Understanding Container

and Closure System

Integrity Testing

Crystal Booth, M.M.

Masters of Microbiology from North Carolina State University
Former Associate Director of Microbiology at Novartis
Understanding Container and Closure System Integrity
Testing
 Application of the Assay
 What is the test used for?
 Regulations governing the test
 Review some regulatory observations regarding Container and Closure System
Integrity Testing (CCIT)
 Examining some of the various testing methods
 Developing and Validating the Method
 Developing a method
 Performing the validation
 Establishing acceptance criteria
 Performing the test
 Stability Testing
 Interactive Exercise
 Using an interactive game, participants review information pertaining to
monitoring microbial metrics.
Application of the Assay
What is the Test Used For?
 Container closure systems should maintain the sterility
and product quality of sterile final pharmaceutical,
biological, and vaccine products throughout their shelf-
life.4
 Contaminants may include microorganisms, reactive gases, and
other substances.13
 Container closure systems consist of primary packaging
components and secondary packaging components.13
 Primary packaging components are those components that come
into direct contact with the product, such as a glass vial or
syringe.
 Secondary packaging components, according to USP <1207> are
those components that are vital to ensure correct package
assembly, such as aluminum caps over stoppers.13
What is the Test Used For?
 What is Container Closure Integrity Testing (CCIT)?
 CCIT is an assay that evaluates the adequacy of
container closure systems to maintain a sterile barrier.

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Some Regulations Governing the Test
 21CFR211.94 Drug Product Containers and Closures2
 (a) Drug product containers and closures shall not be reactive,
additive, or absorptive so as to alter the safety, identity, strength,
quality, or purity of the drug beyond the official or established
requirements.
 (b) Container closure systems shall provide adequate protection
against foreseeable external factors in storage and use that can
cause deterioration or contamination of the drug product.
 (c) Drug product containers and closures shall be clean and, where
indicated by the nature of the drug, sterilized and processed to
remove pyrogenic properties to assure that they are suitable for
their intended use. Such depyrogenation processes shall be
validated.
 (d) Standards or specifications, methods of testing, and, where
indicated, methods of cleaning, sterilizing, and processing to
remove pyrogenic properties shall be written and followed for
drug product containers and closures.
Some Regulations Governing the Test
 European Commission EudraLex- The Rules Governing
Medicinal Products in the European Union
 Annex 1- Manufacture of Sterile Medicinal Products
• 117. “Containers should be closed by appropriately validated
methods. Containers closed by fusion, e.g. glass or plastic
ampoules should be subject to 100% integrity testing. Samples of
other containers should be checked for integrity according to
appropriate procedures.”1
• *Repeated in PIC/S PE 009-12 (Pharmaceutical Inspection
Convention)12
 Volume 4- Part II Basic Requirements for Active Substances
used as Starting Materials
• 9.20. “Containers should provide adequate protection against
deterioration or contamination of the intermediate or API that
may occur during transportation and recommended storage.”3
Some Regulations Governing the Test
 ICH Harmonized Triplicated Guideline, Quality of Biotechnological
Products: Stability Testing of Biotechnological/Biological Products
Q5C10
 Sterility testing or alternatives (e.g. container/closure integrity testing)
should be performed at a minimum initially and at the end of the
proposed shelf-life. 10

Other Guidance Material

 Guidance for the Industry “Container and Closure System Integrity Testing in
Lieu of Sterility Testing as a Component of the Stability Protocol for Sterile
Products.9
 USP <1207> Sterile Product Packaging- Integrity Evaluation, and the new
updated drafts <1207>13, <1207.1>14, <1207.2>15 and <1207.3>16 Series
 PDA Technical Report 27
 PDA White Paper: Container Closure Integrity Control versus Integrity Testing
during Routine Manufacturing4
Some Regulations Governing the Test
 FDA Compliance Program Guidance Manual, Chapter 56-
Drug Quality Assurance Program 7356.002A- 09/11/155
 (5) Verification of Container and Closures. The physical and
chemical characteristics of containers and closures can be critical
to the sterility and stability of the finished product. Many
containers and closures look alike (color and dimensions), but are
made of different materials or have a different surface treatment
such as silicone on stoppers and ammonium sulfate on Type I glass.
 Evaluate the firm’s procedures for assuring containers and closure
consistently meet appropriate specifications.
 Determine what tests and examinations are done to verify the
containers and closures are made of the correct materials with the
correct dimensions (critical to ensuring continuing container-
closure integrity) and are free of critical defects.
 Some Regulations Governing the Test
 FDA Compliance Program Guidance Manual, Chapter 56-Drug Quality Assurance
Program 7356.002A- 09/11/155
 (6) Container / Closure Integrity. The integrity of the container / closure system
is critical to assuring that all units of drug products remain sterile through
shipment, storage and use. Leaking containers or closures lead to product
contamination.
Reference: FDA’s 1994 Guidance for Industry for the Submission of Sterilization
Process Validation in Applications for Human and Veterinary Drug Products.
 Evaluate the tests and studies performed to demonstrate the integrity of
container / closure systems for all sterile drugs, including:
• Verify that all incoming container-closure components meet specifications, including all
appropriate dimensions.
• Determine studies adequately simulate the stress conditions of the sterilization process,
handling and storage.
• Verify that the units tested in validation are appropriate (e.g., for terminally sterilized
drug product, the units selected should be exposed to the maximum sterilization cycles
using the production process).
• Sensitivity of the test is specified.
• Container-closure integrity is demonstrated during validation and as part of the
stability program (in lieu of sterility testing), over the shelf life of the product.
 A Few Regulatory Observations
Regarding Container and Closure
System Integrity Testing

