13
Validation of Terminal Sterilization
Thomas J. Berger and Kevin D. Trupp
Department of Microbiology and Global Process Sterilization Engineering, Hospira, Inc.,
Lake Forest, Illinois, U.S.A.
INTRODUCTION TO PARENTERAL PRODUCT
STERILIZATION
The previous chapter discussed the steam sterilization
approach for the processing of hard goods or porous
loads. This chapter will discuss the sterilization vali-
dation approach that can be used in the processing of
parenteral products by terminal sterilization using moist
heat. The underlying principles of steam sterilization are
applicable to both hard goods and terminal sterilization
of parenteral products, but both have their unique
characteristics.
An organized sequential ow of activities must
occur as new parenteral formulations are developed,
and subsequently processed in the manufacturing facility.
The moist heat sterilization of pharmaceutical solutions is
established and veried through a series of activities that
conrm the product has received a dened thermal
exposure that renders the product sterile. R&D activities
can include sterilization developmental engineering
studies consisting of sterilization cycle development;
container thermal mapping; microbial closure validation,
D- and z-value analysis; containerclosure integrity vali-
dations as well as nal formulation stability studies.
The subsequent production phase activities must
include initial sterilization vessel qualication which
demonstrates that the vessel will deliver the dened
sterilization process in a consistent and reproducible
manner. Also, solution and containerclosure microbial
validation studies must be conducted at subprocess
production sterilization conditions employing heat-
resistant microorganisms. Equipment validation,
ltration studies and assessment of the bioburden on
Abbreviations used in this chapter: AAMI, Association for the
Advancement of Medical Instrumentation; APE, antimicrobial
preservative efcacy; API, active pharmaceutical ingredient;
ASME, American Society of Mechanical Engineers; BET, bacterial
endotoxin testing; BI, biological indicator; BIER, biological
indicator evaluator resistometer; DP, direct plate; EMA, European
Medicinal Agency; F/N, fraction/negative; GAMP, good auto-
mated manufacturing practice; HMI, humanmachine interface;
I/O, input/output; ICH, International Conference on Harmoniza-
tion; ISPE, International Society for Pharmaceutical Engineering;
LAL, limulus amebocyte lysate; LVP, large volume parenterals;
MES, manufacturing execution system; MOS, maintenance of
sterility; P&D, penetration and distribution; PLC, programmable
logic controller; PSLR, predicted spore logarithmic reduction; R&D,
research and development; RTD, resistance temperature detector;
SCADA, supervisory control and data acquisition; SLR, spore
logarithmic reduction; SVP, small volume parenterals; TC,
thermocouple.
component parts, as well as the environment, must also
be ascertained.
The developmental and production phases of ster-
ilization technology activities are then drafted into
documents that are submitted as part of a new drug
application for the particular parenteral formulation.
These reports must follow applicable regulatory require-
ments for products that are terminally sterilized. Such
studies allow one to establish, with a high level
of sterilization assurance, the correct sterilization cycle
(F0, temperature, product time above 1008C, etc.) to be
used for the steam sterilization of a specic parenteral
formulation in a particular containerclosure system.
STERILIZER DESIGN
The validation of a steam sterilization cycle is dependent
on the equipment chosen. The sterilizer and its support
systems must be designed and constructed to deliver the
effective cycles repeatedly and consistently. Qualication
of the sterilizer consists of proper design, installation
according to design, operational testing to ensure that
design criteria and operational requirements are met and
performance qualication to conrm that the product is
sterilized per specication.
Sterilizer design is geared to the type of product or
materials/equipment to be sterilized. All steam steriliza-
tion cycles are based on contact with saturated steam,
steamair mixtures or superheated water. Saturated
steam is water vapor in equilibrium with liquid water.
The values of temperature and pressure at which pure
saturated steam can exist are shown by the phase
diagram in Chapter 12, Figure 3. Saturated steam can
exist only along the phase boundary for liquid and
gaseous water; that is, the relation between its tempera-
ture and pressure is xed. An increase or reduction in the
temperature of saturated steam must result in a corre-
sponding increase or decrease in its pressure and vice
versa. Steamair mixtures can be used when overpressure
is required to maintain product shape or container
integrity. Superheated water cycles require air overpres-
sure and the water is either heated by direct injection of
steam or indirectly via a heat exchanger.
Parts and hard goods are typically steam sterilized
using a saturated steam process whereas the trend for
product sterilization is towards the use of superheated
water or steamair mixture processes. These processes are
needed as a majority of the new products require air
overpressure during the sterilization process to maintain
desired container characteristics and integrity.
188 III: STERILIZATION, SANITIZATION AND STERILITY ASSURANCE
Products in glass containers can utilize the satu-
rated steam processes as described in Chapter 12, but
many products and containers require the use of air
overpressure during the sterilization process. This
section will discuss some of the key design considerations
