Validation of Dry Heat Processes PDA TR-3

9/18/2019
Validation of Dry Heat

Processes Used for
Depyrogenation
And Sterilization
Technical Report No. 3

(2013 Revision)
Validation of Dry Heat Processes
© 2013 Parenteral Drug Association
Task Force Members

• Bruce Bear, Bear Consulting • Brian Jordan, Valsource,
Services LLC
• Stewart Davenport, Pfizer, • Peter Lee, Mattell Inc.
Inc.
• Hans Melgaard, Despatch
• Mike Davies, GlaxoSmithKline Industries
Pharmaceuticals
• Christian Supina, Baxter
• Steve Folio, Althea
Technologies Healthcare Corporation
• Jill Giulianelli, West-Ward • Rita Welser, Boehringer

Pharmaceuticals Ingelheim
• Deborah Havlik, Hospira, Inc. • Robyn Wong, Celgene
Corporation

© 2013 Parenteral Drug Association 2
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Instructor Bio
Deborah Havlik
• DAHavlik Consulting, Greater Chicago Area
• Director, Pharmaceutical Microbiology, R&D,
Hospira, a Pfizer company
• Contract Micro Lab and Ethylene Oxide
Sterilization
• Medical Device Industry, Class III manufacturer
• PDA Task Force
• Convenor of ISO WG 14, Dry Heat; Microbiological
Methods
• AAMI: Radiation, Ethylene Oxide, Dry Heat,
Microbiological Methods, Aseptic Processing, BIs
Class Introductions
• Name
• Company
• Position/Title
• Student objectives/expectations
• Instructor’s learning objectives

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Announcements
• Attendance list
• Course/Instructor evaluations
• ACPE Form
• Disclosure

Course Schedule
• 8:30 a.m. Class Begins

• 10:00 – 10:15 a.m. Morning Break
• 12 noon – 1:00 p.m. Lunch (provided)
• 2:30 – 2:45 p.m. Afternoon Break
• 3:45 – 4:00 p.m. Class Ends

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Course Outline
• Introduction and Scope

• Glossary of Terms
• The Science of Dry Heat Inactivation and
Sterilization
• Equipment Design
• Equipment Verification
• Process Development
• Performance Qualification
• Ongoing Process Control
Section 1
Introduction
and Scope

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Introduction
• Update of PDA’s Technical Report No. 3, Validation of

Dry Heat Sterilization Processes used for Sterilization
and Depyrogenation (issued in 1982)
• Focus
– Microbiology and engineering qualification of dry heat
sterilization and depyrogenation processes
– General approach to sterilization and depyrogenation science
in batch and continuous sterilizers (ovens and tunnels)
Validation: A documented program that provides a high level of scientific

assurance that a manufacturing process will reliably produce acceptable
product. The proof of validation is obtained through rational experimental
design and the evaluation of data, preferably beginning from the process
development phase and continuing through the commercial production phase
Scope (1)
• Provides information to the manufacturers of

pharmaceutical products for validating dry heat
depyrogenation and sterilization processes
• Concepts and methods presented in this
technical report are points to consider during the
validation of dry heat processes
– Not intended to be a regulatory standard
– Other technically equivalent methods may exist and
may be used if they can be supported by sound
scientific methods

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Scope (2)
Use of biological indicators and endotoxin indicators
Points to Equipment design

consider:
Equipment verification
Process development
Performance qualification for new systems
Development and validation of processes for existing

systems.
Ongoing maintenance of the validated process.

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Scope
• Process development in both ovens and tunnels

focusing on:
– Load type
– Loading patterns
– Temperature profiles for depyrogenation and sterilization
• Background sections specific to dry heat
processes
• Applicable to the following:
– Forced hot air dry heat batch processes (chambers)
– Continuous processes (tunnels)
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Out of Scope
• Dry heat processes used for the following:

– Sterilization of oil based and oil products
– Fixed processing streams
– Processes using infrared and microwave heating
media

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Section 2:
Glossary of Terms

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Glossary of Terms
• Current FDA, ICH, and other regulatory definitions are used

except when more clarity is added by the Task Force
• Regulatory guidelines offer other definitions that may be
considered
• Variations in the use of some terms may differ from company
to company, and some may be subject to change in the
future
• However, the terms used in a validation program must be
clearly defined and well understood within the company
• Clearly defined in internal Standard Operating Procedures
(SOPs), standards, and in regulatory filings

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Section 3:
The Science of Dry Heat

Depyrogenation
and Sterilization

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Dry Heat Processes
Sterilization
Dry Heat
OR
Processes
Depyrogenation
and sterilization
The purpose of process will dictate the validation

approach
Depyrogenation: The destruction or removal of bacterial endotoxins.

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Dry Heat Depyrogenation (1)
• One primary method used to inactivate and remove

bacterial endotoxins by thermal destruction
• Ovens or tunnels used for depyrogenation of heat
resistant materials
– Glassware, metal equipment, instruments, containers, and
heat stable chemicals
• Limulus Amebocyte Lysate (LAL) assay provides a
means of assessing the performance of dry heat
endotoxin inactivation on a quantitative basis
Bacterial Endotoxin: Endotoxins are fever producing substances commonly

found in the cell wall of certain Gram negative bacteria.
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Destruction of LPS
“Whereas many • 250 C for 30 minutes
sterilization
processes are
predictable, many
depyrogenation
procedures are Literature references
purely empirical.” • Lack of agreement in
temperature and times
Kevin Williams, Eli Lilly & • Many conditions affect
Co. depyrogenation
parameters

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• Temperature and exposure time

– Validate appropriately
– Demonstrate adequate and reproducible level of endotoxin

reduction when operated routinely within established
tolerances
• Include an endotoxin challenge, where necessary, as

an integral part of the validation program
– Example: by inoculating one or more of the articles to be
treated with 1000 or more USP Endotoxin Units (EU) of
bacterial endotoxin

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Dry Heat Depyrogenation:

Inactivation of Endotoxin
Commonly used for materials that can

withstand high temperatures for a period of
time using incineration in order to achieve
Bacterial Endotoxin inactivation

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Heat Lethality of Incineration
Anticipate
Provides large
microbial
reductions well in
margin of safety with
excess of 10100
regard to sterility
• B. atrophaeus (formerly Therefore
Bacillus subtilis var niger)
Process lethality
spores have D values
of only a few seconds
can be defined
at temperatures used on the basis of
for depyrogenation endotoxin
inactivation

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Validation and Practice
Validation
• Dry heat endotoxin reduction studies are always
conducted rather than microbial inactivation studies
• Inactivation rate of endotoxin is slower than the
inactivation rate of the biological indicator used (e.g.,
spores of Bacillus atropheus)
In Practice
• The reduction of endotoxin challenge by three or more
logs will result in a process that also achieves the
probability of non-sterility substantially less than 10-6

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Inactivation of Endotoxin
Greater than
Inactivation
of Endotoxin
= or = 3 log of
reduction
Assumption: the depyrogenation process will inactivate

bacterial endotoxins that could be presented by any given
component manufacturing process.

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Review Question #1
Dry heat depyrogenation is the only method

to inactivate and remove bacterial
endotoxin by thermal destruction
• True
• False

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Review Question #2
During validation, dry heat endotoxin

reduction studies are always conducted
rather than microbial inactivation studies
because:
A. The inactivation rate of the endotoxin is faster than
the inactivation rate of the biological indicator
B. The inactivation rate of the endotoxin is slower than
the inactivation rate of the biological indicator

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Reduction of Endotoxin
• Thermal incineration
dependent on factors that
Most accurate
affect heat distribution in a assessment of
chamber or tunnel reduction:
– Mass and configuration of the Endotoxin Indicator
least prone to variables
load
associated with
• Chemical nature of endotoxin recovery
components impact techniques
recovery of endotoxin only
– Not reduction
Endotoxin Indicators: An article challenge vial of endotoxin (or a carrier

spiked with endotoxin) designed for use in depyrogenation studies
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Glossary of Terms:
Endotoxin Indicator (EI)
An article challenge vial of endotoxin (or a
carrier spiked with endotoxin) designed for
use in depyrogenation studies.
Image courtesy of rapidmicrobiology.com

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Endotoxin Indicator (EI)

for Depyrogenation
• The endotoxin (a purified lipopolysaccaride) is validated
for use in or on an Endotoxin Indicator
• Carrier is made from a material appropriate for the
intended depyrogenation processes to which it will
be subjected
• The endotoxin on a carrier is added at a
concentration sufficient to allow recovery of a
minimum of 1000 USP Endotoxin Units/carrier
• The Endotoxin Indicator would allow for accurate
indication of at least a 3-log reduction in USP
Endotoxin Units during depyrogenation process
challenges
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Endotoxin Indicators
• Determinants for endotoxin reduction

– Exposure time
– Temperature
• Variables that may affect endotoxin recovery from
suitable carriers or items may include:
– Formulation of the endotoxin
– Surface type, composition and configuration of carrier
– Inoculum (spike) concentration
– Method of inoculation and drying of endotoxin
– Stability and storage of inoculated carriers or items
A detailed guideline for preparing and recovering endotoxin challenges has been published.
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Selecting a Carrier for

Depyrogenation Studies
Two approaches
Carrier Selection Commercial EI
• Inoculate carrier • Heat resistant

• Same chemical • Easily vortexed
and physical
properties

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Preparation and Inoculation
Washing/Rinsing
• Water for Bacterial
Endotoxin Testing (BET) Inoculation (spiking)
• Water for Injection (WFI) • High potency endotoxin
• Minimize intrinsic • Small volume
contamination resulting • Not less than 1,000
in false positives International Units (IU)
units
• One International Unit
(IU) = one Endotoxin
Unit (EU)
Note: the level of endotoxin with which to inoculate a carrier is somewhat controversial. Purified endotoxin, which is
used in depyrogenation studies, binds more thoroughly to the carrier surface and is more difficult to recover than
natural endotoxin
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Bacterial Endotoxin vs LPS
Lipopolysaccharide
Ultrastructure
Of Gram –ve
Bacterial Cell
Wall

© 2013 Parenteral Drug Association
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Glossary of Terms:
Water for Bacterial Endotoxin Test (BET)
Image courtesy of osawaterworks.com
Sterile Water for Injection or

other water that shows no
reaction with the specific
bacterial endotoxin test
reagent with which it is to be
used, at the limit of
sensitivity of such reagent.

