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 component parts, as well as the environment, must also
STERILIZATION be ascertained.
The developmental and production phases of ster-
The previous chapter discussed the steam sterilization ilization technology activities are then drafted into
approach for the processing of hard goods or porous documents that are submitted as part of a new drug
loads. This chapter will discuss the sterilization vali- application for the particular parenteral formulation.
dation approach that can be used in the processing of These reports must follow applicable regulatory require-
parenteral products by terminal sterilization using moist ments for products that are terminally sterilized. Such
heat. The underlying principles of steam sterilization are studies allow one to establish, with a high level
applicable to both hard goods and terminal sterilization of sterilization assurance, the correct sterilization cycle
of parenteral products, but both have their unique (F0, temperature, product time above 1008C, etc.) to be
characteristics. used for the steam sterilization of a specic parenteral
An organized sequential ow of activities must formulation in a particular containerclosure system.
occur as new parenteral formulations are developed,
and subsequently processed in the manufacturing facility.
The moist heat sterilization of pharmaceutical solutions is STERILIZER DESIGN
established and veried through a series of activities that
conrm the product has received a dened thermal The validation of a steam sterilization cycle is dependent
exposure that renders the product sterile. R&D activities on the equipment chosen. The sterilizer and its support
can include sterilization developmental engineering systems must be designed and constructed to deliver the
studies consisting of sterilization cycle development; effective cycles repeatedly and consistently. Qualication
container thermal mapping; microbial closure validation, of the sterilizer consists of proper design, installation
D- and z-value analysis; containerclosure integrity vali- according to design, operational testing to ensure that
dations as well as nal formulation stability studies. design criteria and operational requirements are met and
The subsequent production phase activities must performance qualication to conrm that the product is
include initial sterilization vessel qualication which sterilized per specication.
demonstrates that the vessel will deliver the dened Sterilizer design is geared to the type of product or
sterilization process in a consistent and reproducible materials/equipment to be sterilized. All steam steriliza-
manner. Also, solution and containerclosure microbial tion cycles are based on contact with saturated steam,
validation studies must be conducted at subprocess steamair mixtures or superheated water. Saturated
production sterilization conditions employing heat- steam is water vapor in equilibrium with liquid water.
resistant microorganisms. Equipment validation, The values of temperature and pressure at which pure
ltration studies and assessment of the bioburden on 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
Abbreviations used in this chapter: AAMI, Association for the gaseous water; that is, the relation between its tempera-
Advancement of Medical Instrumentation; APE, antimicrobial
ture and pressure is xed. An increase or reduction in the
preservative efcacy; API, active pharmaceutical ingredient;
ASME, American Society of Mechanical Engineers; BET, bacterial
temperature of saturated steam must result in a corre-
endotoxin testing; BI, biological indicator; BIER, biological sponding increase or decrease in its pressure and vice
indicator evaluator resistometer; DP, direct plate; EMA, European versa. Steamair mixtures can be used when overpressure
Medicinal Agency; F/N, fraction/negative; GAMP, good auto- is required to maintain product shape or container
mated manufacturing practice; HMI, humanmachine interface; integrity. Superheated water cycles require air overpres-
I/O, input/output; ICH, International Conference on Harmoniza- sure and the water is either heated by direct injection of
tion; ISPE, International Society for Pharmaceutical Engineering; steam or indirectly via a heat exchanger.
LAL, limulus amebocyte lysate; LVP, large volume parenterals; Parts and hard goods are typically steam sterilized
MES, manufacturing execution system; MOS, maintenance of
using a saturated steam process whereas the trend for
sterility; P&D, penetration and distribution; PLC, programmable
logic controller; PSLR, predicted spore logarithmic reduction; R&D,
product sterilization is towards the use of superheated
research and development; RTD, resistance temperature detector; water or steamair mixture processes. These processes are
SCADA, supervisory control and data acquisition; SLR, spore needed as a majority of the new products require air
logarithmic reduction; SVP, small volume parenterals; TC, overpressure during the sterilization process to maintain
thermocouple. desired container characteristics and integrity.
188 III: STERILIZATION, SANITIZATION AND STERILITY ASSURANCE

