PERFORMANCE QUALIFICATION OF AUTOCLAVE/ AUTOCLAVE VALIDATION PROTOCOL

 PERFORMANCE QUALIFICATION OF AUTOCLAVE


Equipment I.D No.



1.    OBJECTIVE:

The objective of this protocol is to provide the method to be used for the validation of the sterilizing process using an autoclave meant for sterilization of Media, Glasswares, garments, Pipette tips and Borer etc.

 

2.     PURPOSE:

To establish the Autoclave Cycle, which when operated within specified operating ranges, helps to achieve a predetermined Sterility Assurance Level (SAL).

 

3.     RESPONSIBILITY:

Representatives from the following departments:

4.1  Quality Control – Preparation, co-ordination and documentation of results

4.2 Engineering – Utility Support and rectification if any break down

4.3 Quality Assurance – Verification and Technical Approval.

 

4.     FREQUENCY:

Performance Qualification of Autoclave to be re-qualified on:

4.1   Substitution of existing Autoclave with a new system

4.2  Any major modification to the existing Autoclave since purchase or after the last performance qualification, which must be properly documented through a change control system.

4.3   Periodical Validation after every six months covering:

·         Heat Distribution Studies

·         Heat Penetration Studies

·         Microbial challenge Test

·         Microbial Limit Test

·         Air entrapment Test (By Bowie- Dick)

5.       DEFINITION

5.1       Sterile:

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

5.2       Sterilization:

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

5.3       Bio burden:

The number of viable microorganisms in or on an object or preparation entering a sterilization step (usually expressed as Colony Forming Unit per unit of volume).

5.4       D Value:

The D Value is the time  (in minutes) required to reduce the microbial population by 90% or 1 log cycle.

5.5       Z Value:

The Z Value is the difference in temperature required to change the D Value.

5.6       F0 Value:

The F0 Value is the equivalent amount of time delivered by a thermal process at a           particular temperature, with respect to a reference temperature and a specific Z Value.

5.7       Biological Indicators (Bls)

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 Autoclave, the most resistant microorganism is Bacillus stearothermophilus.

5.8       Sterility Assurance Level (SAL):

A term related to probability of finding a non-sterile unit following a sterilization step. It is usually expressed in the negative power of 10 (i.e. one in million =10-6).

 

6.       STATEMENT OF PURPOSE

The purpose of this protocol is to provide an outline of the performance qualification of the Autoclave, for the following performance attributes.

·                     To establish the time periods required for the various sub cycles.

·                     To establish the uniformity of temperature distribution in the Autoclave chamber  and identify the location of the “coolest point”.

·                     Determine the heat distribution and penetration for maximum Autoclave loads.

·                    To establish that the steam penetration at the “slowest to heat point” of the specific  load, at the established sterilization phase cycle time, meets the specific F0 Value.

·                    To study the destruction of a resistant microbial challenge by a moist heat cycle, at     the Sterilization Cycle time established by thermal F0 studies, and establishment of the Sterility Assurance Level (SAL).

7.0       JUSTIFICATION FOR NO. OF THERMOCOUPLES AND ITS LOCATION INSIDE THE AUTOCLAVE

7.1       Justification for selection of Autoclave is given in table as mentioned below:

S.No.

Locations

Justification

1.

Left side wall (Top)

To cover side wall, chance of less steam penetration

2.

Left side wall (Middle)

To cover side wall, chance of less steam penetration

3.

Left side wall (Bottom)

To cover side wall, chance of less steam penetration

4.

Right side wall (Top)

To cover side wall, chance of less steam penetration

5.

Right side wall (Middle)

To cover side wall, chance of less steam penetration

6.

Right side wall (Bottom)

To cover side wall, chance of less steam penetration

7.

Middle (Top)

To cover the middle area moreover to check chance of air entrapment

8.

Middle of Autoclave

To cover the middle area moreover to check chance of air entrapment

9.

Middle (Bottom)

To cover the middle area moreover to check chance of air entrapment

10.

Near to Drainage

More chance of heat loss

11.

Near to Steam Releasing valve

Chance of maximum heat.

 

7.2       Use 11 Thermocouples to cover entire area of Autoclave and refer the Annexure – I for location of thermocouples. 