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EI Fixation Methods
• Air drying the endotoxin challenge

solution to the sample surface is
recommended
– Representative of how endotoxins are
naturally affixed to items
• Drying EIs under an ambient laminar
flow hood is commonly conducted
– However, studies have demonstrated
differences in endotoxin recovery
depending on the type of glass and the
method of fixation
• Lyophilization
• Vacuum drying

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Sample Processing (1)
Follow standard procedure and

document process of Endotoxin
Indicator samples
Include control samples such as
negative and positive controls in dry
heat depyrogenation studies
Set aside additional inoculated items
to serve as unprocessed endotoxin
indicator positive controls

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Sample Processing (2)
• Positive controls should be handled

in the same manner as the items to
be depyrogenated
– To evaluate the amount of recoverable
endotoxin and calculate log reduction
Image courtesy of abstracts.aapspharmaceutica.com

• Items that have not been inoculated
should also be set aside to serve as
a negative control

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Review Question #3
What determines endotoxin reduction? (Select

all that apply)
A. Formulation of the endotoxin
B. Surface type, composition, and configuration
C. Inoculum concentration
D. Exposure time and temperature

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Review Question #4
Which variables may affect endotoxin recovery?

(Select all that apply)
A. Method of inoculation and drying of endotoxin
B. Stability and storage of inoculated carriers or
items
C. Formulation of the endotoxin
D. Surface type, composition and configuration of
carrier
E. Exposure time and temperature

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Recovery
• The final testing for recovery of endotoxin spike

after treatment will demonstrate whether or not the
quantity of spiked endotoxin was reduced by 3 logs
• Studies demonstrating endotoxin recovery, using
EIs with different surface types, have concluded
that a combination of vortexing and sonicating
improves recovery
– For inoculated surfaces that are not amenable to vortex
mixing or sonication, a previously screened surface active
solution may facilitate the physical removal of endotoxin

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Glossary of Terms:
Bacterial Endotoxin Test (BET)
Assay for measuring active endotoxin by combining a liquid

test sample with Limulus amebocyte lysate (LAL) reagent
and measuring the resulting proportional reaction via visual,
turbidimetric, chromogenic, or other validated means of
detection.

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Recovery: BET Method/Analyses
• Allow endotoxin recovery and quantification of at least a three log

reduction of the EI
– Example:
EI spike = approx. 5000
LAL Reagent w/at least

IU/component
0.125 IU/ml in sensitivity

Gel-clot assay
conducted Photometric assay has a
suitable standard curve
range such as 0.1-to-10
IU/mL or equivalent
• Conduct all BET analyses of positive controls (unprocessed EIs) and

recoveries from processed components using validated test methods
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Result Interpretation/Endotoxin Log

Reduction Calculations
The amount of recoverable endotoxin assayed from the

unprocessed positive control should demonstrate a
3-log reduction of the challenged items.
Depyrogenation
Inactivation of
process will
endotoxin achieves May
Assume sufficiently inactivate
greater than or equal to
endogenous
3 logs of reduction
endotoxins that could
be present

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Equation 3.2.4-1
Provides a calculation to determine the log

reduction of an endotoxin indicator
ELR = Log IU0 – Log IUf
IU0 = Average
ELR = Endotoxin recovered positive IUf = Recovered
Log Reduction control endotoxin spike concentration
concentration after exposure
(unprocessed)

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Example of Equation 3.2.4-1
Log 1225 – Log 0.5 = 3.1 - (-0.3) = 3.4
Average recovered
positive control Test indicators after Endotoxin log
endotoxin indicator heat treatment reduction
• Highly unlikely that an overkill dry heat cycle would yield

detectable endotoxin in extracts from undiluted exposed EIs
• When endotoxin is undetectable, IUf is the sensitivity of the BET
method
• Such as the labeled sensitivity of a gel-clot reagent or the lowest
endotoxin concentration of a photometric standard curve

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Glassware Depyrogenation
• Depyrogenation by washing, rinsing and the
sterilization process is as effective as dry heat
depyrogenation and is cost effective
• Manufacturing process for glass excludes the
potential for bioburden and endotoxin contamination
– Promptly packaged after production in a cleanroom
environment
– Subjected to shrink-wrap for delivery and storage
• Risk of contamination due to exposure to bioburden

is highly unlikely during the handling, packing and
shipping of glass containers during processing and Image courtesy of triforest.com
post cleaning
Note: Not all aspects of this TR are applicable to glassware or other containers that
are used to package products destined for terminal sterilization.
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Glassware Depyrogenation
• For products that are aseptically processed and

terminally sterilized, guides exist to ensure glass
is pyrogen-free through depyrogenation
• The rationale as to why terminally sterilized glass
containers may not need to be depyrogenated is
due to the low risk of bacterial endotoxins
contamination from the glass containers
manufacturing process, which can include
temperatures of 815.6°C to 982.2°C (able to
destroy bacterial endotoxins)

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Quality Risk Management
• Implement a quality risk management

program to ensure patient safety
throughout the pharmaceutical
manufacturing process that includes
glassware
• Conduct a formal risk assessment of the
shipping, handling and storage of glass
containers using recognized tools (e.g.,
FMEA, HACCP)
– Determine areas of potential endotoxin
contamination with recommended mitigation
and post mitigation review

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Assessment Considerations
Packaging to
Quality audit prevent
Handling and
of glass microbial and
shipping
manufacturer endotoxin
contamination
Storage
Storage conditions
conditions prior to and
prior to following
packaging shipping

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Steps to Mitigate Risk (1)

May Include:
Environmental
controls to
Inadvertent in- protect
transit damage glassware once
affecting packed and/or
Inspection of washed
shipping integrity of
containers upon shipping
receipt for water container
damage

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Steps to Mitigate Risk (2)

May Include:
Bacterial endotoxin
testing of each
batch of finished
Daily testing of WFI sterile product
for bacterial
endotoxins and
Control of the rinse microbial counts not
or wash water step to exceed limits that
prior to filling would elicit a
through quality of pyrogenic response
water used (WFI
recommended)

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Review Question #5
Which assessment factors should be

considered when implementing a quality risk
management program? (select all that apply)
A. Quality audit of glass manufacturer
B. Storage conditions prior to packaging
C. Packaging to prevent microbial and endotoxin
contamination
D. Storage conditions prior to and following
shipping
E. All of the above
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Review Question #6
It cannot be assumed that the

depyrogenation process will inactivate
bacterial edotoxins present in any
component even if the inactivation of
endotoxin achieved greater than or equal to
3 logs of reduction
A. True
B. False

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Dry Heat Sterilization
Slow-acting
sterilizing
agent
Higher
temperatures
Longer
Image courtesy of spaulding-rogers.com exposure
times

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Glossary of Terms:
Sterilization Process and Heat
• A process used to render a

Sterilization
Process: product free of viable organisms
with a specified probability
• Energy that is transferred as a

result of a temperature difference
Heat:
between an object and its
surroundings

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Dry Heat Sterilization:

Ovens or Tunnels
• Heated filtered air distributed
uniformly throughout the
chamber by:
– Convection
– Thermal radiation Image courtesy of rpdinc.com
• Blower system and devices for:

– Sensing
– Monitoring
– Controlling the operating
parameters
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Sterilization
Rate of Temperature
microbiological
destruction Uniformity of the heating medium
influenced by:
Conductivity of the containers and/or

equipment
Accessibility of the device or item fluid
pathway
Physiological state of product bioburden

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Sterilization:
Mechanisms of Inactivation (1)
• Typical dry heat temperatures are greater
than or equal to 160°C
– However processes at significantly lower
temperatures have been developed and
validated
• Dry heat inactivates microorganisms
primarily by oxidizing organic compounds

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• Sub-lethal dry heat

temperatures induce
mutations in B. atrophaeus
spores
Image courtesy of llnl.gov
• Primary factors affecting inactivation in

microorganisms include:
Water
Temperature Time
content

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• The death of a homogeneous culture of
microorganisms exposed to constant lethal stress
(survivor curve) has been shown empirically to
follow first-order kinetics
• The rate of microbial lethality is a function of:
– Thermal resistance of the microorganism
– Lethal stress
• Rate of microbial lethality is independent of the
number of microorganisms in the challenge
Survivor Curve: Graphical representation of the inactivation of a population of
microorganisms with increasing exposure to a microbicidal agent under stated
conditions
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The Survivor Curve: Equation 3.3.1-1