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A Few Regulatory Observations Regarding
Container and Closure System Integrity Testing
 FDA Warning Letter Dated 24Jun09:
 This is a repeat violation of the February 2007 inspection.
 Your QCU failed to establish an adequate stability testing
program designed to evaluate the integrity of the container-
closure system. Specifically, SOP-QC-XXXX does not include
the storage orientation for liquid products. The stability
samples for liquid products should be stored in an upright or
inverted orientation in order to test and evaluate the integrity
of the bottle seal.
A Few Regulatory Observations Regarding
Container and Closure System Integrity Testing
 FDA Warning Letter Dated 20Jan11:
 Your firm failed to ensure your container closure system
provided adequate protection against foreseeable external
factors in storage and use that can cause deterioration or
contamination of the drug product [21 C.F.R. § 211.94(b)].
• For example, your firm identified 542 incidences through consumer complaints of
product defects such as, leaks, bursts, and premature activation during the period of
January 2008 to August 2010. These are critical defects that can impact sterility and
stability of your product. Your firm identified that the probable cause was the result of
defective materials used in the manufacture of the container closure system.
• Your response is not adequate since the sampling plans described are
not based on appropriate statistical criteria to sufficiently identify
these known potential defects, especially given the history of the
supplier for this container closure system.
• Furthermore, your final product inspection procedure and use of a (b)(4) does not appear
to be effective in preventing shipments of product with critical defects to the
marketplace. Additionally, our data indicates there may be other cases of foreign
substances in products manufactured at your facility such as an insect found in the
intravenous solution of XXXX and dirt reported inside of (b)(4) and the (b)(4) of XXXXX®.
Please provide an evaluation of the suitability of any of these potentially affected lots.
A Few Regulatory Observations Regarding
Container and Closure System Integrity Testing
 FDA Warning Letter Dated 31May13:
 Your firm failed to ensure your container closure system
provided adequate protection against foreseeable external
factors in storage and use that can cause deterioration or
contamination of the drug product (21 CFR 211.94(b)).
 For example, you received consumer complaints identifying at
least ten (10) membrane leaks and one hundred fifty-five
(155) inadequately-fitting blue caps during the period of
November 2011 to March 2013. These are critical defects that
can impact the sterility and stability of your products.
 This is a repeat violation
A Few Regulatory Observations Regarding
Container and Closure System Integrity Testing
 FDA Warning Letter Dated 02Nov15:
 FDA investigators also noted CGMP violations at your facility,
causing your drug product(s) to be adulterated within the
meaning of section 501(a)(2)(B) of the FDCA. The violations
include, for example:
• Your firm failed to establish adequate written procedures designed to
assure batch uniformity and integrity of drug products that describe
the in-process controls, and tests, or examinations to be conducted on
appropriate samples of in-process materials of each batch (21 CFR
211.110(a)).
• Your firm failed to ensure container closure systems provide protection
against foreseeable external factors in storage and use that can cause
deterioration or contamination of the drug product (21 CFR 211.94(b)).
A Few Regulatory Observations Regarding
Container and Closure System Integrity Testing
 FDA Warning Letter Dated 21APR16:
 Puncturing a container compromises the integrity of the container closure
system, and each puncture increases the chances of contamination.
 In response to our observation regarding (b)(4), you indicated that you
developed a container closure integrity test for the (b)(4) and plan to
demonstrate the sterility of the (b)(4) throughout its expiry. However, this
test only confirms the sampled portion is sterile and does not validate the
integrity of the container. Additionally, the tested portion may not have
detectable microbial contamination after the (b)(4) container loses
integrity, but the amount of bacterial endotoxin may still increase. Your
proposed corrective action is not adequate to demonstrate container
closure integrity. Therefore, your response does not indicate how you will
ensure your finished product is sterile and the amount of endotoxin is
within an acceptable limit after multiple punctures to the (b)(4)
containers.
Examining Some of the Various Testing Methods
 Container closure integrity testing can be performed in
many different ways.
 All methods have pros and cons.8
 Some containers, such as ampoules, require 100% integrity
testing.4
 New proposed changes to USP Chapter <1207> describes
several methods the user and experiment with.
 The proposed update removes the requirement to
compare the results of other container closure integrity
(CCI) assays to the microbial ingress challenge.
 The updated chapter series is expected to become official
in August 2016.
Examining Some of the Various Testing Methods
 The chapter (proposed USP <1207>) divides the tests
into two major categories.
 Deterministic- less subject to error and provide
quantitative results8
 Probabilistic- more uncertainty in assay results,
includes traditional methods8
 Testing methods included in the chapter were chosen
from peer reviewed articles and are supported by the
American Society for Testing and Materials (ASTM) 8
 The user may choose from the methods listed or even
methods not listed as long as the final method is properly
validated and optimized for the closure system.8
Examining Some of the Various Testing Methods
 Deterministic methods include:
 Electrical Conductivity and Capacitance Test
(HVLD)
 Laser-based Gas Headspace Analysis
 Mass Extraction
 Pressure Decay
 Tracer Gas (vacuum mode)
 Vacuum Decay

