12/21/22, 11:08 AM Autoclave (Steam Sterilizer) Validation : Pharmaguideline

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Autoclave (Steam Sterilizer) Validation

Autoclave is used for sterilization of various articles in microbiology laboratory as well in sterile manufacturing. This article has procedure for autoclave validation including
steam penetration, heat distribution and penetration, bio-challenge study, estimation of F0 value and acceptance criteria of steam sterilizer validation in pharmaceutical
industry.
Sterilization process in the pharmaceutical industry in an autoclave will be considered qualified for consistent and reliable performance (validated) on
successful completion of the following tests–
• Bowie-Dick Test for steam penetration (3 trails).
• Empty Chamber Heat distribution studies (3 trails) with temperature mapping probe at different locations of the sterilizer chamber.

• Loaded chamber heat Distribution & penetration studies (3 trails) for each sterilization load of fixed loading pattern –
1) Sterile area garments (20 number Garments packs, Each pack contains 01 Nos. Boilersuit, 01 Nos. Headgear, 02 Nos booties, 01 pairs gloves)
2) Glassware (S.S Manifold holder (06 holders) 02 Nos, Sampling unit of Compressed air 02 Nos, 500 ml sampling bottles 10 Nos, 250 ml sampling
bottles 25 Nos, 04 Nos S.S Bin.)
3) Media (SCDA Medium – 500 ml 09 Nos. Conical flask, SCDM – 100 ml 20 Nos. tubes, FTM – 100 ml 10 Nos. tubes, MSA – 250 ml 01 Nos
Conical flask, CA – 250 ml 01 Nos Conical flask, BGA – 250 ml 01 Nos Conical flask, BSA – 250 ml 01 Nos Conical flask, MCA – 250 ml 01 Nos
Conical flask, Peptone Water – 500 ml 06 Nos. Conical flask.)

With temperature mapping probes along with Biological Indicator (Geobacillus stearothermophillus spore vials containing 10^6 or more spores per
vials) inside the innermost possible layer of the load subjected to sterilization.
• Estimation of the F0 Value achieved during the sterilization hold period at each temperature mapping probe.
To qualify these tests the equipment should fulfill the acceptance criteria described in the individual test procedures. After completion of the
qualification tests, all the data generated will be compiled together to evaluate the ability of the steam sterilizer to sterilize different components at the
set parameters and set loading pattern.
A) Bowie-Dick test for Steam Penetration
Objective
The objective of this test is to ensure that the vacuum pulses applied the sterilization hold period are sufficient to remove the entrapped air so as to
facilitate rapid and even steam penetration into all parts of the load and maintaining these conditions for the specified temperature holding time (17
minutes at 121 °C)

If air is present in the chamber, it will collect within the Bowie-Dick test pack as a bubble. The indicator in the region of the bubble will be of a
different color as compared to the color on the remaining part of the test paper, because of a lower temperature, lower moisture level or both. In this
condition the cycle parameters to be reviewed and the normal sterilization cycles to be modified accordingly.

Bowie-Dick cycle should be normally preceded by a warm-up cycle, as the effectiveness of air removal may depend on all parts of the sterilizer being
at working temperature.

Procedure
1. Record the set parameters for the Bowie-Dick test cycle in The Annexure.
2. Place one Bowie-Dick test pack near the drain of the sterilization chamber.
3. Select cycle Bowie-Dick on the control panel & operate the steam sterilizer.
4. The print out taken during the Bowie-Dick test cycle & the Bowie-Dick test indicator should be preserved.
5. Compile the observation made during the qualification test for complete evaluation of the system.
Acceptance Criteria
The Bowie-Dick Test indicator should show a uniform  color  change, non – uniform change and/or air entrapment (bubble) spot on the pattern
indicates inadequate air removal from the sterilization chamber.
Observation & Results
Record the observation and results in format.

Related: SOP for Steam Penetration Study by Bowie-Dick Test

B) Empty Chamber Heat Distribution Studies

Objective
The objective of this test is to ensure that, The sterilizer is capable of attaining a temperature of 121  °C during the sterilization hold period with the
steam pressure of 1.1 to 1.2 kg/cm2. Ensure FSSAI Label Compliance
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12/21/22, 11:08 AM Autoclave (Steam Sterilizer) Validation : Pharmaguideline

Temperature spread within the range of 121  °C to 124  °C during the sterilization cycle will demonstrate the uniform heat distribution within the
chamber.

Any location where the temperature indicator is placed, not achieving the minimum sterilization temperature of 121°C throughout the sterilization
temperature hold will be considered as a cold spot.

Procedure
1.0 Record the set parameter for the sterilization cycle to be operated during the test for empty chamber heat distribution study, in the Annexure.
2.0 Pass minimum 16 no. Temperature mapping probe into the chamber through the port of the sterilizer. Seal the port with silicone sealant so that
steam leakage does not take place. Suspend the probes in the chamber in the different position so that probes do not touch any metallic. Record the
position of the probes in a respective schematic form.
3.0 Connect the probes to a suitable autoclave data logger, which can scan and print the actual temperature observed at different locations with
respect to time.
4.0 Operate the steam sterilizer and also start the autoclave data logger to record actual temperature within the sterilization chamber with respect to
time.
5.0 When the sterilization cycle completes, 1) Collect printout of the sterilizer and preserve as Annexure. 2) Download the data-analysis and printing.
Record the temperatures observed at different locations in the Annexure.
6.0 If the empty chamber heat distribution study is acceptable perform three consecutive runs to demonstrate cycle and sterilizer reproducibility.
7.0 Compile the data generated during the qualification test for complete evaluation of the system.

