-09 Standard Test
Method for Nondestructive Detection of Leaks in
Packages by Vacuum Decay Method (3). This ap-
proach is nondestructive, simple to perform, and ap-
propriate for packages of lyophilized dosage forms not
requiring a headspace of vacuum or nonreactive gases.
ASTM method precision and bias (P&B) studies sup-
port the sensitivity and reliability of this approach for
parenteral product packages (3, 4). Leak detection
results reported in the ASTM P&B work demonstrate
this method’s ability to detect leaks as small as ap-
proximately 5␮m in nominal diameter in rigid, non-
porous packages. Published studies have shown opti-
mized vacuum decay methods to be more sensitive and
reliable than compendial and ISO standard dye ingress
tests (5).
Prior to each day of testing, the suitability of the
vacuum decay leak test system was confirmed by
introducing a controlled leak into the test system via a
National Institute of Standards and Technology
(NIST)-certified air flowmeter while conducting leak
tests using no-defect master packages. The consistent,
controlled airflow challenge will yield repeatable and
reliable test results given an integral leak test system.
These system suitability checks used a test method
with pass/fail criteria established during instrument
performance qualification. This method is not the final
optimized test method for the finished product package
detailed in the current report.
Development and optimization of the final test method
for the finished product package utilized actual pack-
age test samples representing no-defect product pack-
ages (negative controls) as well as packages with
artificially created defects (positive controls). By test-
ing a population of negative-control, no-defect pack-
ages, final method parameters and acceptance criteria
took into account virtual leakage derived from outgas-
sing of moisture or other volatiles from package com-
ponents and/or release of air trapped under the alumi-
num cap. Positive control packages representing a
variety of possible defect scenarios were included in
method development and optimization to fully chal-
Vol. 65, No. 5, September–October 2011
lenge the method in regard to sensitivity and reliabil-
ity. For instance, leaks located below the product fill
level might clog the flow of leaking gas, preventing
their detection; therefore, this study used product-
filled, with-defect packages to evaluate this possibil-
ity. Large leaks might prove hard to detect, as the
package headspace gas may be lost during a lengthy
test system evacuation, prior to test start. For this
reason, larger defect sizes were included, and with-
defect packages were filled with glass beads to simu-
late typical product headspace volumes. Leaks at the
stopper-vial interface under the cap might require a
longer test time to allow leaking gas to escape from
the package. Therefore, positive controls included
packages with channels cut across the glass vial finish
and with various stopper defects. Finally, vials with
holes laser-drilled through the wall simulating cracks
or chips were also included; each hole was sized for
nominal diameter using a positive air flow measure-
ment technique. Holed vials enabled verification of the
test method’s sensitivity limit.
Once vacuum decay test method parameters were op-
timized, final method validation entailed testing a
sample population of negative-control, no-defect
packages and positive-control packages made using
vials with laser-drilled holes (size certified). All vali-
dation study packages contained lyophilized product.
The test sample population was tested in random order
over three replicate test days.
All research was financed by Bristol Myers Squibb,
Co. (BMS) of New Brunswick, NJ, and was conducted
at Whitehouse Analytical Laboratories (WAL) in
Whitehouse, NJ, under the direction of Dana M.
Guazzo of RxPax, LLC.
A glossary of terms is provided at the conclusion of
this report.
Materials and Methods
1. Test System
All vacuum decay leak tests employed a VeriPac 455
Micro Leak Test System by Packaging Technologies
& Inspection (PTI) of Tuckahoe, NY. The VeriPac
455 system is a non-destructive benchtop leak tester.
The system consists of a 1000 Torr absolute pressure
transducer (measurements in mbar units) plus a 10
Torr differential transducer (measurements in Pa
units), an external vacuum pump, and an external test
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Figure 1
A. PTI VeriPac 455 Micro Leak Test System. B. Microcalibrator airflow meter. C. PTI VeriPac 455 Micro Leak
Test System functional diagram.
chamber specifically designed for the container to be
tested. The design of the leak test system, including
the test chamber, ensures minimal deadspace volume,
so the smallest leaks will trigger maximal pressure rise
readings. (Test chambers designed to closely fit the
given test package system are required to ensure max-
imum test method sensitivity.) A microcalibrator by
Furness Controls, Ltd. (Indian Trail, NC) with an
operational range of 0.001 to 2.000 cm3 䡠 min⫺1
(⫾0.001 cm3 䡠 min⫺1) was used to introduce a con-
trolled leak into the system as part of suitability tests
performed prior to each day of operation, and to
challenge the leak test system during method devel-
opment. Photographs of the test system and the mi-
crocalibrator flowmeter are shown in Figures 1A and
1B, respectively. A test system functional diagram is
included in Figure 1C.
2. Test Method Overview
The test is performed by first placing a test package
inside the test chamber. The test chamber is closed and
the operator activates the test sequence. The test se-
quence consists of three sequentially programmed
time segments discussed below. The length of each
time segment is developed specifically for each prod-
uct-package combination. Two programmed pressure
reference limits, also described below, define each test
as either pass (no leak) or fail (leak). These limits are
also distinct for each product-package system.
2.1. Evacuation Time (Seconds): Prior to test start,
an external vacuum source maintains the VeriPac 455
488
test system under constant vacuum while a closed
valve separates the test chamber from the evacuated
test system (Maintaining the system under a vacuum
between tests provides more consistent, reliable test
results by preventing the accumulation of volatiles in
the test system, stabilizing the absolute pressure trans-
ducer, and shortening the evacuation time segment.)
Upon test sequence activation, this valve opens allow-
ing the rapid establishment of a vacuum in the test
chamber. When the evacuation time is complete, a
second valve closes, isolating the vacuum source from
the fully evacuated test system plus test chamber. The
absolute pressure transducer measures pressure in the
test system plus test chamber (system/chamber)
throughout evacuation.
2.2. Equalization Time (Seconds): An equalization
time segment immediately follows evacuation. This
time segment allows for system/chamber pressure
equilibration (stabilization). The absolute pressure
transducer monitors system/chamber pressure through-
out equalization.
2.3. Test Time (Seconds): A test time segment fol-
lows equalization while the absolute pressure trans-
ducer continues to monitor the pressure rise in the
system/chamber. The differential pressure transducer
simultaneously tracks the net rise in pressure within
the system/chamber from the beginning to end of the
test time. At the completion of this last time segment,
the system/chamber vents allowing test package re-
moval from the test chamber. Immediately post-vent-
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ing, the valve separating the test system from the
test chamber closes, the valve between the vacuum
source and the test system opens, and the test system
returns to its pre-test evacuated state. The test result
is reported as either pass or fail in accordance with
the established pressure reference limits. In addi-
tion, the final pressure readings from the absolute
and differential pressure transducers are displayed
and recorded.
2.4. Pressure Reference Limits: Tests pass or fail
according to two pressure reference limits unique to
each product-package combination. For a test sample
to pass (i.e., to show no leakage), test results must
comply with both pressure reference limit specifica-
tions. A test will fail (i.e., show leakage) if either
pressure reference limit specification is not met.
2.4.1. Absolute Pressure Reference Limit (mbar)
The test will pass if the absolute pressure trans-
ducer reading monitored from equalization start
to the end of the test is less than the absolute
pressure reference limit (APRL). The test will
fail (and the cycle will abort) if the absolute
pressure is equal to or greater than the APRL.
2.4.2. Differential Pressure Reference Limit (Pa)
The test will pass if the differential pressure
transducer reading monitored throughout the test
time segment remains less than the differential
pressure reference limit (DPRL). The test will
fail (and the cycle will abort) if the differential
pressure is equal to or greater than the DPRL.
2.5. System Flush: The vacuum decay leak test in-
strument/chamber itself must be relatively free of
virtual and real leaks in order to detect the smallest
leakage from test packages. To prevent the accumu-
lation of volatiles in the test system that might yield
a virtual leak pressure rise, the test system is pro-
grammed to maintain vacuum conditions between
tests, as previously noted in Section 2.1. Test cham-
ber sealing surfaces are cleaned periodically to pre-
vent debris build-up on test chamber sealing sur-
faces that could cause background leakage.
Cleaning is performed by wiping surfaces with a
low-lint wipe moistened with distilled or purified
water. After cleaning, the chamber is closed and the
test system/chamber is flushed (i.e., evacuated) in
order to dry gasket surfaces. A 60 s flush time was
selected for this study.
Vol. 65, No. 5, September–October 2011
3. Test Sample Population
3.1. Product-Package System: The lyophilized prod-
uct was packaged in a 20 mL capacity glass vial of
Type I tubing with a 20 mm finish diameter. Each vial
was stoppered using a 20 mm lyophilization-style
stopper, and was closed with an aluminum crimp cap
with a plastic flip top. The headspace in each product-
filled package consisted of air at ambient pressure.
3.2. Test Packages: Test packages included master
packages guaranteed free of leaks, negative-control
test packages with no known defect, and positive-
control packages with known package defects, all
briefly described below. Test packages specified for
test system suitability, method development, and method
validation are identified in Table I. Test package and
component illustrations are included in Figures 2–12.
3.2.1. Master packages (test sample ID codes 5-1 to
5-5), guaranteed leak-free, were filled to over-
flow capacity with silicone sealant, stoppered,
and capped. Master packages were used for
system suitability tests and as part of method
development and method validation. (Note:
Masters made of other materials were explored
but were rejected. Vacuum decay results of
masters made of the same vial/stopper/cap com-
ponents as actual product packages more
closely mirrored results of actual product-filled,
no-leak packages.)
3.2.2. Negative-control packages for method develop-
ment included empty vials, stoppered and
capped at WAL (test sample ID codes 2-1 to
2-10), and product-filled vial packages assem-
bled by BMS (test sample ID codes 13-1 to
13-10). Negative-control packages used for
method validation were all product-filled and
assembled by BMS (test sample ID codes N-1
to N-70).
3.2.3. Positive-control packages with defective stop-
pers (test sample ID codes S-1, S-6, and S-7)
used during method development were filled
with glass beads to simulate normal product fill
level. Defective stoppers were either pierced
using a needle or cut with scissors. These pack-
ages were prepared by WAL.
3.2.4. Positive control packages used for method de-
velopment included a variety of vial defect
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TABLE I
Test Sample Identification

