7.

0 LOAD PATTERN FOR VALIDATION OF AUTOCLAVE

7.1 Empty Chamber Heat distribution

studies with temperature mapping probe at different locations of the sterilizer chamber.

7.2 Loaded chamber heat Distribution & penetration studies for each sterilization load of fixed
loading pattern at 121°C.

7.3 Porous Load (Heat Penetration Studies

for Sterilization of Glass wares, Accessories hand gloves etc.)

7.4 Liquid Load (Heat Penetration Studies for Sterilization of media etc.)

8.0 VALIDATION METHODOLOGY FOR AUTOCLAVE

8.1 Heat distribution studies (Empty Lode)

8.1.1 Objective: is to ensure that, the steam sterilizer is capable of attaining a temperature of
121 C during the sterilization hold period with steam pressure of 1.5 bar.

8.1.1.2 Temperature spread within the range of 121 C to 134 C during sterilization cycle will
demonstrate the uniform heat distribution within the chamber.

8.1.2 Procedure

8.1.2.1 Record the set parameter for the sterilization cycle to be operated during the test for empty
chamber heat distribution study.

8.1.2.2 Pass minimum 8 no. Temperature 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.

8.1.2.3 Connect the probes to a suitable data logger, which can scan and print the actual temperature
observed at different locations with respect to time.
 1 2
3
4

5
6
7
8

Figure 1.0- Schematic diagram for Probe Location for Empty Cycle (1-8 are the locations for probe)

8.1.2.4 Operate the autoclave as per SOP and also start the data logger to record actual temperature
within the sterilization chamber with respect to time.

8.1.2.5 When the sterilization cycle completes, Download the data-analysis and printing. Record the
temperatures observed at different locations.

8.1.2.6 If the empty chamber heat distribution study is acceptable perform three consecutive runs to
demonstrate cycle and sterilizer reproducibility.

8.1.3 Acceptance Criteria

8.1.3.1 Throughout the well time all temperature measured in the chamber do not fluctuate by more
than 10 c

8.1.3.2 The minimum accumulated F0 value should be more than biological F0 value for the biological
indicator strips exposed to the bio-challenge studies.

8.2 Loaded Chamber Heat Distribution & Penetration Studies Along with chemical &
Biological Indicator (Porous Load & Liquid Load)

8.2.1.1 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 F0 Value during the complete
sterilization hold period.8.2.1.2 If the minimum F0 Value is not achieved throughout the cycle, load
configuration or size of the load has to be reviewed and cycle to be repeated.
8.2.1.3 Temperature spread within the range of 121 C to 135 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.

8.2.1.4 There could be the possibility of lag period for attaining 121 C during heat penetration runs as
the probes are placed deep into the load. But if minimum F0 Value is achieved at each probe, there is no
need to consider for lag period. But if minimum F0 Value is not achieved at each probe, then cycle time
will have to be increased so as to achieve minimum F0 Value.

8.2.1.5 The location where minimum F0 Value is observed during the sterilization cycle will be
considered as cold spot.

8.2.2 Procedure

8.2.2.1 Record the set parameter for the sterilization cycle to be operated during the test for loaded
chamber heat penetration study in the Annexure.

8.2.2.2 Pass minimum 8 no. Temperature mapping probes 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 each temperature mapping probe during the last cycle of media discard.

Record the position of the probes and biological indicators in a representative schematic form.

8.2.2.3 Connect the probes to a suitable data logger, which can scan and print the actual temperature
with respect to time

4
5
7
1

6
2 3 8

Figure 2.0 Schematic diagram for Probe Location along with Biological Indicator media sterilization
loaded cycle
 = Petri dish

5
6
= Conical flask 2
1

= Test tube

7 3

= Filtration unit

4 8

Figure 3.0 Schematic Location of glassware at the time of sterilization.

8.2.2.4 Operate the steam Sterilizer as per SOP and also start the data logger to record the actual
temperatures within the sterilization chamber with respect to time.

8.2.2.5 When the sterilization cycle completes, Record the temperatures observed at different locations.
Aseptically collect the exposed biological indicators and send the indicators to microbiology lab for
further incubation and observed the results.

8.2.2.6 If the load penetration study is acceptable perform three consecutive runs to demonstrate cycle
and sterilizer reproducibility.

Compile the data generated during the qualification test for complete evaluation of the system.

8.2.3 Acceptance Criteria

8.2.3.1 Throughout the dwell time all temperature measured in the chamber do not fluctuate by more
than 10 C.

8.2.3.2 The calculated minimum FO value should be more than biological FO value for the biological
indicator strips exposed to the bio-challenge studies.

8.2.3.3 No growth should be observed for exposed sterilized indicator & Negative Control (unexposed
indicator) in the SCDM.
8.2.3.4 Growth should be observed in the Positive control tube that was not exposed to the Sterilization
cycle.

8.3 Bio-challenge studies

8.3.1 Objective

8.3.1.1 The steam sterilization process, when challenged with Bacillus stearothermophillus biological
indicator spore strip/ampoule spore population of NLT 105 spores strip, should reduce bacterial load by
mean of Sterility Assurance Level (SAL) 10-5 Biological Indicators should be used as per the load.

