Performance Qualification of High Pressure High Vaccum (HPHV) Steam Sterilizer in Pharmaceuticals : Pharmaguideline

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Performance Qualification of High Pressure High Vaccum (HPHV) Steam Sterilizer in Pharmaceuticals

Performance Qualification Protocol for Steam Sterilizer (Autoclave) and Procedure for Vacuum Leak Test, Steam Quality Test, Bowie-Dick Test, Heat Distribution Test, Hest Penetration Test and F0 Calculation.

Qualification of High Pressure High Vaccum (HPHV) Steam Sterilizer in Pharmaceuticals is done by following procedure.

1.0  CALIBRATION OF TEMPERATURE SENSORS

1.1  PRE & POST CALIBRATION OF TEMPERATURE SENSORS 

Prepare the container with crushed ice and add enough purified water to ensure the proper slush solution allow the temperature to stabilize ensure to add sufficient crushed ice to maintain the equilibrium state of ice & water.

1.2  PROCEDURE

Temperature sensors which are used for qualification study shall be calibrate in ice bath at approximately 0°C and in high temperature reference block at 50,100,121,150°C prior to its usage in qualification.
Record the temperature of all the sensors while putting them in ice bath after one minute of temperature stabilization put the individual sensor to the slot of high temperature reference block which is stabilized at required temperature. Record the reading at least one minute after stabilization of temperature. Record the temperature for five minute by data logger and attach the report with print outs with reports.

1.3  ACCEPTANCE CRITERIA

No temperature sensors vary by 1°C in ice bath from the mean of temperatures shown by the calibrated thermometer during the data logging period.
No temperature sensors vary by 1°C in high temperature reference block from the mean of temperatures Shown by the calibrated thermometer during the data logging period. 

2.0  QUALIFICATION TESTS

The High Pressure High Vacuum Steam Sterilizer will be qualified after validating (As per the methods outlined in this Protocol) the equipment for desired performance and its ability to sterilize different components and/or loads at the set parameters & set loading patterns, repeatably & consistently.
The High Pressure High Vacuum Steam Sterilizer will be considered qualified for consistent and reliable performance (Validated) on successful completion of the following tests.
·  Vacuum Leak test (3 Trials without probe & 3 Trials with probe)
·  Bowie –Dick Test for Steam penetration (3 trials on 3 different days)
·  Steam qualification tests (3 trials on 3 different days)
·  Empty Chamber Heat Distribution studies (3 trials) with temperature mapping probes at different locations of the sterilizer chamber.
·  Loaded Chamber heat penetration studies (3 trials) for each sterilization load of fixed loading pattern, with temperature mapping probes inside the innermost possible layer of the load subjected for sterilization.
·  Bio-Challenge studies using Bacillus stearothemophilus spore strips (containing 106 or more spores per strip) during the heat distribution & heat penetration studies.
·  Estimation of the F0 value achieved during the sterilization hold period at each temperature-mapping probe.
·  Quality of the steam condensate collected from the sampling point provided in the sterilizer chamber condensate drain line for all loads.
·  Vacuum break filter integrity testing.
To qualify these tests the equipment should fulfill the acceptance criteria described in the individual test procedures. After completion of the qualification tests all the data generated will be compiled together to evaluate ability of the High Pressure High Vacuum Steam Sterilizer to sterilize different components at the set parameters and set-loading pattern.

2.1  VACUUM LEAK RATE TEST

Objective

Objective of this test is to ensure that the rate of vacuum drop is within the acceptable limits when the High Pressure High Vacuum Steam Sterilizer chamber is subjected to the vacuum of more than /equal to –0.93 Kg/cm2 on gauge (equivalent to 100 m bar absolute pressure).  3 Trials should be done for this qualification test.