Package
Identification
Code Description Defect Size
System suitability
5-1 to 5-5 Master no-defect packages n/a
Filled with silicone sealant
2-1 to 2-10 Negative-control packages n/a
Empty, assembled at WAL
Method development
5-1 to 5-5 Master no-defect packages n/a
Filled with silicone sealant
2-1 to 2-10 Negative-control packages n/a
Empty, assembled at WAL
13-1 to 13-30 Negative-control packages n/a
Product-filled, supplied by BMS
S-1 Stopper defect, 21gauge needle single pierce 21 gauge puncture
through closure
Glass-bead-filled package
S-6 Stopper defect, single radial cut through flange Closure top radius
across land seal (Not measured)
Glass-bead-filled package
S-7 Stopper defect, land seal flange outer edge removed Flange width
Glass-bead-filled package (Not measured)
V-1 to V-5 Vial defect, land seal surface channel 0.74–89 mm (W) ⫻
Glass-bead-filled packages 0.76–0.96 mm (H)
V-6 to V-8 Vial defect, valve seal surface channel 1.12–1.65 mm (W) ⫻
Glass-bead-filled packages 0.27–1.08 mm (H)
V-9 to V-11 Vial defect, land ⫹ valve seal surfaces channel Valve: 0.13–1.34 mm (W) ⫻
Glass-bead-filled packages 0.41–0.97 mm (H)
Land: 0.88–1.24 mm (W) ⫻
0.16–1.80 mm (H)
27-1-, 27-3- to 27-5- Vial with hole in neck 2.39–3.38 ␮m
27-6- to 27-10- -X: stopper leg blocking hole 12.16–14.14 ␮m
27-11- to 27-15- -O: open hole; no stopper leg blocking hole 4.74–5.58 ␮m
Glass-bead-filled package
Method validation
5-1 to 5-5 Master no-defect packages n/a
Filled with silicone sealant
N-1 to N-70 Negative-control n/a
Product-filled, supplied by BMS
P-1 to P-10 Vial with hole in neck 2.39–3.17 ␮m
Product-filled, hand-assembled at WAL
P-11 to P-20 Vial with hole in neck 4.36–6.02 ␮m
Product-filled, hand-assembled at WAL
P-21 to P-30 Vial with hole in neck 12.08–15.04 ␮m
Product-filled, hand-assembled at WAL