Products in glass containers can utilize the satu- 3. Fan(s) to continuously recirculate the steamair
rated steam processes as described in Chapter 12, but mixture during heat-up and exposure and to recircu-
many products and containers require the use of air late the air during cooling.
overpressure during the sterilization process. This 4. Cooling provisions (e.g., cooling coils) to cool the
section will discuss some of the key design considerations air/product.
for terminal steam sterilizers and provide some specics
for the various type steam sterilization processes utilizing Recirculated Superheated Water Sterilization
air overpressure. Sterilization with recirculating superheated water (some-
times referred to as a water cascade or raining process) is
Typical Design Considerations for Steam Sterilizers more efcient than a steamair mixture and is therefore
more common. There are many types of recirculating
1. A pressure vessel constructed according to the superheated water processes, the most common is a
ASME or equivalent international code. This must process where the bottom portion of the sterilizer
withstand at least 50% in excess of the required (below the product zone) is lled with water and a
internal pressures. recirculation pump is used to continuously recirculate
2. A safety door mechanism to prevent opening while water from the bottom of the sterilizer to spray nozzles
the unit is under pressure: the locking device may be above the product zone. A slight modication to that
actuated directly by internal pressure or indirectly process is the use of a water distribution pan in lieu of
through an automatic switch. The door itself may be spray nozzles. Another version of the recirculating
of the swing-out or sliding type. Separate entry and superheated water process is to completely submerge
exit doors are preferable. the product in water but this process is inefcient from
3. Process control system (typically a PLC for control- a utilities consumption standpoint. All of these recircu-
ling and monitoring the process). lating superheated water processes utilize air
4. Process data recorder or data collection system. overpressure and the overpressure can be controlled
5. Product racks designed to hold/support the sealed during the sterilization process to minimize most types
product containers and to provide adequate of container deformation. There is no limit to the
heating/cooling media ow throughout the maximum overpressure used but it would typically be
product zone. limited by the chamber pressure rating. The minimum
6. Pressure safety relief valves for both the chamber and overpressure will be driven by the temperature being
jacket (if equipped with jacket). used, the pressure needed to maintain the desired
Note. A microbial retentive vent/air lter would not product characteristics and the required overpressure
typically be required for processes used for terminal needed to prevent the recirculation pump from loosing
sterilization as there is no direct contact between the prime. These recirculating processes are typically heated
heating/cooling media and the contents of the containers. and cooled indirectly with external heat exchangers
located in the recirculating water loop but direct injection
Steam Air Mixture Sterilization of steam and cooling water can also be used.
The primary benet to the steamair mixture process over In addition to the typical sterilizer design consider-
a superheated water process is the product is not ations mentioned earlier, a superheated water sterilizer
subjected to direct contact with water (except as conden- would also include a large recirculating water system
sate), which in some cases can cause cosmetic issues with (e.g., pump, pipes, heat exchangers, headers, spray
the container. Steamair mixture processes typically nozzles) including specic water level control valves
utilize large recirculating fans to prevent the formation and monitoring devices.
of cold/hot spots in the sterilizer. The steamair mixture
process typically uses an indirect cooling method such as Rotary and Shaker Sterilization
cooling of the jacket or with cooling coils within the In some cases, certain products (i.e., suspensions and
sterilizer. Because of this indirect cooling method, the emulsions) require agitation during the sterilization
cooling rate of the product is typically much slower and process. For those types of products, it is typical to use a
less efcient than direct exposure of the product rotating rack within the sterilizer but other agitation
containers to cooling water. methods such as an internal shaking device are available.
Some of the specic sterilizer design considerations Refer to Figure 1 for the typical design of a rotary sterilizer
for a steamair mixture process include the following: and Figure 2 for the typical design of a sterilizer using
1. A jacket and insulation: the jacket would utilize steam a shaking mechanism. It is possible to use any of the
during heating and exposure phases of the cycles and sterilization processes listed above with product agitation.
cooling water can be introduced to the jacket during
the cooling phase of the process. Continuous Sterilization
2. A thermostatic steam trap to efciently remove the For this version of the superheated water process,
condensate from the chamber: this is open when cool containers are terminally heat sterilized in a continuous
(in contact with air or condensate) and closed when in sterilizer by a process where the containers move through a
contact with steam. As condensate collects, the trap constantly controlled environment in carriers with individ-
opens owing to the slight temperature reduction and ual compartments. The time, temperature, and pressure
the condensate is discharged. There is also a similar requirements are set to predetermined values and are
steam trap to remove steam condensate from automatically and continuously controlled, monitored,
the jacket. and recorded. Refer to Figure 3 which depicts the pattern
 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
Figure 1 Photo of sterilizer with a rotary mechanism. Source: entering and exiting recirculating water for both heating
Photo Provided by Fedegari Autoclavi SpA, Albuzzano, Italy. and cooling continuously monitors, records, and controls
the temperature of the process water.
Air overpressure is required to protect the con-
that containers (e.g., parenteral exible product containers)
tainer from stress while exposed to the high sterilizing
follow as they move automatically through the continuous
temperatures.
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 STERILIZER CONTROL SYSTEMS
maintained within predetermined limits.
The sterilizing phase begins as the product enters A key to effective sterilizer operation lies in the auto-
the hot water environment within the pressure vessel. mated process control system. By eliminating the
The hot water environment may be a superheated water 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
Suspension Rods 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
Platform Frame 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
Platform Lock Arm
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-
Lock Screw
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
Platform Rails Bridge Plate type data collection system. In many cases the PLC can
also provides batch data logging functionality. The
minimum variables to record for steam sterilization
Figure 2 Photo of sterilizer with a shaking mechanism. processes are typically time, temperature, and pressure.
190 III: STERILIZATION, SANITIZATION AND STERILITY ASSURANCE