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 Examining Some of the Various Testing Methods
Deterministic Test Time
Package Content Package Leak Size Class Effect of Method
Leak Test Measurement Outcome and Data Analysis Order of
Requirements Requirements (Detection Range) on Package
Technologies Magnitude
Electrical Liquid (with no combustion Less electrically 3 to 6 Quantitative measure of electrical current Nondestructive, Seconds
conductivity and risk) must be more conductive than passing through the test sample: provides an although product
capacitance electrically conductive liquid product. Range varies with indirect determination of leak presence as stability studies
(high-voltage leak than package. product–package, shown by a drop in test sample electrical are recommended
detection) instrument, test resistivity, with a resultant increase in voltage to verify effect of
Product must be present sample fixturing, reading above a predetermined pass/fail limit. exposure.
at leak site. and method
parameters.
Laser-based gas Gas volume, path length, Transparent or 1 to 6 Quantitative measure of gas headspace Nondestructive Seconds
headspace and content must be translucent, content of the test sample by laser-based gas
analysis compatible with amber or Detection of analysis, for product requiring a headspace of
instrument’s detection colorless. smallest leakage low oxygen concentration, low moisture vapor
capability. rates is a function concentration, and/or low absolute pressure.
of time span
between analyses. To calculate the leak rate of the test sample,
one can use results compiled as a function of
time.
Mass extraction Gas or liquid must be Rigid, or flexible 3 to 6 Quantitative measure of mass flow rate Nondestructive Seconds to
present at leak site. with package resulting from test sample headspace escape minutes
Presence of liquid at leak restraint Range varies with or liquid product volatilization within an
site requires test mechanism. product–package, evacuated test chamber housing the test
pressures below vapor instrument, test sample.
pressure. Porous package fixtures/chamber,
requires masking and method Leak rate of the test sample may be estimated
Product must not clog leak to limit gas flow parameters. by comparing the mass flow results of the test
path. through porous sample to tests performed using leak rate
material. standards and positive controls.

Quantitative measure of pressure provides an

indication of larger leak presence.
 Examining Some of the Various Testing Methods
Deterministic Test Time
Package Content Package Leak Size Class Effect of Method
Leak Test Measurement Outcome and Data Analysis Order of
Requirements Requirements (Detection Range) on Package
Technologies Magnitude
Pressure Gas must be present Compatible with 3 to 6 Quantitative measure of pressure drop within a Nondestructive, Minutes to
decay at leak site. pressure detection pressurized test sample; pressure drop results although means days,
mode. Range varies with from escape of gas through leak paths. used to access depending
Product (especially product–package, test sample on package
liquids or semi- Rigid, or flexible with instrument, and Results that are compiled as a function of time are interior may volume and
solids) must not package restraint method parameters. indicative of leakage rate of the whole test sample. compromise test required leak
cover potential leak mechanism. sample barrier. limit of
sites. detection
Tracer gas Tracer gas must be Able to tolerate high- 1 to 4 Quantitative measure by spectroscopic analysis of Nondestructive, Seconds to
detection, added to package. vacuum test conditions tracer gas leak rate while tracer gas is being although access minutes
vacuum mode Detection of larger drawn out of a tracer-flooded test sample into a to package interior
Tracer gas must Rigid, or flexible with leaks is a function ofvacuum chamber. for tracer gas
have access to package restraint instrument capability introduction may
package surfaces to mechanism. and test sample Leak rate of the total sample can be calculated by compromise test
test package for fixturing. normalizing the measured tracer leak rate by sample barrier.
leaks. Limited tracer gas tracer concentration in the test sample.
permeability
Vacuum decay Gas or liquid must Rigid, or flexible with 3 to 6 Quantitative measure of pressure rise (vacuum Nondestructive Seconds to
be present at leak package restraint decay) within an evacuated test chamber housing minutes
site. mechanism Range varies with the test sample; vacuum decay results from
product–package, headspace escape from the test sample, or liquid
Presence of liquid at Porous package instrument, test product volatilization.
leak site requires requires masking to sample chamber,
test pressures below limit gas flow through and method Leak rate of the test sample may be estimated by
vapor pressure. porous material. parameters. comparing vacuum decay results for the test
Product must not sample to results of tests performed using leak
clog leak path. rate standards and positive controls.
a The leak size class detection range cited for each technology allows for method-to-method comparison. The leak sizes actually detected by a fully developed and validated

deterministic method may deviate from these ranges, as noted.