for terminal steam sterilizers and provide some specics
for the various type steam sterilization processes utilizing
air overpressure.
Typical Design Considerations for Steam Sterilizers
1. A pressure vessel constructed according to the
ASME or equivalent international code. This must
withstand at least 50% in excess of the required
internal pressures.
2. A safety door mechanism to prevent opening while
the unit is under pressure: the locking device may be
actuated directly by internal pressure or indirectly
through an automatic switch. The door itself may be
of the swing-out or sliding type. Separate entry and
exit doors are preferable.
3. Process control system (typically a PLC for control-
ling and monitoring the process).
4. Process data recorder or data collection system.
5. Product racks designed to hold/support the sealed
product containers and to provide adequate
heating/cooling media ow throughout the
product zone.
6. Pressure safety relief valves for both the chamber and
jacket (if equipped with jacket).
Note. A microbial retentive vent/air lter would not
typically be required for processes used for terminal
sterilization as there is no direct contact between the
heating/cooling media and the contents of the containers.
Steam Air Mixture Sterilization
The primary benet to the steamair mixture process over
a superheated water process is the product is not
subjected to direct contact with water (except as conden-
sate), which in some cases can cause cosmetic issues with
the container. Steamair mixture processes typically
utilize large recirculating fans to prevent the formation
of cold/hot spots in the sterilizer. The steamair mixture
process typically uses an indirect cooling method such as
cooling of the jacket or with cooling coils within the
sterilizer. Because of this indirect cooling method, the
cooling rate of the product is typically much slower and
less efcient than direct exposure of the product
containers to cooling water.
Some of the specic sterilizer design considerations
for a steamair mixture process include the following:
1. A jacket and insulation: the jacket would utilize steam
during heating and exposure phases of the cycles and
cooling water can be introduced to the jacket during
the cooling phase of the process.
2. A thermostatic steam trap to efciently remove the
condensate from the chamber: this is open when cool
(in contact with air or condensate) and closed when in
contact with steam. As condensate collects, the trap
opens owing to the slight temperature reduction and
the condensate is discharged. There is also a similar
steam trap to remove steam condensate from
the jacket.
3. Fan(s) to continuously recirculate the steamair
mixture during heat-up and exposure and to recircu-
late the air during cooling.
4. Cooling provisions (e.g., cooling coils) to cool the
air/product.
Recirculated Superheated Water Sterilization
Sterilization with recirculating superheated water (some-
times referred to as a water cascade or raining process) is
more efcient than a steamair mixture and is therefore
more common. There are many types of recirculating
superheated water processes, the most common is a
process where the bottom portion of the sterilizer
(below the product zone) is lled with water and a
recirculation pump is used to continuously recirculate
water from the bottom of the sterilizer to spray nozzles
above the product zone. A slight modication to that
process is the use of a water distribution pan in lieu of
spray nozzles. Another version of the recirculating
superheated water process is to completely submerge
the product in water but this process is inefcient from
a utilities consumption standpoint. All of these recircu-
lating superheated water processes utilize air
overpressure and the overpressure can be controlled
during the sterilization process to minimize most types
of container deformation. There is no limit to the
maximum overpressure used but it would typically be
limited by the chamber pressure rating. The minimum
overpressure will be driven by the temperature being
used, the pressure needed to maintain the desired
product characteristics and the required overpressure
needed to prevent the recirculation pump from loosing
prime. These recirculating processes are typically heated
and cooled indirectly with external heat exchangers
located in the recirculating water loop but direct injection
of steam and cooling water can also be used.
In addition to the typical sterilizer design consider-
ations mentioned earlier, a superheated water sterilizer
would also include a large recirculating water system
(e.g., pump, pipes, heat exchangers, headers, spray
nozzles) including specic water level control valves
and monitoring devices.
Rotary and Shaker Sterilization
In some cases, certain products (i.e., suspensions and
emulsions) require agitation during the sterilization
process. For those types of products, it is typical to use a
rotating rack within the sterilizer but other agitation
methods such as an internal shaking device are available.
Refer to Figure 1 for the typical design of a rotary sterilizer
and Figure 2 for the typical design of a sterilizer using
a shaking mechanism. It is possible to use any of the
sterilization processes listed above with product agitation.
Continuous Sterilization
For this version of the superheated water process,
containers are terminally heat sterilized in a continuous
sterilizer by a process where the containers move through a
constantly controlled environment in carriers with individ-
ual compartments. The time, temperature, and pressure
requirements are set to predetermined values and are
automatically and continuously controlled, monitored,
and recorded. Refer to Figure 3 which depicts the pattern