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Figure 2
Master test package.
types, all prepared by WAL. First, vials with
channels cut across the finish area and filled
with glass beads were used (test sample ID
codes V-1 to V-11). Second, vials with laser-
drilled holes in the vial neck, filled with glass
beads, were stoppered so that the hole was
either blocked by the inserted lyophilization
stopper leg (test sample ID codes 27-1-X to
Figure 3
Test package components: glass vial, elastomeric
stoppers, aluminum caps with plastic flip tops.
Vol. 65, No. 5, September–October 2011
Figure 4
Package fill variations. Left to right: Glass-bead-
filled packages (2), lyophilized product-filled pack-
age supplied by BMS, lyophilized product manu-
ally transferred from original vial to with-hole vial
(used in method validation).
27-15-X) or was between the stopper legs and
therefore unblocked (test sample ID codes 27-
1-O to 27-15-O). Method validation positive
control packages having vial defects only in-
cluded vials with laser-drilled holes, hand-filled
with lyo-powder, stoppered, and capped (test
sample ID codes P-1 to P-30). All laser-drilled
holes were created by Lenox Laser of Glen
Arm, MD. Hole target sizes were approximately
3 ␮m, 5 ␮m, and 15 ␮m in nominal diameter.
Lenox Laser certified the size of each manufac-
tured hole to within an uncertainty of ⫾2.5%,
with an orifice size tolerance of ⫾10%.
4. Test Parameter Selection Procedure
The vacuum decay leak test for each product-package
system is defined by a unique combination of time and
pressure parameter limits (refer to Materials and
Methods, Section 2). Accurate and repeatable identi-
fication of with-leak and without-leak packages re-
quires appropriate selection of evacuation, equaliza-
tion, and test time limits, as well as pressure limits,
APRL and DPRL. These parameter limits are deter-
mined by performing a series of method development
and optimization experiments.
Evacuation time should be just long enough to estab-
lish consistent system/chamber target pressure read-
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Figure 7

Stopper defect S-7. Flange edge removed.

ings for no-leak packages. (The VeriPac test system

employed during this study used a target pressure of
about 0.1 mbar.) Conversely, evacuation times that are
too long will remove essentially all headspace gas
from largely leaking packages; without headspace gas,
no pressure rise will occur and the leak will go unde-
tected. Equalization time should be long enough to
allow the system to stabilize, yielding consistent ab-
solute pressure readings for no-leak packages. Test
Figure 5 time should be long enough for packages with the
smallest leaks to yield significantly greater pressure
Closure defect S-1. Single 21 gauge needle punc- readings (in mbar and/or Pa units) than no-leak pack-
ture. Bottom figure: 25ⴛ (Magellan V200 ages. Absolute and differential pressure reference lim-
Videomicroscope). its are established by observing the pressure rise in the
system/chamber for no-leak packages versus known-

Figure 8
Figure 6
Glass vial defect V-1. Land seal surface channel.
Stopper defect S-6. Single radial cut. 25ⴛ (Magellan V200 Videomicroscope).

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Figure 9
Glass vial defect V-6. Valve seal surface channel.
Top view: 25ⴛ; Bottom view: 115ⴛ (Magellan
V200 Videomicroscope).
leaking packages when tested according to the pro-
posed time segment limits (prior to final pressure
reference limits selection, programmed pressure limit
values should be quite high to prevent test cycle
abortion). The final time segment lengths and pressure
reference limits chosen should permit no-leak pack-
ages to pass, while detecting with-leak packages.
The current study considered package-to-package and
day-to-day differences when selecting time and pres-
sure limits. Varying time gaps between tests were also
explored. ASTM F2338-09 P&B studies (3) also in-
vestigated variability among instruments and test sites.
In the present study, research was limited to one
instrument/test chamber and one test site.
5. Package Leak Test Method Validation
After method optimization and pressure limit accep-
tance criteria were defined, a validation study was
Vol. 65, No. 5, September–October 2011
Figure 10
Glass vial defect V-9. Land ⴙ valve seal surfaces
channel. 25ⴛ (Magellan V200 Videomicroscope).
performed to demonstrate the detection limit of the
final vacuum decay leak test method for testing BMS
product-filled packages with and without laser-drilled
hole defects, using the PTI VeriPac 455 Micro Leak
Test System. A random-order test sample population
of 70 negative-control, no-defect packages and 30
positive-control, with-defect packages were tested in
replicate over 3 days. Method validation acceptance
criteria required all negative-control packages to pass
(no leak detected), and all positive-control packages
made with laser-drilled holes in the vial neck 5 ␮m
and larger in nominal diameter to fail (leak detected).
Results and Discussion
1. System Suitability Tests
Suitability tests were performed each day of operation
to verify the VeriPac 455 test system’s functionality
and to confirm the absence of system/chamber leaks.
System suitability checks used a test method with
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Figure 11
Glass vial defect V-10. Land ⴙ valve seal surfaces
channel. 25ⴛ (Magellan V200 Videomicroscope).
pass/fail criteria established during instrument perfor-
mance qualification. This method is not the final op-
timized test method for the finished product package
detailed in the current report. Test acceptance criteria
for system suitability were
● All 10 leak tests of master packages must be
passed.
● All five leak tests of master packages with a NIST
airflow challenge of 0.100 ⫾ 0.005 (cm3 䡠 min⫺1)
introduced into the test system must show failure.
The time parameter limits for system suitability tests
were established using a set of five master packages
and 10 negative-control, product-filled packages to
represent no-leak tests, and using the NIST microcali-
brator airflow meter to introduce a controlled leak into
the test system. The test parameter selection procedure
outlined in Materials and Methods, Section 4, was
followed. The absolute and differential pressure ref-
494
Figure 12
Laser-drilled hole in vial neck. Top image, left to
right: Vial 27-3—Hole in “O” closure position,
Hole in “X” closure position. Bottom image: 2.93
␮m nominal hole diameter, by Lenox Laser, of Glen
Arm, MD. 25X (Magellan V200 Videomicroscope).
erence limits were set based on data from no-leak
mean absolute and differential pressure readings mea-
sured over multiple days of testing. Table II outlines
the parameter limits of the final system suitability test
method. All current study system suitability tests per-
formed met the acceptance criteria, namely, all master
TABLE II
System Suitability Test Method Parameter
Settings
Parameter Setting
Evacuation 6.00 s
Equalization 20.00 s
Test 10.00 s
Final absolute pressure reference limit 2.7 mbar
Final differential pressure reference limit 30 Pa
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tests passed and all with-leak tests (0.100 cm3 䡠 min⫺1)
failed.
2. Package Leak Test Method Development
The final package integrity test method was required
to meet the following acceptance criteria.
● All packages with no known defect must pass (i.e.,
no leakage detected).
● All packages with a hole in the vial of greater than
or equal to 5 ␮m in nominal diameter must fail
(i.e., leakage detected).
Method time and pressure parameter limits were op-
timized according to the test parameter selection pro-
cedure presented in Materials and Methods, Section 4,
and described in detail in the following sections. Pack-
ages used for this work included master packages and
negative-control packages filled with lyophilized
product. Positive-control packages were used to inves-
tigate the method’s ability to find various defect types.
Positive-control packages included vials with laser-
drilled holes, vials with channel defects cut across
finish surfaces, and artificially defective closures (re-
fer to Table I for a list of negative and positive
controls).
2.1. Evacuation Time Optimization:Negative-con-
trol, product-filled packages supplied by BMS (13-1 to
13-10) were exposed to evacuation times ranging from
3 to 7 s (Tables IIIa, IIIb). An evacuation time of 6 s
was sufficiently long to ensure consistent 0.1 mbar
absolute pressure readings at the end of evacuation
time for all negative-control packages tested. Positive-
control packages with vial channel defects (V-1 to
V-11) were then tested with an evacuation time of 6 s
(Table IIIc). Two vials with land surface channel
TABLE III
Test Method Evacuation Time Optimization
IIIa. Test Parameter Settings
Parameter
Evacuation
Equalization
Test
Absolute pressure reference limit
Differential pressure reference limit
1
Differential pressure reference limit is irrelevant as test time ⫽ 0.00s.
Vol. 65, No. 5, September–October 2011
defects (V-2, V-4) and all vials with channels cut
across both valve and land seal surfaces (V-9 to V-11)
gave absolute pressure readings much greater than
those of the no-defect vials. Therefore, with appropri-
ate APRL settings, packages with such large defects
should be detectable following a 6 s evacuation time.
The other vials with either land channels or valve
channels (V-1, V-3, V-5 to V-8) exhibited absolute
pressure readings matching the negative control pack-
ages (0.1 mbar) after 6 s evacuation; these vials either
leaked more slowly or the closures effectively sealed
the channel paths. During all evacuation optimization
tests the system suitability test method APRL of 2.7
mbar was used; the DPRL was irrelevant, as tests did
not proceed beyond initial evacuation.
2.2. Equalization Time Optimization:Negative-con-
trol, product-filled BMS packages (13-1 to 13-10)
were tested both with and without the addition of a
NIST airflow artificial leak (0.100 cm3 䡠 min⫺1), using
a 6 s evacuation period, followed by variable equal-
ization times (Tables IVa, IVb). A 20 s equalization
time produced consistent absolute pressure readings
for negative-control packages. During the equalization
time segment, noticeable differences could be ob-
served between no-leak-added and with-leak-added
pressure readings.
Masters and negative-control and positive-control
packages were subsequently tested using a 20 s equal-
ization time segment, preceded by either 6 s or 7 s
evacuation (Table IVc). Tests using 6 s evacuation
yielded pressure readings 10 fold greater for bigger
channel defect packages (V-2, V-4, V-9, V-10, V-11),
and for packages with larger 12 to 14 ␮m holes
(27-6-O to 27-10-O), as compared to no-defect pack-
ages (masters, negative controls). Some smaller chan-
nel defect packages (V-5 to V-8) and vials with holes
about 5 ␮m in nominal diameter (27-11-O to 27-15-O)
Setting
Variable
0.00 s
0.00 s
2.7 mbar
N/A1
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IIIb. Negative-Control Tests