Examining Some of the Various Testing Methods
 Electrical Conductivity and Capacitance Test (also known as High
Voltage Leak Detection HVLD)
 Leak detection in walls of nonporous, rigid or flexible packaging
containing liquid or semi-liquid product (e.g. ampoules) 15, 17
• High voltage/high frequency charge is applied across the container-closure
system17
• A leak will cause an increase in current across the high voltage electrodes,
triggering the reject mechanism for the leak detector17
 Key factors include voltage level, probe positioning, container-closure
system geometry and wall thickness, and product formulation17
Advantages Disadvantages
• Automation of the integrity test17 • High voltage/high voltage field could
• Rapidity of the test procedure potentially affect potency and stability
(seconds) 17 of some protein-based therapeutics. 17
• Can be used online17 • Requires conductive liquid fills11
• Nondestructive15
• Could potentially detect leak
locations15
• Quantitative Electrical Conductance
Measurements15
• Testing under normal atmosphere11
 Examining Some of the Various Testing Methods
 Laser-based Gas Headspace Analysis
 Typically performed using non-contact methods, such as frequency
modulation spectroscopy17
• Near infrared diode laser light passes through the gas headspace region15
• Light is absorbed as a function of gas concentration and pressure15
• Absorption information is processed using phase-sensitive detection
techniques15
• A microprocessor analyzes the data and yields the test results15
 Can be used for lyophilized products or oxygen-sensitive liquid
products17
 Headspace gas analysis is rapid (seconds)—allows 100% inspection of
oxygen sensitive products or products packaged under vacuum17
 Nondestructive and provides quantitative results15
 Key parameters: Headspace Volume, Package Temperature, Headspace
Pressure and Vacuum, Sensitivity of the Headspace Analysis Instrumentation17
Examining Some of the Various Testing Methods
 Mass Extraction
 Nondestructive and Quantitative15
Advantages
 Can be used for detecting leakage in nonporous, rigid or flexible packages15
• Rapidity of the test
• Packages with a porous component can be tested by masking the porous package procedure 17
component15 • Nondestructive15
 The test sample is placed inside a test chamber that is pneumatically connected • Quantitiative15
to a mass extraction leak test system equipped with a vacuum generator Disadvantages
package15
• Requires fixtures for
• The chamber is quickly evacuated for a predetermined time to reach a each container
predetermined vacuum level. A series of evacuation cycles are performed, each closure system 17
intended to identify smaller leakage rates15 • Proteinaceous
• After each cycle, the test system is isolated from the vacuum source and products may
measurements of absolute pressure, pressure decay rate, and/or gas mass flow interfere with defect
rate are captured15 detection11
• Readings greater than predetermined limits that were established using negative
controls are indicative of container leakage, triggering test cycle abort15
• For those test samples passing all previous larger leak vacuum cycles, a final
vacuum is drawn15
• With all flow from the test chamber directed through the mass flow sensor, the
mass flow rate is measured. Mass flow above a predetermined limit established
using negative controls is indicative of container leakage15
Examining Some of the Various Testing Methods
 Pressure Decay
 Intended for integrity testing of the gas headspace region of the
test sample (nonporous, rigid, or flexible packages) 17
• For this test, the container-closure system is placed in a test fixture
that is either pressurized or evacuated17
• The test chamber is allowed to stabilize, and then the change in
pressure or vacuum is measured over time17
• Pressure or vacuum can be measured directly or by differential
pressure between the test chamber and a reference chamber17
 Key test parameters include temperature, package geometry, test
fixture geometry, volume of package headspace, water vapor
pressure inside the package, stabilization time, and test time17
Advantages Disadvantages
• Rapidity of the test • Requires fixtures for each
procedure 17 container closure system 17
• Can be used online17 • Proteinaceous products
• Nondestructive15 may interfere with defect
• Quantitiative15 detection11
Examining Some of the Various Testing Methods
Advantages
 Tracer Gas Detection (Vacuum Mode) • Rapidity of the test
 Detects leakage from nonporous, rigid or flexible packages15 procedure 17
• Nondestructive15
 The test requires the presence of tracer gas inside the test sample • Quantitiative15
package15 • Sensitivity11
• Helium is the most commonly used tracer gas, and hydrogen is also
used15
Disadvantages
 The leakage rate of tracer gas is quantitatively measured using a
spectrometric analytical instrument specific for the tracer gas15 • Requires helium-
containing
 To perform the vacuum-mode test, test samples that have been fully or headspace (for
partially flooded with tracer gas are placed inside an evacuation helium leak
chamber15 detection) or
modified atmosphere
• The instrument's vacuum pump evacuates the test chamber or fixture,
drawing any leaking tracer gas through the analyzer15 packaging (for
headspace oxygen
• The absolute leak rate of the test sample may be calculated by testing)11
normalizing test results by the partial pressure of the tracer gas within
the test sample at the time of test15
• Test sample leakage is judged acceptable if the absolute leak rate is
below that which has been reported to put product quality at risk15
Examining Some of the Various Testing Methods
Advantages
 Vacuum Decay • Rapidity of the test
 Nondestructive and quantitative15 procedure 17
• Nondestructive15
 Detects leaks in nonporous, rigid or flexible packages. Packages • Quantitiative15
with a porous component can be tested by masking the porous
package component. 15 Disadvantages
• Requires fixtures for
• The test sample is placed in a closely fitting evacuation test each container
chamber, which is equipped with an external vacuum source. 15 closure system 17
• The test chamber plus test system dead space are evacuated for a • Proteinaceous
products may
predetermined period of time. 15 interfere with defect
• The targeted vacuum level chosen for the test is predetermined on detection11
the basis of the test sample type under evaluation. 15
• The rise in dead space pressure (i.e., vacuum decay) is monitored
for a predetermined length of time using absolute and/or
differential pressure transducers. 15
• A pressure increase that exceeds a predetermined pass/fail limit
established using negative controls indicates container leakage. 15
Examining Some of the Various Testing Methods
 Probabilistic Methods Include:
 Microbial Challenge by Immersion
 Tracer Liquid Tests (e.g. Dye Ingress)
 Bubble Tests
 Tracer Gas (Sniffer Mode)

