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Confidential
Autoclaves and Autoclave
Validation
TJ Stough
June 8, 2010
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Confidential
Presentation Overview
References
Autoclaves Defined
Types of Autoclaves
Cycle Types
Validation
Lethality and Sterility Assurance Level
Revalidation
Safety

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Confidential
References
PDA Technical Report #1, August 2007
Guidance for Industry
For the Submission of Documentation for Sterilization
Process Validation in Applications for Human and
Veterinary Drug Products (CDER 1994)
The Validation and Periodic Testing of Benchtop Vacuum
Steam Sterilizers (MHRA DB 9804)
Autoclaves Defined
Autoclaves are sterilizers which, using the
physical properties of steam as a sterilant,
combine the use of pressure and vacuum to
ensure that the maximum benefit is gained from
the sterilant in the shortest amount of time
Steam is superheated to provide the most
efficient heat transfer
Why Autoclaves?
Autoclaves provide the most cost-effective and
efficient form of sterilization for products and
production components that are not heat or
moisture sensitive.
Autoclaves leave no chemical residue compared
to other forms of sterilization
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Moist or Dry? It matters.
Steam or moist heat in an autoclave kills by:
Coagulating and denaturing enzymes and structural protein
Penetrates cell walls
Does it in 15-30 minutes at 121C
Dry heat Convection or oven heat kills by:
destructive oxidation of essential cell constituents
Does it in 300 minutes at 121C or ~ 60 minutes at 160C
Cannot be used for liquids
Usually only used for glass and metal.
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Types of Autoclaves
Benchtop Autoclaves (gravity)
Found in dental offices, tattoo shops, etc.
Advantages
Easy to operate
Generally no external utilities required
Disadvantages
No air removal
Can only sterilize non-volumetric items
(tools)
Gravity Displacement
Uses steam (lighter than air) to force the air
out of the chamber
No vacuum
No separate steam generator
Generally only good for solid, unwrapped items
Cannot remove air*
Air can puddle making cold spots in load
Can be used for liquid loads**
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Positive Pressure Displacement
Similar to gravity displacement in principle, but steam is
supplied by external steam generator
Advantages
Better for air removal since steam is pulsed in,
disturbing air, which mixes and purges more
chamber/load air out.
Disadvantages
Higher cost than gravity displacement autoclaves
Generally smaller chamber than gravity due to other
components like the steam generator
Vacuum (Vacuum Displacement)
Vacuum pump removes air from the chamber
Vacuum is broken with steam
Advantages
Best for air removal
Can sterilize all types of hard goods, including
bottles, cylinders, and other volumetric cont.
Disadvantages
Most expensive
Load items must be able to withstand vacuum
Standard Autoclave Cycles
Vacuum cycles
Used for porous or hard goods,
Volumetric containers
Removes air from loads
Gravity cycles
Used for liquid loads
No vacuum
Air overpressure
Used for small volume liquid (terminal sterilization)
Leak Test
Bowie-Dick, Daily Air Removal Test (DART)
Definition of Terms
Dwell /Exposure phase
Start of the actual sterilization cycle (dry goods)
All points in load are at or above 121C
Prevac Pulse/Vacuum Pulse
Part of the heat-up phase in vacuum autoclaves where the chamber is put
under vacuum then the vacuum is broken with steam
Load Probe
In some autoclaves, an internal probe used to control the liquid load cycle.
Placed into the center of mass of an equal mass of water. Only used for liquids
Slow exhaust phase
In a liquid cycle following exposure, the chamber is kept under pressure until
the liquid temperature is below 100C to prevent the rapid boil-off of the liquids
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Definitions (continued)
Clean Steam
Steam generated from a DI or Purified-quality source water
Pure Steam
Steam generated from a WFI-quality (pyrogen-free) source water
Plant Steam
Steam generated from a non-tested or city water-quality source water
Can be used as the heat source for the jacket since there is no product
contact
Can be used as the heat source to generate clean steam
Jacket
surrounds autoclave chamber and is kept heated to a few degrees below
sterilization temperature as an efficiency so that the sterilant is not also used as
the heat source
Sterile
To make free from live bacteria or other microorganisms.
To render free of microorganisms: decontaminate, disinfect, sanitize.
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Validation
What needs to be validated?
New equipment (IQ/OQ/PQ)
New load configurations (PQ)
Equipment with new major components (OQ,PQ)
Relocated equipment (IQ,OQ,PQ)
Equipment with new utilities (IQ, OQ)
And how often?
Industry standard for autoclaves is yearly or
When one of the above conditions is true
Stages of validation
Design Validation
Not always required
Installation Qualification
Operational Qualification
Performance Qualification
A Cycle development phase should normally precede
PQ to get the most out of the validation effort
Design Qualification
Should be done prior to purchase
Considers user requirements when deciding on
the specific design of a system or equipment
Ideally, selects a suitable, preferred vendor for
the appropriate system
Installation Qualification
Requires Protocol
Ensures that equipment is installed properly
Per manufacturers utility requirements
Per manufacturers space requirements*
Identification and verification of all system
elements, parts, services, controls, gauges and
other components
Operational Qualification
Requires protocol
Tests alarms
Tests unit at all high and low limits
Worst case conditions
Tests software limits
Very intensive testing
Requires draft of SOP at this point
Must be approved prior to start of PQ
Cycle Development
Should be done prior to PQ
Uses thermocouples (TCs) and a datalogger
Figures out worst-case load configuration
Finds best parameters/set points to use
Lets you know if your autoclave will sterilize
your goods
Finds minimum load
Find any other autoclave limitations
Planning a Load Configuration
Some things to consider when planning your load:
How many do you need at once? How often?
How many do you have?
How many will fit? Is it packed too tightly?
Does your autoclave have the capacity?
Do you need to reallocate/re-aliquot into smaller
containers?
Is efficiency worth failure? (no)
Should you individual wrap/package or can you put
several items in the same pouch?
Is it overpackaged?
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Performance Qualification
Shows consistency of performance over time
3x Empty Chamber per cycle type
Validates the established worst-case load from cycle
development runs
Also can have a minimum load* (generally just for liquid
cycles)
Requires a worst-case load per cycle type
Liquids
Hard goods, etc.
Load configurations must be documented in SOP
Lethality and Sterility Assurance Level
Lethality, or F
0
, is a mathematical representation of the amount of
kill per unit time
At 121.1C, you get 1.0 F
0
per min, or 1 log reduction in a theoretical population
of organisms
The industry standard Sterility Assurance Level (SAL) target is 10
-6
,
which requires a 12-log reduction to attain
If you get 12 F
0
during your exposure, then you effectively have a 1 in a million
chance of contamination.
F
0
Formula: 10^((temp-121.1)/10) (calculated by Kaye or manually)
F
H
Formula: 10^((temp-170.0)/20) (calculated by Kaye or manually)
SAL Calculation: LOG B = LOG A - (F
0
/D121)
Log B = SAL, Log A= Spore population, F
0
= total for run, D121=1