Absolute Pressure (mbar)

Evacuation Evacuation Evacuation Evacuation Evacuation
Package ID Description 3.00 s 4.00 s 5.00 s 6.00 s 7.00 s
13-1 to 13-10 Negative controls 0.8–1.3 0.1–0.8 0.1–0.3 0.1 0.1
BMS product
IIIc. Positive-Control Tests

Absolute Pressure (mbar)

Package ID Description Evacuation 6.00s
V-1 Vial land channel 0.1
V-2 Vial land channel 3.3 (cycle aborted 8.74s)1
V-3 Vial land channel 0.1
V-4 Vial land channel 4.5 (cycle aborted 8.75s)1
V-5 Vial land channel 0.1
V-6 Vial valve channel 0.1
V-7 Vial valve channel 0.1
V-8 Vial valve channel 0.1
V-9 Vial land ⫹ valve channel 2.3
V-10 Vial land ⫹ valve channel 3.4 (cycle aborted 8.75s)1
V-11 Vial land ⫹ valve channel 8.2 (cycle aborted 8.75s)1
1
Absolute pressure reference limit was exceeded and test cycle was aborted at test cycle time indicated.

demonstrated absolute pressure readings about 2 or 3
fold greater than no-defect packages. There was no
difference in pressure readings between negative-con-
trol packages and packages with holes smaller than 5
␮m. Increasing evacuation time to 7 s lowered abso-
lute pressure readings for several of the known-leak-
ing packages tested, likely due to more package head-
space gas loss from these larger leak packages during
test cycle evacuation (Table IVc). Therefore, a 6 s
evacuation time rather than 7 s, coupled with 20 s for
equalization, was preferred to ensure detection of the
largest package leaks.
Extending equalization time from 20 s to 60 s was
attempted to see if very long equalization may produce
lower, more consistent test time differential pressure
readings for negative-control packages, making it pos-
sible to detect the smallest vial-hole leaks during the
final test time segment (Table V). A 20 s test time
segment and a DPRL of 100 Pa was chosen for these
tests (Table Va). Positive controls included packages
having 2.4 to 3.4 ␮m holes blocked by the closure leg
(27-1-X to 27-5-X). A 60 s equalization time did result
in lower differential pressure readings, but no signif-
icant difference in differential pressures was observed

TABLE IV
Test Method Equalization Time Optimization, 0 –20 s
IVa. Test Parameter Settings

Parameter Setting
Evacuation 6.00 s or 7.00 s
Equalization Variable
Test 0.00 s
Absolute pressure reference limit 50 mbar
Differential pressure reference limit N/A1
1
Differential pressure reference limit is irrelevant as test time ⫽ 0.00s.