 Microbial
Challenge by Immersion and Dye Ingress
are the most recognized leak test methods.
 USP<1207> Series is encouraging a move toward
the deterministic methods.
 Examining Some of the Various Testing Methods
Probabilistic
Package Content
Leak Test
Requirements
Technologies
Bubble emission Gas must be present at
leak site.
Product (especially liquids
or semi-solids) must not
cover package surfaces to
be leak tested.
Microbial Growth-supportive media
challenge, or product.
immersion
exposure Presence of liquid at the
leak site required for
method reliability.
Test Time
Package Leak Size Class Measurement Outcome and Data Effect of Method
Order of
Requirements (Detection Range) Analysis on Package
Magnitude
Rigid, or flexible 4 to 6 Qualitative measure by visual inspection of Destructive Minutes
with package bubble emission caused by escape of test
restraint Range varies with sample headspace while sample is
mechanism. product–package, submerged and exposed to differential
test sample fixturing pressure conditions. Alternatively, sample
and positioning, surfaces may be exposed to surfactant.
method parameters,
and analyst technique Continuous bubble emission indicates
and skill. presence of a leak, its location, and its
relative size.
Able to tolerate 4 to 6 Qualitative measure by visual inspection of Destructive Weeks
pressure and microorganism growth inside test samples
immersion Range varies with filled with growth-supportive media or
challenge. container-closure, product, post immersion in heavily
test sample fixturing contaminated challenge media while
Rigid, or flexible and positioning, exposed to differential pressure conditions,
with package challenge condition followed by incubation to encourage
restraint severity, and inherent microbial growth.
mechanism. biological variability.
Growth in the test sample indicates the
presence of test sample leak site(s)
capable of allowing passive or active entry
of microbes.
 Examining Some of the Various Testing Methods
Probabilistic Test Time
Package Content Package Leak Size Class Measurement Outcome and Data Effect of Method
Leak Test Order of
Requirements Requirements (Detection Range) Analysis on Package
Technologies Magnitude
Tracer gas Tracer gas must be added Leak site 3 to 6 Quantitative measure by spectroscopic Nondestructive, Seconds to
detection, sniffer to package. accessible to analysis of tracer gas near the outer although access to minutes
mode probe. Range varies with test surfaces of the tracer-flooded test package interior for
Tracer gas must have sample, method sample, sampled using a sniffer probe. tracer gas
access to package surfaces Limited tracer gas parameters, test sample introduction may
to be tested for leaks. permeability. fixturing, and analyst Tracer presence above a pass/fail limit compromise test
technique and skill. indicates presence and location of a leak sample barrier.
Smaller leak detection site.
may be possible under
optimum test conditions.
Tracer liquid Contents must be Rigid, or flexible 4 to 6 Measure of tracer in test sample Destructive Minutes to
compatible with liquid tracer. with package previously submerged in tracer-charged hours
restraint Able to tolerate liquid liquid while exposed to differential
Product must not clog leak mechanism. immersion. pressure conditions. Alternatively, tracer-
path. charged test samples may be submerged
Compatible with Smaller leak detection in tracer-free collection fluid.
liquid tracer may be possible under
detection mode. optimal test conditions Tracer presence indicates leak site(s)
employing chemical capable of allowing tracer passage, and
analysis tracer detection. tracer magnitude may indicate relative
leak size (assuming a single-leak
Range varies with pathway).
container-closure, test
sample fixturing and Tracer migration measurement may be
positioning, challenge quantitative (by chemical analysis;
condition severity, and preferred approach for small leak
tracer liquid content. detection) or qualitative (by visual
inspection).
aThe leak size class detection range cited for each technology allows for method-to-method comparison. The leak sizes actually detected by a fully developed and validated deterministic method
may deviate from these ranges, as noted.
Examining Some of the Various Testing Methods
Advantages
 Microbial Challenge by Immersion • Widely used for
decades11
 Suitable for any container-closure system that can • Industry and
withstand immersion and pressure changes.17 regulatory
familiarity11
• Readily incorporated
 Test article is immersed in a broth containing the test into media fills11
organism. 17
Disadvantages
• Brevundimonas diminuta, Serratia marcescens, • Less sensitive11
Escherichia coli and other organisms have been used • Detection
probabilistic for
for this test. 17 small-size defects
(i.e. <20 µm) 11
 This test may be static (no pressure or vacuum) or • Destructive11
dynamic (pressure and vacuum are applied, typically to
simulate air transport) 17
 Key test factors include: bacterial size and motility,
differential pressure, challenge media, exposure time,
and viable count in the challenge media. 17
Examining Some of the Various Testing Methods
Advantages
 Tracer Liquid Tests (e.g. Dye Ingress) • Widely used for
decades11
 The dye leak test is the most common liquid tracer • Industry and
test.15 regulatory
familiarity11
• The container is immersed in a methylene blue solution
and pressure and vacuum are applied to the container. 15 Disadvantages
• Less sensitive11
• The container inspected visually or via • Detection
spectrophotometry (preferred method). 15 probabilistic for
small-size defects
 Key factors include differential pressure, compatibility (i.e. <20 µm) 11
• Destructive11
of the dye with the product, liquid viscosity and
surface tension, inspector training and experience (for
visual inspection) or assay sensitivity (for
spectrophotometry). 15
 Qualitative and destructive17
Examining Some of the Various Testing Methods