Steam Flowrate
Sensor

Heaters
Surge Tank

Pump
Indexer
Sterilizing Tank

Entry
Lock
Take-Up
Shuffle

Temperature
Sensor

Cooling Tank Exit

Lock Unloading

Tower Water Temperature

Heat Exchangers Sensor
Surge Tank
Mixing
Tank
Pump Tower Water Surge Tank

Pump Pump
Ozone

Figure 3 Schematic of a sterilizer for continuous processing of exible containers.

Typical sensors include temperature measurement Sterilizers that rely on recirculated water as part of
devices (RTDs or TCs), pressure measurement devices, the sterilization process can include a ow sensor. The
and where applicable level measurement devices and ow sensor may be a direct measurement such as a ow
ow measurement devices. It is customary that the meter (i.e., coriolis, ultrasonic, magnetic, etc.) or an
temperature sensor used to control the process tempera- indirect measurement such as differential pressure
ture not be used to provide the batch record process data. sensor across the recirculation pump. Direct measure-
An independent/secondary temperature sensor for batch ments are always preferred.
reporting provides a high degree of insurance that the For I/O devices, there are analog types and discrete
cycle actually ran within its dened limits. Heavy wall types. The analog inputs are typically from process
thermowells should not be used, as this will affect the sensors and the analog outputs are typically for control
time response of the measurement. Thin-walled thermo- of proportional valves. The discrete inputs are typically
wells or temperature elements with stainless steel sheaths from switch type (operator and process) devices and the
should be used for temperature measurement. discrete outputs are typically for activating hardware
The pressure sensor should be equipped with a such as valves, pumps, lights, etc.
sanitary-type diaphragm and connected to the sterilizer The design and development of the sterilizer
using a sanitary tting. A sanitary diaphragm can intro- control system software should follow the principles of
duce errors to the pressure measurement due to the ISPE GAMP 4 Guide for Validation of Automated
stiffness of the diaphragm. This stiffness is related to the Systems (1). This guideline details a software life cycle
size of the diaphragm. The impact is negligible for from conception thru decommissioning.
diaphragms above 3 inches diameter. This should be
considered when sizing the connection to the sterilizer.
STERILIZATION CYCLES
Sterilizers that maintain a specic water level (i.e.,
recirculated water process) should be equipped with liquid The type of steam sterilization cycle to be utilized is
level sensors. These sensors may be in the form of single- dependent on product needs and equipment availability.
point-level-type probe or a continuous level sensor. Regard- As discussed in Chapter 12, the sterilization of hard goods
less of what type sensor is used a separate high-level sensor or porous loads typically require the use of a pulsed pre-
must also be provided. The separate high-level sensor vacuum cycle as it is preferable to remove the air from the
provides greater assurance that the collected water at the porous materials being sterilized whereas in the terminal
bottom of the vessel remains below the product level. sterilization of aqueous solutions in sealed containers,
 13: VALIDATION OF TERMINAL STERILIZATION 191