Acceptance Criteria
There should be the uniform distribution of heat in the sterilizer chamber during the sterilization hold period and the temperature at each temperature
mapping probes should be within the range of 121 °C to 124 °C during the sterilization hold period.
Observations and Results
Record the observations and results in formats.

Related: Low Temperature Sterilization Process (115°C)

C) Loaded Chamber Heat Distribution & Penetration Studies

Objective
The objective of this test is to ensure that, the steam is sufficiently penetrating into the innermost portions of the load subjected for sterilization to
achieve the desired temperature of 121 °C during the complete sterilization hold period with the steam pressure of 1.1 to 1.2 kg.cm2.

If Sterilization temperature (121  °C) is not achieved throughout the cycle, load configuration or size of the load has to be reviewed and cycle to be
repeated.

Temperature spread within the range of 121  °C to 124  °C during sterilization hold period indicate that the uniform heating process which is achieved
in the empty chamber heat distribution study is not affected by load. There could be the possibility of the lag period for attaining 121  °C during heat
penetration runs as the probes are placed deep into the load.

Any location where the temperature indicator is placed, not achieving the minimum sterilization temperature of 121°C during sterilization temperature
hold period will be considered as a cold spot.

Procedure
1.0 Record the set parameter for the sterilization cycle to be operated during the test for loaded chamber heat penetration study in the Annexure.
2.0 Pass minimum 16 no. Temperature mapping probe into the chamber through the port provided. Seal the port with silicone sealant so that steam
leakage does not take place. Place the probes inside the load components, which are supported to be the most difficult points for steam penetration,
also place biological indicator along with temperature mapping probe (12 Nos.). Record the position of the probes and biological indicators in a
representative schematic form.
3.0 Connect the probes to a suitable data logger, which can scan and print the actual temperature with respect to time.
4.0 Operate the steam Sterilizer and also start the data logger to record the actual temperatures within the sterilization chamber with respect to time.
5.0 When the sterilization cycle completes, 1) Collect printout of the sterilizer and preserve as Annexure. 2) Download the data-analysis and printing.
Record the temperatures observed at different locations in the Annexure. 3) Aseptically collect the exposed biological indicators and send the
indicators to the microbiology lab for further incubation and observed the results.
6.0 If the load penetration study is acceptable perform three consecutive runs to demonstrate cycle and sterilizer reproducibility.
7.0 Compile the data generated during the qualification test for complete evaluation of the system.
Acceptance Criteria

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12/21/22, 11:08 AM Autoclave (Steam Sterilizer) Validation : Pharmaguideline

There should be uniform distribution & penetration of heat in the load subjected for sterilization during the sterilization hold period and the
temperature at each temperature mapping probe should be within the range of 121 °C to 124 °C during the complete sterilization hold period.

Observations and Results

Record the observations and results in formats enclosed as Attachment
D) Bio-challenge studies
Objective
The steam sterilization process in the pharmaceutical industry, when challenged with Geobacillus stearothermophillus Biological indicator spore vial,
spore population of NLT 106 spores/vial, should reduce bacterial load by mean of Sterility Assurance Level (SAL) 106

On incubation of the loaded biological indicator, if growth is observed, then the sterilization cycle parameters to be reviewed.

Procedure
1.0 Determine the initial counts of the biological indicator.
2.0 Collect the exposed indicator (during the loaded chamber heat distribution & heat penetration studies) by using sterile forceps and scissors in a
100 ml beaker and then send to microbiology lab for incubation (Incubate the vial at 55 to 60 °C for 48 hours)
3.0 Keep one vial as a negative control provided by the Mfg of biological indicator as well as one vial as a positive control (unexposed vial biological
indicator).
4.0 Observe any growth (purple color – sterile, yellow color – Non-sterile) in the vial daily. Record the observations on daily basis in the Annexure.
5.0 Compile the data generated during the qualification test for complete evaluation of the system.
Acceptance Criteria
No bacterial growth should be observed during the incubation period of 48 hours at 55 to 60 °C.
Observations and Results
Record the observations and results in formats enclosed as Attachment.

E) Estimation of F0 Value
Objective
The calculated F0 value should not be less than the biological F0 value at all temperature mapping locations during the sterilization hold period.

Procedure
1.0 Record the temperature at all temperature mapping probes during the sterilization hold period in the Annexure.
2.0 Calculate the F0 value at each temperature mapping probe by using the equation as below.
3.0 Record the F0 value (Results) in the Annexure.
4.0 Compile the data generated during the qualification test for complete evaluation of the system.

Calculation
F0 = dt S10(T-121)/z
Where
dt = Time interval between two following temperature measurements (1 minutes).
T = The observed Temperature at that particular time.
Z = The change in the heat resistance of Geobacillus stearothermophillus spores as the temperature is changed (10 °C).
Acceptance Criteria
The calculated minimum F0 value (by equation) should be more than biological F0 value for the biological indicator vial exposed for the bio-challenge
studies.
- The biological F0 value for the specific biological indicator spore vial is calculated as per the following equation
F0 = D121 (Log A – Log B)
Where
D121 = D value of the of the biological indicator at 121 °C.
A = Biological indicator concentration or spore population.
B = Desired level of non – sterility. (10 °C)

Related: Steam Sterilization (Heating in an Autoclave)

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of pharmaguideline.com, a widely-read pharmaceutical
blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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