Procedure

Record the set parameters for the vacuum leak test in the Annexure-1 of Attachment –1.
Operate the empty Steam Sterilizer as per the SOP. Allow the sterilization chamber temperature to stabilize.
When the chamber temperature is stabilized, the vacuum pump starts.
Achieve chamber pressure (on gauge) equivalent to <- 100 mbar absolute pressure. Observe channel No.6 of the multi point recorder for vacuum).
When desire level of pressure is achieved in the sterilizer chamber (Pressure P1) record the observation in Annexure-2 of Attachment-1.
Wait for 5 minutes to stabilize the chamber pressure (This time allowance is provided to allow evaporation of condensate in the chamber).
After 5 minutes record the observation (Pressure P2) wait for 10 minutes, after 10 minutes record the observations (Pressure P3)
Calculate rate of Vacuum drop in the sterilizer chamber by using the equation given below and record the results in Annexure-2 Attachment-1.
The printout taken during the vacuum leak test should be preserved as per the Annexure-3 of attachment-1
Compile the data generated during the qualification test for complete evaluation of the system.
Prepare summary and conclusion of the performance qualification studies, which will be finally approved by the Head – Manufacturing & Head – Quality Assurance.
Remark – The vacuum leak test should be performed only when the sterilization chamber is empty, dry and at room temperature.

Basis of Calculation

The actual observations obtained during the vacuum leak test should be compiled in the Annexure-2 of Attachment – 1 and the rate of Vacuum drop should be calculated by using the following formula.
Rate of vacuum of drop per minute = (P3-P2) / 10
Where,
P2 = Pressure (vacuum) observed after 5 minutes of achieving the desired level of pressure.
P3 = Pressure (vacuum) observed after 10 minutes of achieving pressure P2  

Acceptance Criteria

When the Chamber pressure (on gauge) equivalent to <- 100 mBar absolute pressure is applied, the rate of vacuum drop at the end of 10 minutes holding time should not be more than 13 mBar i.e., equivalent to NMT 1.3 mBar per minute.

Observation & Results

Record the observations and results in formats enclosed as Attachment-1

2.2 STEAM QUALITY TESTS

A continuous supply of saturated steam is required for steam sterilization. Too high a level of non-condensable gases will prevent the attainment of sterilizing conditions; too little moisture carried in suspension may allow the steam to become superheated during expansion into the chamber, while excess moisture may cause damp loads.
For all these tests, the steam should be sampled from the steam service pipe. The measurements are taken during a period of maximum steam demand, when steam is first admitted to the sterilizer chamber.
Presence of non-condensable gases more than the desired level might get accumulated within the sterilization chamber in the form of air pockets, which might affect steam penetration this leading to probable non-sterile unit.
NOTE: Silicone rubber tubing is porous to steam and should not be used to carry steam in these tests.

2.2.1 Non-Condensable Gas Test

Objective
Objective of this test is to ensure that, The pure steam supply of the High Pressure High Vacuum Steam Sterilizer does not contain non-condensable gases more than the desire level (NMT 3.5%) when measured on-line during the standard sterilization cycle.
Procedure
This test is used to demonstrate that the level of non-condensable gases in the steam will not prevent the attainment of sterilization conditions in any part of the load. The method described should be regarded not as measuring the exact level of non-condensable gas, but a method by which the provision of acceptable steam quality can be demonstrated.
The apparatus is shown and described in Figure No.1 (all sizes are nominal). Connect the needle valve to the steam service pipe as shown in Figure No.1.
Assemble the apparatus so that condensate will drain freely from the long rubber tube into the sampling pipe. If the tube is too short, copper or stainless steel tubing may also be used.
Fill the container with cold water until it overflows. Fill the burette and funnel with cold water, invert them and place them in the container. Draw out any air that has collected in the burette. With the steam sampling pipe out of the container, open the needle valve and allow steam to purge the air form the pipe. Place the pipe in the container, locate the end within the funnel, and add more cold water until it flows through the overflow pipe. Place the empty measuring cylinder under the container overflow.
Adjust the needle valve to allow a continuous sample of steam into the funnel sufficient to cause a small amount of “Steam Hammer” to be heard. Ensure that all the steam is discharged into the funnel and does not bubble out into the container. Note the setting of the needle valve. Close the valve.
Ensure that the container is topped up with cold water and that the measuring cylinder is empty. Draw out any air present in the burette.
Ensure that the sterilizer chamber is empty except for the usual chamber furniture. Select and start the operating cycle.
When the steam supply to the chamber first opens, open the needle valve to the previously noted setting, allowing a continuous sample of steam into the funnel sufficient to cause a small amount of steam hammer to be heard.
Allow the steam sample to condense in the funnel. Any non-condensable gases will rise to the top of the burette. Overspill formed by the condensate and the water displaced by the gases will collect in the measuring cylinder.
When the temperature of the water in the container reaches 70-75 °C close the needle valve. Note the volume of gas collected in the burette (Vb) and the volume of water collected in the measuring cylinder (Vc).
Calculate the fraction of non-condensable gases as a percentage as follows.
When the temperature of the water in the container reaches 70-75 °C close the needle valve. Note the volume of gas collected in the burette (Vb) and the volume of water collected in the measuring cylinder (Vc).
Calculate the fraction of non-condensable gases as a percentage as follows.
Fraction of non-condensable gases = 100 x (Vb/Vc).
The test should be considered satisfactory if the fraction of non-condensable gases does not exceed 3.5 %.
The test should be done two more times to check consistency. If the results of the three tests differ significantly, then the cause should be investigated before proceeding further.