Log NF = −F(T,z)/DT + Log N0
NF = Number of
DT = Thermal
microorganisms
resistance value, in
after exposure
minutes, of the
of F equivalent
microorganism at a
minutes
specific
temperature
F(T,z) = Equivalent lethality Note: This specific temperature
must be the same as the
of a process calculated as reference temperature used for
minutes at a reference calculating F-value
temperature (T), using a
defined temperature N0 = Number of
coefficient (z) microorganisms
prior to exposure
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Glossary of Terms:
F-Value and z-value
F-Value (Lethality Factor):
• A measurement of process effectiveness

• F is the calculated equivalent lethality (using a
specified z-value) for a sterilization process, in terms
of minutes at a reference temperature (Tref),
delivered by a sterilization process to an item
z-value:
• The number of degrees of temperature change to

reduce the lethality by a factor of 10
• The z-value allows integration of the lethal effects of
heat as the temperature changes during the heating
and cooling phases of a dry heat process
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FH-Value for Sterilization (1)
• A measure of heat input

• The concept is comparable to the F0 concept for
moist heat sterilization
• References lethality to equivalent times at
160°C
– Other reference temperatures can also be considered,
but 160°C is primarily used
• FH values are shown in units of minutes or
seconds
• Calculations of FH use the same equations as
the calculations of F0
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FH-Value for Sterilization (2)
• FH- is a term used to model exposure time to

dry heat
• Routine operational processes are not
generally square wave processes
– Therefore the z-value, or temperature coefficient, is
used in the model to calculate the equivalent
lethality at different temperatures such as during
the heat-up and cool down phases
FH: Expressed by a reference temperature so that it truly represents the

equivalent exposure time, in terms of lethality, at that reference temperature

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Glossary of Terms:
Routine Operational Process
• Parameters that are specified for ongoing

operations
• Typically controlled to produce additional
lethality over the qualified minimum parameters
(i.e., time and temperature) in order to provide
increased sterility assurance

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Glossary of Terms:
Heat-Up Phase
• The phase of a process that

occurs prior to the exposure
phase
• Process parameters are
developed for this phase in
order to meet applicable
user requirements for load
conditioning (e.g., pre-heating)

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FH-Value for Depyrogenation
• May be used in dry heat

depyrogenation processes to
calculate the time in minutes
equivalent to a lethality or
endotoxin destruction effect
delivered by dry heat at 250°C
– F-value reference temperature is set
at 250°C
– z-value minimum is set at 46.4°C.

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FH Values:
Equation 3.3.1.1-1
Theoretical FH values z-value = 20°C
can be calculated
using the following
parameters: Tref-value = 160°C
The FH values can be determined using the calculation below
n  

T i - T ref 
 
FH  t 10  
 z 
  
 i 
i 1  
 
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FH Values (2)
n  

T i - T ref 
 
FH  t 10  
 z 
  
 i 
i 1  
 
FH • Equivalent lethality in minutes
Tref • Reference temperature equal to 160.0 C
z • Spore lethality z-value equal to 20.0 C
Ti • Measured temperature in C during the time

interval “ti”
ti • Time interval of data recording “i” in minutes
n • The number of data recordings
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z-Value and D-Value
D-Value z-Value
• The decimal reduction • The number of

time is the amount of degrees of
time (in minutes) temperature change
required at a reference necessary to change
temperature for a one- the D-value by a factor
log, or 90%, reduction of 10
of a microbial • Dry heat sterilization
population under calculations = 20°C
specified lethal
conditions.
Both D-value and z-value are determined experimentally.

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D-Value for Dry Heat Sterilization
Always specify with a reference temperature (DT)
Example
a Biological Indicator (BI) challenge

system with a D160oC= 1.9 minutes,
requires 1.9 minutes at 160oC to
reduce the population by one logarithm

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Review Question #7
Dry heat sterilization requires which of the

following:
A. Slow-acting sterilizing agent
B. Higher temperatures
C. Longer exposure times
D. All of the above

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Review Question #8
The rate of microbiological destruction

associated with devices and items sterilized
by dry heat is influenced by: (select all that
apply)
A. Temperature
B. Uniformity of the heating medium
C. Carrier
D. Conductivity of the containers
E. Pathway to the heating medium
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Review Question #9
The primary factors affecting inactivation of

the microorganism include:
A. Time and temperature
B. Water content, temperature, and time
C. Air and temperature
D. Temperature and number of
microorganisms

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Biological Indicators (BI)

• Synonyms Note: Important to
− BI Challenge System evaluate the dry heat
resistance of the BI in
− Microbial Challenge the actual
− Microbiological Challenge System configuration to be
tested for validation as
• Characterized by the following: resistance of the
− Name of the species of bacterium used microorganisms may
− Number of the strain in the original be affected by the
substrate upon which
collection
they are placed
− Number of viable spores per carrier
− D-value
Biological Indicator Challenge System (BI): A standardized preparation of
selected microorganisms used to assess the effectiveness of a sterilization
procedure during process development and validation.
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Dry Heat Sterilization: BIs
• Use microbiological challenges specific to

dry heat sterilization processes
• Spore preparations
– Clean
– Well characterized with respect to their thermal
responsiveness
– “Parent” cultures obtained from recognized
culture collections
• Some sources:
– American Type Culture Collection (ATTC)
– National Collection of Industrial, Marine, and
Food Bacteria (NCIMB) Image courtesy of aiu.edu

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BI Selection and Type of Carrier (1)
• Available in a variety of configurations

including:
– Spore suspensions in solutions
– On paper carriers (i.e., spore strips or
discs)
– Other types of carriers
• The most common BI microorganism
used is Bacillus atrophaeus
– For qualification of dry heat sterilization
Image courtesy of krackeler.com
processes using the overkill design
approach
– However, other dry heat sterilization
resistant biological indicators could be
acceptable
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Biological Indicator Selection

and Type of Carrier (2)
Considerations Resistance of the test strain to the
when selecting particular sterilization method is greater
indicator organisms: than the resistance of microorganisms
potentially contaminating the item or
component
Test strain is non-pathogenic
Test strain is easy to culture

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Biological Indicators:
Sterilization Process
• The sterilization process can be
evaluated in one of two ways
– The BI can be exposed to a partial
process and the kill rate is calculated
– The BI can be exposed to an overkill
process
• Perform a determination of the stability of the
population and resistance of the BI
• Base the actual population and resistance on the
desired lethality and the design approach (e.g., overkill or
product specific approach)

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BI Vendor/Shipping and Handling

If the reliability of a vendor’s
Certificate of Analysis is
Supplied values for resistance of established through a supplier
a biological indicator can be used qualification program
in lieu of confirmatory testing of
each lot
And the BI is not modified
• Confirmatory spore population testing should be conducted upon

receipt of biological indicators due to potential shipping and
handling concerns
• Shipping and handling procedures should be assessed to ensure
no BI adulteration has occurred (e.g., x-ray)
• It is important to use the same enumeration methodology that the
vendor used in order to minimize variables that could lead to
differences in spore count
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BIs and Dry Heat Sterilization
• Compendia (e.g., USP, EP, JP) contain monographs

defining BIs that may be used in evaluating dry heat
sterilization
• It is recommended that consideration is given to
applicable regulatory expectations
Image courtesy of tinhangtech.com

81
Review Question #10
A BI is characterized by the following:

A. Name of the species of bacterium
B. Number of the strain in the original
collection
C. Number of viable spores per carrier
D. All of the above

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Review Question #11
The sterilization process can be evaluated in

one of two ways:
A. Calculate FH-value and survivor curve
B. Expose BI to a partial process and
calculate the kill rate or expose BI to an
overkill process
C. Vortexing and sonication

83
Section 4.0:
Equipment Design

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Equipment Design
• Validation of dry heat depyrogenation and
sterilization cycles is dependent upon
– Equipment
– Load to be processed
• Goal is to deliver effective processes that are
consistent and reproducible
• Design and construction of the equipment governed
by User Requirement Specifications (URS)
• Successful qualification
– Formal verification of proper design
– Installation and operational testing
• Ensures design criteria and specifications are met
85
Overview
Design
Equipment Qualification
User
FAT SAT Installation Operational Performance
Requirements
Physical (Micro)Biological

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Glossary of Terms:
Sterilization Cycle
A sequence of defined
operating parameters
(e.g., time and
temperature) and
conditions required to
render an item sterile.

87
Equipment Design: (1)

User Requirements Specifications (URS)
• Identify the following:

– Oven and tunnel requirements
– Type of load to be processed
• Provides basis for:
– Specification
– Design
– Verification

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Equipment Design: URS (2)
Installation and process requirements

Performance
Temperature requirements Environmental
ranges (e.g., production classifications
capacity)
Installation
location Safety
Control strategy
requirements considerations
and conditions
Utility
requirements

89
Equipment Design:
Dry Heat Process
Predominant
method
Forced
Accomplished Convection
by transferring Focus of
heat to the tunnel and
load through oven design
several means Conduction
Convection: The transfer of heat by the circulation or movement of the

heated liquid or gas.

