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.

Ankur ChoudharyPrintQuestion Forum 12 comments

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.

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

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)

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