Figure No.1


Acceptance Criteria:
The measured non-condensable gases in the pure steam should not cross 3.5%
Observations & Results
Record the observations and results in formats enclosed as Attachment-2, Annexure-1

2.2.2 Superheat

Objective
This test is used to demonstrate that the amount of moisture in suspension with steam from the service supply is sufficient to prevent the steam from becoming superheated during expansion into the chamber.
The method described here uses a low-volume sample, continuously taken from the center of the steam service pipe. The level of superheat determined by this method cannot be regarded as indicative of the true dryness of the steam in the pipe since condensate flowing along the inner surface is not collected. However, devices designed to separate free condensate are incorporated into the steam delivery system to the chamber and therefore the level determined by this method is representative of steam conditions likely to prevail within the chamber during the plateau period.
Procedure
This test should normally follow a satisfactory test for non-condensable gases.
This test, and the subsequent dryness value test, requires a pitot tube as shown in figure No.2. The rest of the apparatus is shown and described in figure No.3. All sizes are nominal.
Fit the Pitot tube concentrically within the steam service pipe as shown in figure No.3.
Fit the sensor entry gland to the steam service pipe. Insert one of the sensors through the gland and position the axis of the pipe.
Insert the second sensor through the gland in the expansion tube and position it on the axis of the pipe. Wrap lagging around the expansion tube. Push the tube onto the pitot.

Figure No.2.

Ensure that the sterilizer chamber is empty except for the usual chamber furniture. Select and start the operating cycle.
From the measured temperatures, note the temperature in the steam service pipe (for use in the dryness test) and in the expansion tube (Te) when the steam supply to the chamber first opens. Calculate the superheat in oC from the following equation:
Superheat  = Te - To
Where:
To is the boiling point of water at local atmospheric pressure.
The test should be considered satisfactory if the superheat measured in the expansion tube does not exceed 25°C.
Record the observations and results in formats enclosed as Attachment-2, Annexure-2