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Equipment Design:
Convection Heating
Method of transferring heat

through a medium by motion of
its parts
Image courtesy of proprofs.com
• Facilitated by the action of a mechanical

Forced
device (typically a fan), with air passed
Convection
through HEPA or ULPA filters to provide a
Heating
low particulate environment
• A result of the buoyancy forces

Natural
generated by differences in density
convection
caused by temperature gradients in the
Heating
fluid mass
91
Equipment Design:
Dry Heat Process
Forced
Accomplished
Convection
by transferring
Molecular
heat to the
Interaction
load through
several means Conduction
“Free”
Electrons

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Equipment Design:
Conduction Heating
Image courtesy of www.vtaide.com
• Molecules at higher energy levels

Molecular
impart energy to adjacent molecules
Interaction
at lower energy levels
• The ability of solids to conduct heat

depends on the “free” electron
“Free” concentration
Electrons • Pure metallic solids contain the highest
concentration (best conductors of heat)
• Non-metals contain the lowest (poorest
conductors of heat)
93
URS: HEPA Filters
• Heated air forced through these filters

provides an environment with an appropriate
level of particulates suitable for components
being processed
• Heated air is provided to the clean zone that
heats the glassware to depyrogenation
temperatures for the time required for
endotoxin reduction
Note: A later section will discuss design and test parameters for HEPA
filters used in dry heat ovens and tunnels.
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URS:
Oven and Tunnel Considerations
• Non-reactive interior surfaces with smooth

seams and corners
• High temperature, non-shedding gaskets
• Sealed fan bearings
• Sealed control or access penetrations into the
chamber
• Minimize sharp corners and expansion joints

95
Equipment Design:
HEPA or ULPA Filters (1) Image courtesy of techrite.com
• High continuous temperature

design
– Composite filters – 350°C
– Ceramic filters – 400°C
• Temperature limit dependent on sealant type
and filter construction
• Define leak testing at process temperature
for on-going assurance
• Organic challenge agents discouraged

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Equipment Design:
HEPA and ULPA Filters (2)
• Filters should be constructed of materials that

minimize particulate shedding due to temperature
changes
• Items to consider for high temperature HEPA or
ULPA filters include:
– Non-ferrous metal frame
– High temperature glass fiber media
– High temperature sealant
– Non-shedding filter packing
– High temperature gaskets Image courtesy of thermalproductsolutions.com

97
Equipment Design:
HEPA and ULPA Filters (3)
• After installation follow filter manufacturer’s

instructions for curing (burn-off) the filters
at high temperature
• Binders will outgas (burn-off) from the
media
• Install filters with vertical frames with pleats
in vertical position to prevent media sag

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Glossary of Terms
Image courtesy of epsovens.com Batch Oven:

• A convection oven with a chamber or chambers where items
are dry heat sterilized or depyrogenated as a single load in a
discontinuous process
• An oven that uses fans to circulate High Efficiency Particulate
Air (HEPA or Ultra Low Particulate Air (ULPA) filtered heated
air around the articles to be sterilized or depyrogenated
99
Equipment Design:
Batch Convection Oven
• Typically controlled and monitored
by a programmable temperature
control system
– Provide appropriate temperature ramp,
exposure and cool down
Image courtesy of despatch.com • Monitor air flow and pressure
differential provided by the oven fan
• Control door interlocks during the
process and until the processed
load is unloaded to prevent ingress
into the oven

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Equipment Design:
Interlock System on a Double Door Batch
Convection Oven
• Mechanism to prevent:
– Opening of either door out of sequence
– Or both doors at the same time
• Maintain pressure gradient in an
environmentally controlled area:
Unloading Loading
Chamber
Area Area
Highest
pressure

101
Equipment Design:
Batch Convection Oven
Measure
Filter air differential
intake and pressure
exhaust vents across filters
Conduct risk
assessment to
determine the
type of filter
needed

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Example of Batch
Figure 4.1.2-1:
Convection Oven Showing Airflow

103
Equipment Design:
Continuous Convection Tunnel (1)
• Conveyor system moves load through heated
tunnel
– Predetermined load
– Predetermined rate
• May include three zones: Image courtesy of www.enceko.pl
Load/Preheat • Pre-warm glassware

Zone
• Glassware exposed to process temperature
Hot Zone for sufficient time to effect sterilization and
depyrogenation
• Cool glassware to room temperature prior to

Cool Zone exiting sterilizer
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Equipment Design:
• Correlates to exposure time

Conveyor
• Document
speed is
critical • Variable speed accommodates vial
sizes
• Temperature control of hot zone is

required
Temperature
• Temperature distribution can vary with
load
Image courtesy of bio-world.com
• Temperature high limit controls are
required for continuous processes

105
Figure 4.1.3-1:
Continuous Convection Tunnel

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Equipment Design:
• Differential pressure ranges across zones

minimize cool zone air entry into discharge
area
• Set and document position of gates between
zones
• Monitor cool zone to assure items are cooled
to appropriate temperature
– Sanitized by a qualified sanitization process
– Frequency of sanitization determined by risk

107
Equipment Design:
Cool Zone Decontamination Methods
Cool Zone self- Exposure to

decontamination disinfectants/ Manual cleaning
by thermal sterilants and sanitization
means (vaporized hydrogen
peroxide)
• Incorporate controls into tunnel design

• Disinfectants/sterilants could also be drawn from
the room into cool zone

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Equipment Design:
Continuous Tunnels
• Typically include data recorders to monitor the
process and print process reports
• Monitor physical parameters
– Conveyor speed
– Temperature of the pre-heat zone
– Heat zone and cool down zones
– Forced air fan alarms
– Zone pressure
• System alarm indicates deviation from process
specifications
109
Review Question #12
Which of the following are considered in the

specification, design, and verification of the
equipment: (select all that apply)
A. Type of load to be processed
B. Temperature requirements
C. Selection of HEPA filters
D. Environmental classifications

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Review Question #13
The predominant method of the dry heat

process includes which of the following:
(select all that apply)
A. “Free” electrons
B. Forced convection heating
C. Molecular interaction
D. Natural convection heating

111
Review Question #14
What factor affects the temperature

distribution in a continuous convection
tunnel?
A. Conveyor speed
B. Full, empty, or partially loaded tunnel
C. Different size containers

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Section 5.0:
Equipment Qualification

113
Equipment Qualification:
Process Equipment
 Check process equipment and associated

systems for proper operation
• Factory acceptance testing
• Commissioning
• Installation qualification
 Verify materials of construction
• URS
• Manufacturer’s specification

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Glossary of Terms
Commissioning:
• A well planned, documented and managed engineering
approach to the start-up and transfer of facilities, systems and
equipment to the end-user that results in a safe and functional
environment that meets established design and user
requirement specifications.
• Commissioning precedes Qualification and includes three
phases:
2) Adjustment
1) Inspection and 3) Functional/
and Setting to
Testing, Regulation Run Testing
Work

115
Equipment Qualification (1)

Qualification of equipment
controls typically includes:
• Verification of alarms
• Set points
• Temperature control
• Interlocks
• Sequence of operations
Operational Qualification
• Functional testing of all process steps

• Ensures correct sequence of operation
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Glossary of Terms: Verification
• A systematic approach to verify that

manufacturing systems, acting singly or in
combination, are fit for intended use, have
been properly installed, and are operating
correctly
• An umbrella term that encompasses all types
of approaches to assuring equipment is fit for
use such as qualification, commissioning and
qualification, verification, system validation, or
other
117
Additional Functionality Testing (2)
• Focus on critical process parameter alarms

• Ensures equipment can reliably indicate
non-conforming and unsafe operating
conditions
• Completed as a separate test during OQ or
included in the testing of overall operation of
the unit
Critical Alarm Testing: A series of tests to determine if the electro/mechanical
operations described in the equipment specification perform as stated

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Table 5.0 –1: Example of Equipment

Qualification Checklist (1)
Checklist Item Batch Continuous
(Oven) (Tunnel)
Electrical Program Logic – ensure that each step is in X X
the correct sequence and that it is repeatable
Verification of alarms – ensure alarms operate as X X
specified (e.g., system overloads, interlocks)
Item Interlocks Overload – must work correctly, not X
allowing excess item buildup during processing
Door Interlocks – must work correctly, not allowing X
access during the process
Gasket Integrity – X X
Ovens: check for positive/negative pressure seal of all
door gaskets
Tunnels: check that the leakage rate does not exceed a
predetermined value at all panel gaskets from zone to
zone and to the external environment

119

(Oven) (Tunnel)
Air Balance – check that the ∆P meets defined requirements X X
respect to the preparation section through the tunnel.
Vibration Analysis – check blowers for correct dynamic X X

balancing to minimize vibration
Louver Balance Ability – check that louver/linkage X
mechanisms can be actuated and adjusted for balance
Louver Position- document position of manually adjusted X X
Louvers (as applicable)
Gate Balance Ability – check that gate control mechanisms X
can be actuated and adjusted for balance
Blower Rotation – check that blowers rotate in the specified X X
direction