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IVb. Negative Control Tests, with and without NIST Airflow Leak

Absolute Pressure (mbar) With 6.00s Evacuation

Equalization Equalization Equalization Equalization
Package ID Description 0.00 s 2.00 s 10.00 s 20.00 s
13-1 to 13-10 Negative controls, BMS product 0.1 0.1–0.4 0.5–1.1 1.3–1.9
13-1 to 13-10 Negative controls ⫹ 0.1cc/min Not tested 0.3–0.8 1.1–1.6 1.7–2.8
NIST airflow
IVc. Master, Negative, and Positive Control Tests

Absolute Pressure (mbar)

Evacuation 6.00s Evacuation 7.00s
Package ID Description Equalization 20.00s Equalization 20.00s
5-1 to 5-5 Masters 0.4–0.8 0.1–0.8
2-1 to 2-10 Negative controls, empty packages 0.5–1.5 Not tested
S-1 Closure needle puncture 1.2 Not tested
S-6 Closure flange radial cut 0.7 Not tested
S-7 Closure flange edge removed 0.8 Not tested
V-1 Vial land channel 1.2 0.7
1
V-2 Vial land channel 50.0 (cycle aborted 26.61s) 45.3
V-3 Vial land channel 1.3 0.7
V-4 Vial land channel 50.2 (cycle aborted 50.0 (cycle aborted 17.89s)1
16.77s)1
V-5 Vial land channel 1.9 0.9
V-6 Vial valve channel 2.1 0.9
V-7 Vial valve channel 1.9 1.1
V-8 Vial valve channel 1.9 1.0
V-9 Vial land ⫹ valve channel 7.6 6.2
V-10 Vial land ⫹ valve channel 11.6 14.0
V-11 Vial land ⫹ valve channel 50.2 (cycle aborted 46.2
13.04 s)1
27-1-O Vial 3.38 ␮m hole, open to vial 1.5 Not tested
27-3-O Vial 2.93 ␮m hole, open to vial 1.2 Not tested
27-4-O Vial 2.39 ␮m hole, open to vial 1.0 Not tested
27-5-O Vial 2.67 ␮m hole, open to vial 1.1 Not tested
27-11-O Vial 4.92 ␮m hole, open to vial 3.0 Not tested
27-12-O Vial 5.09 ␮m hole, open to vial 2.8 Not tested
27-13-O Vial 4.92 ␮m hole, open to vial 2.9 Not tested
27-14-O Vial 4.74 ␮m hole, open to vial 2.2 Not tested
27-15-O Vial 5.58 ␮m hole, open to vial 3.6 Not tested
27-6-O Vial 12.85 ␮m hole, open to vial 11.5 Not tested
27-7-O Vial 12.16 ␮m hole, open to vial 12.0 Not tested
27-8-O Vial 13.18 ␮m hole, open to vial 11.5 Not tested
27-9-O Vial 13.18 ␮m hole, open to vial 11.6 Not tested
27-10-O Vial 14.14 ␮m hole, open to vial 13.7 Not tested
1
Absolute pressure reference limit was exceeded and test cycle was aborted at test cycle time indicated.

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TABLE V
Test Method Equalization Time Optimization, 20 s versus 60 s
Va. Test Parameter Settings

Parameter Setting
Evacuation 6.00 s or 7.00 s
Equalization Variable
Test 20.00 s
Absolute pressure reference limit 50 mbar
Differential pressure reference limit 100 Pa
Vb. Negative and Positive Control Tests

Evacuation 6.00 s Evacuation 6.00 s

Equalization 20.00 s Equalization 60.00 s
Abs P Diff P Abs P Diff P
Package ID Description (mbar) (Pa) (mbar) (Pa)
2-1 to 2-10 Negative controls, empty packages 1.2–1.5 31–37 2.0–2.3 19–24
2-1 Negative control, empty package ⫹ 0.050 Not tested Not tested 3.3 44
cc/min NIST airflow
27-1-X Vial 3.38 ␮m hole, blocked by stopper leg 2.2 64 4.5 53
27-3-X Vial 2.93 ␮m hole, blocked by stopper leg 1.2 40 3.0 35
27-4-X Vial 2.39 ␮m hole, blocked y stopper leg 1.2 40 3.0 34
27-5-X Vial 2.67 ␮m hole, blocked by stopper leg 1.6 45 3.0 35

between negative controls and the smallest-hole pack-
ages (Table Vb). Therefore, lengthening equalization
beyond 20 s proved unbeneficial.
2.3. Test Time Optimization: Test time optimization
trials included test time segments of 10 s, 20 s, and
30 s (evacuation and equalization times were kept
constant at 6 s and 20 s, respectively) (Table VI).
Absolute and differential pressure reference limits
were set high (50 mbar and 100 Pa) to prevent pre-
mature test cycle abortion.
As summarized in Table VIb, the shortest 10 s time
segment easily permitted reliable detection of vial
holes greater than or equal to about 5 ␮m, producing
absolute and differential pressure readings well above
master or no-defect packages. Lengthening the test
time to 20 s or 30 s made it possible to reliably detect
vials with holes of about 3 ␮m, as differential pressure
readings of with-hole packages were as much as 2 fold
those of nonleaking packages. Upright and inverted
packages tested for 30 s demonstrated no difference in
results, implying that powdered product, simulated by
glass beads, would have no impact on test results (the
hole in test package 27-2-O appears to have clogged;
results duplicate no-defect packages for all tests at-
tempted).
A microcalibrator was used to introduce a NIST trace-
able airflow leak into the test system/chamber during
negative-control package leak tests to evaluate a 30 s
test time leak test method (with 6 s evacuation and
20 s equalization, Table VII). Absolute and differen-
tial pressure reference limits were set high (50 mbar
and 100 Pa) to avert test cycle abortion. Tests with a
0.050 cm3 䡠 min⫺1 microcalibrator leak yielded similar
results to packages with holes about 3 ␮m in nominal
diameter, with pressure readings about twice those of
negative-control packages (refer to Table VIb, 30 s
tests). With-leak data on day 1 versus day 2 were
essentially identical. Thus a test time of 30 s supports
the required leak size detection limit of 5 ␮m and
larger with a comfortable safety margin.
2.4. Test Method Repeatability, and Proposed
APRL and DPRL Determination: The proposed leak
test method— 6 s evacuation, 20 s equalization and
30 s test—was used to perform 3 day replicate tests of
a random order population set of positive- and nega-
tive-control packages in order to verify the method’s

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TABLE VI
Test Method Test Time Optimization
VIa. Test Parameter Settings

Parameter Setting
Evacuation 6.00 s
Equalization 20.00 s
Test Variable
Absolute pressure reference limit 50 mbar
Differential pressure reference limit 100 Pa
VIb. Master, Negative and Positive Control Packages Tests