Figure 1 Photo of sterilizer with a rotary mechanism. Source:
Photo Provided by Fedegari Autoclavi SpA, Albuzzano, Italy.
that containers (e.g., parenteral exible product containers)
follow as they move automatically through the continuous
sterilizer. The water lock of the pressure vessel is used to
provide product entrance into and out of the overpressure
environment. The overpressure environment is constantly
maintained within predetermined limits.
The sterilizing phase begins as the product enters
the hot water environment within the pressure vessel.
The hot water environment may be a superheated water
Suspension Rods
Platform Frame
Platform Lock Arm
Lock Screw
Platform Rails Bridge Plate
Figure 2 Photo of sterilizer with a shaking mechanism.
13: VALIDATION OF TERMINAL STERILIZATION 189
spray, which is circulated over the top of the continuously
moving carriers. The residence time of the product within
this sterilizing environment and the water temperature
are controlled within predetermined limits to assure the
required heat input.
Cooling begins as the product transfers from the
sterilizing environment and enters the cooling environ-
ment, which is also within the pressure vessel. The
cooling water environment is a cool water spray that is
circulated over the top of the continuously moving
carriers. The temperature of this water is controlled
within predetermined limits to assure that the required
degree of cooling is achieved before the product leaves
the cooling environment.
A system of xed temperature sensors located in the
entering and exiting recirculating water for both heating
and cooling continuously monitors, records, and controls
the temperature of the process water.
Air overpressure is required to protect the con-
tainer from stress while exposed to the high sterilizing
temperatures.
STERILIZER CONTROL SYSTEMS
A key to effective sterilizer operation lies in the auto-
mated process control system. By eliminating the
dependence on operator intervention and data recording,
automatic temperature and sequential control provides
assurance that the validated sterilization cycle is
consistently and repeatedly delivered. A typical control
system for a new sterilizer includes the following hard-
ware components:
& PLC
& Operator interface panel(s)
& Data recorder/data collection system
& Process Variable Sensors
& I/O devices
The PLC is most commonly used as the primary
component of the automated process control system as it
provides sequential control of the process, provides
control of all proportional valves, controls all devices,
receives operator input via the operator interface panels
and provides process information (such as process vari-
able information and alarms) to the operator via displays
and/or operator interface panels. The PLC typically
contains specic recipe information for the various
cycles to be utilized. In some cases the PLC can be used
for data collection, but it is much more common to use a
separate data recorder/data collection system.
The operator interface panel can be as simple as
switches and displays or as complex as a stand-alone PC
running a SCADA with a HMI software package. These
devices are typically used to select the recipe, start the
cycle and display process information during the cycle.
The higher level PC based SCADA type operator inter-
face panels can provide detailed cycle reports and
trending information.
The data recorder/data collection system can range
from a simple strip chart recorder to a full-blown MES
type data collection system. In many cases the PLC can
also provides batch data logging functionality. The
minimum variables to record for steam sterilization
processes are typically time, temperature, and pressure.
190 III: STERILIZATION, SANITIZATION AND STERILITY ASSURANCE
Steam Flowrate
Sensor
Heaters
Surge Tank
Pump
Sterilizing Tank
Temperature
Sensor
Cooling Tank
Tower Water
Heat Exchangers
Surge Tank
Mixing
Tank
Pump Tower Water
Ozone
Figure 3 Schematic of a sterilizer for continuous processing of exible containers.
Typical sensors include temperature measurement
devices (RTDs or TCs), pressure measurement devices,
and where applicable level measurement devices and
ow measurement devices. It is customary that the
temperature sensor used to control the process tempera-
ture not be used to provide the batch record process data.
An independent/secondary temperature sensor for batch
reporting provides a high degree of insurance that the
cycle actually ran within its dened limits. Heavy wall
thermowells should not be used, as this will affect the
time response of the measurement. Thin-walled thermo-
wells or temperature elements with stainless steel sheaths
should be used for temperature measurement.
The pressure sensor should be equipped with a
sanitary-type diaphragm and connected to the sterilizer
using a sanitary tting. A sanitary diaphragm can intro-
duce errors to the pressure measurement due to the
stiffness of the diaphragm. This stiffness is related to the
size of the diaphragm. The impact is negligible for
diaphragms above 3 inches diameter. This should be
considered when sizing the connection to the sterilizer.
Sterilizers that maintain a specic water level (i.e.,
recirculated water process) should be equipped with liquid
level sensors. These sensors may be in the form of single-
point-level-type probe or a continuous level sensor. Regard-
less of what type sensor is used a separate high-level sensor
must also be provided. The separate high-level sensor
provides greater assurance that the collected water at the
bottom of the vessel remains below the product level.
Indexer
Entry
Lock
Take-Up
Shuffle
Exit
Lock Unloading
Temperature
Sensor
Surge Tank
Pump Pump
Sterilizers that rely on recirculated water as part of
the sterilization process can include a ow sensor. The
ow sensor may be a direct measurement such as a ow
meter (i.e., coriolis, ultrasonic, magnetic, etc.) or an
indirect measurement such as differential pressure
sensor across the recirculation pump. Direct measure-
ments are always preferred.
For I/O devices, there are analog types and discrete
types. The analog inputs are typically from process
sensors and the analog outputs are typically for control
of proportional valves. The discrete inputs are typically
from switch type (operator and process) devices and the
discrete outputs are typically for activating hardware
such as valves, pumps, lights, etc.
The design and development of the sterilizer
control system software should follow the principles of
ISPE GAMP 4 Guide for Validation of Automated
Systems (1). This guideline details a software life cycle
from conception thru decommissioning.
STERILIZATION CYCLES
The type of steam sterilization cycle to be utilized is
dependent on product needs and equipment availability.
As discussed in Chapter 12, the sterilization of hard goods
or porous loads typically require the use of a pulsed pre-
vacuum cycle as it is preferable to remove the air from the
porous materials being sterilized whereas in the terminal
sterilization of aqueous solutions in sealed containers,