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(Oven) (Tunnel)
Blower Revolutions Per Minute (RPM) – verify that X X
the correct blower RPM is achieved
Heater Elements - check that all heater elements X X
operate
Room Balance – check that the ∆P balance is positive X X
from the sterile core to the preparation area when the
tunnel or double door batch oven is in operation
Verify HEPA Filter Integrity X X
Temperature Sensor – ensure temperature sensors are X X
installed appropriately (location, orientation).
Belt Speed – verification of correct belt, speed X
controller and recorder

121
Final Check
• A series of processes to verify each system
component
– Interacts correctly
– In the programmed sequence of events is
repeatable
• Use the appropriate standards for calibration
tolerance and calibration frequency for
instruments used to:
– Monitor critical operational parameters
– Monitor critical quality attributes
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Environmental Qualification (1)

• Control of particulates with HEPA or (ULPA) filters
• Pressurization of clean zones and chambers
• Verify particulate levels, clean zone
pressurization and air flow and balance meet
design specifications
• Particulate quality appropriate for exiting
environment classification
• Establish airflow parameters to maintain
temperature uniformity and air quality under
dynamic conditions
• Use airflow velocity readings to determine airflow
uniformity across HEPA filters

123
Environmental Qualification (2)
• Operational testing verifies aseptic

environment is maintained
• Perform particulate testing at operating
temperature as well as during heat-up
and cool down phases
• Draw samples from worst case locations
• Meet applicable standards for particulate
concentration at a given location

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Equipment Verification:
Room Differential Pressure
• Establish requirements for oven or tunnel
• Impacts unit air flow and temperature distribution
• Verify air pressure differentials in operational testing of
sterilizer
• Minimum pressure differential established is dependent
on the system design
• Verify air balance between sterilizer and outside
environment

125
Review Question #15
Qualification of equipment controls typically

includes:
A. Verification of alarms, set points,
temperature control, interlocks, and
sequence of operations
B. Functionality testing of all process
steps
C. Factory acceptance testing,
commissioning, and Installation

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Review Question #16
It is important to verify that each system

component interacts correctly and
repeatability in the programmed sequence
of events.
A. True
B. False

127
Uniformity of Heat
• Perform temperature distribution studies

to establish appropriate air flow which
ensures thermocouple accuracy:
– Batch Ovens
– Tunnels
Image courtesy of www.enginebuildermag.com
Temperature Distribution: Temperature measurement of the heating

medium (e.g., forced hot air) across the chamber load zone
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Uniformity of Heating Media:

Batch Ovens
• Adjust discharge louvers

• Document position
• Obtain uniform temperature
profile
• Monitor the particulate quality of discharge air
at process temperature
• Ensure that it falls within pre-established
acceptable limits

129
Uniformity of Heating Media:

Continuous Tunnels
• Adjust air supply

– Controlled flow
– Change fan speed or damper position
• Ensure air quality at process temperature
• Data used as basis for future flow
pattern modifications
• Determine number and type of tests
necessary

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Empty Chamber Temperature

Distribution (Ovens and Tunnels)
• Confirms air balance and heated air
supply provides even heating
• Typically multiple runs to confirm
reproducibility of:
Heating
Cool
Heat up time medium
locations
uniformity
• Establish process temperature set point

– Minimum temperature specification

131

Distribution: Batch Oven
• Distribute thermocouples throughout the

chamber load zone
• Represent horizontal, vertical, and lateral planes
• Thermocouples should not contact internal
surfaces
• Place thermocouple close to control
thermocouple and correlate average temperature
Load Zone: Area within in the chamber where materials to be sterilized or

depyrogenated may be placed.

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Distribution: Continuous Tunnel
• Distribute thermocouples across belt width

– Affix to belt or mounting bar
– Measure at entrance, exit, load zone
• Thermocouples should not contact internal
surfaces
• Placement height representative of
anticipated load
• Place thermocouple close to control
thermocouple (where possible) and correlate
133
Temperature Distribution Qualification
• Confirm critical and key operating

parameters
• Document
• Tunnels at nominal set points
– Belt speed
– Hot zone temperature
• Define equilibration time
• Evaluate temperature variability
Image courtesy of www.comsol.com

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Review Question #17
Temperature distribution studies are performed

to establish:
A. Proper calibration tolerance and
calibration frequency
B. Appropriate air flow to ensure equipment
performance
C. Room differential pressure requirements
for the oven or tunnel

135
Review Question #18
Multiple empty chamber distribution tests are

performed to confirm: (select all that apply)
A. The air balance and heated air supply
will provide even heating
B. Reproducibility of heat-up and cool
locations
C. Heating medium uniformity

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Section 6:
Process
Development

137
Process Development
Goal: Identify critical and key operating

parameters
Select items constituting load
Determine thermal dynamics of

chamber
Determine thermal profile of the

items and loading patterns
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Process Development
• Represent actual
manufacturing
conditions (e.g.,
spraying vials with water
to simulate vial washing)
• Document
Image courtesy of www.pharmaceutical-int.com development process

139
Process Development:
Process Design Approaches
Overkill Design Product Specific

Approach Approach
Both approaches provide a process that delivers

the appropriate level of sterility assurance or
endotoxin reduction to the items being
depyrogenated or sterilized

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Overkill Design Approach
• Depyrogenation Process
– Demonstrate a minimum of a 3 log reduction of endotoxin
at the coolest location in the worst load
• Sterilization Process
• Biological indicators used to demonstrate lethality
− Known population
− Resistance
• Bacillus atrophaeus generally used
• Deliver a probability of a non-sterile unit (PNSU) that is
less than or equal to 10-6
Note: Consider the potential for thermal degradation of items when overkill used
Probability of a Non-Sterile Unit (PNSU): The number that expresses the
probability of occurrence of a non-sterile unit after exposure to a sterilization
process
141
PNSU Formula
The probability of a non-sterile unit at the minimum FH delivered in

the load can be calculated using the following equation:
Log PSNU = -FH/D + Log No
FH = Lowest FH No = initial
D = D160C-value of the BI
calculated within load population of BI
D160C-value = 2 minutes
FH = 30 minutes N0 = 1 x 106
Log PSNU = -30 minutes/2minutes + 1 x 106

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Calculation of PNSU
Log PSNU = -30 minutes/2minutes + 1 x 106
Log PSNU = -30/2 + 6

Log PSNU = -15 +6
Log PSNU = -7
PSNU = 10-7
Therefore, the predicted probability of a non-sterile
unit at the coolest area within the load (lowest FH
value) is 10-7 (not more than 1 non-sterile unit in 107
units) which is well in excess of the accepted
standard of 10-6.
143
Product-Specific Approach (1)
• Used to develop sterilization

cycles for heat labile items
• Determine population and heat
resistance of microorganisms
in/on product
• Design process to result in a
PNSU of 10–6
Product-Specific Design Approach: A sterilization design approach that is

based on the characteristics of the bioburden (on or in the load) and the heat
sensitivity of the product that delivers the lethality needed to achieve a PNSU ≤ 10-
6 on or in the items to be sterilized.

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Product-Specific Approach (2)
• Inactivation of microbial challenge to a

predetermined level demonstrates that the
desired probability of survival of product
bioburden is achieved
• Bioburden must be monitored
• Frequency of monitoring determined through
risk assessment
Note: For additional detail on the product specific design approach

to sterilization, see PDA Technical Report No. 1.
145
Defining Operating Parameters
• Critical operating parameters

– Temperature
– Exposure time (conveyor speed)
• Additional key parameters
– Time and temperature during heat up and
cool down phases
– Pressure differentials
Operating Parameters: Values (e.g., time, temperature, air-flow) that are

controlled and/or measured that collectively define each phase of a process
(e.g., heat-up, exposure, cool-down).
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Critical Process Parameters

and Key Parameters
Critical Process
Key Parameters
Parameters (CPP)
• Values that are • Values that are
controlled and/or controlled and/or
measured measured
• Linked to safety and • Used to assure the on-
efficacy of a product or going “state of control”
the process and consistency of
• Reject load if failure to runs
meet a critical • Investigate failure to
parameter meet a key process
parameter

147
Operational Parameters (1)
 Develop operational parameters that ensure
 Sterilization: minimum PNSU of a least 10-6
 Depyrogenation: minimum 3 log reduction in endotoxin
 Achieve parameters within the loads or
components
 Use as a starting point for process or process
development
 Perform temperature studies to determine worst
case conditions in load

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Review Question #19
The goal of process development is to identify

critical and key operating parameters that will
result in a load meeting the minimum
acceptance criteria for depyrogenation/
sterilization
A. True
B. False

149
Review Question #20
The two process design approaches include:

A. Sterilization and Depyrogenation
B. Overkill and Product-Specific
C. Sterility Assurance and Endotoxin
Reduction

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Review Question #21
Operational parameters for depyrogentation

should: (select all that apply)
A. Achieve a minimum PNSU of at least 10-6
B. Achieve a minimum 3 log reduction in
endotoxin
C. Be achieved within the loads or
components being processed

151
Batch Oven Process Development
• Identification of loading patterns and worst case

load
• Temperature distribution studies verify heating
uniformity of the load zone
• Heat penetration studies demonstrate
temperature being delivered to items in load for
the appropriate time
• Typically determined using temperature
measurements
• Confirmed with BI or EI challenge testing

