Steam Sterilizer Validation Requirements Per The New

Standard ISO 17665-1:2006

For decades, steam sterilization (autoclaving) has been an integral part in the manufacturing,
cleanroom, and laboratory processes for the medical device, pharmaceutical, biologics, and human
tissue/HCTP industries. It has been a common industry practice to validate steam sterilizers using
the published guideline ISO 11134 Sterilization of health care products — Requirements for
validation and routine control - Industrial moist heat sterilization,1 issued in 1994. In late 2006,
AAMI released the document intended to supersede 11134, with ANSI/AAMI/ISO 17665-1:2006
Sterilization of health care products — Moist heat — Part 1: Requirements for the development,
validation, and routine control of a sterilization process for medical devices.2 While other steam
sterilizer guidance documents do exist,3,4 it is anticipated that the new 17665 standard will be
recognized by the FDA and will be commonly employed to validate autoclave processes. The good
news to manufacturers or other users of these guidelines is that many of the current validation
practices are the same in the new document. This article will outline the basic requirements for
steam sterilizer validation via the halfcycle overkill method, and list some of the differences between
the two documents.

REQUIREMENTS PRIOR TO VALIDATION

The 17665 document makes it clear in numerous locations that the user’s quality system must adhere
to ISO 13485:2003 Medical devices — Quality management system — Requirements for regulatory
purposes.5 So if a user wishes to claim full compliance with the new 17665 steam standard, then
their quality system must also be in compliance with ISO 13485, including items such as
preventive/periodic maintenance and regular calibration for the sterilizer, documentation, change
control, purchasing, etc. When compared with the previous steam document, the new 17665 also has
more information on product and process characterization, sterilizing agent characterization,
installation qualification/IQ, and operational qualification/OQ. The new document also states more
clearly that a fully compliant validation is not just a series of successful halfcycles, but is the full
complement of successful IQ, OQ, and PQ.

Sterilization agent characterization will be simple for most users — moist heat/steam at 121 or 132
°C, and cycle selection (gravity, prevacuum, etc.). Process and equipment characterization means
defining and documenting items like the sterilizer cycle parameters, products (or product families) to
be sterilized, load configurations and limits, placement of biological indicators or chemical
indicators (BIs/CIs), process tolerances, and equipment identification. Much of this type of
information would be recorded in well-written validation protocols or validation final reports.
Biological indicators often use spores of the bacterial species Geobacillus stearothermophilus at a
titer of greater than 106per BI, although other species or titers are sometimes used.

The new 17665 document also has more information on IQ and OQ. It defines IQ as “obtaining and
documenting evidence that equipment has been provided and installed in accordance with its
specification.” Autoclave installations commonly document items such as the sterilizer identification
numbers, location, line voltage and amperage, water supply piping and pressure limits, steam line
requirements, filtration, chamber size, structure and support, piping materials, software certification,
manuals, drawings and documentation, and calibrations (temperature, pressure, and timer). The
sterilizer must be installed in such a manner to facilitate any necessary maintenance, repair,
adjustment, cleaning, and calibration.
OQ is defined as “obtaining and documenting evidence that the installed equipment operates within
predetermined limits when used in accordance with its operational procedures.” Autoclave OQs
commonly test or verify items such as cycle operation and programming instructions, safety and
alarm testing, error reporting, empty chamber temperature profiling and chamber temperature
limits/specifications, air removal testing, leak testing, temperature control anomalies, full cycle full-
load temperature profiles (if proposed fullcycle exposure time is known), and determination of any
hot or cold spots within the chamber.

The product definition and process definition sections of the new document list things such as
product specifications, product families, packaging, re-sterilization issues, package moisture,
stability and potency of container products, re-usable container systems, process challenge
devices/PCDs, sterility assurance level/SAL, BIs and CIs, and bioburden determination if necessary.
PCDs are described as products or items that provide a known resistance to the sterilization process.
They are commercially available or may also be created from the user’s product line by inserting
spore strips, spore dots, inoculated threads, etc. into items or locations that are determined to be the
most-difficult-to-sterilize product or location in the load.

There are many other activities or decisions to be made prior to or during the IQ/OQ, that are not
necessarily detailed in either standard. Items such as:

● Obtaining calibrated temperature recording devices or thermocouples

● Ordering supplies such as BIs, CIs, Bowie-Dick test packs, packaging materials, etc. and noting if
adequate laboratory facilities are available

● Determining worst-case validation load and worst-case test product or PCD. The protocol or final
report should contain a written rationale describing how the loads and product(s) were selected

● Selecting cycle type: 121 or 132 °C, gravity or prevacuum cycle, etc.; and determining if drying
time needs to be qualified

● Is product bioburden testing necessary?

● Is product resterilization to be allowed and what are the requirements for resterilization?

● Is product stability or shelf life testing necessary for the user’s products?

● Does packaging testing or packaging validation need to be included with the protocol?