the major concern is to provide rapid heat transfer to the
wall of the lled product containers and air removal is not
required (nor even desirable as the hydrating moisture is
contained within each container). Parenteral products may
be lled into rigid or exible containers. In either there is
typically air or nitrogen present in the headspace above the
liquid. As the solution is heated, this gas expands and adds
to the internal pressure increase resulting from the
evolution of water vapor from the aqueous vehicle within
the heated container. Thus, the pressure within the
container will exceed the chamber pressure during steam
process for sealed containers.
Glass vials can be sealed with special closures to
withstand this pressure. As long as the pressure differ-
ential between the chamber and the containers does not
become too great during the steam exhaust portion of the
cycle, the vials will not burst. If rapid cooling of the load
is desired, the pressure differential might become signi-
cant enough to cause closure integrity to be lost.
Plastic bags, semi-rigid containers and syringes
present a greater problem because they do not have the
inherent strength of glass and may burst or deform as the
pressure differential increases. To prevent this, air must be
injected into the chamber to raise the pressure above the
saturation pressure of the steam. This is particularly
important during the cooling cycle, when the chamber
pressure is reduced at a much faster rate than that within
the container.
The following section provides a description of the
various steam sterilization cycles used for parenteral
products in sealed containers.
Saturated Steam Pre-Vacuum Cycle
For a saturated steam process, the most common (and
perhaps most effective) method to remove the entrapped
air from the sterilizer is to remove it mechanically before
the actual sterilization begins. This is done by means of a
mechanical vacuum pump or steam eductor. This cycle
can be used for products in glass containers. A sketch of a
typical pre-vacuum cycle is shown in Chapter 12,
Figure 7.
Saturated Steam Gravity Displacement
or Steam Purge Cycle
Other means for eliminating air without a vacuum
source include the use of a gravity displacement cycle
or a steam purge cycle. For the gravity displacement
cycle, steam is introduced on the side or top of the
vessel and the cold air is forced out via the drain. The
steam purge cycle uses large quantities of steam distrib-
uted via headers under the entire product zone with
numerous large vents located at the top of the vessel.
For these types of cycles, the appropriate vents or drains
should be fully open and the large steam supply valve
fully open for an extended time and temperature to
ensure that the air is adequately removed from the
sterilizer. Once the vents and drains close, the process
runs like a traditional saturated steam process. It is
important to determine that the measured temperature
and pressure are consistent with the steam saturation
curve in Chapter 12, Figure 3. This process can be used
with glass containers.
13: VALIDATION OF TERMINAL STERILIZATION 191
Steam Air Mixture Cycle
It is important to understand the physical principle
involved in a mixture of steam and air. The xed rela-
tionship between temperature and pressure seen in
Chapter 12, Figure 3 no longer applies. Daltons law
states that the pressure of an ideal mixture of gases is
equal to the sum of the partial pressure of the gases, or
P Z PA C PB C PC :
Raoults law further states that, for ideal mixtures,
the partial pressure of the gas is equal to its vapor pressure
multiplied by the mole fraction in the liquid. For steam in
equilibrium with pure condensate, this reduces to
PA Z pA
where PA is the partial pressure of steam and pA is the
vapor pressure of the condensate. The difference between
the observed chamber pressure P and PA is the partial
pressure of air.
The presence of air, although necessary for the
maintenance of container integrity, can reduce the heat
transfer efciency. The objective of the design in the
air overpressure cycle is to maintain a well-mixed
chamber. This assures that the heat transfer to the load
will be uniform regardless of the presence of air. Mixing
may be accomplished in several ways. The air may be
injected directly into the incoming steam. Usually,
though, some mechanical means is selected.
Most steamair sterilizers use a fan built into the top
or end of the chamber, which circulates and mixes the air
and the steam (Chapter 12, Fig. 9). Some steamair
sterilizers are capable of using water during the cool-
down process to cool the containers more rapidly. This
rapid cooling may also be necessary for product stability.
Various methods (i.e., direct injection, recirculation
through a heat exchanger, etc.) for introducing the
cooling media can be utilized.
Recirculating Superheated Water Cycle
The typical recirculating superheated water process
(sometimes referred to as a water cascade or raining
process) begins by the addition of water to the sterilizer
to a predened level (below the product zone). Then a
water recirculation pump is started to continuously
recirculate water from the bottom of the sterilizer to
spray nozzles or a water distribution pan above the
product zone. The recirculation pump is on throughout
the heat-up exposure and cool-down phases. During
heat-up, the water is heated at a pre-dened rate via a
heat exchanger in the recirculation loop or with the direct
injection of steam. Also during heat-up, compressed air is
added to the chamber to attain the desired overpressure
levels. Once the temperature set point is achieved, the
controller steps into the hold portion of the cycle and the
temperature and pressures are maintained at the desired
levels. For cooling, the steam supply is shut off and the
recirculating water is cooled at a controlled rate by
introducing cooling media to a heat exchanger installed
in the water recirculation loop or by the direct injection of
cooling water into the recirculating loop. This type of
process does not require the use of a jacket but does
require specic water level controls.
192 III: STERILIZATION, SANITIZATION AND STERILITY ASSURANCE
The recirculating superheated water process is very
efcient and the temperatures and pressures can be
tightly controlled during the entire process, thus mini-
mizing container stresses.
STERILIZATION CYCLE DEVELOPMENT
This section will address sterilization and associated
microbiological activities that occur in R&D areas as
well as the production environment when using the
BI/bioburden approach in support of a parenteral
product. The list below depicts some of the sterilization
engineering and microbiological activities associated
with a parenteral product as it moves through develop-
ment. These studies or similar ones are ordinarily
conducted in developmental sterilizers or may occur as
investigative engineering studies in a production steri-
lizer as appropriate. The overkill method can be used for
some of the more stable parenteral formulations, and its
validation is accomplished as described in Chapter 12.
Sterilization development activity Activity statement
Cycle development Develop preliminary
container sterilization
specications with
engineering parameters
such as temperature,
time and F0
Container thermal mapping Determine cold spot and
assess heat penetration
within nished container
Formulation development Perform analytical
feasibility studies prior to
product nalization with
methods validations
Parenteral solution microbiological
evaluations:
Moist heat D- and z-value analysis Perform triplicate D-value
analysis on each
parenteral formulation at
three temperatures,
e.g., 1128C, 1188C and
1218C and then
calculate the z-value
APE Perform on nal product if
it contains a
preservative or if there is
a multidose claim for the
container
In-process bioburden analysis Perform studies with a
panel of microorganisms
to validate 70% recovery
for the ltration process
Spike hold time studies Inoculate parenteral
product with bioburden
and growth promotion
compendia organisms to
evaluate the products
ability to support
microbial growth
Container closure evaluations:
Microbial closure inactivation Perform kill curve kinetics
using bioburden and BI
(spores) inoculated onto
the worse case closure
site
Container integrity Perform dye ingress,
microbial challenge or
physical integrity tests
following exposure to
maximum sterilization
conditions that stress
the container
Stability runs Perform analytical
chemistry and
microbiological
evaluations at various
temperatures and times
per ICH and or
compendia
requirements
BET Perform test method
validation of API,
excipients and nal
product per compendia
requirements
Sterilization engineering personnel primarily focus
their efforts in determining whether a parenteral formu-
lation packaged in a particular container conguration can
be sterilized in a current cycle or whether a new cycle must
be developed. The referenced EMEA (2) decision tree is
followed when evaluating a new parenteral product in an
LVP or SVP container. Sterilization feasibility studies are
conducted in a sterilizer to ascertain the physical effects of
the cycle on the product in question. Product attributes
that can be affected by a cycle are closure integrity, product
potency, pH, color, shelf-life stability, visible, and subvi-
sible particulates as well as nal product sterility. Once the
basic engineering parameters (e.g., temperature, time and
F0) are established, then engineering thermal container
mapping studies can be performed (3,4).
Container Thermal Mapping Validation Studies
An R&D sterilizer is smaller than a production facility
sterilizer, but can simulate the sterilization cycles
conducted in the larger production vessels. Container
thermal mapping studies (when applicable) are typically
performed in a laboratory sterilizer:
1. To locate the coldest zone or area inside a container.
2. To determine the cold zone in the container and its
relationship to the location monitored during
validation studies.
3. To generate data that may be used during the setting of
production sterilization control parameters.
When conducting thermal mapping studies, there
are various factors to be considered, and these are
dependent upon the:
1. Type of container (exible or rigid)
2. Container orientation, size and ll volume
3. Cycle type and temperature
4. Viscosity
5. Autoclave trays/design/surface contact
6. Autoclave spray patterns/water ow.
Typical container mapping data obtained for lipid
emulsions contained within a 1000 mL glass container are
shown as an example in Tables 1 and 2. The following
summarizes the process for obtaining heat map data from
the glass intravenous container lled with approximately
1000 mL of lipid emulsion.
 13: VALIDATION OF TERMINAL STERILIZATION 193