the major concern is to provide rapid heat transfer to the Steam Air Mixture Cycle
wall of the lled product containers and air removal is not It is important to understand the physical principle
required (nor even desirable as the hydrating moisture is involved in a mixture of steam and air. The xed rela-
contained within each container). Parenteral products may tionship between temperature and pressure seen in
be lled into rigid or exible containers. In either there is Chapter 12, Figure 3 no longer applies. Daltons law
typically air or nitrogen present in the headspace above the states that the pressure of an ideal mixture of gases is
liquid. As the solution is heated, this gas expands and adds equal to the sum of the partial pressure of the gases, or
to the internal pressure increase resulting from the
evolution of water vapor from the aqueous vehicle within P Z PA C PB C PC :
the heated container. Thus, the pressure within the Raoults law further states that, for ideal mixtures,
container will exceed the chamber pressure during steam the partial pressure of the gas is equal to its vapor pressure
process for sealed containers. multiplied by the mole fraction in the liquid. For steam in
Glass vials can be sealed with special closures to equilibrium with pure condensate, this reduces to
withstand this pressure. As long as the pressure differ-
ential between the chamber and the containers does not PA Z pA
become too great during the steam exhaust portion of the
cycle, the vials will not burst. If rapid cooling of the load where PA is the partial pressure of steam and pA is the
is desired, the pressure differential might become signi- vapor pressure of the condensate. The difference between
cant enough to cause closure integrity to be lost. the observed chamber pressure P and PA is the partial
Plastic bags, semi-rigid containers and syringes pressure of air.
present a greater problem because they do not have the The presence of air, although necessary for the
inherent strength of glass and may burst or deform as the maintenance of container integrity, can reduce the heat
pressure differential increases. To prevent this, air must be transfer efciency. The objective of the design in the
injected into the chamber to raise the pressure above the air overpressure cycle is to maintain a well-mixed
saturation pressure of the steam. This is particularly chamber. This assures that the heat transfer to the load
important during the cooling cycle, when the chamber will be uniform regardless of the presence of air. Mixing
pressure is reduced at a much faster rate than that within may be accomplished in several ways. The air may be
the container. injected directly into the incoming steam. Usually,
The following section provides a description of the though, some mechanical means is selected.
various steam sterilization cycles used for parenteral Most steamair sterilizers use a fan built into the top
products in sealed containers. 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-
Saturated Steam Pre-Vacuum Cycle down process to cool the containers more rapidly. This
For a saturated steam process, the most common (and rapid cooling may also be necessary for product stability.
perhaps most effective) method to remove the entrapped Various methods (i.e., direct injection, recirculation
air from the sterilizer is to remove it mechanically before through a heat exchanger, etc.) for introducing the
the actual sterilization begins. This is done by means of a cooling media can be utilized.
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, Recirculating Superheated Water Cycle
Figure 7. 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
Saturated Steam Gravity Displacement to a predened level (below the product zone). Then a
or Steam Purge Cycle water recirculation pump is started to continuously
Other means for eliminating air without a vacuum recirculate water from the bottom of the sterilizer to
source include the use of a gravity displacement cycle spray nozzles or a water distribution pan above the
or a steam purge cycle. For the gravity displacement product zone. The recirculation pump is on throughout
cycle, steam is introduced on the side or top of the the heat-up exposure and cool-down phases. During
vessel and the cold air is forced out via the drain. The heat-up, the water is heated at a pre-dened rate via a
steam purge cycle uses large quantities of steam distrib- heat exchanger in the recirculation loop or with the direct
uted via headers under the entire product zone with injection of steam. Also during heat-up, compressed air is
numerous large vents located at the top of the vessel. added to the chamber to attain the desired overpressure
For these types of cycles, the appropriate vents or drains levels. Once the temperature set point is achieved, the
should be fully open and the large steam supply valve controller steps into the hold portion of the cycle and the
fully open for an extended time and temperature to temperature and pressures are maintained at the desired
ensure that the air is adequately removed from the levels. For cooling, the steam supply is shut off and the
sterilizer. Once the vents and drains close, the process recirculating water is cooled at a controlled rate by
runs like a traditional saturated steam process. It is introducing cooling media to a heat exchanger installed
important to determine that the measured temperature in the water recirculation loop or by the direct injection of
and pressure are consistent with the steam saturation cooling water into the recirculating loop. This type of
curve in Chapter 12, Figure 3. This process can be used process does not require the use of a jacket but does
with glass containers. 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 Container integrity Perform dye ingress,
microbial challenge or
tightly controlled during the entire process, thus mini-
physical integrity tests
mizing container stresses. following exposure to
maximum sterilization
conditions that stress
STERILIZATION CYCLE DEVELOPMENT the container
