sop for Validation protocol of steam sterilizer autoclave

TABLE OF CONTENTS
Sr. No. Title Page No.
1 PROTOCOL APPROVAL SHEET…………………………..……………..
2 OBJECTIVE…………………………………………………………………..
3 SCOPE…………………………………………………………………..……
4 RESPONSIBILITY……………………………………………………..…….
5 EQUIPMENT DESCRIPTION………………………………………………
6 REQUIREMENT……………………………………………………………..
7 LOAD PATTERN FOR VALIDATION OF AUTICLAVE………………….
8 VALIDATION METHODOLOGY FOR AUTOCLAVE………..……..……
9 SET PARAMETER…………………………………………..…………..….
10 REVALIDATION CRITERIA…………………………………………………
11 FREQUENCY OF VALIDATION……………………………………………
12 DEFINITION…………………………………………………………………..
13 REFERENCE…………………………………………………………………
14 DOCUMENTS……………………………………………………….……….
15 ABBREVIATIONS…………………………………………………………….

1.0 PROTOCOL APPROVAL
This Validation Protocol has been prepared and Reviewed and Approved by following:
DEPARTMENT NAME DESIGNATION SIGNATURE DATE
PREPARED BY
Executive – QA
CHECKED BY
Head
Quality Control
APPROVED BY
Head
Quality Assurance

2.0 OBJECTIVE:
The objective of this protocol is designed to establish sufficient data to assure that the Autoclave Supplied by

M/s. ABC equipment is suitable for sterilizing the loads. In addition, this validation protocol is intended to

assure the sterility of the items, when the equipment is operated in accordance with the established standard

operating procedure to maintain reliability and repeatability.

3.0 SCOPE:
This protocol is applicable For Vertical Autoclave, which is installed in Quality Control deptt. Microbiology section

4.0 RESPONSIBILITY:

Department	Responsibilities
Microbiology	Development of Validation protocol and assembling of data into a final report. Placing of Biological indicator & chemical indicator(strips) Collection & analysis of Biological indicator
Quality Control	Review of this protocol.
Quality Assurance	Review and Approval of protocol and of the final report.

5.0 EQUIPMENT DESCRIPTION:
5.1 Autoclave Ist
Name of the Equipment : Steam Sterilizer (Autoclave)
Make : ABC
Equipment I D No. :
Equipment Location. : Autoclave (Microbiology Department)
5.2 Autoclave IInd
Name of the Equipment : Steam Sterilizer (Autoclave)
Make : ABC
Equipment I D No. :
Equipment Location. : Autoclave (Microbiology Department)

5.3 USE OF EQUIPMENT:
5.3.1 The Autoclave is used for decontamination of culture dilution tubes & slants, Environmental monitoring plates & tubes,

water testing plates & tubes, Raw material MLT plates & tubes, Finished product MLT plates & tubes, disinfectant testing

media tubes, used micropipette tips, positive and negative cultures plates and tubes, Environmental isolates plates & tubes

at 121° to 124°C for decontamination of the media is accomplished by heat transfer from steam to media.
6.0 REQUIREMENT:
6.1 In order to efficiently conduct validation of an Autoclave, the following requirements must be fulfilled
6.1.1 Calibrated Data logger with PT-100 sensors.
6.1.2 Biological Indicator for Pours & Liquid Load (106 spores of Bacillus stearothermophilus in
strips.

6.1.3 Culture media used for validation purpose
6.1.4 All instruments used for validation must be calibrated.
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 121ºC.
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:
8.1.1.1 Objective of this test is to ensure that, the steam sterilizer is capable of attaining a temperature of 1210C during the

sterilization hold period with steam pressure of 1.1 to 1.2 kg/cm2.
8.1.1.2 Temperature spread within the range of 1210C to 1240C 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.

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 10C
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 Objective
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 Fo Value during the complete sterilization hold period.

8.2.1.2 If the minimum Fo 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 1210C to 1240C 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 1210C during heat penetration runs as the probes are placed

deep into the load. But if minimum Fo Value is achieved at each probe, there is no need to consider for lag period. But if minimum Fo Value is not achieved at each probe, then cycle time will have to be increased so as to achieve minimum Fo Value.

8.2.1.5 The location where minimum Fo 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.

Figure 2.0 – Schematic diagram for Probe Location along with Biological Indicator media sterilization loaded cycle

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 10C.

8.2.3.2 The calculated minimum F0 value should be more than biological F0 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 121ºC – 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 Observe 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 F0 Value
8.4.1 Objective
8.4.1.1 The calculated F0 value should not be less than the biological F0 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 F0 value (Results) in the Annexure.
8.4.2.4 Compile the data generated during the qualification test for complete evaluation of the system.

9.0 SET PARAMETER:

Load description	Sterilization Temperature	No. of cycle	Sterilization Hold time (Minute)
Heat Distribution Empty cycle	121^oC	1	20
Porous Load- Minimum Load: Sterilization of Glassware (25 Nos Petri plates, 5 Nos sampling bottles), Accessories (5 Nos spatulas, 5 Nos forceps, 5 pairs Hand gloves, 4 Nos Measuring Cylinder, 4 Nos Filtration Assembly.	121^oC	1	20
Maximum Load: Sterilization of Glassware (100 Nos Petri plates, 8 Nos sampling bottles), Accessories (8 Nos spatulas, 8 Nos forceps, 10 pairs Hand gloves, 8 Nos Measuring Cylinder, 6 Nos Filtration Assembly.	121^oC	1	20

Liquid Load

Minimum Load: Sterilization of Media (2x 100 ml SCDM, 200 ml SCDA, 100 ml SDA 100 ml CA, 100 ml MSA, 100 ml MCA, 2X100 ml MCB, 2x90ml Buffered Sodium chloride Peptone).

121^oC

Maximum Load: Sterilization of Media

(10 x100 ml SCDM, 3x400ml SCDA, 10 x 90 ml Buffered Sodium chloride Peptone, 500 ml SDA, 300 ml CA, 300 ml MCA, 300 ml MSA, 10 x100 ml MCB).

121^oC

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 non compliance 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 ºC) required for one-log reduction.
12.6 F0 VALUE: The F0 Value is the equivalent time for which a monitored article is exposed to the temperature of 1210 C.

12.7 BIOLOGICAL INDICATORS (BIs): BIs 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 are 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 for Pharmaceutical Products.”

14.0 DOCUMENTS
14.1 Any Change in the protocol and activity shall be documented and supported with change control.
14.1.1 Protocol No. along with Annexure
14.1.2 The report shall be prepared by Microbiologist, Reviewed by Head-QC and approved by Head-QA. The report shell have following as its Annexure
14.1.3 Calibration certificates.
14.1.4 Certificates of biological indicator.
14.1.5 Chemical indicator.
14.1.6 Any other relevant documents (if any).

15.0 ABBREVIATIONS
15.1 MHS : Moist Heat Sterilizer
15.2 NA : Not Applicable
15.3 RTD : Resistant Temperature Device
15.4 QA : Quality Assurance
15.5 QC : Quality Control
15.6 SAL : Sterility Assurance Level
15.7 °C : Degree Centigrade
15.8 SOP : Standard Operating Procedure
15.9 NMT : Not More Than
15.10 VMP : Validation Master Plan
15.11 BI : Biological Indicators
15.12 A.R. : Analytical Report
15.13 BP : British Pharmacopoeia
15.14 WHO : World Health Organization
15.15 cGMP : Current Good Manufacturing Practice