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Batch Oven:
Developing Load Patterns (1)
• Determine ‘worst case’ load based on
– Load mass
– Configuration
– Other parameters
• Verify choice
• Develop process parameters to achieve required
conditions of time and temperature
• Ovens: industry experience demonstrates validation of
maximum load for a given load configuration is considered
sufficient to validate loads of less mass (i.e. minimum
loads)

153
Glossary of Terms:
Worst Case, Maximum, and Minimum Loads
Worst Case Load: The load configuration that is

determined to be most difficult to sterilize or depyrogenate.
This is a function of the process control strategy and load
item characteristics (e.g., mass, configuration)
Minimum Load: The minimum quantity or mass of items

permitted in a depyrogenation or sterilization load
Maximum Load: The maximum quantity or mass of items

permitted in a depyrogenation or sterilization load

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Batch Oven:
• Allow adequate space in load configuration

– Air circulation
– Penetration by heat
– Removal of moisture
• Consider wrapping or container type
− Protects against contamination (microbial and
particulate)
− Ensures adequate air movement and moisture
removal

155
Batch Oven:
• Raise large items (not on cart) off of floor to
allow air circulation
• Trolley position within the oven must be
reproducible and included in load pattern
documentation
• Load orientation can affect item heat up
• Trays, racks, carts
• Should not generate particulates
• Must withstand heating process without
degradation
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Batch Oven:
Identification of worst case

Heat penetration studies
location or hardest to
using FH values may be
sterilize/depyrogenate
useful to identify hardest to
locations in the load should
sterilize/depyrogenate
be performed by analysis of
areas.
heat penetration studies.

157
Loaded Batch Oven

Temperature Distribution Studies (1)
• Primary purpose is to verify temperature

distribution of the heating medium across the
load
• May be conducted concurrently with heat
Image courtesy of www.despatch.com
penetration studies
• Place temperature thermocouples in load
– Not in contact with the items or oven hardware
(e.g., carts, shelves, trays)
• Document sensor locations for each load Image courtesy of picotech.com
Thermocouple: A device for measuring temperature in which a pair of wires of

dissimilar metals (such as copper and iron) are joined and the free ends of the
wires are connected to an instrument (such as a voltmeter) that measures the
difference in potential created at the junction of the two metals.
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Loaded Batch Oven

Temperature Distribution Studies (2)
• During temperature distribution qualification

confirm and document operating parameters
• Exposure phase acceptance criteria may
include the maximum
– Variation in the temperature measured by
each probe
– Variation in the temperature measured from
probe to probe
– Difference in temperature between the
probes and the control temperature set point

159
Glossary of Terms:
Exposure Phase/Dwell Time
Exposure Phase: The phase of the process in which the

appropriate parameters are maintained within defined
ranges for the time (exposure time or dwell period) and
temperature determined to be necessary to achieve the
desired lethality
Dwell Time: The period that items are subjected to a

given processing condition. Synonym:
• Synonym: Residence Time

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Loaded Batch Oven

Heat Penetration Studies (1)
Prior to heat penetration studies

• Load characterization through item-
mapping studies may be necessary
• Identifies appropriate monitoring locations
within individual load items
Item-temperature mapping: Determines the location within the item or

package most difficult to heat

161
Loaded Batch Oven

• Data may be used to calculate F-values for each
probe location
• Position temperature probes in the load to determine
the slowest-to-heat location(s) within the load
– Represents most difficult to depyrogenate/ sterilize
– Generally conducted under worst case operating
conditions
• Minimum time and temperature set point
Heat Penetration: Heat penetration testing is a temperature measurement that

is used to evaluate the amount of energy that has been transferred to the
materials within the load. For measurements of heat penetration, the probes
should be placed in the load with the tips of the sensors in contact with the items
being evaluated.
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Loaded Batch Oven

• For loads with items that have
different heat penetration
characteristics, place probes in
representatives of each item type
• Document Image courtesy of directindustry.com
• Temperature sensor locations for each

qualification load
• Rationale for selecting locations
Penetration Probe: A thermocouple placed in contact with the load item to

measure the temperature of the load item.

163
Figure 6.3.3-1 Load Profile:

Batch Oven

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Loaded Batch Oven

• Evaluate effectiveness of process in the

heat penetration study
– Analyze calculated FH values for all temperature
probes
• Use data to determine cold spots which
provides worst case location in PQ
• BI or EI challenge studies can be
conducted concurrently
• Calculate lethality and/or endotoxin
reduction
165
Continuous Convection
Tunnel Process Development (1)
• Typically used to sterilize and depyrogenate

containers before filling with final product
• The following constitutes an integrated
continuous flow operation
Container Depyrogenation Filling

washer tunnel machines

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Continuous Convection
Tunnel Process Development (2)
• Tunnels may operate at a fixed belt speed and the number of
containers varied according to the demand from the filling
machine
• A variable speed belt offers greater operating flexibility and is
more common in newer installations
• The period during which the containers remain within the hot
zone, also referred to as the “sterilization / depyrogenation
dwell time”, is set by conveyor belt speed
• Required dwell time in the tunnel hot zone can be empirically
derived by thermal analysis during empty and loaded chamber
studies
Dwell Time: The period that items are subjected to a given processing condition.
[Synonym: Residence Time]
167
Continuous Tunnels:
Developing Loading Patterns (1)
• The ‘worst case’ load determined on the basis of the
following
– Load mass
– Configuration
– Other parameters
• Develop process parameters to achieve the required

conditions of time and temperature for the load
• Place load items into the tunnel so they remain upright
• To maintain this load
– The belt speed must be consistent with the washer speed, or
– The belt speed is constant and the width of the load is varied
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Continuous Tunnels:
• Exposure time controlled by:

– Belt speed
– Set-point temperature
• Temperature of glass is affected by:
– Exposure temperature
– Airflow
– Mass of glassware
• Exposure temperature maintained by a feedback
control loop
– Controls temperature at a constant steady rate
– May vary as the load conditions change

169
Continuous Tunnels:
Example
• When glass first enters the tunnel, the

introduction of cold containers may cause the
tunnel temperature to drop.
• The severity of the drop is dependent on the
response characteristics of the control system.
• Airflow should be regulated and relatively
constant so not to impact temperature.

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Continuous Tunnels:
• Establish relationship between
Drying/ heating/
Washing Speed cooling zone belt Filling speed
speed
• Load the conveyor belt in a uniform manner to provide

reproducible thermal conditions within the heating zones
• Irregular gaps in the components will produce non-
standard but typically more effective thermal conditions
• These situations may occur in production operation and
are of no consequence
− However they should be avoided during qualification studies
• The process must provide components from the cooling
zone into the aseptic area at a suitable temperature for
filling Validation of Dry Heat Processes
171
Continuous Tunnels:
• Introduce loads into the heating zone in a manner
representative of actual manufacturing conditions
• Determine thermometric conditions when the load
enters and exits the hot zone
• The largest load mass/unit area may be validated
as representative of the range of items to be
qualified
• Selection of the worst-case challenge(s)
conducted using scientific rationale and/or
determined through heat penetration or challenge
studies
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Continuous Tunnels:
Dry Heat The ‘worst case’ load for each temperature
setting must be determined by calculating the
Tunnels throughput of items (expressed in mass/time)
Consider belt speed/item passage time
Consider number and weight of items being

processed in a unit of time (tunnel capacity)
Set belt speed to ensure that dwell time within

hot zone provides required biological
inactivation or endotoxin reduction

173
Continuous Tunnels:
Developing Loading Patterns Summary (1)
Selection of a “worst case” load simplifies the
validation approach to tunnel depyrogenation by
running studies under conditions which produce a
“worst case” challenge seen in production for the
following three categories:
Mass Rate of
Belt
per unit mass
speed
area loading

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Continuous Tunnels:
Developing Loading Patterns Summary (2)
The steps can be summarized as follows:
Load the tunnel

at the maximum
Set the belt rate
speed to the
Select the maximum for
container that any container
produces the
maximum mass
per unit area

175
Loaded Tunnel
Temperature Distribution Studies
• Assess the impact of
the load on the ability
of the hot zone to
maintain temperature
uniformity
• May be conducted
concurrently with heat
penetration studies
Image courtesy of www.bausch-stroebel.com

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Loaded Tunnel Temperature

Distribution Measurement
Measuring Attach sensors to the load near the probed
loaded containers to allow free flow of air around sensor
temperature
distribution Sensing element should not touch the containers
Measures temperature uniformity across width and

length of load
When placing temperature sensors account for cool

areas found during the empty belt chamber studies
Document location of temperature sensor

177
Loaded Tunnel Temperature

Distribution Performance
• Confirm and document operating parameters
during qualification runs
– Minimum and maximum temperature measured at
each probe position
– Variation in the temperature between probes
– Difference between the probes and set point
temperature
• Operate tunnel at nominal set points for belt
speed and hot zone set point temperature
• Define steady state or equilibration time
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Loaded Tunnel Heat

Penetration Studies
• Verifies the worst case load under worst case

operating conditions reaches and maintains
depyrogenation temperatures
• Endotoxin studies can be conducted
concurrently
• Separate EI glassware and glassware for
temperature sensors
– Ex.: Do not use inoculated glassware for the
temperature sensors
• Perform studies in triplicate
179
Loaded Tunnel Heat Penetration

Studies: Glass Loads
• Replicate residual water left by the normal
washing process when preparing glass loads
– Add representative amounts of water in the items
containing sensors
• Ensure the load is
received into the hot
zone as dry as
possible during line set
up
Image courtesy of penntech-corp.com

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Loaded Tunnel Heat

Penetration Studies: Probes
• Use sufficient number of probes
to provide adequate profile of
temperature penetration of the
load
• Place probes in contact with
interior of individual items
• Account for any cool areas found
in temperature distribution studies
when placing probes
• Provide diagrams with specific
temperature sensor locations
181
Figure 6.4.3-1
Glass Vial Load Heat Penetration Profile:
Continuous Convection Oven

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Typical Qualification
Acceptance Criteria
Minimum and
maximum
FH-values
temperatures
achieved
Minimum dwell
Variation in the
time achieved
temperature
above a defined
between probes
temperature
183
FH-Values
Calculation of an F-value at each

probe location helps:
• Assess process comparability
• Evaluate process repeatability
• Locate cool areas
Note: There is no minimum FH-value acceptance criterion for

depyrogenation. Endotoxin inactivation efficacies cannot be
accurately correlated with standard dry heat lethality conventions
(FH) which rely upon a linear destruction model.