VALIDATION – PERFORMANCE QUALIFICATION

AAMI TIR #13 states “Sterilization process validation is a documented procedure for obtaining,
recording, and interpreting the results required to establish that a process will consistently yield
product complying with its predetermined specifications.” For the purposes of this article, the
primary specification will be sterility. The performance qualification/PQ or microbiological
qualification is a series of tests that establishes that the installed and properly operating sterilizer will
process the users desired chamber loads to achieve the specified sterility assurance level/SAL. It
must be remembered that the load is part of the validation — that is, if the user makes significant
changes to the load at any point in the future — then re-validation may be necessary. The previous
ISO 11134 document gave relatively little guidance information and few specifications for
conducting the test cycles necessary to qualify the user’s proposed fullcycle exposure time(s). The
 new 17665 steam document varies little from the previous standard in respect to the minimal PQ
information that is provided. The 17665 describes bioburden validation methods and the more
commonly used halfcycle “overkill” method. It should be noted that at the time this article was
prepared, the proposed guidance document that is to accompany ISO 17665-1 was not yet available.
This guidance document may provide more advice on microbiological qualification issues (ISO
17665-2 Sterilization of health care products — Moist heat —Part 2: Guidance on the application of
ISO 17665-1). For this article, the general requirements for an overkill cycle PQ will be reviewed.

While many activities are required to complete the PQ, the primary goal for the commonly employed
overkill validation is this: the user needs to complete three consecutive successful halfcycles in order
to qualify their proposed fullcycle exposure for routine processing of sterilization loads. In our case,
successful means all BIs are killed (no growth upon incubation) for the three consecutive halfcycles.
If, for example, there was no BI growth for the three test cycles at ten minutes exposure at 121 °C,
then a 20-minute exposure at the same temperature would be adequate for routine daily processing,
assuming all other aspects or requirements of the IQ/OQ/PQ are successful, documented, reviewed,
and approved.

But a description of the PQ needs much more detail than this. Validation protocols vary in format
from company to company, but most will capture similar information for the final report. An
example of validation protocol and final report sections would be:

Title page with approval signatures

• Purpose, background information, or general goal(s) of validation

• Scope with more specifics about methods, cycles, facility, SAL, products and load, exclusions, etc.
• References with published standards and company SOPs
• Equipment, supplies, validation loads, BIs, etc.
• Rationale for selection of products, load, cycles, PCDs, etc.
• Procedure or methods (more details on this below)
• Acceptance criteria which list the pass/fail requirements
• Deviation report which lists any unexpected results, with potential effects on the validation,
along with accept/reject rationale
• Results and conclusions which assign a pass/fail decision to each acceptance criteria,
summarize study, and include any requirements for revalidation
• Attachment which lists any data sheets, diagrams, certificates, temperature records, etc., for
inclusion with final report
• Approvals section for final report.

To conduct the halfcycles, the user assembles the worst-case validation test load, temperature
loggers, BIs/PCDs, and CIs if necessary. The temperature loggers and BIs are seeded throughout the
load to represent various chamber locations, keeping in mind any cold spots or previously
determined most-difficult-to-sterilize locations. For small chambers, as few as five or six BIs and
temperature loggers may be needed. Ten is a common sample size for many chambers. Large, multi-
pallet-sized chambers may require many more samples per run. The sterilizer is programmed for
one-half of the proposed full-cycle exposure time. Upon completion of the test cycle, the BIs are
immediately removed and incubated, and the test load must be allowed to return to normal
temperature prior to starting another test cycle. Temperature recorder data is downloaded and printed
immediately to determine if any unusual temperature conditions existed. Information is entered on
the data sheets (data sheets that would have been one of the attachments to the written protocol), and
all temperature records and data sheets are retained for the final report. BIs are checked regularly
throughout the http://www.cemag.us/Article_Print.asp?pid=709 (5 of 7) [13/8/2008 11:56:50 AM]
Controlled Environments® | Articles | Steam Sterilizer Validation Requirements Per The New
Standard ISO 17665-1:2006 incubation period, and include positive control (unprocessed) BIs which
must show growth. As stated before, all processed BIs must show no growth in order for the
validation runs to be considered successful.

Final reports should contain: 1) all sterilizer run data or recorder charts, signed and reviewed; 2) all
temperature recorder data, signed and reviewed; 3) all data sheets with BI, CI, or any other test
results, reviewed and signed; 4) any deviations recorded and investigated, with final disposition; 5)
results, conclusions, and discussion; 6) calibration documents for any measuring instruments used
during the study; 7) the approved full-cycle parameters and acceptable placement locations for BIs
for normal processing; and 8) manufacturers’ certificates of analysis for any items such as BIs,
growth media, growth promotion test cultures, etc. Including digital photographs of sterilizer, load,
PCD, etc. can be quite helpful for an auditor who may be reviewing the report at a later date. The
completed final report packet must then be routed for review and signed for approval.

POST-VALIDATION

There are still issues to be addressed when all activities seem to have been completed. The sterilizer
must be added to a regular and documented calibration program. The sterilizer must be included in a
regular and documented periodic/preventive maintenance program. And the sterilizer must be added
to the validation schedule for its annual requalification. The user needs to verify that all personnel
that will be using the autoclave are trained using applicable operation and safety SOPs. Untrained
staff should not be allowed to run the sterilizer. Approved products, loads, cycles, and load limit
information must be readily available to all operators. SOPs for daily processing must list all
requirements for data that is to be reviewed and retained from the sterilizer runs, with logbook, filing
system, or archive for run records. SOPs must also address items such as 1) segregation of processed
and non-processed product, 2) storage requirements for processed products if necessary, 3)
notification of management or maintenance if sterilizer malfunctions or if recorder chart lists any
errors, cautions, or warnings, 4) immediate notification of management for BI test failure, including
investigation and product quarantine procedure as appropriate, and 5) resterilization requirements if
resteril-ization is to be allowed.