Test 30.00 s
Test 10.00 s Test 20.00 s Test 30.00 s Inverted
Diff Diff Diff Diff
Package Abs P P Abs P P Abs P P Abs P P
ID Description (mbar) (Pa) (mbar) (Pa) (mbar) (Pa) (mbar) (Pa)
5-1 to 5-3 Masters 0.7–0.8 20–22 0.8–1.2 28–30 0.9–1.4 43–45 Not Not
tested tested
2-1 to 2-10 Negative controls, 1.0–1.6 15–19 1.4–1.8 24–30 1.5–1.9 36–40 1.5–2.0 40–46
empty packages
27-1-O Vial 3.38 ␮m 2.1 37 2.8 66 3.3 97 3.7 97
hole, open
27-3-O Vial 2.93 ␮m 1.7 29 2.0 44 2.9 55 2.6 66
hole, open
27-4-O Vial 2.39 ␮m 1.4 25 2.1 51 2.7 72 2.9 75
hole, open
27-5-O Vial 2.67 ␮m 1.7 32 2.5 57 3.1 84 3.4 80
hole, open
27-11-O Vial 4.92 ␮m 4.1 73 Not Not Not Not Not Not
hole, open tested tested tested tested tested tested
27-12-O Vial 5.09 ␮m 4.1 78 Not Not Not Not Not Not
hole, open tested tested tested tested tested tested
27-13-O Vial 4.92 ␮m 3.8 67 Not Not Not Not Not Not
hole, open tested tested tested tested tested tested
27-14-O Vial 4.74 ␮m 3.4 61 Not Not Not Not Not Not
hole, open tested tested tested tested tested tested
27-15-O Vial 5.58 ␮m 5.5 98 Not Not Not Not Not Not
hole, open tested tested tested tested tested tested

day-to-day repeatability. For these tests, the absolute
and differential pressure reference limits were set in-
tentionally high at the time of testing in order to
prevent test cycle abortion, enabling end-of-test pres-
sure readings for all but the largest leaks (APRL ⫽ 50
mbar, DPRL ⫽ 100 Pa). Absolute and differential
pressure readings were necessary so that appropriate
APRL and DPRL values could be calculated for the
final test method, taking into account normal day-to-
day and sample-to-sample pressure reading variations.
The test sample population included 40 negative-con-
trol packages, five negative-control packages with
NIST airflow leak added, and 24 positive-control,
with-defect packages. Thirty negative-control pack-
ages included BMS-manufactured product packages
and 10 WAL hand-assembled empty packages. Posi-
tive-control packages included vials with laser-drilled
holes ranging in size from 2.39 to 14.14 ␮m, all with
stoppers positioned so that the inserted stopper leg
blocked the vial neck hole. Other positive controls

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TABLE VII
Test Method Sensitivity Verification Using NIST Airflow Leaks
VIIa. Test Parameter Settings

Parameter Setting
Evacuation 6.00 s
Equalization 20.00 s
Test 30.00 s
Absolute pressure reference limit 50 mbar
Differential pressure reference limit 100 Pa
VIIb. Negative Control Tests, with and without NIST Airflow Leaks

Package Package Test Microcalibrator Abs P

ID Description Day Leak (cc/min) (mbar) Diff P (Pa)
2-1 to 2-5 Negative controls, 1 0.000 1.4–1.9 34–46
empty packages 2 0.000 1.0–1.6 32–44
1 0.050 2.3–2.9 70–72
2 0.050 2.0–2.6 67–74
2-1 Negative control, 1 0.100 3.3 100 (cycle aborted
empty package 56.42 s)1
1
Differential pressure reference limit was exceeded and test cycle was aborted at test cycle time indicated.

included stoppers with a single needle puncture (S-1),
a radius cut (S-6), and a missing flange edge (S-7).
Vials with channel defects included those with a chan-
nel at the land surface only (V-2, V-4), with a channel
along the valve or neck only (V-6 to V-8), and with a
channel along both valve and land surfaces (V-9 to
V-11).
Test results from the no-defect packages tested ac-
cording to the parameter settings listed in Table VIIIa

TABLE VIII
Proposed Final Method Repeatability Tests
VIIIa. Test Parameter Settings

Parameter Setting
Evacuation 6.00 s
Equalization 20.00 s
Test 30.00 s
VIIIb. Negative-Control Packages1 Statistical Analysis

Absolute Pressure Differential Pressure

Analysis (mbar) (Pa)
Mean (n ⫽ 140) 1.9 37
Minimum 1.1 10
Maximum 3.3 55
Standard deviation 0.3 5
Mean ⫹ 6 (std dev) 3.7 67
(Absolute pressure (Differential pressure
reference limit) reference limit)
1
Packages included in analysis: Client-supplied, product-filled packages (13-1 to 13-30), and WAL hand-assembled
empty packages (2-1 to 2-10).