Table 1 Heat Input (F0 Units)

1000 mL glass I.V. containers-heat mapping study (lipid emulsion)
Run CLHK00.049 Run CLHK01.050
TC number btl 1 btl 2 btl 1 btl 2 Average (SD)
1,12 7.91 C7.28 8.13 C7.36 7.67 (0.415)
2,13 (PC) 7.79 7.49 8.02 7.64 7.74 (0.226)
3,14 C7.46 7.40 C7.71 7.47 C7.51 (0.137)
4,15 7.64 7.80 7.87 7.96 7.82 (0.135)
5,16 12.66 12.90 12.95 12.91 12.86 (0.132)
6,17 12.73 12.46 12.77 12.68 12.66 (0.138)
7,18 12.78 12.69 12.95 12.91 12.83 (0.120)
8,19 13.32 13.33 13.42 13.78 13.46 (0.223)
9,20 14.21 14.33 14.03 14.56 14.28 (0.222)
10,21 H15.87 H17.24 H15.18 H16.09 H16.10 (0.856)
11,22 15.47 16.56 14.77 16.07 15.72 (0.773)
HC 8.41 9.96 7.47 8.73 8.64 (1.028)
PCC 0.33 0.21 0.31 0.28 0.28 (0.053)
Note: H denotes hottest TC location; C denotes coldest TC location; PC denotes approximate location of the production prole TC; Data from TC#9 used with a
postcalibration variance of C0.258C at 1008C; All heat input values are calibration corrected.

TC probes (Copper Constantan, type T,
0.005 in. diameter) were used to monitor 11 locations
within the 1000 mL container. The TC probes were posi-
tioned at various distances (in inches) as depicted (Fig. 4).
Each container was lled with approximately 1000 mL
of the lipid emulsion, evacuated to 20 in. of mercury and
sealed with an aluminum overseal.
A at perforated rack on a reciprocating shaker cart
was used in the autoclave. The cycles target temperature
was 1238C, recirculating water spray cycle with 70 rpm of
axial agitation and 30 psig (pounds per square inch) of air
overpressure.
When the sterilization cycle was controlled to give a
heat input of approximately 7.5 F0 minutes in the coldest
emulsion area, the average coldest emulsion area was
found to be measured by TC number (TC#) 3,14. The
average hottest emulsion area was measured by TC# 10,
21. The difference between the hottest and coldest emul-
sion areas ranged from 7.5 to 10.0 F0 minutes with an
average of 8.6 F0 minutes. Therefore, when the coldest
emulsion area registered 7.5 F0 minutes, the hottest
emulsion area would average 16.1 F0 minutes.
The emulsion area approximating the validation TC
location was measured by TC # 2,13 and averaged 7.7 F0
minutes when the coldest emulsion was approximately
7.5 F0 minutes (Fig. 5).
Solution/Product Moist Heat Resistance D- and
z-Value Analysis
A BIER vessel meets specic performance requirements
for the assessment of BIs per American National Stan-
dards developed and published by AAMI (5). One
important requirement for a BIER steam vessel is the
capability of monitoring a square wave heating prole.
Refer to Figure 6 for a schematic of the steam BIER
vessel used to generate the D- and z-value data. D-value
is the time in minutes required for a one log or 90%
reduction in microbial population (Refer to Chapter 12,
Fig. 1). The z-value is the number of degrees of

Table 2 Solution Heat Rates (Minutes)

1000 mL glass I.V. containers-heat mapping study (lipid emulsion)
Run CLHK00.49 Run CLHK01.050

btl 1 btl 2 btl 1 btl 2 Average (SD)

Coldest location
Thermocouple number 3 12 3 12
Time to 1008C 19.0 19.0 19.0 19.0 19.00 (0.000)
Time R1008C 21.0 21.0 21.0 21.0 21.00 (0.577)
Time R1208C 4.0 3.0 4.0 3.0 3.50 (0.577)
Time R120K1008C 4.0 5.0 4.0 5.0 4.50 (0.577)
Maximum temperature (8C) 120.82 120.77 120.92 120.77 120.82 (0.071)
Heat input (F0) 7.46 7.28 7.71 7.36 7.45 (0.187)
Production prole TC location
Thermocouple number 2 13 2 13
Time to 1008C 19.0 19.0 19.0 19.0 19.00 (0.000)
Time R1008C 22.0 21.0 22.0 21.0 21.50 (0.577)
Time R1208C 4.0 3.0 4.0 3.0 3.50 (0.577)
Time R120K1008C 5.0 5.0 5.0 5.0 5.00 (0.000)
Maximum temperature (8C) 120.91 120.82 120.91 120.92 120.89 (0.047)
Heat input (F0) 7.79 7.49 8.02 7.64 7.74 (0.226)
Note: H denotes hottest TC location; C denotes coldest TC location; PC denotes approximate location of the production prole TC; Data from TC#9 used with a
postcalibration variance of C0.258C at 1008C; All heat input values are calibration corrected.
194 III: STERILIZATION, SANITIZATION AND STERILITY ASSURANCE

PSLR Values
1000 mL Glass I.V. Container Lipid emulsion moist heat resistance values (D1218C and
- Heat Mapping Study z-values) were generated in the steam BIER vessel using
Thermocouple Locations the BI C. sporogenes as shown in Table 3. The columns in
the Table list the representative code or list number of the
product, the emulsion or product name, its average D1218C
11/12 11/2 11/2 11/2
value and z-value and nally the PSLR value. Those
parenteral formulations with the lowest PSLR value(s)
are those that should be used for the microbial validation
Fill Level
at subprocess conditions, since these provide the most
10,21 11,22
3/4 microbial resistance (6).
5,16 6,17 7,18 8,19 9,20