In summary, there seem to be no drastic or revolutionary changes in making the transition from ISO
11134 to ISO 17665. The new 17665 steam document provides more information and more guidance
in some areas, while leaving other areas (such as PQ) relatively unchanged. While users would be
advised to obtain the 17665-2 guidance document when it becomes available, it is anticipated that
manufacturers will not find any great difficulties in applying the new standard.
This is the html version of the file http://www.aapspharmaceutica.com/meetings/files/139/James
%20Gallagher.pdf.
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Page 1

Validation of Steam
Sterilizers
Arden House 2009
James Gallagher
Kalypsys Inc
1
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 2

Introduction to Validation of
Steam Sterilizers
Goals of Presentation
• Overview of basic principles for steam
sterilization
and microbiology
• Review key aspects of EN 285 and
PDA Technical
Report #1
• Facilitate decision making for
Pharmaceutical
Scientists looking to Contract
Manufacturing
Organization (CMO) for aseptic
processing that
includes steam sterilization
2
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 3

Introduction
Outline
• Introduction
• Sterilizer Design Aspects
• Steam / Thermo
• Micro Aspects
• Validating Sterilization Cycles
• Checklist / Troubleshooting
• Regulatory
3
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 4

Introduction to Validation of
Steam Sterilizers
Regulatory Drivers and Guidance
Source
Document
FDA
FDA, Guidance for Industry
Sterile Drug Products Produced by Aseptic Processing —
Current Good Manufacturing Practice , Sept 2004
CFR
Relevant Sections on Sterilization, purity and control
EN285
Sterilization – Steam Sterilizers – Large Sterilizers,
Amendment 1, March 2008
USP <55>
Biological Indicators, Resistance Performance Tests
PDA
Technical Report #1: Validation of Moist Heat
Sterilization Processes: Cycle Design, Development and
Ongoing Control
Validation of Steam Sterilizers
AAPS Arden House 2009
4
Page 5

Introduction to Validation of
Steam Sterilizers
Working with Contract Manufacturing
Organizations (CMO)
• Sponsor needs to partner with CMO
• Assume aseptic process is defined, and
the RFP has taken
into account the level of validation
needed
• Review the assumptions and risk
analysis on the required
steam sterilization processes are
included; confirm
assumptions in original risk assessment
are still valid
• Consider including an engineering
(pilot) batch in the RFP
• Due Diligence: site visit, audit → Input
on CMO selection
• Transferring Aseptic Process
Technology to the CMO
5
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 6

Introduction to Validation of
Steam Sterilizers
Three Examples of Contract
Services
Validation of Steam Sterilizers
AAPS Arden House 2009
6
Example of Project
Typical Sterilization Needs
Driving Forces
Early Phase Clinical
Program in US
Receiving Tank / Bag
Documentation for IND
Speed to Clinic -- Leverage
CMO’s existing cycles /
programs
WW Phase III Clinical
Program
Filter Assembly
Filling Line Parts
Documentation to support
NDA/ CTD and PAI
Scale-up
Secondary source for
Commercial Product
Stopper processing
Filling Manifold
Supplement to NDA / CTD
New facility Qualification
Equivalency with existing
product / package/ process
Optimal cycles
Page 7

Sterilizer Design Aspects

Sterilizer interface with aseptic
process
• Sterilizers are a
critical means to
provide access to an
aseptic process
• Components are
prepped /cleaned
prior to sterilization
• Dedicated,
disposable or
multiple use
Validation of Steam Sterilizers
AAPS Arden House 2009
7
Prep Area (non sterile)
Entry
Sterilizer
#1
Sterilizer
#2
Sterilizer
#3
Aseptic Processing Suite
Aseptic Corridor
Page 8

Sterilizer Design Aspects

Autoclave Diagram
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 9

Sterilizer Design Aspects

Sterilizer Design Features
• Jacketed vessels, internal volume,
steam trap,
insulation
• Filtered air (< 0.3um) with steam
backflow device
• Temperature sensors = Pt resistance
types
• 2 independent temperature sensors
• Failed cycle can be vented and loading
door opened,
standard door interlocks
• Condensate trap within 2 meters of the
connection
• May have air breaks on drains to
prevent backflow
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 10

Sterilizer Design Aspects

Control of a sterilizer
• Critical parameters are steam quality,
temperature and
time
• Temperature sensor is normally in the
drain
• Automated cycles, sensors monitoring
and alarms
• Key measurements are temperature,
pressure and time
10
Validation of Steam Sterilizers
AAPS Arden House 2009
Common Indicators on Sterilizer Controller
Door lock (both ends)
Cycle in progress / Cycle Complete
Fault
Cycle selected
Cycle counter
Cycle Stage Indicator
Page 11

Sterilizer Design Aspects

Temp and Pressure Measurements
on Sterilizer
• Temperature Measurements
– Jacket, Chamber, Drain, Load Probes and
Recorders
• Pressure Measurements
– Jacket, Chamber and Recorders
• Time
– Exposure time, heat up time
Validation of Steam Sterilizers
AAPS Arden House 2009
11
Parameter
Temperature
Pressure
Accuracy of Range
1% over the 50 – 150˚C
range
< 1.6% over range
0 to 400kPa / (-1 to 3 bar)
Accuracy at Sterilization 0.5˚C at sterilization temp
+/-5 kPa (0.05 bar)
Resolution
0.1˚C for digital
1kPa (0.01 bar)
Page 12