Calculating Lethality
Notated as F
0
for steam sterilization or
The equivalent sterilization time spent at the base
temperature, which is 121C
Lethality Rate : 10
(T-Tb)/Z

F
0
= t x Lethality Rate
t : Cycle time
T : Actual Cycle temperature
Tb : Base Temperature
Z : Microbial Death Rate Constant
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Thermal resistance of microbes
What is the D-Value? (decimal reduction value)
The D-Value is the time, in minutes, at a specific
temperature, required to reduce the surviving
microbial population by 1-log (90%).
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What is the Z-value?
The Z-value is the temperature change required to result in
a 1-log reduction in D-value
To determine Z-value, one must determine the D-value at 3
different temperatures then plot the value.
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Lethality Table
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Biological Indicators
PQ uses biological indicators to prove efficacy
Besides Lethality and Sterility Assurance Level, there
is a necessity to show a PHYSICAL kill, which is
achieved using Biological Indicators.
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population of Geobacillus stearothermophilus is
generally used due to its robustness
Ampoule BIs are used for liquid loads
Spore strips are used for hard goods loads
Placed in cold spots found during cycle development
adjacent to thermocouples
**Ampoule BIs CANNOT be used for dry goods, and
strips CANNOT be used for liquid loads**
More about Biological Indicators
Spore Strip BIs require a separate growth medium
Strips must be placed in the growth medium using aseptic
technique and incubated at 55-60C in a validated incubator.
Ampoule BIs are self-contained and need only be placed into
incubator. If they are contaminated, they will become turbid and
the medium will change color
BIs must be properly stored and should be inventory controlled.
Some applications use different organisms other than Geobacillus
stearothermophilus, but autoclaves use this organism due to its
thermal robustness
Spore suspension indicators must be sterile filtered, plated, then
counted to measure any viable growth
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Other Species of BIs - overview
Bacillus pumilus is used for gamma irradiation and e-Beam
due to its resistance to radiation
Bacillus subtilus is used for EtOH, H
2
O
2
, and Dry heat
sterilization due to its resistance to alkylating agents,
oxidizing chemicals, and heat.
Aspergillus niger is used for UV sterilization due to its
Ultraviolet resistance
NOTE: depending on the application, these will be spore
suspensions of various populations, not strips OR ampoules
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Revalidation
Performed Annually on maximum and minimum load
(no exceptions)
Performed if repairs done on equipment
(change control assessment)
Performed if load configuration is changed
Investigative runs (PQ addenda) can be performed
here in lieu of full revalidation
Safety (General)
High temperatures!
Autoclaves operate at very high
temperatures
DO NOT reach into an autoclave with bare
hands
Jacket is usually heated and will burn you
Always wear proper PPE, minimally high
temperature gloves
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Safety (Liquids)
Wear glasses or face shields for liquid loads as
liquids can splatter
Allow sufficient cool down time after removing
load, prior to handling TCs
Ensure that any caps are loose prior to loading
autoclave
Allow liquids to cool prior to removing
Biological Indicators
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Questions?

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You Made It!

Thank you for your attention!

Call me at 303-884-8175 or stop me anytime
if you think of anything later
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