Stability runs Perform analytical
This section will address sterilization and associated chemistry and
microbiological activities that occur in R&D areas as microbiological
well as the production environment when using the evaluations at various
BI/bioburden approach in support of a parenteral temperatures and times
product. The list below depicts some of the sterilization per ICH and or
compendia
engineering and microbiological activities associated
requirements
with a parenteral product as it moves through develop- BET Perform test method
ment. These studies or similar ones are ordinarily validation of API,
conducted in developmental sterilizers or may occur as excipients and nal
investigative engineering studies in a production steri- product per compendia
lizer as appropriate. The overkill method can be used for requirements
some of the more stable parenteral formulations, and its
validation is accomplished as described in Chapter 12.
Sterilization engineering personnel primarily focus
their efforts in determining whether a parenteral formu-
Sterilization development activity Activity statement lation packaged in a particular container conguration can
Cycle development Develop preliminary be sterilized in a current cycle or whether a new cycle must
container sterilization be developed. The referenced EMEA (2) decision tree is
specications with followed when evaluating a new parenteral product in an
engineering parameters
LVP or SVP container. Sterilization feasibility studies are
such as temperature,
time and F0
conducted in a sterilizer to ascertain the physical effects of
Container thermal mapping Determine cold spot and the cycle on the product in question. Product attributes
assess heat penetration that can be affected by a cycle are closure integrity, product
within nished container potency, pH, color, shelf-life stability, visible, and subvi-
Formulation development Perform analytical sible particulates as well as nal product sterility. Once the
feasibility studies prior to basic engineering parameters (e.g., temperature, time and
product nalization with F0) are established, then engineering thermal container
methods validations mapping studies can be performed (3,4).
Parenteral solution microbiological
evaluations:
Moist heat D- and z-value analysis Perform triplicate D-value Container Thermal Mapping Validation Studies
analysis on each An R&D sterilizer is smaller than a production facility
parenteral formulation at sterilizer, but can simulate the sterilization cycles
three temperatures, conducted in the larger production vessels. Container
e.g., 1128C, 1188C and thermal mapping studies (when applicable) are typically
1218C and then performed in a laboratory sterilizer:
calculate the z-value 1. To locate the coldest zone or area inside a container.
APE Perform on nal product if 2. To determine the cold zone in the container and its
it contains a
relationship to the location monitored during
preservative or if there is
a multidose claim for the
validation studies.
container 3. To generate data that may be used during the setting of
In-process bioburden analysis Perform studies with a production sterilization control parameters.
panel of microorganisms When conducting thermal mapping studies, there
to validate 70% recovery are various factors to be considered, and these are
for the ltration process dependent upon the:
Spike hold time studies Inoculate parenteral 1. Type of container (exible or rigid)
product with bioburden 2. Container orientation, size and ll volume
and growth promotion 3. Cycle type and temperature
compendia organisms to
4. Viscosity
evaluate the products
ability to support
5. Autoclave trays/design/surface contact
microbial growth 6. Autoclave spray patterns/water ow.
Container closure evaluations: Typical container mapping data obtained for lipid
Microbial closure inactivation Perform kill curve kinetics emulsions contained within a 1000 mL glass container are
using bioburden and BI shown as an example in Tables 1 and 2. The following
(spores) inoculated onto summarizes the process for obtaining heat map data from
the worse case closure the glass intravenous container lled with approximately
site 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, emulsion area registered 7.5 F0 minutes, the hottest
0.005 in. diameter) were used to monitor 11 locations emulsion area would average 16.1 F0 minutes.
within the 1000 mL container. The TC probes were posi- The emulsion area approximating the validation TC
tioned at various distances (in inches) as depicted (Fig. 4). location was measured by TC # 2,13 and averaged 7.7 F0
Each container was lled with approximately 1000 mL minutes when the coldest emulsion was approximately
of the lipid emulsion, evacuated to 20 in. of mercury and 7.5 F0 minutes (Fig. 5).
sealed with an aluminum overseal.
A at perforated rack on a reciprocating shaker cart
was used in the autoclave. The cycles target temperature Solution/Product Moist Heat Resistance D- and
was 1238C, recirculating water spray cycle with 70 rpm of z-Value Analysis
axial agitation and 30 psig (pounds per square inch) of air A BIER vessel meets specic performance requirements
overpressure. for the assessment of BIs per American National Stan-
When the sterilization cycle was controlled to give a dards developed and published by AAMI (5). One
heat input of approximately 7.5 F0 minutes in the coldest important requirement for a BIER steam vessel is the
emulsion area, the average coldest emulsion area was capability of monitoring a square wave heating prole.
found to be measured by TC number (TC#) 3,14. The Refer to Figure 6 for a schematic of the steam BIER
average hottest emulsion area was measured by TC# 10, vessel used to generate the D- and z-value data. D-value
21. The difference between the hottest and coldest emul- is the time in minutes required for a one log or 90%
sion areas ranged from 7.5 to 10.0 F0 minutes with an reduction in microbial population (Refer to Chapter 12,