Steam / Thermo
Steam Overview– why is steam
so effective?
• Condenses, collapses as wet film of
condensation
increases the heat flow to sterilized item
• Volume of steam ( ~6 cu ft / lb); 350x
volume of water
• 50 – 100 lbs of steam used in a typical
cycle
• The “killing power” of steam is due to
its latent heat of
vaporization
– 1 L water to boiling = 80 cal
– 1 L boiling water to steam = 540 cal
12
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 13

Steam / Thermo
Steam Enthalpy
13
Validation of Steam Sterilizers
AAPS Arden House 2009
Temperature
(°C )
Pressure
(bar) (psig)
Enthalpy of Steam
(J/g)
(BTU/lb)
100˚ C
1.013 14.7
2,675
1,150
121° C
2.048 29.7
2,707
1,164
126˚C
2.392 34.7
2,715
1,167
134°C
3.039 44.1
2,725
1,171
Page 14

Steam / Thermo
Steam Quality / Testing
• Steam quality = how much water is
contained in the
steam % by weight / % by volume
• Dryness value = 1.0 for dry saturated
steam; less
latent heat capacity for lower steam
quality
• Pharma sterilization cycles use
saturated steam with
no superheat, minimal NCG
• Clean steam used in Pharma
applications;
condensate complies with WFI
monograph
• Understand CMO’s limitations for
steam production
14
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 15

Steam / Thermo
Causes of poor steam quality
• Issues with clean steam generator
• Water hammer – water slug moving
through pipes
resulting in a banging sound
• Piping Insulation– prevents steam
from condensing
• Times of higher steam demand:
winter, startup
• Condensate in piping: in AM, after a
shutdown
Validation of Steam Sterilizers
AAPS Arden House 2009
15
Page 16

Steam / Thermo
Non-condensable gases(NCG)
• Gases that cannot be liquefied by
compression under
the conditions used in a sterilization
cycle
• NCG do not contract / expand like
steam, move to an
area of lower velocity (the sterilizer);
steam/gas mix
• Sources: Air: open door, piping, steam
supply
• Lower temperature, can insulate items
→ impact cycles
– i.e. 10% air will lower incoming steam
temp by 7°F
• CO
2
can dissolve in the condensate →
carbonic acid;
corrosive to metal pipes
• Oxidation from dissolved 0
2
Validation of Steam Sterilizers
AAPS Arden House 2009
16
Page 17

Steam / Thermo
Steam Trap
• Automatic valve that drains water,
vents air, but traps steam in the
system, located on drain legs and
steam filters
• Located at the bottom of the
sterilizer, drains condensate from
the jacket and the chamber
• Steam traps also used on air vents
• Failure mechanisms for steam traps
Validation of Steam Sterilizers
AAPS Arden House 2009
17
Page 18

Micro Aspects
Overview
Validation of Steam Sterilizers
AAPS Arden House 2009
18

SAL = 10-6
Accumulated
Lethality, F0
Biological
Indicators
Overkill or
Bioburden
Cycle?
D, z and F
values
Microbiological
Aspects of
Sterilization
 Page 19

Micro Aspects
Sterility Assurance Level (SAL)
• The probability of a single viable
microorganism
being present on a sterilized unit is one
in one million
after the item has undergone a
sterilization process;
often called a six log reduction
• PNSU (Probability of a Non-Sterile
Unit)
• Cannot directly measured this
objective
• For parenteral products, desire a SAL
of 10-6
19
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 20

Micro Aspects
Biological Indicators (BI)
• Population of microorganisms (usually
spores)
inoculated onto a suitable medium
• Placed in sterilizer load locations to
determine the
sterilization cycle efficacy by
deactivating BI
• The challenge microorganism
is selected based upon its
resistance to the given
process
• Quality of BI defined by
microbiological count and
D-Value
Validation of Steam Sterilizers
AAPS Arden House 2009
20
Page 21

Micro Aspects
G Stearothermophilus
• Geobacillus stearothermophilus
for use in steam sterilization
at 121.1°C to 135°C (275°F)
• Incubate at 55˚- 60°C
• Thermophiles found in hot springs
areas such as
Yellowstone NP; highly resistant to heat
• Most spore forming microbes have D-
value < 0.5 min;
commercial spore strips have D-value
1.5 - 2 min
• Desire a population of spores on a strip
of ~105 or more
• Direct inoculation onto test substrates
(closures etc)
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 22

Micro Aspects
Inoculation of BI / Positive
Controls
• After the sterilization cycle, the
retrieved BI is placed in
a tube of growth medium and incubated
per USP <55>
• A color and/or turbidity change
indicates the results of
the sterilization process, no change in
indicates
sterilization conditions were achieved,
otherwise the
growth of the spores indicates that the
sterilization
process has not been met.
“It also should be noted that the resistance of
microorganisms can vary widely
depending on the material to be sterilized. For this reason,
careful consideration
should be given during sterilization validation to the nature
or type of material
chosen as the carrier of the biological indicator to ensure an
appropriately
representative study” Sept 2004 FDA GUIDANCE.
Validation of Steam Sterilizers
AAPS Arden House 2009
22
Page 23

Micro Aspects
D-value
• D value is the thermal
resistance value (min) of a
target organism
• D value is the time in
minutes at a specific
temperature to reduce the
surviving microbial
population by 1-log, or 90%
reduction in population.
• Typical D-values for
commercial spore strip lots
are ~1.5 - 2 min
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 24