Figure No. 3

2.2.3 DRYNESS TEST

The accurate measurement of the percentage of moisture content in the steam is difficult, and the traditional methods where constant steam flow is required are not suitable for sterilizers. This test should be regarded not as measuring the true content of moisture in the steam, but as a method by which the provision of acceptable steam quality can be demonstrated. 
Objective
a)  This test is used to demonstrate that the dryness value is not less than 0.90 (if metal loads are to be processed, the dryness value should not be less than 0.95);
b)  Throughout the operating cycle, the temperature measured in the steam service pipe is within 3°C of that measured during the superheat test.
Procedure
The test is conveniently carried out immediately after the superheat test.
This test requires a pitot tube as shown in Figure No.2. The apparatus is shown and described in figure No.4. All sizes are nominal. A laboratory balance is also required, capable of weighing a load up to 2 kg with an accuracy of 0.1g or better.
If it is not already fitted, fit the Pitot tube concentrically within the steam service pipe as shown in figure No.3.
If it is not already fitted, fit the sensor entry gland to the steam service pipe. Insert a temperature sensor through the gland and position it on the axis of the pipe.
Connect the rubber tube to the longer of the pipes in the stopper, place the stopper in the neck of the vacuum flask, weigh the whole assembly and note the mass (M1).
Remove the stopper and tube assembly and pour 650 + 50 ml of cold water (below 27°C) in to the flask. Replace the stopper and tube assembly, weigh the flask and record the mass (M2).
Support the flask close to the pitot, and ensure that the rubber tube and flask are protected from excess heat and draughts, do not connect it to the pitot tube yet.
Introduce the second temperature sensor through the shorter of the two pipes in the stopper and into the water in the flask. Note the temperature of the water in the flask (To).
Ensure that the sterilizer chamber is empty except for the usual chamber furniture. Select and start the operating cycle.
When the steam supply to the chamber first opens, connect the rubber tube to the pitot discharge and wrap lagging around it. Arrange the rubber tube to permit condensate to drain freely into the flask. Not the temperature in the steam service pipe (TS).
When the temperature of the water in the flask is approximately 80°C, disconnect the rubber tube from the pitot, agitate the flask so that the contents are thoroughly mixed, and note the temperature of the water (T1).
Weigh the flask and stopper assembly and note the mass (M3).
The initial mass of water in the flask is given by Mw = M2 – M1
The mass of condensate collected is given by MC = M3 - M2.  
Calculate the dryness value of the steam from the following equation:
D= (T1-T0) (4.18MW + 0.24) / LMC - 4.18 (TS-T1) / L
Where:
T0 = Initial temperature of the water in the flask (°C);
T1 = Final temperature of the water and condensate in the flask (°C);
TS = Average temperature of the steam delivered to the sterilizer (°C);
MW = Initial mass of water in the flask (Kg);
MC= Mass of condensate collected (Kg);
L= latent heat of dry saturated steam at temperature TS (kJ Kg-1).
Acceptance Criteria
The test should be considered satisfactory if the following requirements are met.
A) The dryness value is not less than 0.90 (if metal loads are to be processed, the dryness value should not be less than 0.95);
B) Throughout the operating cycle, the temperature measured in the steam service pipe is within 3°C of that measured during the superheat test.
Observation and results
Record the observations and results in formats enclosed as Attachment-2, Annexure-3.

2.3 BOWIE – DICK TEST FOR STEAM PENETRATION

Objective

Objective of this test is to ensure that the vacuum pulses applied before the sterilization hold period are sufficient to remove the entrapped air or noncondensable gases 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 {8 minutes (500 seconds Approx) 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 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

Procedure

Record the set parameters for the Bowie-Dick test cycle in the Annexure-1 of Attachment-3
Place one Bowie-dick Test Pack in the center of the sterilization chamber supported approximately 100 to 200 mm above the sterilization chamber base.
Select cycle no. 4 on the Control panel & operate the High Pressure High Vacuum Steam Sterilizer as per the SOP.
The printout taken during the Bowie-Dick test cycle & the Bowie-Dick test indicator should be preserved as per the Annexure-3 Attachment-3.

Test pack size

Remarks – Bowie-Dick cycle should be normally preceded by a warm-up cycle as the effectiveness of air removal may depend on all the parts of the sterilizer being at working temperature.

Acceptance Criteria

The Bowie-Dick Test indicator should show a uniform color change. No change, non-uniform change and/ or air entrapment (bubble) spot on the pattern indicates inadequate air removal from the sterilization chamber.

Observations & Results

Record the observations and results in formats enclosed as Attachment-3

2.4 HEAT DISTRIBUTION EMPTY CHAMBER

Objective:

Objective of this test is to ensure that,
·  The High Pressure High Vacuum Steam Sterilizer is capable of attaining a temperature of 121ºC during the sterilization hold period with steam pressure of 1.2 kg/Cm2.
·  Temperature spread within the range of 121.1°C to 124°C during sterilization cycle will demonstrate the uniform heat distribution within the chamber.
·  Any Location(s) where the temperature sensor is placed, not achieving minimum sterilization temperature of 121.1°C throughout the sterilization temperature hold will be considered as cold spot.
·  team condensate collected during the sterilization cycle from the sample collection port should meet the requirements for Water for Injection USP.