 Bubble Tests
 The item under test is pressurized to about 3 psig and
immersed in a bath containing water or water and
surfactant (e.g., polysorbate 80).15
• This test can detect leaks as small as 10-5 mbar-L/sec.15
 Key test factors include differential pressure, test
time, immersion fluid surface tension, visible
inspection conditions (e.g., light intensity,
magnification, and background), and visual
inspector training and experience.15
 Qualitative and destructive17
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Examining Some of the Various Testing Methods
 Tracer Gas (Sniffer Mode)
 Test samples are flooded completely or partially with the
tracer gas via one of several options. 17
• Piercing a closed test sample to introduce pressurized tracer
gas (sealant is applied to close the puncture site) 17
• Flooding the test sample before package closure17
• “Soaking” a closed test sample by pressurizing with tracer gas
(most applicable to larger leak detection) 17
 Test samples are checked for leakage by scanning the outer
package surfaces using a vacuum wand17
 The use of negative and positive controls along with the test
samples provides evidence of test method limit of
detection.17
 The sniffer mode is generally chosen when the leak location
must be identified. 17
 Developing and
Validating the Method
 Developing a Method
 Package integrity verification occurs during three product
life cycle phases:14
1. The development and validation of the product-
package system
2. Product manufacturing
3. Commercial product shelf-life stability assessments
 Testing needs to show the complete picture of container
CCI over the lifecycle of the product.
 Any leak test requires optimization for each product-
package application.13
 Developing a Method
 All methods have limitations, but the following aspects
should be considered.11
 Methods must be suitable for its intended use
 Methods must be applicable to the specific drug product- Other Criteria
package (e.g. drug products can interact with CCI defects) • Liquid or solid product?
• Headspace gas?
 Methods must detect leaks effectively • Headspace vacuum?
• No Headspace?
 Non-destructive CCI testing • Package design?
•
 Choose a preliminary method following vendor’s •
Rigid or flexible package?
Moveable components?
recommendations or literature research. • Polymeric materials?
• Metallic materials?
 Choice of method depends on specific desired outcomes14 • Transparent or Opaque
1. Detecting the presence of leak paths materials?
• Porous materials?
2. Determining the location of leak paths • Multi-dose systems?

3. Measuring leak rate for the whole package

4. Evaluating the potential for microbial ingress
Developing a Method
 Positive and negative controls need to be created
for the assay.
 Positive controls are needed to simulate defects.
• Leaks that occur naturally are rarely uniform holes or
channels. They are generally complex tortuous paths.13
 The controls will be tested along with intact
samples under different testing parameters. 11
 Negative and positive controls are designed and
assembled appropriately with consideration of
container–closure design, materials of
construction, characteristics of anticipated
package leaks, and impact of product contents on
test results.14
Developing a Method
 Many methods are available to create positive controls
Positive Control Type Advantages
 Easy sample
Micro-pipettes11 preparation
 Sample geometry can
remain unchanged
Laser drilled
 Better resemble natural
holes11
defects in glass (cracks)
and polymer (pinholes)
 Easy sample
preparation
Microtubes11  Robust
 Easy to use
 Easy to prepare
 Easy to use
Wire
Disadvantages
 Fragile
 Broken tips may not be
easily detected
 Cost
 “Hole” size of laser-
drilled effects needs to
be calibrated
 The length of the
microtube defects
usually longer than that
of the typical “real-world”
defects
 Can create a tenting
effect where the size of
the control is not
accurate
Developing a Method
 In addition to choosing a preliminary method, creating
positive and negative control samples, acceptance
criteria must be predetermined.
• All negative controls must pass
• All positive controls with leaks at or above the claimed
limit of detection must fail
• A lower limit of detection must be established
• An upper limit of detection should be established

http://www.rxpax.com/training-services/
Developing a Method
 USP <1207.1> and ICH Q2 (R1) describes how to
determine and establish a limit of detection (LOD) for
the method chosen. 14
 The LOD is the smallest leakage rate or leak size that the
method can reliably detect, given the product-package of
interest.14
 During method development trials all negative controls must
pass and all positive controls with leaks at or above the
claimed limit of detection must fail. 14
 The LOD for a given method is defined as the smallest-leak
positive control subset that consistently demonstrates
leakage in 100% of the positive-control subset units at that
defect size and larger. 14
• The percentage of positive controls to be detected must be specified
in the validation study protocol by the end user. 14
 Multiple methods are available to determine limit of
detection criteria.
Developing a Method
 USP <1207.1> also describes the need to verify the
largest leak detection capability or upper limit of
detection.
 All analytical methods have optimum measurement ranges. 14
 All leak test method selection should also take into account the largest
leak sizes likely to occur in the sample population. 14