Micro Aspects
Z Value
• Z value is the heat resistance
of a spore as a function of
temperature (°C)
• Z value is the temperature
change required to result in a
1-log reduction in D-value
• Generally used standard
value is Z= 10˚C
• Z = (T
2
–T
1
) / (log D
1
– log D
2
)
Thermal Resistance Curve
Validation of Steam Sterilizers
AAPS Arden House 2009
24
D
121
1.6 min
D
131
0.16 min
D
111
16 min
Page 25

Micro Aspects
F value, Accumulated Lethality
• Accumulated Lethality is the F value
•F
0
is the equivalent time that a microbial
population
with a z value of 10 has been held at
121˚C
•1F
0
= the equivalent of 1 minute at 121˚C
• Equation; F = Σ 10(T-121.1)/z x t
Where T = Temperature
F
0
= equivalent sterilization time (min)
• Z = 10˚C is generally used
Validation of Steam Sterilizers
AAPS Arden House 2009
25
Page 26

Micro Aspects
F, D and Z values and their
relationships
• Equation #1: Log N
F
= -F
(T,z)
/D
T
+ log N
0
• Equation #2: F
(T,z)
= (Log N
0
– Log N
F
)xD
T
• Typical D-values are ~1.5 - 2 min;
natural is <0.5 min
26
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 27

Micro Aspects
Product Specific (Bioburden)
Approach
• Quality attributes impacted by high
thermal input
• Collect detailed bioburden and D-value
data
• Example: Liquid Loads, terminal
sterilization with a
bioburden of 100 CFU and D value =
0.5 min
• Equation #2: F
(T,z)
= (Log N
0
– Log N
F
)xD
T
–F
0
= (Log 102 – Log 10-6 ) x 0.5 min = 4.0 min at
121˚C
27
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 28

Micro Aspects
Overkill Approach
• Many definitions and process
requirements for
Overkill Cycles
• Provides a minimum 12 log reduction
of
microorganisms having a D-value of at
least 1 min
• Avoids collecting bioburden and D-
value data by
assuming extreme case conditions:
– Bioburden level is 106
– D-value is 2.5 minutes
• Equation #2: F
(T,z)
= (Log N
0
– Log N
F
)xD
T
F
0
= 12 log (2.5 min/log) = 30 minutes at 121.1˚C
28
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 29

Micro Aspects
Compare F
PHY
and F
BIO
•F
PHY
determined from thermocouple data
during heat
penetration study
•F
BIO
is the delivered lethality calculated by
the actual
kill of microorganisms in a BI system
•F
BIO
=D
T
x LR
D is the D value
LR is the log reduction of BI population during
a cycle
• Agreement between F
PHY
and F
BIO
Validation of Steam Sterilizers
AAPS Arden House 2009
29
Page 30

Micro Aspects
BIER vessel
• BIER (Biological Indicator Evaluator
Resistometer)
Systems are designed to provide
environmental
conditions to evaluate the resistance of
microbial
populations to sterilization
• Confirm population of spores on strip;
purity
• Confirms D-values from commercial
lots of spore
strips
– Fractional Negative or Direct enumeration
methods
– Repeat value or confirm by survivor kill
“The microbial count of a biological indicator should be
confirmed. Biological
indicators should be stored under appropriate conditions.”
FDA, Sept 2004
30
Validation of Steam Sterilizers
AAPS Arden House 2009
Page 31
Validating Sterilization Cycles
Overview
Validation of Steam Sterilizers
AAPS Arden House 2009
31
Thermal
Validation
System
Type of
Goods
Worst Case
Load
Assessment
Load
Configuration
Thermocouples
Overkill or
Bioburden?
Page 32

Validating Sterilization Cycles

Aspects of Thermal Validation
System
• System that meets international cGMP
requirements
• Performs pre and post calibration of
thermocouples
• Consider IRTD for reduced setup time,
minimal
sensor handling and automated sensor
calibration
• High Temperature range -195 to 420°C
• 21 CFR Part 11 Compliant
• Calibration Traceable to NIST
• Facilitates study data and generates
regulatory
required reports
32
Validation of Steam Sterilizers
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Page 33

Validating Sterilization Cycles

Thermocouples
• Number must be documented and
justified; watch
acceptance criteria!
• Accuracy of thermocouples = +/- 0.5˚C
• Type T Class 1 thermocouple wire
• Use of telemetry sensors
• TCs threaded through gland to the
chamber
• Pre and post calibration verification
“The sensing devices used for validation studies should be
calibrated before and
after validation runs” FDA Sept 2004 Guidance”.
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Validating Sterilization Cycles

Type T Thermocouples
• Type T Thermocouple
• Wire insulation color:
+ = Blue
- = Red
Wire material:
+ = Copper
- = Constantan
Properties:
+ = Copper color
Validation of Steam Sterilizers
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Junction
Exposed
Un-Grounded
Grounded
Tip
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Conducting Validation
More on Thermocouples
35
Validation of Steam Sterilizers
AAPS Arden House 2009

• Number / access may result in failed

test cycle
• Can be easily damaged by autoclave
cart wheels
• Essential that the thermocouples do not
affect air
removal or steam penetration
• Label thermocouples
• Conducting verification regularly (not
necessarily
after each run)
• Place one near the sterilizer’s
temperature sensor
“In general, the biological indicator should be placed
adjacent to the temperature
sensor so as to assess the correlation between microbial
lethality and predicted
lethality based on thermal input” FDA Sept 2004 Guidance.
Page 36