11/12 Accumulated Fbio for Lipid Emulsions

1,12 2,13 3,14 4,15
Figure 4 Heat mapping study using a 1000 mL glass container.
temperature required for a 10-fold change in the D-value.
(Refer to Chapter 11 for additional details on F-, D- and
z-values.)
Master Solution/Product Concept
The family category of lipid emulsions and their respective
D1218C and z values as well as classication in terms of
microbial resistance is shown in Table 3. A categorization
of parenteral formulations with associated D1218C and
z-values and their potential impact on microbial resistance
using the BI, Clostridium sporogenes were previously
reported (6). In addition, the methodologies used for D-
and z-value analysis were likewise cited. The data in
Table 3 indicate that the # 1 emulsion is at the top of the
list, since it affords the most microbial moist heat resist-
ance. It is therefore the emulsion that should be
microbiologically challenged (inoculated with spores) as
part of the emulsion validation scheme. D- and z-value
data have been reported for other BIs such as Geobacillus
stearothermophilus (68) and Bacillus subtilis 5230 (9). There
are many factors that can affect moist heat resistance
including a BIs age, sporulation media used, as well as
the particular spore strain employed (10).
Accumulated Fbio and z-values (Table 4) were used to
construct the PSLR ranking for lipid emulsions as pre-
viously discussed for Table 3. The Fbio is the heat input for
the biological solution based on the emulsions moist heat
D- and z-values. By inputting the sterilizer temperatures
from the coldest TC of an engineering run for a particular
container/sterilization cycle, the emulsion can be ranked
according to PSLR values. The combined D1218C and
z-value allows comparison of moist heat rankings
between emulsions.
The data in Table 4 demonstrate that the #1 Emul-
sion has the lowest PSLR (7.105), thereby affording the
highest moist heat resistance upon inoculation. Gener-
ation of this table allows prediction of which emulsion to
microbiologically challenge as part of validation in the
production sterilizer.
Microbial Closure Inactivation Validation in a
Developmental Sterilizer
In lieu of using the large type steam sterilizers in the
production environment, microbial inactivation at the
closure/bottle interface of an emulsion container can be
assessed in a developmental sterilizer. The closure micro-
bial inactivation (kinetic) studies can determine how the
size of the container, type of closure compound used as
well as closure preparatory processes (e.g., leaching,
washing, siliconizing, autoclaving) inuence microbial
inactivation. Microbial closure kinetic studies are
conducted at various time intervals in a given steriliza-
tion cycle. DP count or F/N methodologies are used to

1000 mL Glass I.V. Container

- Heat Mapping Study

Average Heat Input (F0) at Various Locations

Fill Level
16.1 15.7
12.9 12.7 12.8 13.5 14.3

7.7 7.7 7.5 7.8

Figure 5 Heat mapping study with average
heat input (F0) at various locations in a
1000 mL glass container.
 13: VALIDATION OF TERMINAL STERILIZATION 195

Schematic of the Steam B.I.E.R Vessel

Top Instrument
Chamber Well Vent
Vent Thermocouples Pressure
to Digital Display Recording
Sample and Datalogger Controller
Chamber Steam
Control
Valve

Steam Reservoir

Drain/Lower
Main
Figure 6 Schematic of a steam biological
Chamber indicator evaluator resistometer vessel used
Vent Steam
Supply for generating moist heat resistance D- and z-
values for inoculated parenteral solutions or for
biological indicators.

Table 3 IV Lipid Emulsions Ranking

Predicted spore log
List # Solution D121 z-value reduction
1 20% Emulsion 0.7 10.6 7.1
2 10% Emulsion w/increased linolenate 0.7 11.4 7.5
3 10% Emulsion w/100% soybean oil 0.6 10.1 8.0
4 20% Emulsion w/100% soybean oil 0.7 12.8 8.2
5 20% Emulsion w/increased linolenate 0.6 10.6 8.3
6 10% Emulsion w/50% safower & 50% soybean oil 0.6 10.7 8.4
7 20% Emulsion w/50% safower & 50% soybean oil 0.6 12.7 9.5
8 10% Emulsion 0.4 11.1 12.9

Table 4 Accumulated Fbio by List Number and z Value

Solution
Temperature Time F (PHY) 1 2 3 4 5 6 7 8
(8C) (min) zZ10.0 zZ10.6 zZ11.4 zZ10.1 zZ12.8 zZ10.6 zZ10.7 zZ12.7 zZ11.1
105.4 1 0.0269 0.0330 0.0419 0.0278 0.0592 0.0330 0.0340 0.0579 0.0384
110.1 1 0.0793 0.0915 0.1082 0.0813 0.1380 0.0915 0.0935 0.1359 0.1019
114.1 1 0.1991 0.2181 0.2427 0.2023 0.2834 0.2181 0.2212 0.2806 0.2336
116.2 1 0.3228 0.3442 0.3709 0.3265 0.4134 0.3442 0.3476 0.4106 0.3611
118.1 1 0.5000 0.5200 0.5445 0.5035 0.5819 0.5200 0.5232 0.5794 0.5356
119.1 1 0.6295 0.6462 0.6663 0.6324 0.6966 0.6462 0.6489 0.6946 0.6591
119.4 1 0.6745 0.6897 0.7079 0.6772 0.7352 0.6897 0.6921 0.7334 0.7014
119.2 1 0.6442 0.6604 0.6799 0.6470 0.7092 0.6604 0.6630 0.7073 0.6729
118.5 1 0.5483 0.5672 0.5903 0.5515 0.6253 0.5672 0.5703 0.6230 0.5819
117.8 1 0.4667 0.4872 0.5124 0.4702 0.5513 0.4872 0.4905 0.5487 0.5033
116.2 1 0.3228 0.3442 0.3709 0.3265 0.4134 0.3442 0.3476 0.4106 0.3611
114.1 1 0.1991 0.2181 0.2427 0.2023 0.2834 0.2181 0.2212 0.2806 0.2336
110.6 1 0.0889 0.1020 0.1197 0.0911 0.1510 0.1020 0.1042 0.1487 0.1130
105.9 1 0.0301 0.0367 0.0463 0.0312 0.0648 0.0367 0.0379 0.0634 0.0426
101.7 1 0.0115 0.0148 0.0198 0.0120 0.0305 0.0148 0.0153 0.0296 0.0178
Total F 4.7436 4.9734 5.2646 4.7826 5.7366 4.9734 5.0107 5.7044 5.1573
D value 0.70 0.70 0.60 0.70 0.60 0.60 0.60 0.40
PSLR 7.105 7.521 7.971 8.195 8.289 8.351 9.507 12.893
196 III: STERILIZATION, SANITIZATION AND STERILITY ASSURANCE