 Once an optimized method is created, multiple lots

should be tested representing the package integrity at
the extremes of finished product-package profiles. 14
 e.g. relevant product variations, including various packaging component
sources/lots, drug products batches, as well as packaging sites and
lines.11
 The quantities to be tested should be sufficient to provide adequate
assurance of package integrity. 14
Developing a Method
 At this point
 A method has been chosen
 Target acceptance criteria has been established
 Positive and negative controls have been created
 Leak test method parameters have been developed
and optimized through experimentation to ensure
sensitive, accurate, robust, and reproducible leak
detection for specific product–package systems.14
 And successful qualification(s) have been performed
 A validation protocol should be written outlining
the successful trials and parameters created
during the method develop phase.
 Performing the Validation
 Container closure integrity methods need to be validated for
specific drug-product packages
 Various components, such as the drug product, can affect the
testing outcome.
 Validation of the final leak test method is required to
demonstrate test method detection limit, accuracy, range,
robustness, and precision.11, 13
 Method accuracy is the ability of a leak test to correctly identify or size
leaks (as per the intended leak test outcome).14
 Method precision is a measure of test result reproducibility and is proven
during method validation by testing a randomly mixed population of
negative and positive controls over multiple days by multiple operators
and, when possible, using multiple test instruments.14
 USP <1225> Validation of Compendial Procedures and ICH Q2(R1)
provide good guidance on method validations
 Performing the Validation
 The established validation protocol should be followed when
performing the validation.
 Validations are typically performed in triplicate by performing
the method created in the method development phase.
 USP <1207.1> recommends randomly mixing negative and positive
control populations over multiple days by multiple operators and, when
possible, using multiple test instruments.14
 Quantities of samples to be tested must be sufficient to
provide adequate assurance of package integrity and will likely
vary on the basis of:
1. The complexity of the product–package 14
2. The specifics of the user specification requirements14
3. The prior experience of the producer. 14
 Performing the Validation
 The method validation protocol should be written to describe New USP <1207>
series removes the
introducing a number of defects of known size or leak rate as requirement to
controls compare to microbial
ingress
 Acceptance criteria for leak test method validation should
include the following:
1. All negative controls pass (no leaks are identified) 14
2. All positive controls with leaks at or above the designated limit of
detection fail (leaks are detected). 14
3. An integral package is one that conforms to specific product-package
maximum allowable leakage limits.13
 For some test methods (e.g. liquid tracer leak detection by
mass spectrophotometric analysis), test blanks are also
included as part of method validation and routine testing. 14
 Blanks are not equivalent to, and should not substitute for, negative
controls. 14
 Performing the Validation
 After the successful validation, a report should be written
describing the acceptable parameters, acceptance criteria,
validation results, and the method to be used for routine
testing and stability testing.
 Establishing Acceptance Criteria
 Acceptance criteria was established in the method development
phase.
 A lower limit of detection was established and demonstrated
 An upper limit of detection was established and
demonstrated
 Positive and negative controls were created
• All positive controls must fail
• All negative controls must pass
 Leak test method parameters were developed and optimized
through experimentation (e.g. amount of vacuum to pull)
 Units that test to be non-integral need to be investigated
 Performing the Test
 After the method is develop and validated, a routine testing SOP
(standard operating procedure or test method) should be
established.
 The routine SOP should lock down all the parameters and
acceptance criteria for the specific product/container-closure
system so that the test is performed the same way every time.
 “The manufacturer should be able to justify the amount of testing
required on the basis of statistical process control results generated
during the validation phase, and later, on the basis of routine
manufacturing product-quality trending analyses.”14
 Quantities of samples to be tested must be sufficient to provide adequate
assurance of package integrity.14
 Package integrity should be re-evaluated when changes are
required in package design, package materials, or
manufacturing/processing conditions.14
Stability Testing
 The routine testing SOP should be utilized when performing
CCIT for stability testing.
 Container-closure integrity should be demonstrated as part of
the stability program over the shelf life of the product for
new and existing products.5
 Sterility testing or alternatives (e.g. container/closure
integrity testing) should be performed at a minimum
initially and at the end of the proposed shelf-life. 10
 Container-closure integrity testing can be used to replace
sterility testing in stability protocols.4, 9
 CCIT may not replace the sterility test for release
testing.4, 9
Stability Testing
 If a non-destructive test has been validated for the specific
container-closure system, it is useful during stability studies.
 The same container can be used throughout the stability
period.
 This saves money and allows for more meaningful profiles
of container-closure integrity.17
 Where the same strength and exact container/closure system