Validating Sterilization Cycles

3 Classes of Goods to be sterilized
Type
Goods Sterilized
Typical Pre-
Vac
Air Removal
Hard Goods
Equipment, Piping,
Glassware
1 or more
(3 typically)
Easy air removal and
steam penetration
Wrapped Goods Hoses, Gowns, Filters,
Vessels with vents
3 or more
More difficult air
removal/ steam
penetration
Liquids
Media, Product
(F
0
> 15 min)
None
Air overpressure
process. Heat and
cool without vacuum
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AAPS Arden House 2009
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Validating Sterilization Cycles

Establishing Load Patterns
“The specific load configurations, as well as biological
indicator and temperature
sensor locations, should be documented in validation
records. Batch production
records should subsequently document adherence to the
validated load
patterns” Sept 2004 guidelines.
37
Validation of Steam Sterilizers
AAPS Arden House 2009
Load
Position
Comment
Fixed
Fixed
Identical for all processing runs
Instructions list items and position in diagram
Fixed
Variable
Location can vary
Validate positional equivalency during runs
Instructions reference list of items
Variable Variable
Location and position can vary
Validate minimum and maximum loads
Demonstrate min / max are adequate in validation
Flexible instructions for Operations
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Validating Sterilization Cycles

Types of Saturated Steam
Processes
• Pre-vacuum process is most
commonly used
saturated steam process
– Removes air in vacuum pulses
– Multiple pulses allow pre-conditioning
of goods,
reducing the equilibration time
• Gravity Displacement
– Steam displaces the heavier air
– Air pushed out drain through steam
trap
– Steam distribution is critical
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AAPS Arden House 2009
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Validating Sterilization Cycles

Cycle development for new item
• Establishing a cycle prior to validation
• Assessment of the item / current cycle
adequate?
– Class of goods, type of load, OK or bio?
• Conduct heat penetration study
• Determine equilibration time: time T
REF
– time T
slow
• Drying studies if needed
• Correlate F
PHY
and F
BIO
for cycle
Validation of Steam Sterilizers
AAPS Arden House 2009
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Validating Sterilization Cycles

Determination of Worst Case Load
• Determine locations that are worst case
with steam
integrators and/or Thermocouples
• Determine most difficult to sterilize
items in load
– Hoses – cut, insert, seal
– Bottles – center, just above bottom
– Filters – air removal
• Fixed load or variable load -- degree of
flexibility
desired in CMO’s Operations
• May be a destructive test ; pass through
expense
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 41
Validation of Steam Sterilizers
AAPS Arden House 2009
41

Validating Sterilization Cycles

Definition of Loads – Contract
Service Examples
Contract Service Goods Type
Load
OK or Bio?
Cycle
#1 Vessel
Wrapped
Fixed
Overkill
121˚C saturated
steam
#2 Filter
Wrapped
Fixed
Overkill
121˚C saturated
steam
#3 RTS Closure
Wrapped
Variable
Overkill
121˚C saturated
steam
Page 42

Validating Sterilization Cycles

Example Cycle Overview
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AAPS Arden House 2009
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Validating Sterilization Cycles

Types of Studies
Cycle Description
Standard
Key Aspects
Pressure Rise
(Leak Test)
< 1.3mbar/min
Check prior to thermal studies
Monitors rise in pressure under
vacuum
Temperature
Distribution
Empty Chamber
Often performed in requal programs
Verifies uniform distribution of steam
Temperature variation: each probe,
probe to probe, probes to set point
Item Cool Point
Min load
Multiple runs if needed
Identifies the most difficult to sterilize
point in a test article
Validation of Steam Sterilizers
AAPS Arden House 2009
43
“These uniformity or mapping studies should be conducted
with
calibrated measurement devices.” FDA Sept 2004 Guidance
Page 44

Validating Sterilization Cycles

Types of Studies
Cycle Description
Standard
Key Aspects
Temperature
Distribution
Min / Max Load Verifies uniform distribution of steam
Temperature variation: each probe, probe to
probe, probes to set point
Heat Penetration
Max Loads
multiple runs
Map temperature with TCs
Temps as above
Equivalency of variable loads using same cycle
Maximum equilibration time
Used to calculate F
PHY
Can be combined with BI challenge study
Process Lethality
Biological Qualification
(BI challenge)
Full Load
3 consecutive
runs
Map temperature with TCs
Place BI at probed locations, including most
difficult to heat
Used to calculate F
BIO
Incubate BI post cycle
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Validation of Steam Sterilizers
AAPS Arden House 2009
Page 45

Validating Sterilization Cycles

Minimum Acceptable Cycle
• Qualified MAC cycles confirmed
biologically
and physically
• Safety margin through use of
higher exposure
times or temperatures
• Total Dwell Time is additional
lethality +
demonstrated lethality from
process validation
• Half cycle methods
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AAPS Arden House 2009
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Page 46

Validating Sterilization Cycles

Acceptance Criteria Guide
• Thermal Systems
• Process Cycles
• Reference Tests
• EN285 Steam quality items
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 47

Validating Sterilization Cycles

Acceptance Criteria–Thermal
Systems
Aspect
Standard
TC Temperatures during
Dwell Time
All temps during dwell time within 3˚C (-1˚C /+2˚C) of SP
TC Temperatures during
Dwell Time
Fluctuation of TCs within chamber NMT 1˚C
TC Temperatures during
Dwell Time
All temps measured in chamber do not differ from each
other by 2˚C
Steam Temperature
Corresponds to its vapor pressure measurement
Equilibration Time
Lag between hottest / coldest thermocouples is NMT 30
sec (15 sec for smaller chamber)
Timer Accuracy
+/- 1%
Pre and post calibration
check
Temp measurement system is accurate to +/-0.5˚C
Validation of Steam Sterilizers
AAPS Arden House 2009
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Validating Sterilization Cycles

