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 0^oC, 100^oC, 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)