8.       PERFORMANCE QUALIFICATION PROCEDURE

The procedures to conduct the experiments are as follows:

8.1       Verify the following as per Instrument Operating procedure and calibration certificate kept in place before validation

8.2       Temperature Display of Autoclave:

The temperature display should be calibrated within the period of Autoclave Validation

            8.3       Compound pressure gauge of Autoclave:

The pressure gauge should be calibrated within the period of Autoclave Validation

          8.4       Calibration of Thermocouples:

·         Calibrate all the thermocouples of data logger before and after the validation using standard thermometer and also made available party’s calibration certificates.

·         Calibrate all the thermocouples of data logger using standard thermometer at 0C, 100 0C, 1210C, and 150 0C.

               8.5      Empty Chamber Heat Distribution Studies:

·         Conduct the test three times and ensure that it has a cold start each time.

·         Keep the chamber empty for each cycle.

·         Fix 11 RTDS in the chamber, through the access port as shown in the Annexure – I.

·         Use Teflon tape to secure probes in position. Ensure that tips do not touch any metallic surface.

·         Attach the probes to the multiple probe temperature data logger

·         Close the doors of Autoclave.

·         Set the Sterilization Time for 15 minutes.

·         Set the data logger to display the temperature of all the probes, after every 1-minute and record it manually.

·         Operate the Autoclave as per respective SOP.

·         Start the sterilization cycle and the data logger at the same time.

·         Record the temperature of all the probes after attaining the set temperature in the specimen format of Annexure – III (a).

·         Show the graphical representation of temperature as per the specimen format of Annexure – III (b).

·         Analyze the results and calculate the F0 value for all the locations by the following formula

F0 = t1 t2 10 (T-T0) /Z dt.

Where t= time interval of temperature measurement (1 minute here)

                                               T = Exposure temperature at any given instant

                                               T0= process reference temperature (121 0C)

 Z = a resistance value of 10 0C assumed for Steam                              Sterilizer

·         For calculation of F0 value, consider the temperature of more than 100°C only.

·         Use specimen format of Annexure – IV (a) for F0 value.

·         Locate the coolest point as the position in the proximity of the probe, showing the minimum F0 value.

·         Show the graphical representation of F0 value as per the specimen format of Annexure – IV (b).

8.6       Loaded Chamber Heat Penetration Studies:                                               

·         Carry out the test using the maximum load intended to be sterilized using the Autoclave.

·         Set the operation values of parameters as established during cycle development studies, and the sterilization time as 20 minutes.

·         Load the Autoclave Chamber for different materials as per the pattern shown in Annexure – II (a), (b) and (c).

·         Fix 11 RTDS in the chamber, through the access port as shown in the Annexure – I.

·         Fix the RTDs such that the sensor is in contact with the material to be sterilized

·         Use Teflon tape to secure RTD probes in position.

·         The mapping of probes should be done to ensure proper representation of full load.

·         Allocate a reference number to each probe and mention in the diagram as described in the Annexure – II (a), (b) and (c).

·         Attach the RTD probes to the multiple probe data logger.

·         Close the door of Autoclave.

·         Set the data logger to display the temperature of all the probes after every 1-minute and record the same.

·         Operate the Autoclave as per latest version of SOP .

·         Start the sterilization cycle and the data logger at the same time.

·         Record the temperature of all the probes after attaining the set temperature in the specimen format of Annexure – III (a).

·         Show the graphical representation of temperature as per the specimen format of Annexure – III (b).

·         Analyze the results and calculate the F0 value for all the locations by the following formula

F0 = t1 t2 10 (T-T0) /Z dt.

Where t= time interval of temperature measurement (1 minute here)

T = Exposure temperature at any given instant

T0= process reference temperature (121 0C)

Z = a resistance value of 10 0C assumed for Steam Sterilizer

·         For calculation of F0 value, consider the temperature of more than 100°C only.

·         Locate the coolest point as the position in the proximity of the probe, showing the minimum F0 value.

·         Show the graphical representation of F0 value as per the specimen format of Annexure – IV (b).

·         Repeat the experiment three times to ensure reproducibility of location of slowest to heat point, and F0 value in the specific load

·         On the basis of the results of the calculated F0 value increase or decrease the Sterilization Cycle Time to achieve a desired F0 value.

8.7       Microbial Challenge Test:

·         Carry out the test using the loads for which F0 (thermal) has been established.

·         Prepare suspension of Bacillus stearothermophilus at concentration of 102 to 107 CFU/ml, in increment of multiples of 10 (i.e. 6 different concentrations).