evaluate the surviving organisms. Test data has been
generated demonstrating the value of using both a
moist heat organism (C. sporogenes) and a dry heat
organism (B. subtilis now known as Bacillus atrophaeus)
as BIs for the sterilization validation of closure systems
(11,12). A typical graphic representation of the inacti-
vation kinetics is illustrated in Figure 7. The above
studies may also be performed in a production sterilizer
as engineering or feasibility studies.
Container Closure Integrity Validation
Containerclosure integrity or MOS validations are run
on all moist heat terminally sterilized products with
closure systems of a parenteral container. This validation
is performed to demonstrate that the closure system of a
container is capable of maintaining the emulsion and
uid path in a sterile condition throughout the shelf life
of the product.
In a typical MOS study, the product container is
sterilized at a temperature which is higher than the
upper temperature limit of the chosen sterilization
cycle and for a time that is greater than the maximum
time limit for the cycle or producing an F0 subzero level
greater than the maximum F0 level for the cycle. The
rationale for the selection of the maximum temperature
and heat input level for the prechallenge sterilization is

1.0E+06
Study 1
Study 2
Linear Regression

1.0E+05

1.0E+04

1.0E+03

C. sporogenes

1.0E+02
Survivors

Fraction of
1.0E+01 Negative
Fraction/Negative Zone Units
0.01
0.05
0.10
B. subtilis 0.20
0.30
1.0E+00 0.40
0.50
0.60
Enhanced Bioburden 0.70
0.80

1.0E01 0.90

0.95

1.0E02 0.99

1.0E03
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0
Figure 7 Microbial kinetic inacti-
vation of bioburden as compared
Time Above 100 C
Average Count of 0 for Direct Plate Testing is Given a Value of 1.00E01
to biological indicators, Bacillus
atrophaeus (formerly named
Average Count of 0 for Fraction/Negative Testing is Given a Value of 1.00E02
Bacillus subtilis) and Clostridium
sporogenes.
 13: VALIDATION OF TERMINAL STERILIZATION 197

that rubber and plastic closures are subjected to thermal PRODUCTION FACILITY STERILIZATION
stresses during sterilization and those stresses are maxi- DEVELOPMENT
mized at the highest temperature and the longest time
allowed. The production list below depicts some of the sterilization
In some cases, the closures, e.g., administration or engineering and microbiological activities associated
additive port are claimed to be sterile by a radiation with a parenteral product as it moves into the production
process. In such cases, the closures are sterilized in bulk environment. These studies occur in a production
exceeding the maximum end of the radiation process environment as appropriate.
e.g., 40 kGy, then fabricated to the exible container
and exposed to steam sterilization cycle conditions Production facility activity Activity statement
exceeding the maximum temperature end of the cycle. Heat P&Ds Perform triplicate studies for
Thus, the closures are stressed by a joint process of minimum and maximum
radiation as well as steam prior to performing the loading conditions using
closure integrity test. temperature probes within
the product containers and
outside the containers to
Product Validation for Endotoxin measure the sterilizer
Endotoxins are lipopolysaccharides from the outer cell heating medium
membrane of gram-negative bacteria. Endotoxins can be temperature
detected by the manual gel-clot method known as the Solution (master) Perform microbial validation
LAL test. There are also various quantitative methods microchallenge validation of a parenteral solution or
(turbidimetric and chromogenic) which use more rapid master solution at sub-
automated methodologies. All nal product formulations process conditions in the
production sterilizer
have regulatory requirements to be tested for endotoxins
Containerclosure microchallenge Perform microbial validation
and the method must be validated using three different validation as applicable of the containerclosure
lots of nal product. LAL testing should be performed on system at sub-process
nal product formulations per FDA Guidelines and other conditions in the
regulatory compendia. Emulsion formulations, if colored production sterilizer
or opaque cannot be tested by the turbidimetric method Hold time studies Microbial, chemical and
and therefore may use a comparable test e.g., LAL, endotoxin studies are
chromogenic or kinetic. performed to establish the
The LAL test is for products other than oral and topical longest time that a product
can be held following
products (e.g., parenteral solutions, some devices, etc). Endo-
manufacture but prior to
toxin testing is usually required at three different times in lling and sterilization
the cycle of the product. First, endotoxin testing should be
performed on the lot of drug being used in clinical studies to
ensure that the product is safe for the patients with respect to Engineering P&D Validation
endotoxin. Second, in the developmental stages, endotoxin Perform triplicate studies with minimum and maximum
testing is usually required at the beginning and end of the loading congurations with temperature probes pene-
stability studies. Finally, once the product is ready to be trating the product containers as well as temperature
marketed, each lot of the product requires endotoxin testing probes distributed outside the product containers in a
prior to release. production sterilizer at nominal operating process
To improve in-process control, a process should parameters.
also be in place to decide if endotoxin testing should be
performed on the APIs and/or excipients used in the Microbial Solution Validation in a Production
product. In order to determine this, the ICH guidelines Sterilizer
for quality should be used; i.e., Q7A Good Manufac- Table 5 shows the microbial solution validation con-
turing Practice Guidance for APIs. ducted at subprocess conditions in a fully loaded

Table 5 Lipid Emulsion Microbial Solution Validation

Fraction negative method
No. positivea/ No. positivea/ Sporeb
a
Average no. no. positive no. negative No. positive / logarithmic
Organism Code spores/bottle controls controls no. test samples reduction
C. sporogenes 5C6 4.8!105 2/2 0/4 0/20 O7.0
C. sporogenes 15C6 6.4!105 2/2 0/4 0/20 O7.1
G. stearothermophilus 5B2 7.6!101 2/2 0/4 0/20 O3.2
G. stearothermophilus 15B2 7.7!101 2/2 0/4 0/20 O3.1
F0 Rangec: 5.87.6; Temperature Range: 1201258C; Agitation: 6773cpm
a
Positive for the indicator microorganism.
b
Spore logarithmic reductionZlog aKlog b; where a, initial population of spores; b, 2.303 log (N/q)Zln (N/q); where N, total number of units tested; q, number of
sterile units.
c
F, integrated lethality or equivalent minutes at 121.18C for hottest and coldest thermocoupled containers.
198 III: STERILIZATION, SANITIZATION AND STERILITY ASSURANCE