Acceptance Criteria– Process
Cycles
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AAPS Arden House 2009
48
Aspect
Standard
Concerns
Temperature Distribution
Minimum F
0
met for TCs
Correlate T and P
Acceptable number of TCs
Heat Penetration
Determine most difficult to
sterilize item / desired load
Air removal, large mass,
length of hoses
F
0
Range
Min F
0
at end of exposure
Max equilibration time
Microbial Inactivation
during BI challenge
No BI growth
Growth with + control
SAL = 10-6
Positive control of BI
SOPs for handling BI
Comparison of F
PHY
and
F
BIO
Agreement for minimal
cycle
Page 49

Validating Sterilization Cycles

Acceptance Criteria – Reference
Tests
Aspect
Standard
Load Dryness
Mass increase < 1% Textiles test pack
Mass increase < 0.2% Metal test pack
Dynamic Pressure Test
Average pressure change NMT 10 bar/min for any 3
second interval
Sound Power
Sound level meter reading NMT 3dB change from
original operating level
Validation of Steam Sterilizers
AAPS Arden House 2009
49

• Reference tests performed during

validation of cycle,
revalidation and in periodic / routine
tests
• Most test are done on an empty
chamber
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Validating Sterilization Cycles

Acceptance Criteria – Reference
Tests II
Aspect
Standard
Thermometric Tests
(Full Load)
Equilibration time NMT 30 sec (large)
TCs within +3°C of sterilization temp
Minimum hold time at sterilization temp
Temps within 2°C during hold time
Chamber steam temp corresponds to pressure
Bowie and Dick Test
Uniform change of indicator color
Air Leakage Flow Rate
NMT 1.3mbar/min
Air Detector (if present)
Alarms if <2°C temperature difference at start
of equilibration time
Hollow Load Test
Process challenge device reaches endpoint
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AAPS Arden House 2009
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Validating Sterilization Cycles

Acceptance Criteria -- EN285
Steam Quality
Item
Description
Limits
Non condensable
gases (NCG)
Air and other gases, which do not condense
under the conditions of steam sterilization and
prevent the attainment of sterilization
conditions in any part of the load
< 3.5%
Superheat
Steam whose temperature, at any given
pressure, is higher than that indicated by the
equilibration curve for the vaporization of water
≤ 25°C
Dryness Value
The dryness fraction is a measure of the
amount of moisture carried by the steam being
supplied and used for sterilization
0.90 – 0.95
Contaminants
Clean steam condensate tests per EP
EN285 Table E.2
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AAPS Arden House 2009
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Validating Sterilization Cycles

Bowie-Dick Test / Steam
Penetration
• Test is designed to test air removal, the
absence of
air leaks and steam penetration into a
porous load
• Test Sheet consists of chemical
indicators on two test
sheets positioned inside porous materials
and sealed
inside a disposable outer wrap
• Single use packs available
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Validation Sterilization Cycles

More on Bowie and Dick Test
• Successful test indicates rapid and even
penetration
of steam into the test pack. Retention of
air within
the pack due to:
– adequacy of pre-vacuum
– air leak
– presence of NCG in steam supply
• Bowie –Dick cycles: to be carried out
periodically per
EN285
• Test pack can also be used for load
dryness test
(textiles)
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 54

Validating Sterilization Cycles

Assessing Limitations /
Restrictions
• Qualification of multiple autoclaves,
– Initial qualification
– demonstration of equivalency
– bracketing approach
– supported by risk assessment
• Ongoing monitoring of cycles
• PQ studies + operational efficiencies if
equivalent.
Otherwise pick coolest one
• Drying studies, end of cycle
adjustments as needed
with post vacs, heating
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Validation of Steam Sterilizers
AAPS Arden House 2009
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Validating Sterilization Cycles

Contract Service Example #1: Post
Sterile
Filtration Vessel (Tank / Bag)
• Assumes an early phase clinical
program in US
• Will an existing cycle provide
sufficient assurance that
the new load will achieve SAL?
Bracketing strategy?
– Overkill Cycle, Wrapped Good
– 121˚C sat steam cycle with pre-vac
– Fixed Load Configuration (min load)
– Thermal mapping Study
– Heat Penetration Study / BI Challenge Study
• Include documentation for
sterilization process in IND
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 56

Validating Sterilization Cycles

Contract Services Example #2:
Filter Assembly
• Assumes a late phase WW clinical
program / CTD filing
• Steam sterilizer cycles will be held to
EN285 standard
for Regulatory filings and PAI
inspections
– Overkill Cycle, wrapped good
– 121˚C sat steam cycle with pre-vac
– Fixed load configuration
– Thermal mapping study
– Heat Penetration
– BI Challenge
• Include documentation for
sterilization process in CTD
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AAPS Arden House 2009
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Page 57
Validating Sterilization Cycles
Contract Service Example #3:
RTS Closures
Validation of Steam Sterilizers
AAPS Arden House 2009
57

• Assumes sponsor is partnering with

CMO to source
existing product; closures cycle not
currently qualified
• WW sourcing --Steam sterilizer cycles
will be held to
EN285 standard for Regulatory filings
and PAI inspections
– OK, but may consider bioburden based cycle
– Variable load, wrapped good
– Thermal mapping study
– Heat Penetration
– BI Challenge: direct inoculation on closures
– Drying studies may be needed
• Include documentation for
sterilization process in CTD
Page 58