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Review Question #22
Batch oven process development consists of:

(select all that apply)
A. Utilities Analysis
B. Identification of loading patterns and worst
case load
C. Temperature distribution studies
D. An integrated continuous flow
E. Heat penetration studies

185
Review Question #23
During a loaded batch oven heat penetration

study temperature probes should be
positioned in the load to determine the
fastest-to-heat locations
A. True
B. False

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Review Question #24
In order to maintain a “tight” load in a

continuous tunnel, so that items that may be
distributed will be held upright by the
neighboring items the following must exist:
A. Belt speed must be consistent with the
washer speed
B. Belt speed must remain constant and the
width of the load must be varied
C. All of the above
D. A or B
187
Review Question #25
Selection of the worst case load for tunnel

depyrogenation
A. Simplifies the validation approach
B. Is conducted through heat penetration
studies
C. All of the above
D. None of the above

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Section 7:
Performance
Qualification
Performance Qualification (PQ): Documented verification that a system
is capable of consistently performing or controlling the activities of the
processes it is required to perform or control, according to written and
pre-approved specifications, while operating in its specified operating
environment

189
Performance Qualification
• Performance Qualification
– Process of obtaining and documenting evidence that the
equipment, as installed, consistently operates in
accordance with predetermined criteria
Components of Performance Qualification
Biological
Physical Qualification
Qualification

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Performance Qualification:
Physical Qualification (1)
• Ensures the depyrogenation/ sterilization
process as developed is reproducible
– Physical Qualification
– Endotoxin/endotoxin qualification
• Verifies temperature profiles within the oven or
tunnel
• Ensure items reach required temperature for
specified time
Physical Qualification: A component of performance qualification that
demonstrates that pre-determined physical requirements including
temperature distribution and heat penetration are achieved consistently
throughout the load.
191
Physical Qualifications (2)
• Confirm reproducibility of temperature
profiles developed in process development
by performing temperature distribution and
heat penetration qualification with ‘worst’
case loads
• Place calibrated temperature probes in the
coolest locations established during
process development temperature
distribution and heat penetration studies
• Confirm during heat penetration
qualification that items exiting the tunnel to
the filling suite are cooled to a product-safe
temperature Validation of Dry Heat Processes
192
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Biological Qualification
• Demonstrates that the required endotoxin
inactivation or lethality is achieved
consistently throughout load
– Endotoxin
– Biological Indicators
• Place indicators in numerous locations in
the load, including the most difficult to
depyrogenate or sterilize locations
Biological Qualification: A component of performance qualification that
demonstrates, by use of biological indicators, that the required lethality is
achieved consistently throughout the load.
193
Biological Qualification:
Dry Heat Depyrogenation
Validation data demonstrates the process consistently
reduces endotoxin by three (3) logs
• Inoculate articles to be treated with a minimum of 1,000
International Units (IU) of endotoxin per article
• Inoculated articles tested for endotoxin to demonstrate at
least 3 log reduction from original concentration after
treatment in the dry heat depyrogenation process
− Limulus Amebocyte Lysate (LAL) test
− Bacterial Endotoxins Test (BET)
• Where depyrogenation has been demonstrated,
sterilization is assured due to the lower resistance of
microorganisms to dry heat (as compared to bacterial endotoxin).
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Biological Qualification:
Dry Heat Sterilization
Validation data demonstrates the process
consistently delivers a microbial survivor
probability for the challenge organism of not less
than 10-6
• Select number of BIs and processes for
performance qualification (PQ) to cover
range of conditions in load during routine
processing
• Placement of BIs based on data from
process development
• Place each heat penetration probe in the
load adjacent to a BI location

195
Biological Indicator Testing (1)
• Follow manufacturer’s
directions for use
– Medium and conditions to
be used for the recovery of
microorganisms after
exposure to the sterilization
process
• Aseptic technique when
testing BIs
• Investigate positive BIs
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Biological Indicator Testing (2)
If a Determine the spore population, confirm purity
suspension and identity of the BI
is used for
direct or Stock suspension should be predominantly
carrier spores in a non-nutritive liquid
inoculation,
consider the Determine the D-value (This is done on the
following item being sterilized if the BI is inoculated onto
the item)
points:
Preparation of the BI needs to be carefully
controlled for purity, protecting the D-value,
protecting the count, and so forth
Determine worst case inoculation site on the
item to be inoculated based on slowest to heat
location.
197
Endotoxin Indicator Testing (1)
• Widely accepted method that demonstrates overkill or a
three log reduction of endotoxin
– Inoculation of endotoxin onto materials to be depyrogenated
– Recovery of endotoxin spike from treated components
• The stages of such testing via dry

heat depyrogenation process are:
– Preparation and inoculation/
endotoxin spike application and
controls
– Dry heat treatment process
– Recovery
Image courtesy of brass-asiapacific.com
– Endotoxin log reduction calculation
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• Use actual items or representative coupon of
surface type to be depyrogenated
– Heat resistance and effectiveness of the
depyrogenation process may be influenced by
carrier type
• Adsorption of endotoxin spike to carrier surface is
biggest obstacle affecting recovery
• Use commercially available EIs or endotoxin
challenge vials in place of actual production items
or representative coupons
– Must demonstrate equivalent or worst-case
challenge to the depyrogenation process
199
Conduct preliminary studies with the selected

endotoxin indicator, the inoculation, drying
and recovery methodology prior to
performing depyrogenation validation studies
Image courtesy of gen-biotech.com

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Process Equivalency (1)
• Possible to establish the equivalence of:
– Two or more dry heat batch processes (chambers)

OR
– Continuous processes (tunnels) similar in design
(including utilities being supplied)
• Recommend a robust risk analysis process to

make this determination
• Analysis includes, but is not limited to, a design

and engineering evaluation

201
Some Chamber or tunnel size and configuration

factors to
consider Airflow dynamics
include:
Temperature come-up and uniformity
Conveyor speed
Temperature equilibration time
Materials of construction

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• Regulatory approval may
be required to support a
reduction in qualification
testing
• In order to demonstrate equivalency all

processes must be qualified and meet
operating parameter acceptance criteria

203
Operational Equivalence
• Established by an analysis of all process data
associated with validated process in new
equipment
• Compare chamber and load profiles using the same
critical process parameters
• Confirm the specifications, acceptance criteria, and
load configuration are the same as those used in
established sterilization validation process of
existing equipment

204
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Performance Qualification
Comparable criteria include the following

parameters such as:
Heat
Load Temperature
penetration, FH
configurations distribution
value range
Heat-up and
BI or EI cool down times
inactivation of the
product/item

205
Review Question #26
Dry heat depyrogenation validation data

should demonstrate that the process
consistently delivers a microbial survivor
probability for the challenge organism of not
less than 10-6
A. True
B. False

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Review Question #27
What is the biggest obstacle affecting recovery

for a Endotoxin Indicator Test?
A. Adsorption of the endotoxin spike to the
carrier surface
B. Heat resistance and effectiveness of the
depyrogenation process
C. All of the above
D. None of the above

207
Review Question #28
In order to establish process equivalency the

following is recommended to make this
determination:
A. An analysis of all the process data associated
with a validated process in the new equipment
B. A robust risk analysis process including a design
and engineering evaluation
C. A comparison of chamber and load profiles using
the same critical process parameters

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Section 8:
Ongoing Process
Control

209
Ongoing Process Control
Conduct ongoing control and monitoring of

the process
After
After release of
On a continual completion of
the equipment
basis performance
for use
qualification

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Ongoing Process Control:

Important Elements
• Review of critical operating parameters for routine
release
• Evaluation of changes
• Periodic revalidation of equipment
• An effective preventative maintenance program to
include filter integrity testing
• Instrument calibration
• Inspection of component wear (i.e., belts)
• Periodic revalidation of the process as necessary
to confirm effectiveness