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VIIIc. Positive and Negative Control Tests Summary
Package Description
Negative controls
Negative controls ⫹ 0.050 ccm NIST airflow leak
Packages with holes 2.39–2.93 ␮m
Packages with holes ⱖ3.38 ␮m
Package with vial land channels (V-2, V-4)
Packages with vial valve channels (V-6, V-7, V-8)
Packages with vial land⫹valve channels (V-10, V-11)
Packages with stopper defects (S-1, S-6, S-7)
2
Acceptance criteria (pass/fail) based on 3.7 mbar and 67 Pa, absolute and differential pressure reference limits,
respectively.
are summarized in Table VIIIb. Final test method
APRL and DPRL were calculated by adding six stan-
dard deviations to the no-defect package absolute and
differential pressure reading means, respectively. The
calculated final APRL was 3.7 mbar, and the DPRL
was 67 Pa. With these limits as leak test pass/fail
criteria, the 3 day replicate test results were re-exam-
ined, and the following observations summarized in
Table VIIIc were made.
● Packages that failed (i.e., leakage was detected)
included
䡩 all packages with vial laser-drilled holes greater
than or equal to 3.38 ␮m in nominal diameter
䡩 all negative control packages with a NIST 0.050
cm3 䡠 min⫺1airflow leak
䡩 all packages with vial land seal surface channels
only (V-2, V-4)
䡩 all packages with vial land plus valve seal sur-
face channels (V-10, V-11)
● Packages that passed (i.e., no leakage detected)
included
䡩 all negative-control, no-leak packages
䡩 all packages with vial laser-drilled holes less
than 3.38 ␮m in nominal diameter
䡩 all stopper defect packages (S-1, S-6, S-7)
Vol. 65, No. 5, September–October 2011
Test Count Test Count
Test Passed2 Failed2
Count (No Leak (Leak
Total Detected) Detected)
140 140 0
15 0 15
9 9 0
33 0 33
6 0 6
9 9 0
6 0 6
9 9 0
䡩 all packages with vial valve land seal channels
only (V-6, V-7, V-8)
Based on these results, the final test method with
proposed pressure reference limits met the required
package integrity leak test acceptance criteria.
Namely, all packages with no known defect passed,
and all packages with a hole in the vial greater than
or equal to 5 ␮m in nominal diameter failed. The
additional positive control tests demonstrated the
method’s ability to detect various defect types and
sizes. For instance, the method detected an air leak
of 0.050 cm 3 䡠 min ⫺1 introduced into the test sys-
tem/chamber. The method detected vial hole defects
as small as 3.38 ␮m in nominal diameter. The four
hole defects missed were 2.39 –2.93 ␮m in diameter,
with differential pressure measurements of 54 – 61
Pa, closely approaching the 67 Pa DPRL limit. The
method also found vial channel defects that permit
package headspace gas leakage at the stopper/vial
land seal surface interface. Vial neck defects not
affecting the land sealing surface did not cause
package leakage. Stopper defects not detected in-
cluded a single 21-gauge needle puncture, a single
radius cut, and a missing closure flange edge. The
method was unable to identify these defects likely
due to the viscoelastic nature of the closure formu-
lation making it possible for the closure to re-close
when punctured or cleanly cut, and to conform to
vial surfaces when compressed and capped into
place. The inability to find certain stopper defects
by vacuum decay, and the lack of leakage from vials
having only neck defects, both illustrate the point
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that no leak test method exists that can prevent or
detect all package defect types. Other means are
necessary to preclude defects such as these from the
package assembly line.
3. Package Leak Test Method Validation
3.1. Purpose:A validation study was performed to
demonstrate the detection limit of the final vacuum
decay leak test method for testing BMS product-filled
packages with and without laser-drilled hole defects,
using the PTI VeriPac 455 Micro Leak Test System.
3.2. Test System Suitability:Prior to each test day,
the VeriPac 455 test system was shown suitable for
use by successfully meeting the system suitability
criteria (refer to Table II for system suitability test
method parameters). Specifically, five master pack-
ages were leak-tested using a test cycle consisting of
6 s evacuation, 20 s equalization, and 10 s test, with
APRL and DPRL of 2.7 mbar and 30 Pa, respectively.
Each master package was tested once with the NIST-
traceable microcalibrator closed (no leak). Then each
master was tested again with the microcalibrator open
to introduce an airflow rate of 0.100 cm3 䡠 min⫺1 into
the test system/chamber. Finally, each master was
tested again with the microcalibrator closed. The test
system was suitable for use if all 10 tests with micro-
calibrator closed passed (no leak detected), and all five
tests with microcalibrator open failed (leak detected).
3.3. Validation Protocol:The optimized leak test
method was used to test a random-order test sample
population of negative- and positive-control packages
on each of three replicate test days. The final method
consisted of 6 s evacuation, 20 s equalization, and 30 s
test time, with an APRL and DPRL of 3.7 mbar and 67
Pa, respectively. The refrigerated test samples were
tested after coming to ambient room temperature to
avoid false-positive results caused by package surface
condensation.
Test samples included 70 negative-control, no-defect
lyophilized product packages, filled and assembled by
BMS. Test samples also included 30 positive-control,
with-defect packages prepared at WAL by transferring
lyophilized product from BMS-supplied packages to
vials with a single laser-drilled hole in the vial neck,
hand-stoppered and capped using a hand-crimping
tool. Three groups of 10 holed vials were included,
representing nominal hole diameters of about 3 ␮m
(range, 2.39 –3.17 ␮m), 5 ␮m (range, 4.36 – 6.02 ␮m),
502
and 15 ␮m (range, 12.08 –15.04 ␮m). Validation study
test samples are listed in Table I.
To avoid risk of false-positive results due to test
chamber gasket debris, test chamber sealing surfaces
were wiped clean followed by a 60 s system flush at
regular intervals throughout validation. Nevertheless,
the protocol included the following procedure to ad-
dress possible false-positive test results.
● If a test package result was questionable (e.g., a
no-defect package fails), the test package was re-
moved from the test chamber, the chamber sealing
surfaces were cleaned, and a 60 s system flush was
performed. A master was tested. Once the master
test passed, confirming test system performance
and test chamber integrity, the questionable pack-
age was retested.
● If the test package retest result was pass, then the
initial result was considered invalid due to debris
on the test chamber sealing surfaces or operator
error (e.g., the test chamber was improperly
closed). The repeat test result was the final result.
The test package passed.
● If the test package retest result showed failure,
both the original and the repeat tests were consid-
ered final. The test package failed.
3.4. Acceptance Criteria:The following acceptance
criteria were applied to test results on each day of
validation.
● All 70 negative-control packages must pass.
● All 10 positive-control packages with holes ap-
proximately 5 ␮m in nominal diameter must fail.
● All 10 positive-control packages with holes ap-
proximately 15 ␮m in nominal diameter must fail.
Test results obtained with the 10 positive-control
packages with holes approximately 3 ␮m in nominal
diameter were included in the validation study for
information only.
3.5. Results:
3.5.1. Test System Suitability Tests
Test system suitability criteria were success-
fully met each day of validation. All 10 tests
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TABLE IX
Final Method Validation Tests
IXa. Test Parameter Settings

Parameter Setting
Evacuation 6.00 s
Equalization 20.00 s
Test 30.00s
Absolute pressure reference limit 3.7 mbar
Differential pressure reference limit 67 Pa
IXb. Positive and Negative Control Tests Summary

Test Count Test Count

Test Passed (No Failed
Packages Total Test Leak (Leak
Package Description (n) Count Detected) Detected)
Negative controls (N1 to N70) 70 210 210 0
Positive controls, holes 2.39– 10 30 21 9
3.17 ␮m (P-1 to P-10)
Positive controls, holes 4.36– 10 30 0 30
6.02 ␮m (P-11 to P-20)
Positive controls, holes 12.08– 10 30 0 30
15.04 ␮m (P-21 to P-30)

with microcalibrator closed passed (no leak de-
tected), and all five tests with microcalibrator
open to deliver a 0.100 cm3 䡠 min⫺1 leak failed
(leak detected).
3.5.2. Package Leak Test Method Validation Tests
Test method results are summarized in Table
IX. In short, test method validation criteria
were successfully met on each of day of vali-
dation. All 70 negative control packages (N-1
to N-70) passed (leak detected). All 10 positive
control packages with holes approximately 5
␮m in nominal diameter (P-11 to P-20) failed
(leak detected). All 10 positive-control pack-
ages with holes approximately 15 ␮m in nom-
inal diameter (P-21 to P-30) also failed.
The 10 positive-control packages with holes
approximately 3 ␮m in nominal diameter (P-1
to P-10) were included in the validation study
for information only. Three of 10 packages in
this hole size group failed (were detected),
seven of 10 passed (were not detected). Inter-
estingly, each of the three holes detected on all
three days were 3.17 ␮m in nominal diameter
(P-2, P-9, P-10). The remaining undetected
holes were smaller, ranging in size from 2.39 to
2.67 ␮m in nominal diameter. Creating and
sizing such tiny holes is challenging. These
holes are prone to clogging with environmental
debris, making their viability questionable. In
addition, variability in pressure rise readings
observed with negative control packages, com-
bined with test system degassing, make reliable
detection of the minuscule pressure rise from
leaks much smaller than 5 ␮m difficult.
Conclusion
A vacuum decay leak test method was successfully
developed and validated for nondestructive container
closure integrity testing a BMS lyophilized product
packaged in a glass vial, stoppered, and closed with an
aluminum seal. This nondestructive leak test method is
intended for use in manufacturing as an in-process
package integrity check, and for testing product stored
on stability in lieu of sterility tests.
The method is performed according to ASTM
F2338-09 Standard Test Method for Nondestructive
Detection of Leaks in Packages by Vacuum Decay
Method. The instrument used for all work was the PTI

