Autoclave(SteamSterlizer)Validation

Procedure for autoclave validation including steam penetration, heat distribution and penetration, biochallenge study, estimation of F0 value and acceptance
criteria of steam sterilizer validation in pharmaceutical industry.
Sterilization process in 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. Boiler suit, 01 Nos.
Headgear, 02 Nos booties, 01 pairs gloves
2 Glassware S.S Mannifold holder 06 holder 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 for 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 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
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
deg.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
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 BowieDick Test

B Empty Chamber Heat Distribution Studies

Objective
Objective of this test is to ensure that, The sterilizer is capable of attaining a temperature of 121 deg.C during the sterilization hold period with steam
pressure of 1.1 to 1.2 kg/cm2.
Temperature spread with in the range of 121 deg.C to 124 deg.C during sterilization cycle will demonstrate the uniform heat distribution within the
chamber.
Any location where the temperature indicator is placed, not achieving minimum sterilization temperature of 121deg.C through out the sterilization
temperature hold will be considered as 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. Temoerature mapping probe into 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 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 dataanalysis 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 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 deg.C to 124 deg.C during the sterilization hold period.
Observations and Results
Record the observations and results in formats.

Related: Low Temperature Sterilization Process 115C

C Loaded Chamber Heat Distribution & Penetration Studies
Objective
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
desired temperature of 121 deg.C during the complete sterilization hold period with steam pressure of 1.1 to 1.2 kg.cm2.
If Sterilization temperature 121 deg.C is not achieved through out 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 deg.C to 124 deg.C during sterilization hold period indicate that, uniform heating process which is achieved in
the empty chamber heat distribution study is not affected by load. There could be the possibility of lag period for attaining 121 deg.C during heat
penetration runs as the probes are placed deep into the load.
Any location where the temperature indicator is placed, not achieving minimum sterilization temperature of 121deg.C during sterilization temperature hold
period will be considered as 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 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 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 dataanalysis and printing. Record
the temperatures observed at different locations in the Annexure. 3 Aseptically collect the exposed biological indicators and send the indicators to
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 deg.C to 124 deg.C during the complete sterilization hold period.
Observations and Results
Record the observations and results in formats enclosed as Attachement
D Biochallenge studies
Objective
The steam sterilization process in 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 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 deg.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 basic in the Annexture.
5.0 Compile the data generated during the qualification test for complete evaluation of the system.
Acceptance Criteria
No bacterial growth should observed during the incubation period of 48 hours at 55 to 60 deg.C.
Observations and Results
Record the observations and results in formats enclosed as Attachement.
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 S10T121/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 temperature is changed 10 deg.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 biochallenge
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 deg.C.
A = Biological indicator concentration or spore population.
B = Desired level of non sterility. 10 deg.C
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