8.3.1.2 For Porous & Liquid Load at 121 C -Biological Indicator for Pours & Liquid Load (105 spores of
Bacillus stearothermophillus should be used.

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

8.3.2 Procedure

8.3.2.1 Collect the exposed indicator (during the loaded chamber heat distribution & heat penetration
studies) and then send to microbiology lab for incubation (Incubate the Geobacillus stearothermophillus

indicator at 550C to 600C for 7 days).

8.3.2.2 Keep one indicator as a negative control provided by the Mfg. of biological indicator as well as
one indicator as a positive control (unexposed biological indicator).

8.3.2.3 0bserve any growth for sterility of media & Glassware sterilization cycle.

Record the observations on daily basis in the Annexure. Compile the data generated during the
qualification test for complete evaluation of the system.

8.4 Estimation of FO Value

8.4.1 Objective

8.4.1.1 The calculated F0 value should not be less than the biological FO value at all temperature
mapping locations during the sterilization hold period.

8.4.2 Procedure

8.4.2.1 Record the temperature at all temperature mapping probes during the sterilization hold period
in the Annexure.

8.4.2.2 Calculate the F0 value at each temperature mapping probe by using the equation as below.

8.4.2.3 Record the FO value (Results) in the Annexure.

8.4.2.4 Compile the data generated during the qualification test for complete.

Calculation

Were:

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 stearothenophillus spores as temperature is changed
(10°C).

The biological F0 value for the specific biological indicator spore vial is calculated as per the |

following equation:

F0= D121 (Log No - Log Nt)

Were

D121=D value of the biological indicator at 121°C.

No= Biological indicator concentration or spore population.

Nt = Minimum sterility Assurance Level Required

9.0 SET PARAMETER:

Sterilization hold
Sterilization No. of
Load Description Temperature cycle
time
(minutes)
Heat Distribution Empty cycle 121°C 1 20
A) Porous Load
Minimum Load: Sterilization of
glassware (25Nos Petri plates, 5 Nos
sampling bottle),
Accessories (5 Nos forceps, 5 pairs of
hand gloves, 4 Nos measuring
cylinder, 4 Nos filtration assembly.
121°C 1 20
Maximum Load: Sterilization of
glassware (100 Nos Petri plates, 8 Nos
sampling bottle,)
Accessories (8 Nos Spatula, 8 Nos
forceps, 10 pairs of hand gloves, 8 Nos
measuring cylinder, 6 Nos filtration
assembly.

B) Liquid Load
Minimum Load: Sterilization of Media
(2x 100 ml SCDA, 200 ML SCDA, 100
ml SDA, 100 ml MCA, 2X 100 ml MCB,
2X 90 ml Buffered sodium chloride
peptone)
121°C 1 15
Maximum Load: Sterilization of Media
(10x 100 ml SCDA, 3X 400 ml SCDA,
10x 90 ml buffered sodium chloride
peptone, 500 ml SDA, 300 ml MCA,
10x 100 ml MCB)

10.0 REVALIDATION CRITERIA:

10.1 Any major modification to any part/ process of the existing autoclave which must be documented
through a change control system.

10.2 Adjustments made in the equipment, as a rectification measure to counter the noncompliance of
the Results.
11.0 FREQUENCY OF VALIDATION:

11.1 The frequency of Periodic Validation for is yearly basis (+15 Days) for existing systems.

12.0 DEFINITIONS:

12.1 STERILE: In strict sense a specimen would be deemed sterile only when there is complete absence
of viable microorganisms from it.

12.2 STERILIZATION: A process, by which all viable microorganisms are removed or destroyed, based on
a probability function.

12.3 BIO BURDEN: The number of viable microorganisms in or an object or preparation entering a
sterilization step (usually expressed as colony forming unit per unit volume).

12.4 D VALUE: The D value is the time (in minutes) required to reduce the microbial

population by 90% or 1 log cycle (i.e., to a surviving fraction of 1/10).

12.5 Z VALUE: The Z Value is the temp. (in C) required for one-log reduction.

12.6 FO VALUE: The FO Value is the equivalent time for which a monitored article is exposed to the
temperature of 1210 C.

12.7 BIOLOGICAL INDICATORS (Bls): Bls

are live spore forms of microorganisms known to be the most resistant living organisms to the lethal
effects of the particular sterilization process. For steam sterilization, the most resistant microorganism is
Bacillus stearothermophilus.

12.8 STERILITY ASSURANCE LEVEL (SAL): A term related to the probability of finding a nonsterile unit
following a sterilization step.

It is usually expressed in the negative power of 10 (i.e., one in one million = 10-5).

12.9 PULSE: A sub cycle at the start of Sterilization Cycle in which Vacuum and Steam is alternatively
supplied to a steam sterilizer chamber, for the purpose of removal of air from the chamber.

13.0 REFERENCES:

Schedule- M- "Good Manufacturing Practices and Requirements of Premises, plant and Equipment of
pharmaceutical products.