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VeriPac 455 Micro Leak Test System. The time re-
quired for performing the test is less than 1 min per
package. The leak test method was evaluated for de-
tection limit as defined by USP General Chapter
具1225典 Validation of Compendial Procedures. The de-
tection limit of the VeriPac 455 leak test method was
proven greater than or equal to 5 ␮m in nominal hole
size. Development studies investigated the ability of
the method to detect other package defect types, in-
cluding vial channel defects and stopper defects.
Glossary of Terms
Absolute pressure. Pressure measurement expressed in
true, or absolute, pressure units.
Absolute pressure reference limit (mbar). The vacuum
decay leak test pass/fail limit determined by an abso-
lute pressure transducer.
Absolute pressure transducer. A tool that provides a
measure of “true” pressure above absolute zero pres-
sure. For example, the absolute pressure transducer in
the VeriPac 455 measures the absolute pressure in the
test system throughout the test cycle in mbar units.
Differential pressure. Pressure measurement ex-
pressed as pressure units greater or lesser than a given
reference pressure.
Differential pressure reference limit (Pa). The vacuum
decay leak test pass/fail limit determined by a differ-
ential pressure transducer.
Differential pressure transducer. A tool that provides
a measure of pressure above or below the chosen
reference pressure. For example, the differential pres-
sure transducer in the VeriPac 455 measures the dif-
ferential pressure (i.e., pressure change) starting from
equalization time end (reference pressure ⫽ 0 Pa), to
test time end, in Pa units.
Leak. A gap or defect in the package wall capable of
permitting gas mass flow into or out of the package.
Leaks are rarely, if ever, precise holes or channels of
defined dimension; therefore, leak size is typically
expressed in leakage rate units. The nominal hole size
dimensions of the glass vial laser-drilled hole defects
cited in the report were determined using flow meters
with a laser-drilled orifice in a metal plate as calibra-
tion standards.
Leakage rate. The rate of fluid flow through a leak
path, without regard to the physical size of the hole
504
through which the flow occurs. Fluid denotes any
liquid or gas that can flow. Factors that affect leakage
rate include the fluid itself, the leak path dimensions,
and the pressure difference across the leak path. Gas
mass flow leakage rate is expressed in SI units of
Pascal cubic meters per second (Pa 䡠 m3 䡠 s⫺1). The
current study reports air flow measured in units of
cubic centimeters per minute (cm3 䡠 min⫺1), taken at
ambient (not standard) temperature and pressure con-
ditions.
Pressure measurement terms. One standard atmo-
sphere (atm) pressure is equivalent to 760 Torr,
1.01325 ⫻ 105 Pascal (Pa), or 1000 mbar.
Vial finish. The top of a vial consisting of the vial
mouth opening and the flange lip.
Vial flange. The lip of the vial extending around the
vial mouth. An aluminum cap is sealed onto a vial by
tucking the cap skirt under the vial flange.
Vial land seal channel. A channel extending horizon-
tally across the land seal surface from the vial mouth
opening to the outer finish edge.
Vial land seal surface. The horizontal surface on top
of a vial. An elastomeric closure sufficiently com-
pressed onto the top of a vial effects a seal across the
land seal surface.
Vial valve seal channel. A channel extending verti-
cally from the vial mouth opening into the vial neck.
Vial valve seal surface. The vertical surface along the
inner neck of the vial. The plug of an elastomeric
closure may affect a valve seal when the closure is
inserted into the vial mouth.
Virtual leak. A virtual leak is caused by the emission
of gases and vapors present within the test system that
falsely mimics actual leakage. Virtual leaks may result
from vapors or gases sorbed into or onto test system
surfaces, or trapped between package components.
Acknowledgments
Bristol Myers Squibb staff: Madhav S. Kamat, Re-
search Fellow, Drug Product Science and Technology.
Whitehouse Analytical Laboratories staff: Therese
Abrenica, Laboratory Manager; Sandra Metroka,
Quality Assurance Manager; Eric Creveling, Senior
Laboratory Analyst.
PDA Journal of Pharmaceutical Science and Technology
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Conflict of Interest Declaration
Author Heinz Wolf is an employee of Packaging
Technologies & Inspection, LLC, the manufacturer of
the PTI VeriPac 455 Micro Leak Test System. All
other authors declare they have no competing inter-
ests.
References
1. Department of Health and Human Services. Con-
tainer Closure Systems for Packaging Human
Drugs and Biologics, CMC Documentation, Guid-
ance for Industry; U.S. Food and Drug Adminis-
tration, Center for Drug Evaluation and Research
and Center for Biologics Evaluation and Research:
Rockville, MD, 1999.
2. Department of Health and Human Services. Con-
tainer and Closure System Integrity Testing in Lieu
of Sterility Testing as a Component of the Stability
Protocol for Sterile Products. Guidance for Indus-
try; U.S. Food and Drug Administration, Center for
Vol. 65, No. 5, September–October 2011
Biologics Evaluation and Research, Center for
Drug Evaluation and Research, Center for Devices
and Radiological Health, Center for Veterinary
Medicine: Rockville, MD, 2008.
3. ASTM F2338-09. Standard Test Method for Non-
destructive Detection of Leaks in Packages by Vac-
uum Decay Method; ASTM International: West
Conshohocken, PA, 2009.
4. Wolf, H.; Stauffer, T.; Chen, S.-C. Y.; Lee, Y.;
Forster, R.; Ludzinski, M.; Kamat, M.; Godorov,
P.; Guazzo, D. M. Vacuum decay container/closure
integrity testing technology. Part 1. ASTM
F2338-09 precision and bias studies. PDA
J. Pharm. Sci. Technol. 2009, 63 (5), 472– 488.
5. Wolf, H.; Stauffer, T.; Chen, S.-C. Y.; Lee, Y.;
Forster, R.; Ludzinski, M.; Kamat, M.; Mulhall, B.;
Guazzo, D. M. Vacuum decay container/closure
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