Procedure

Record the set parameters for the sterilization cycle to be operated during the test for empty chamber heat distribution study, in the Annexure-1 of Attachment-4
Pass minimum 16 no. Temperature mapping probes 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 surface, also place biological indicators along with locations of temperature mapping probes in the sterilizer chamber.
Record the position of the probes and the biological indicators in a representative schematic form in the Annexure-2 of Attachment-4
TEMPERATURE SENSOR PLACEMENT IN THE EMPTY CHAMBER
(NOTE: The temperature sensors shall be placed in the pre determined locations with predetermined sensor numbers corresponding to the data logger channels).
SENSOR NUMBER
LOCATION IN THE CHAMBER
SENSOR NUMBER
LOCATION IN THE CHAMBER
1
Chamber Drain
9
Near RTD two
2
Steam inlet right
10
Near RTD three
3
Steam inlet left
11
Near RTD four
4
Sterile side door top back side
12
Near RTD five
5
Sterile side door bottom back
13
Non-aseptic door top Left corner
6
Non-aseptic door top front side
14
Non-aseptic door bottom Right corner
7
Non-aseptic door bottom front side
15
Aseptic door top left corner
8
Near RTD one
16
Aseptic door bottom Right corner

Justification for the temperature sensor location of choice

Sensor no.1-Control sensor of the equipment is located in the condensate drain. This is very important to know the temperature of the drain point to evaluate the cycle set parameters.
Sensor no. 2&3-To verify the probability of excess temperature at the steam inlet.
Sensor no.4, 5, 6 &7- Any conduction of heat from the door which may cause temperature drop at that particular point.
Sensor nos.8, 9, 10, 11, &12- As these are temperature recording probes at different points- it is necessary to verify/compare the temperatures obtained with strip chart of the equipment.
Sensor nos.13, 14, 15, 16- Any conduction of heat from the door which may cause temperature drop at that particular point.
Connect the probes to a suitable data logger, which can scan and print the actual temperature observed at different locations with respect to time.
Select cycle No.2 on control panel.
Operate the High Pressure High Vacuum Steam Sterilizer as per SOP, and also start the data logger to record actual temperatures within the sterilization chamber with respect to time
Collect sufficient sample of steam condensate as per the SOP, for analysis in a clean and sterile sample bottle from the sampling point provided in the steam condensate drain line.
When the sterilization cycle completes;
1.  Collect Strip chart from the Strip chart Recorder of the sterilizer and preserve as Annexure-3 of Attachment-4
2.  Download the data from data logger into the computer for data-analysis and printing. Enclose the data printouts as per the Attachment-4
3.  Aseptically collect the exposed biological indicators and send the indicators after wrapping in a sterile enclosure to microbiology lab. With necessary identification and the observation sheet as per Annexure-6 of Attachment-4
If the empty chamber heat distribution studies are acceptable, perform three consecutive replicate runs to demonstrate cycle and sterilizer reproducibility.
Compile the data generated during the qualification test for complete evaluations of the system.
Acceptance Criteria
There should be uniform distribution of heat in the sterilizer chamber during the sterilization hold period and the temperature at each temperature mapping probe should be within the range of 121.1°C to 124°C during the sterilization hold period.
Observations and Results
Record the observations and results in formats enclosed as Attachment-4.

2.5 HEAT PENETRATION STUDY – GARMENTS (MAXIMUM LOAD)

Objective
Objective of this test is to ensure that,
·  The steam is sufficiently penetrating into the innermost portions of the Garments with maximum load subjected for sterilization to achieve desired temperature 121.1°C during the complete Sterilization hold period with steam pressure of 1.2 Kg/cm2.
·  If sterilization temperature (121.1°C) is not achieved throughout the cycle, load configuration or size of the load has to be reviewed and cycles to be repeated.
·  Temperature spread within the range of 121.1°C to 124°C during sterilization hold period indicate that, uniform heating process which is achieved in the Loaded chamber heat penetration study is not affected by load. There could be the possibility of lag period for attaining 121.1°C during heat penetration trials as the probes are placed deep into the load any location(s) where the temperature indicator is placed, not achieving minimum sterilization temperature of 121.1°C during sterilization temperature hold period will be considered as cold spot.
·  Steam condensate collected during the sterilization cycle from the sample collection port should meet the requirements for Water for Injection USP.
Load details
·  Boiler Suit – 12 pairs
·  Booties – 12 pairs
·  Gloves (14 inch) in perforated box – 15 pairs
·  Gloves (7 inch) in perforated box – 15 pairs
·  Moping dusters – 15 Nos

Procedure

Record the set parameters for the sterilization cycle to be operated during the test for the loaded chamber heat penetration study in the Annexure-1 of Attachment-5.
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 supposed to most difficult points for steam penetration, also place biological indicator strip along with each temperature mapping probe. Record the position of the probes and the biological indicators in a representative schematic form the Annexure-2 of Attachment-5.
Connect the probes to calibrated data logger, which can scan and print the actual temperature with respect to time.
Operate the High Pressure High Vacuum Steam Sterilizer as per SOP, and also start the data logger to record actual temperatures within the sterilization chamber with respect to time.