average of 8.6 F0 minutes. Therefore, when the coldest 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 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
Figure 4 Heat mapping study using a 1000 mL glass container. 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
temperature required for a 10-fold change in the D-value. according to PSLR values. The combined D1218C and
(Refer to Chapter 11 for additional details on F-, D- and z-value allows comparison of moist heat rankings
z-values.) between emulsions.
The data in Table 4 demonstrate that the #1 Emul-
Master Solution/Product Concept sion has the lowest PSLR (7.105), thereby affording the
The family category of lipid emulsions and their respective highest moist heat resistance upon inoculation. Gener-
D1218C and z values as well as classication in terms of ation of this table allows prediction of which emulsion to
microbial resistance is shown in Table 3. A categorization microbiologically challenge as part of validation in the
of parenteral formulations with associated D1218C and production sterilizer.
z-values and their potential impact on microbial resistance
using the BI, Clostridium sporogenes were previously Microbial Closure Inactivation Validation in a
reported (6). In addition, the methodologies used for D- Developmental Sterilizer
and z-value analysis were likewise cited. The data in In lieu of using the large type steam sterilizers in the
Table 3 indicate that the # 1 emulsion is at the top of the production environment, microbial inactivation at the
list, since it affords the most microbial moist heat resist- closure/bottle interface of an emulsion container can be
ance. It is therefore the emulsion that should be assessed in a developmental sterilizer. The closure micro-
microbiologically challenged (inoculated with spores) as bial inactivation (kinetic) studies can determine how the
part of the emulsion validation scheme. D- and z-value size of the container, type of closure compound used as
data have been reported for other BIs such as Geobacillus well as closure preparatory processes (e.g., leaching,
stearothermophilus (68) and Bacillus subtilis 5230 (9). There washing, siliconizing, autoclaving) inuence microbial
are many factors that can affect moist heat resistance inactivation. Microbial closure kinetic studies are
including a BIs age, sporulation media used, as well as conducted at various time intervals in a given steriliza-
the particular spore strain employed (10). 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 closure systems of a parenteral container. This validation
generated demonstrating the value of using both a is performed to demonstrate that the closure system of a
moist heat organism (C. sporogenes) and a dry heat container is capable of maintaining the emulsion and
organism (B. subtilis now known as Bacillus atrophaeus) uid path in a sterile condition throughout the shelf life
as BIs for the sterilization validation of closure systems of the product.
(11,12). A typical graphic representation of the inacti- In a typical MOS study, the product container is
vation kinetics is illustrated in Figure 7. The above sterilized at a temperature which is higher than the
studies may also be performed in a production sterilizer upper temperature limit of the chosen sterilization
as engineering or feasibility studies. cycle and for a time that is greater than the maximum
time limit for the cycle or producing an F0 subzero level
Container Closure Integrity Validation greater than the maximum F0 level for the cycle. The
Containerclosure integrity or MOS validations are run rationale for the selection of the maximum temperature
on all moist heat terminally sterilized products with 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 conditions in the production sterilizer and subsequently
setup for the BI C. sporogenes and a 3 SLR for the higher tested in the lab by the F/N test method. The data
moist heat-resistant BI, G. stearothermophilus. Each emul- demonstrate that a O3 SLR was achieved at subminimal
sion (20 containers) is inoculated with the appropriate BI process conditions. Replicate test samples (e.g., 3 to 5)
at a target level of 1.0!106 and 1.0!102 for C. sporogenes should be considered for use to verify microbial kill in cold
and G. stearothermophilus, respectively. The 20 inoculated zone locations.
containers are distributed throughout the production
sterilizer for sterilization at subprocess conditions. The
test containers are then returned to the lab for testing by ANCILLARY SUPPORT PROCESS TESTING
the F/N test method. Bioburden Analysis for Closures and Commodities
Determine microbial load on closures and commodities
Microbial Closure Validation in a Production as well as their moist heat resistance analysis.
Sterilizer As part of the microbiological quality control
Table 6 shows the microbial closure validation at sub- program, products and commodities are routinely
process conditions in a fully loaded production sterilizer. sampled during the production process in order to
The BIs used were C. sporogenes and B. subtilis. Acceptance assess the microbial load. This assessment is performed
criteria of three SLR must be achieved for moist heat via the bioburden test for terminally sterilized product.
(C. sporogenes) and dry heat (B. subtilis indicators). The The bioburden test method is developed during the
surface of the stopper that comes into direct contact with product development stage prior to transfer to the pro-
the sidewall of the bottle was inoculated with the appro- duction plant. This test assesses the microbial load of a
priate BI, dried and then a few drops of emulsion were solution prior to terminal sterilization (In-Process
placed over the inoculum to simulate manufacturing Bioburden Test). Micro R&D is responsible for the vali-
conditions. The inoculated closure was assembled to the dation of the bioburden method prior to transfer to the
nished container, exposed to subprocess steam 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.
 13: VALIDATION OF TERMINAL STERILIZATION 199