is used for 3 or more fill contents, the manufacturer may
elect to place only the smallest and largest container size into
the stability program, i.e., bracketing.10
 Summary
 Container Closure Integrity Testing evaluates the adequacy of
container closure barrier systems to maintain a sterile barrier.
 Some guidance documents and regulations that discuss CCIT include
 FDA 21CFR211.94
 European Commission EudraLex Annex 1 and Volume 4- Part II
 ICH Q5CC
 Guidance for the Industry “Container and Closure System Integrity Testing in
Lieu of Sterility Testing as a Component of the Stability Protocol for Sterile
Products”
 USP <1207>
 PDA Technical Report 27
 PDA White Paper- “Container Closure Integrity Control versus Integrity Testing
during Routing Manufacturing”
 FDA Compliance Program Guidance Manual, Chapter 56- Drug Quality
Assurance Program 7356.002A
 Summary
 Container Closure Integrity Testing is currently reviewed by
regulators
 Many different methods are available to perform CCIT.
 The proposed USP <1207> update divides the methods into deterministic
and probabilistic.
 There is a move to move toward more deterministic methods.
 The USP <1207> revised guidance series is expected to become
effective in August 2016 and removes the recommendation to
compare CCIT methods to microbial ingress testing
 Methods described in the proposed USP <1207> update include:
 Electrical Conductivity  Microbial Challenge by
and Capacity Test Immersion
(HVLD)  Tracer Liquid Tests (e.g.
 Mass Extraction Dye Ingress)
 Pressure Decay  Bubble Tests
 Tracer Gas (vacuum  Tracer Gas (Sniffer
mode) Mode)
 Vacuum Decay
 Summary
 Packageintegrity verification occurs during
three product life cycle phases:14
1. The development and validation of the product-package
system
2. Product manufacturing
3. Commercial product shelf-life stability assessments
 CCIT needs to show container integrity over the
life cycle of the product.
 CCITmethods require optimization for each
product-container/closure application.
 All methods have limitations
 Summary
 During method development, a preliminary method is
chosen, optimized, and qualified.
 Target acceptance criteria will be established
 Positive and negative controls will be created
 Leak test method parameters will be developed and
optimized through experimentation.14
 Qualification(s) trials will be performed
 Quantities for testing should be sufficient to provide
adequate assurance of package integrity. 14
 Summary
 Container closure integrity needs to be validated for
each specific drug-product package.
 Validations are typically performed in triplicate.
 USP <1207.1> recommends to randomly mix negative
and positive control populations over multiple days,
with multiple operators, and if possible, using multiple
test instruments.
 Acceptance criteria should include the following:
1. All negative controls pass (no leaks are identified) 14
2. All positive controls with leaks at or above the designated limit of
detection fail (leaks are detected). 14
3. An integral package is one that conforms to specific product-package
maximum allowable leakage limits.13
4. Upper and lower limits of detection should be established as well as any
key testing parameters.
 Summary
 Routine and stability testing should be performed per
an approved standard operating procedure or
method.
 Package integrity should be re-evaluated when
changes are required in package design, package
materials, or manufacturing/processing conditions.14
 CCIT can be used in place of sterility testing during
stability studies.
 Non-destructive CCIT methods save product and
money.
 Bracketing strategies can be utilized during stability
studies when appropriate.10
Using an interactive game, participants review information pertaining to
monitoring microbial metrics.
BINGO
1. Container closure systems should maintain the ___________and
product quality of sterile finished products.
 Sterility
2. All methods have pros and __________.
 Cons
3. Some container, such as ________, require 100% integrity testing.
 Ampoules
4. The proposed USP <1207> revisions remove the requirement to
compare CCIT results to the ___________ingress challenge.
 Microbial
5. The proposed USP <1207> divides the test into _______ major
categories.
 Two
BINGO
6. _____________ assays are less prone to error.
 Deterministic
7. The microbial ingress challenge is a ______________ assay.
 Probabilistic
8. The _______ leak test is the most common liquid tracer
test.
 Dye
9. Testing should show the complete CCI picture over the
___________ of the product
 Lifecycle
10. All __________ controls with leaks at or above the claimed
limit of detection must fail.
 Positive
BINGO
11. A lower limit of _______________ must be established.
 Detection
12. Quantities of samples to be tested must be ____________ to provide
adequate assurance of package integrity.
 Adequate
13. Package integrity should be ______________ when changes are
required in package design.
 Re-evaluated
14. All __________ controls must pass.
 Negative
15. CCIT can be used to replace sterility testing in ___________
protocols
 Stability
BINGO
16. Container __________ integrity methods needs to be validated for
specific drug product packages.
 Closure
17. Revisions to USP <1207> series are expected to become official in
__________ 2016.
 August
18. Package integrity verification occurs during ________ product life
cycle phases.
 Three
19. ____________ drilled holes can be used to create positive controls.
 Laser
20. Choose a preliminary method following vendor’s recommendations or
____________ research.
 Literature
BINGO
21. The vacuum decay method is _____________ and quantitative.
 Nondestructive
22. The dye leak test is destructive and _______________.
 Qualitative
23. CCIT can be performed in ___________ different ways.
 Many
24. _____________ may not replace the sterility test for release testing.
 CCIT