TEMPERATURE SENSOR PLACEMENT IN THE GARMENTS LOAD (MAXIMUM)

(NOTE: The temperature sensors shall be placed in the pre determined locations with predetermined sensor numbers corresponding to the data logger channels).
SENSOR NUMBER
LOCATION IN THE CHAMBER
SENSOR NUMBER
LOCATION IN THE CHAMBER
1
Chamber drain
9
Approx. center garment bag -1
2
Glove Pack (7”) - 1
10
Approx. center garment bag –3
3
Glove Pack (7”) - 2
11
Approx. center garment bag –6
4
Near RTD-1
12
Approx. center garment bag -10
5
Near RTD-2
13
Between 2&3 mopping duster
6
Near RTD-3
14
Between 5&6 mopping duster
7
Near RTD-4
15
Glove Pack (14”) - 1
8
Near RTD-5
16
Glove Pack (14”) - 2
Justification for the temperature sensor location of choice
Sensor no.1-Control sensor of the equipment is located in the condensate drain. This is very important to know the temperature of the drain point to evaluate the cycle set parameters.
Sensor no. 2, &3 – To verify the heat penetration of the innermost portion of the small size gloves.
Sensor no.4, 5, 6, 7 & 8- As these are temperature-recording probes at different points- it is necessary to verify/compare the temperatures obtained with strip chart of the equipment.
Sensor no.9, 10, 11& 12- To verify the heat penetration into the innermost portion of the garment load in the garment pack at different position.
Sensor no.13 & 14- – To verify the heat penetration of the innermost portion of the mopping duster load in the box.
Sensor no. 15 & 16 – To verify the heat penetration of the innermost portion of the small size Gloves.
When the sterilization cycle completes;
1.  Collect thermograph from the multipoint temperature recorder of the sterilizer and preserve as Annexure-3 of Attachment-5
2.  Download the data from data logger into the computer for data-analysis and printing. Record the temperature observed at different locations in the Annexure-4 of Attachment-5 
3.  Aseptically collect the exposed biological indicators and send the indicators after wrapping in a sterile enclosure to microbiology lab with necessary identification and the observation sheet as per Annexure-6 of Attachment-5
If the heat penetration study is acceptable perform three consecutive replicate runs to demonstrate cycle and sterilizer reproducibility.
Compile the data generated during the qualification test for complete evaluation of the system.

Acceptance Criteria

There should be uniform 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 121.1°C to 124°C during the complete sterilization hold period.

Observations and Results

Record the observations and results in formats enclosed as Attachment-5.

2.6 HEAT PENETRATION STUDY – GARMENTS (MINIMUM LOAD)

Objective

Objective of this test is to ensure that,
·  The steam is sufficiently penetrating into the innermost portions of the garments with minimum load subjected for sterilization to achieve desired temperature 121.1°C during the complete Sterilization hold period with steam pressure of 1.2 Kg/cm2.
·  If sterilization temperature (121.1°C) is not achieved throughout the cycle, load configuration or size of the load has to be reviewed and cycles to be repeated.
·  Temperature spread within the range of 121.1°C to 124°C during sterilization hold period indicate that, uniform heating process which is achieved in the Loaded chamber heat penetration study is not affected by load. There could be the possibility of lag period for attaining 121.1°C during heat penetration trials as the probes are placed deep into the load
·  Any Location(s) where the temperature indicator is placed, not achieving minimum sterilization temperature of 121.1°C during sterilization temperature hold period will be considered as cold spot.
·  Steam condensate collected during the sterilization cycle from the sample collection port should meet the requirements for Water for Injection USP.
Load details
·  Boiler Suit – 3 pairs
·  Booties – 3 pairs
·  Gloves (14 inch) in perforated box – 5 pairs
·  Gloves (7 inch) in perforated box – 5 pairs
·  Moping dusters – 15 Nos

Procedure

Record the set parameters for the sterilization cycle to be operated during the test for the loaded chamber heat penetration study in the Annexure-1 of Attachment-6.
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 supposed to most difficult points for steam penetration, also place biological indicator strip along with each temperature mapping probe. Record the position of the probes and the biological indicators in a representative schematic form the Annexure-2 of Attachment-6.
Connect the probes to calibrated data logger, which can scan and print the actual temperature with respect to time.
Operate the High Pressure High Vacuum Steam Sterilizer as per SOP and also start the data logger to record actual temperatures within the sterilization chamber with respect to time.

