by 1.0 OBJECTIVE:
To lay down a procedure for validation of autoclave.
2.0 SCOPE:
This procedure is applicable for procedure for validation of autoclave.
3.0 RESPONSIBILITY:
Microbiologist
4.0 ACCOUNTABILITY:
QC Manager.
5.0 PROCEDURE:
5.1 Validation of an Autoclave for pharmaceutical use will be considered qualified for consistent and reliable performance (validated) on successful completion of the following –
5.2 Bowie – Dick test for Steam Penetration
5.3 Objective
5.4 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 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.
5.5 Procedure: Record the set parameters for the Bowie – Dick test cycle in The Annexure. Place one Bowie – Dick test pack near the drain of the sterilization chamber.Select cycle Bowie – Dick on the control panel & operate the steam sterilizer.The print out taken during the Bowie – Dick test cycle & the Bowie – Dick test indicator should be preserved.
5.6 Compile the observation made during the qualification test for complete evaluation of the system.
5.7 Acceptance Criteria
5.8 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.
5.9 Observation & Results
5.10 Record the observation and results in format.
5.11 Empty Chamber Heat Distribution Studies
5.12 Objective
5.13 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 steam pressure of 1.1 to 1.2 kg/cm2.
5.14 Temperature spread within the range of 121°C to 124°C during sterilization cycle will demonstrate the uniform heat distribution within the chamber.
5.15 Any location where the temperature indicator is placed, not achieving minimum sterilization temperature of 121°C throughout the sterilization temperature hold will be considered as cold spot.
5.16 Procedure
5.17 Record the set parameter for the sterilization cycle to be operated during the test for empty chamber heat distribution study, in the Annexure.
5.18 Pass minimum 16 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.
5.19 Connect the probes to a suitable data logger, which can scan and print the actual temperature observed at different locations with respect to time.
5.20 Operate the steam sterilizer and also start the data logger to record actual temperature within the sterilization chamber with respect to time.
5.21 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.
5.22 If the empty chamber heat distribution study is acceptable perform three consecutive runs to demonstrate cycle and sterilizer reproducibility.
5.23 Compile the data generated during the qualification test for complete evaluation of the system.
5.24 Acceptance Criteria
5.25 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°C to 124 °C during the sterilization hold period.
5.26 Observations and Results
5.27 Record the observations and results in formats.
5.28 Loaded Chamber Heat Distribution & Penetration Studies
5.29 Objective
5.30 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°C during the complete sterilization hold period with steam pressure of 1.1 to 1.2 kg.cm2.
5.31 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.
5.32 Temperature spread within the range of 121°C to 124 °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 °C during heat penetration runs as the probes are placed deep into the load.
5.33 Any location where the temperature indicator is placed, not achieving minimum sterilization temperature of 121°C during sterilization temperature hold period will be considered as cold spot.
5.34 Procedure
5.35 Record the set parameter for the sterilization cycle to be operated during the test for loaded chamber heat penetration study in the Annexure.
5.36 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.
5.37 Connect the probes to a suitable data logger, which can scan and print the actual temperature with respect to time.
5.38 Operate the steam Sterilizer and also start the data logger to record the actual temperatures within the sterilization chamber with respect to time.
5.39 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 microbiology lab for further incubation and observed the results.
5.40 If the load penetration study is acceptable perform three consecutive runs to demonstrate cycle and sterilizer reproducibility.
5.41 Compile the data generated during the qualification test for complete evaluation of the system.
5.42 Acceptance Criteria
5.43 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.
5.44 Observations and Results
5.45 Record the observations and results in formats enclosed as Attachment.
5.46 Bio-challenge studies
5.47 Objective
5.48 The steam sterilization process, 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.
5.49 On incubation of the loaded biological indicator, if growth is observed, then the sterilization cycle parameters to be reviewed.
5.50 Procedure
5.51 Determine the initial counts of biological indicator.
5.52 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°C to 60°C for 48 hours)
5.53 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).
5.54 Observe any growth (purple color – sterile, yellow color – Non sterile) in the vial daily. Record the observations on daily basic in the Annexure.
5.55 Compile the data generated during the qualification test for complete evaluation of the system.
5.56 Acceptance Criteria No bacterial growth should observed during the incubation period of 48 hours at 55° to 60°C.
5.57 Observations and Results
5.58 Record the observations and results in formats enclosed as Attachment.
5.59 Estimation of F0 Value
5.60 Objective
5.61 The calculated F0 value should not be less than the biological F0 value at all temperature mapping locations during the sterilization hold period.
5.62 Procedure
5.63 Record the temperature at all temperature mapping probes during the sterilization hold period in the Annexure.
5.64 Calculate the F0 value at each temperature mapping probe by using the equation as below.
5.65 Record the F0 value (Results) in the Annexure.
5.66 Compile the data generated during the qualification test for complete evaluation of the system.
5.67 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 temperature is changed (10°C).
5.68 Acceptance Criteria
5.69 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.
5.70 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).
6.0 TRAINING:
Trainer : Manager – Quality Control
Trainees : Staff of the microbiology departments
7.0 DISTRIBUTION:
Master Copy : QA Department
Controlled Copy : Microbiology Department
Display Copy : Microbiology Department (If Required)
8.0 ATTACHMENTS:
| Sr. No. | Title | Annexure No. |
| 01. | Protocol for Validation of Autoclave | F-SOP/MB/003/01-01 |
9.0 REFERENCES:
In-House
10.0 ABBREVIATIONS:
| Abbreviations | Extended Form |
| SOP | Standard Operating Procedure |
| NA | Not Applicable |
| MB | Microbiology |
| Sr. No. | Serial Number |
| QA | Quality Assurance |
| DEPT. | Department |
| IPA | Isopropyl Alcohol |
| % | Percentage |
11.0 REVISION HISTORY OF CHANGE:
| Sr. No. | Date | Revision Details | Revision No. |
| 01 | NEW SOP | 00 |