recovery of microbial load at a relatively low level can EMEA decision tree to determine if the product can be
be achieved. sterilized at 1218C for 15 minutes minimum. If it cannot,
The Microbial Limits Test is essentially a bioburden then a justication is documented explaining the reason
test of raw materials used to make the nal product. for selection of an alternate sterilization cycle. Personnel
The test method and validation are conducted in much perform the applicable studies in a developmental steri-
the same manner as the bioburden test. The limit for the lizer as detailed in this chapter, as well as feasibility
microbial limits test is calculated as follows: nal Product studies in development or production sterilizers to
Action Level/maximum concentration of API in the nal monitor and test the physical attributes of the nal
product. This limit is then normalized: by dividing by designed container. Once the parenteral products
the total amount of APIs in the nal product. designs as well as sterilization processes have been
In addition, the production bulk solution is moni- nalized, then the plant/site can perform their standard
tored for total bioburden load including spore formers. penetration, distribution and microbiological studies in
The screening allows the plant quality lab to ascertain if the production sterilizer.
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). REFERENCES
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and those container congurations that have a multidose Decision Trees for the Selection of Sterilisation Methods
claim. This validation is performed per compendial (CPMP/QWP/054/98), Annex to Note for Guidance on
requirements. Development Pharmaceutics, EMEA 2000, 13.
3. Young JH. Sterilization with steam under pressure. In:
Morrissey RF, Phillips GB, eds. Sterilization Technology.
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American National Standard for the Advancement of
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