Checklist
Sponsor’s Checklist of CMO
supplied documents
• Explanation and justification of
method of sterilization
– Diagram showing location of load items, BI
locations, TC
locations, printouts
– Summary of micro results
– Pre and post calibration of thermocouples
• Information on preparation, cleaning
sterilization and
storage of specified equipment and
materials
– Confirming cycle parameters are listed on
Master
Production Records
– Material and Personnel flow diagrams
Validation of Steam Sterilizers
AAPS Arden House 2009
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Page 59

Checklist
Quality and Micro
QUALITY AND MICRO CHECKLIST
Change control System; Cycle Description Form (CDF)
for load configuration
changes covered, impact to utility system changes
assessed?
Type, source, concentration, D-value, Z, plant
environmental isolates
How is micro data reviewed and approved?
Requalification program in place
NIST traceable standards used, review how sensors are
calibrated
Compare process record, SOPs, MBR for indicated
product, spec for time and
temperature requirements
Control Software and documentation should be fully
traceable through the
project with all documentation accurately reflecting the
changes and
developments made throughout the project lifecycle
Filing Documentation : Reports, MBR with documents
for the sterilization
process
Experience of key personnel and staff
Validation of Steam Sterilizers
AAPS Arden House 2009
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Checklist
Sterilizer Design
STERILIZER DESIGN ITEMS
Understanding of sterilizer control system and
parameters that will be included
on batch documentation
Material Flow / Building: floor plan, placement of
autoclaves and critical items
Utilities: clean steam, compressed gasses free of
particulates and oil vapor
Mfg of sterilizer, internal volume, jacket pressure, temp,
filters used, control
system, materials of construction, location of controller
sensor, cold spot
monitoring, alarms, and warning alarms
Review PM schedule; How often is steam trap checked
Adequate generation and distribution of Clean steam;
any limitations
identified
Integrity testing and sterilization of filters for vacuum
break
Validation of Steam Sterilizers
AAPS Arden House 2009
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Checklist
Sterilization Cycle Items
STERILIZATION CYCLE ITEMS
Protocols and data summaries that justify steam cycle,
demonstrate uniformity
reproducibility, conformance to specifications
Sterilization process description: Validation info for
BI, loading patterns, heat
penetration, results for positive controls + tested BI
Heat penetration cycles: load pattern, # of runs, cold spot
for each pattern
Min / Max load configurations: hi/low avg Temp during
dwell, min/ max F0,
dwell time, run date/time, ID
Time limits established, hold times, sampling
instructions, compare load
patterns with SOP and cycle forms, periodic leak tests
Program to check / monitor Steam Quality; ensure that
saturated steam is used
Methods and controls to monitor routine cycles
Any adjustment to cycles for equilibration times
Empty chamber cycles: # runs, cold spot, allowable
variation
Validation of Steam Sterilizers
AAPS Arden House 2009
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Checklist
Business and SOP Checklist
BUSINESS AND SOPS CHECKLIST
Documentation to support Regulatory Filings: supplied in
reports or referenced
in a DMF with authorization letters; MBRs on pivotal
batches
Gap Analysis or Documentation that sterilizer is capable
of meeting EN285
List of registrations and licenses
Site inspection history
Prompt delivery of reports and data
Confirm that sterile prep area has documented SOPs
Procedural controls: SOP or Cycle sheets to include: load
pattern, training,
logbooks
Validation SOP, IQ, OQ, PQ, revalidation, Micro
programs
Number and distribution of thermal monitors listed in
SOP
Prep Team for Pre-Approval Inspection
Training: SOPs and Operator training records
Validation of Steam Sterilizers
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Regulatory
Some FDA Observations to
Sponsors
Validation of Steam Sterilizers
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63
According to FDA documents, the firm also was written
up because equipment
and supplies used to work on, or exposed to pathogenic
and potentially
pathogenic agents, were not kept separate from the
supplies used in product
manufacturing as necessary to prevent cross
contamination"
San Diego-based _____________ has been warned by
the FDA for not
adhering to procedures to prevent microbial
contamination of sterile
pharmaceuticals. “These deviations raise significant
concerns with sterility
assurance of products that were produced under these
conditions,” said the
agency
Connecticut-based _______ for its sterile manufacturing
practices by the FDA.
The agency says the firm has not verified and validated
its production
processes, including sterilization and packaging of
devices. The violations “may
be symptomatic of serious problems in your firm's
manufacturing and quality
assurance systems,” according to the letter.
• Validation of Moist Heat Sterilization Processes: Cycle
Design, Development,
Qualification and Ongoing Control, Technical Report No. 1
Revised 2007, PDA
Journal of Pharmaceutical Science and Technology, Vol 61, No
S-1
• Lewis, Raymond G, Practical Guide to Autoclave Validation,
Pharmaceutical
Engineering, July/ Aug 2002
• EN285:2006+A1, Sterilization – Steam Sterilizers – Large
Sterilizers, Amendment 1,
March 2008
• FDA, Guidance for Industry: Sterile Drug Products Produced
by Aseptic
Processing — Current Good Manufacturing Practice, Sept 2004
• FDA, Guidance for Industry: Submission Documentation for
Sterilization Process
Validation in Applications for Human and Veterinary Drug
Products,
• Agalloco, James, Understanding Overkill Sterilization,
Pharmaceutical Technology