·         Under Laminar Air Flow, spike 0.1 ml of each of the above suspension in 7 depyrogenated vials, i.e. 6X 7 vials and seal the vials using presterilised rubber plugs and aluminums seals. Mark the vials with the intended location reference numbers, as stated in the following steps.

·         Place three sets of vials of each concentration, at the coolest point in the load (as identified during Thermal Heat Penetration studies)

·         Retain one set of vials of each concentration as positive control.

·         Run a complete Autoclave cycle at the cycle parameters established during F0 studies for the particular load.

·         After the cycle, take the vials under the Laminar Air Flow unit.

·         One by one, open the seal and remove the rubber plug. Add 1 ml of sterile peptone water in each of the vials, spiked with Bacillus stearothermophilus suspension and rinse the entire inner surface of each vial, including the controls

·         Prepare two-fold serial dilution of peptone water from the vials and use the pour plate technique to find the microbial load (in CFU) in each set, using Soyabean Casein Digest Agar Medium.

8.8       Microbial Limit Test:

·         Incubate the sterilized media flask or tubes from the load of Microbial Heat Penetration studies and observe for nutritive properties by streaking Bacillus subtilis for Soyabean Casein Digest Agar Medium and Candida albicans for Sabouraud’s Dextrose Agar (As per SOP ).

·         Bacteria: 32.5 ± 2.5 0C for 24 to 48 hrs and Fungi: 25 ± 2 0C for 5 days.

8.9       Air Entrapment Test (By Bowie- Dick):

·         Place ready to use packet of Bowie-Dick in the middle of Autoclave.

·         Keep the Bowie-Dick over container so that middle position can be set.

·         Set the Time of Autoclave for sterilization for 20 minutes.

·         After completion of cycle, take out the Bowie- Dick and open it for observation.

·         If the observation meets with acceptance criteria that will sign that there is no air entrapment

·         Attach the Bowie Dick with report.

·         If the observation does not meet with acceptance criteria (wrinkle with incomplete circle), inform to engineering department for its rectification.

·         Air entrapment can be due to leakage of gasket

·         After rectification, again follow the above-mentioned procedures.

 

9.     ACCEPTANCE CRITERIA:

      9.1     Performance Qualification of Autoclave shall be considered to be complete when all the under mentioned acceptance criteria are met:

  •  All probes must reach the temperature 121 – 124 oC and pressure must be within 15 to 18 lbs for 15 min cycle.
  • The difference in temperature between the coolest spot and the mean chamber temperature should be not greater than +2.5 oC.
  •  Thermocouples should be calibrated before and after a validation experiment at 0oC, 100oC, 121 oC, and 150 oC.
  • Any thermocouple that senses temperature more than 0.5 oC away from standard calibrated thermometer should be discarded.
  • Temperature recorder should be capable of printing temperature data in 0.1 oC.
  • The desired F0 value achieved at the “slowest to heat point” for a specific loads is a minimum of 25 minutes. F0 value at any cycle shall not fall below 25 minutes.
  •  A sterility Assurance level of 10-6 is achieved.
  • No Microbial growth should be observed i.e. Negative control and Nutritive properties of Media must pass (growth must appear)
  • Autoclaved ampoules containing Bacillus stearothermophilus spores suspension ampoules should not show any colour change after 5 days of Incubation.
  • In Air Entrapment Test, Bowie- Dick should show a complete and smoothed circle. 

10.     PERFORMANCE QUALIFICATION REPORT

               The performance qualification report shall consist of a summary of documents, in narrative form, which briefly describes the work as well as conclusion certification regarding acceptability of the Autoclave. 

11.     APPROVALS

The representatives of the departments listed in 4.0 above shall do approvals of the performance qualification. 

12.     CONCLUSION

            Conclusion shall be made based on the results obtained. 

13.          ANNEXURE

Annexure – I         :     Drawing showing the location of Probes.

Annexure – II (a)  :     Drawing showing the pattern of loading of petri dishes.

Annexure – II (b)  :     Drawing showing the pattern of loading of Garments.

Annexure – II (c)  :     Drawing showing the pattern of loading of Media, Glassware and Other Accessories.

Annexure – III (a) :     Sheet for Temperature and Pressure (Heat Distribution / Penetration Study)

Annexure – III (b) :    Graphical presentation of Temperature (Heat Distribution / Penetration Study)

Annexure – IV (a) :     Sheet for Fo Value (Heat Distribution / Penetration Study)

Annexure – IV (b) :       Graphical presentation of Fo Value (Heat Distribution / Penetration Study)

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