211

Routine Release
• Develop, perform and document
procedures for on-going monitoring of
routine operational processes
• Document, review, and maintain
critical and key parameters for each
process
• Reject processes that have not met
minimum defined critical parameters Image courtesy of gbengaadebayo.com
• Investigate key parameters not met

and determine load disposition
following investigation results

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Operational Parameters
Oven: Tunnel:
• Time at temperature • Temperature
(such as minimum heat up
time, minimum exposure or • Conveyor belt
dwell time and minimum cool- speed
down)
• Temperature set- • Differential
point exposure pressure
(between tunnel and clean
• Differential pressure room/cold zone/ tunnel room)
as applicable (e.g.,
between double door batch
oven and a room)
Note: Compendial sterility or biological indicator testing may be required to

support product release.
213

Preventive Maintenance
• Establish program to
assure that ovens and
tunnels remain
mechanically sound and
capable of continuously
meeting process
requirements
• Consider vendor
recommendations

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Points to Consider for PM Program (1)
 Check the oven/tunnel blowers for excessive
vibration
 Visually inspect and test all critical filters
 Lubricate motors
 Visually inspect the conveyer and belts for
unusual wear (tunnel)
 Inspect for unusual wear
– Motors of the conveyor
– Recirculation fan
– Fresh air fan
– Exhaust air

215

Points to Consider for PM Program (2)
 Inspect the cooling water system control valves
 Inspect pneumatic lines for leaks, cracking or
moisture
 Define replacement frequency of HEPA or
ULPA filters
 Inspect door seals
 Inspect tubing of the differential pressure
transducers
 Check and inspect the tunnel and oven
screens for debris and broken glass
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Change Control / Revalidation (1)
A change control program maintains the state of
control
• Document equipment, process, and product changes
• Include documentation of any testing required to
ensure the validated state of control
• Evaluate equipment or process changes
– Determines potential effects of those changes
– Demonstrates modified equipment or process performs as
intended and meets established acceptance criteria
Revalidation: Repeating partial or full validation of a process after a

process change is implemented. Change-based, not time based

217

Change Control / Revalidation (2)
Change control program continued

• Review changes made to product/item to identify
potential impact the change has on existing
validation
• Consider
− Worst case product/item selected for
use in validation testing
− Impact to the thermal conductivity of the
product/item
• Determine assessment of
equipment, process, and Image courtesy of www.ipmglobal.net
product/item changes for revalidation

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Change Control Requests
• Identifies documents that may be affected by the
change and includes:
– Description of the proposed change
– Documented reason/rationale for proposed change
– A description of the tests needed to revalidate the process
– Technical rationale supporting no impact on process
efficacy (minor change)
– Supporting documentation for tests performed,
interpretation of results and conclusions
– Confirmation that documents affected have been updated
– Approval of the change control package by the Quality Unit
219

Periodic Requalification of Equipment (1)
• Re-qualify equipment on a routine schedule to

ensure there has not been an undetected
change
• Perform and document requalification using
qualified operational parameters and
acceptance criteria
• Results should demonstrate that the
equipment’s performance has not changed from
its qualified state
Requalification: Periodic confirmation to demonstrate that equipment
performance has not changed from its qualified state.

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Periodic Requalification of Equipment (2)
Include the following:
• Worst case load configuration in requalification
runs
• Review of the following:
– Change control documentation
– Deviations
– Preventative maintenance records
– Routine process data
– Software, set points and control system operations (to
assure no unexpected changes have occurred)
Note: Requalification could also include an empty chamber run to assess
temperature distribution Validation of Dry Heat Processes
221

Parametric Release Definition
Parametric Release: A sterility release

system based upon effective control,
monitoring, documentation, and batch
records review of a validated sterilization
process in lieu of release procedures based
on end-product sterility testing, such as,
without having to perform Compendial
sterility testing. This program typically
requires prior regulatory approval

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Parametric Release (1)
The elements of a parametric release program build on a
foundation of the entire quality system including:
Change Written
Training
control procedures
Planned Failure
Prevention of
preventative mode/risk
human error
maintenance analysis
Product
Validation Calibration release
requirements

223

Parametric Release (2)
• Applies only to products terminally sterilized

in their final containers or packaging
• Regional regulatory approval is required
prior to use
– Once approved for parametric release, a
product cannot be released based on a sterility
test

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Ongoing Process Control: (1)

Considerations for Parametric Release
• Quality Risk Management: Risk assessments that
identify risks and take corrective and preventative
measures to mitigate and control those risks to an
acceptable level
• Personnel: Competency in engineering and microbiology
specifically trained in sterilization practices
• Package Integrity Data: Validated container closure
testing after exposure to worst-case sterilization
processing to ensure that the sterilized product maintain its
sterility over the shelf life of the product
• Bioburden Control of pre-sterilized Bioburden: Properly
designed manufacturing facility and processes as well as
an established bioburden monitoring program
225

• Handling Products: Ensure that non-sterile products
are segregated from sterilized product
• Sterilization System: Designed and calibrated to
monitor and control the critical operational parameters
and performance attributes
• Biological Indicators: Well characterized and
appropriately selected to provide worst case
challenge to process
• Physical Parameters: Must be defined, reproducible,
and measurable and should be recorded

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• Product Specific Approach for Heat Labile Products:
Biorburden should be well-characterized
• Process Development: Includes load definition, load pattern,
and determination of operational parameters. Ensure required
physical and biological lethality are delivered to the product
resulting in the achievement of less than or equal to 10-6 PNSU
• Perform Equipment Qualification and Process Validation
• Ongoing Process Monitoring: Ensures the validated state of
sterilization process
– Load release procedures and criteria
– Requalification and revalidation requirements
– Preventative maintenance
– Change control programs

227
Review Question #29
When and how often should process

monitoring occur?
A. Every week until product is released
B. On a continuous basis after performance
qualification and release of equipment for
use
C. Once every six months after performance
qualification and release of equipment for
use

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Review Question #30
Revalidation is a periodic confirmation to

demonstrate that equipment performance has
not changed from its qualified state.
A. True
B. False

229
Review Question #31
• A change control program maintains the

state of control and includes the following:
A. Documentation of changes to the
equipment, process, or product
B. Documentation of any testing required
C. Evaluation of equipment, process, and
product changes
D. A and C
E. All of the above
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Review Question #32
Parametric release is a sterility release

program for product based on: (select all that
apply)
A. Effective control, monitoring and
documentation of a validated sterility
product manufacturing process
B. Achievement of critical operational
parameters and performance attributes
C. End-product sterility testing
D. A foundation of the entire quality system
231
Conclusions (1)
• Dry heat processes can provide sterilization or both
depyrogenation and sterilization
– The purpose of the process will dictate the validation
approach
• The dry heat equipment and its support systems
should be designed and constructed to deliver
effective processes in a consistent and reproducible
manner
• The goal of process development is to identify critical
and key operating parameters that will result in a load
meeting the minimum acceptance criteria for
depyrogenation / sterilization
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Conclusions (2)
• Performance qualification is the process of obtaining

and documenting evidence that the equipment, as
installed, consistently operates in accordance with
predetermined criteria
• Ongoing control and monitoring of the process is
conducted on a continuous basis after completion of
performance qualification and release of the equipment
for use

233
Questions

234
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Thank You

235
118

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9/18/2019

Validation of Dry Heat

Technical Report No. 3

Task Force Members

• Jill Giulianelli, West-Ward • Rita Welser, Boehringer

Validation of Dry Heat Processes

Validation of Dry Heat Processes

Validation of Dry Heat Processes

• 8:30 a.m. Class Begins

Validation of Dry Heat Processes

• Introduction and Scope

Validation of Dry Heat Processes

• Update of PDA’s Technical Report No. 3, Validation of

Validation: A documented program that provides a high level of scientific

• Provides information to the manufacturers of

Validation of Dry Heat Processes

Points to Equipment design

Performance qualification for new systems

Development and validation of processes for existing

Validation of Dry Heat Processes

• Process development in both ovens and tunnels

• Dry heat processes used for the following:

Validation of Dry Heat Processes

Validation of Dry Heat Processes

• Current FDA, ICH, and other regulatory definitions are used

Validation of Dry Heat Processes

The Science of Dry Heat

Validation of Dry Heat Processes

Dry Heat Processes

The purpose of process will dictate the validation

Depyrogenation: The destruction or removal of bacterial endotoxins.

Dry Heat Depyrogenation (1)

• One primary method used to inactivate and remove

Bacterial Endotoxin: Endotoxins are fever producing substances commonly

Dry Heat Depyrogenation (2)

Validation of Dry Heat Processes

Dry Heat Depyrogenation (3)

• Temperature and exposure time

– Demonstrate adequate and reproducible level of endotoxin

• Include an endotoxin challenge, where necessary, as

Validation of Dry Heat Processes

Dry Heat Depyrogenation:

Commonly used for materials that can

Validation of Dry Heat Processes

Dry Heat Depyrogenation:

Validation of Dry Heat Processes

Dry Heat Depyrogenation:

Validation of Dry Heat Processes

Dry Heat Depyrogenation:

Assumption: the depyrogenation process will inactivate

Validation of Dry Heat Processes

Dry heat depyrogenation is the only method

Validation of Dry Heat Processes

During validation, dry heat endotoxin

Validation of Dry Heat Processes

Dry Heat Depyrogenation:

Endotoxin Indicators: An article challenge vial of endotoxin (or a carrier

Image courtesy of rapidmicrobiology.com