Table 6 Lipid Emulsion Microbial Closure validation

Sporeb
Initial No. positivea/ No. positivea/ No. positivea/ logarithmic
Microorganism population/stopper no. positive controls no. negative controls test samples reduction
C. sporogenes 8.4!103 2/2 0/4 0/20 O5.2
B. subtillis 3.0!104 2/2 0/4 0/20 O5.8
F0 Rangec: 5.87.6; Temperature Range: 1201258C; Agitation: 6773 cpm
Sterilization validation of 200 mL bottle inoculated closure surface coated with I.V. fat emulsion in cycle with agitation.
a
Positive for the indicator microorganism.
b
Spore logarithmic reductionZlog aKlog b; where a, initial population of spores; b, 2.303 log (N/q)ZIn (N/q); where N, total number of units tested; q, number of sterile
units.
c
F, integrated lethality of equivalent minutes at 121.18C for hottest and coldest thermocoupled containers.

production sterilizer. The acceptance criteria of 6 SLR was
setup for the BI C. sporogenes and a 3 SLR for the higher
moist heat-resistant BI, G. stearothermophilus. Each emul-
sion (20 containers) is inoculated with the appropriate BI
at a target level of 1.0!106 and 1.0!102 for C. sporogenes
and G. stearothermophilus, respectively. The 20 inoculated
containers are distributed throughout the production
sterilizer for sterilization at subprocess conditions. The
test containers are then returned to the lab for testing by
the F/N test method.
Microbial Closure Validation in a Production
Sterilizer
Table 6 shows the microbial closure validation at sub-
process conditions in a fully loaded production sterilizer.
The BIs used were C. sporogenes and B. subtilis. Acceptance
criteria of three SLR must be achieved for moist heat
(C. sporogenes) and dry heat (B. subtilis indicators). The
surface of the stopper that comes into direct contact with
the sidewall of the bottle was inoculated with the appro-
priate BI, dried and then a few drops of emulsion were
placed over the inoculum to simulate manufacturing
conditions. The inoculated closure was assembled to the
nished container, exposed to subprocess steam
conditions in the production sterilizer and subsequently
tested in the lab by the F/N test method. The data
demonstrate that a O3 SLR was achieved at subminimal
process conditions. Replicate test samples (e.g., 3 to 5)
should be considered for use to verify microbial kill in cold
zone locations.
ANCILLARY SUPPORT PROCESS TESTING
Bioburden Analysis for Closures and Commodities
Determine microbial load on closures and commodities
as well as their moist heat resistance analysis.
As part of the microbiological quality control
program, products and commodities are routinely
sampled during the production process in order to
assess the microbial load. This assessment is performed
via the bioburden test for terminally sterilized product.
The bioburden test method is developed during the
product development stage prior to transfer to the pro-
duction plant. This test assesses the microbial load of a
solution prior to terminal sterilization (In-Process
Bioburden Test). Micro R&D is responsible for the vali-
dation of the bioburden method prior to transfer to the
production plant. The validation will demonstrate that

Production Environment Bioburden

Screening Program

Heat Shock Positives

Gram Stain/Plate Morphology

Gram Positive Rods

Yeasts, Molds
Gram Negative Rods
Cocci Second Heat Shock

Retesting with 30-min

Heat Shock not
Routinely Required
10 min (+) 10 min (+)
30 min () 30 min (+)

Calculate Moist Heat

Probability Resistance Analysis
Figure 8 Representative pro-
of Non-Sterility Required
duction environment bioburden
screening program.

recovery of microbial load at a relatively low level can
be achieved.
The Microbial Limits Test is essentially a bioburden
test of raw materials used to make the nal product.
The test method and validation are conducted in much
the same manner as the bioburden test. The limit for the
microbial limits test is calculated as follows: nal Product
Action Level/maximum concentration of API in the nal
product. This limit is then normalized: by dividing by
the total amount of APIs in the nal product.
In addition, the production bulk solution is moni-
tored for total bioburden load including spore formers.
The screening allows the plant quality lab to ascertain if
there are any moist heat-resistant microora present in
the bulk solution prior to the terminal sterilization of the
parenteral solution in its nished container (Fig. 8).
Antimicrobial Preservative Efficacy
Perform on those formulations containing a preservative
and those container congurations that have a multidose
claim. This validation is performed per compendial
requirements.
Sterility Testing (if Required)
Once parametric release is approved by regulatory
authorities, then sterility testing is no longer required
nor can it be used as an alternative in case parametric
release parameters are not used.
Biological Testing Support of R&D and Marketed
Product Stability Programs
There are a number of analytical and microbiological tests
performed over the shelf life of a product. A number of
microbiological tests include BET, ContainerClosure
Integrity and APE if applicable.
CONCLUSION
As one reviews the nal conguration that a terminally
sterilized parenteral product is packaged in, it is not
surprising that a similar evaluation occurred when one
was contemplating how to present the new product as
being sterile and non-pyrogenic. The product develop-
ment team focused on the various designs of sterilizers
and the various manufacturing site locations for the
support of currently marketed products. Once the team
decided the appropriate facility for manufacture, then the
various sterilization cycles discussed in this chapter were
evaluated in order to select the appropriate one best
suited for that parenteral product in its nal container
conguration. If a product is destined for the inter-
national market, then R&D personnel will follow the
13: VALIDATION OF TERMINAL STERILIZATION 199
EMEA decision tree to determine if the product can be
sterilized at 1218C for 15 minutes minimum. If it cannot,
then a justication is documented explaining the reason
for selection of an alternate sterilization cycle. Personnel
perform the applicable studies in a developmental steri-
lizer as detailed in this chapter, as well as feasibility
studies in development or production sterilizers to
monitor and test the physical attributes of the nal
designed container. Once the parenteral products
designs as well as sterilization processes have been
nalized, then the plant/site can perform their standard
penetration, distribution and microbiological studies in
the production sterilizer.
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