TEMPERATURE SENSOR PLACEMENT IN THE GARMENTS LOAD (MINIMUM)

(NOTE: The temperature sensors shall be placed in the pre determined locations with predetermined sensor numbers corresponding to the data logger channels).
SENSOR NUMBER
LOCATION IN THE CHAMBER
SENSOR NUMBER
LOCATION IN THE CHAMBER
1
Chamber drain
9
Approx. center garment bag -1
2
Glove Pack (7”) - 1
10
Approx. center garment bag –2
3
Glove Pack (7”) - 2
11
Approx. center garment bag –3
4
Near RTD-1
12
Between 1&2 mopping duster
5
Near RTD-2
13
Between 2&3 mopping duster
6
Near RTD-3
14
Between 4&5 mopping duster
7
Near RTD-4
15
Glove Pack (14”) - 1
8
Near RTD-5
16
Glove Pack (14”) - 2
Justification for the temperature sensor location of choice
Sensor no.1-Control sensor of the equipment is located in the condensate drain. This is very important to know the temperature of the drain point to evaluate the cycle set parameters.
Sensor nos. 2, &3 – To verify the heat penetration of the innermost portion of the small size Gloves.
 Sensor no.4, 5,6 ,7 & 8- As these are temperature-recording probes at different points- it is necessary to verify/compare the temperatures obtained with strip chart of the equipment.
Sensor nos.9, 10 & 11- To verify the heat penetration into the innermost portion of the garment load in the garment pack at different position.
Sensor no. 12, 13 & 14- To verify the heat penetration of the innermost portion of the mopping duster load in the box.
Sensor no. 15 & 16 – To verify the heat penetration of the innermost portion of the small size Gloves.
When the sterilization cycle completes;
1.  Collect thermograph from the multipoint temperature recorder of the sterilizer and preserve as Annexure-3 of Attachment-6
2.  Download the data from data logger into the computer for data-analysis and printing. Record the temperature observed at different locations in the Annexure-4 of Attachment-6
3.  Aseptically collect the exposed biological indicators and send the indicators after wrapping in a sterile enclosure to microbiology lab with necessary identification and the observation sheet as per Annexure-6 of Attachment-6
If the heat penetration study is acceptable perform three consecutive replicate runs to demonstrate cycle and sterilizer reproducibility.
Compile the data generated during the qualification test for complete evaluation of the system.

Acceptance Criteria

There should be uniform 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 121ºC to 124ºC during the complete sterilization hold period.

Observations and Results

Record the observations and results in formats enclosed as Attachement-6.

2.7 HEAT PENETRATION STUDY- CRITICAL AREA FILTERS

Objective
Objective of this test is to ensure that,
·  The steam is sufficiently penetrating into the innermost portions of the Filtration accessories load (to be used in Critical area) subjected for sterilization to achieve desired temperature 121.1°C during the complete Sterilization hold period with steam pressure of 1.2 Kg/cm2.
·  If sterilization temperature (121.1°C) is not achieved throughout the cycle, load configuration or size of the load has to be reviewed and cycles to be repeated.
·  Temperature spread within the range of 121.1°C to 124°C during sterilization hold period indicate that, uniform heating process which is achieved in the Loaded chamber heat penetration study is not affected by load. There could be the possibility of lag period for attaining 121.1°C during heat penetration trials as the probes are placed deep into the load
·  Any Location (S) where the temperature indicator is placed, not achieving minimum sterilization temperature of 121.1°C during sterilization temperature hold period will be considered as cold spot.
·  Steam condensate collected during the sterilization cycle from the sample collection port should meet the requirements for Water for Injection USP.
Load details
·  0.2m , 10” Housings – 1 Nos. for Disinfectant filtration
·  4” Air vent Filters – 5 Nos
·  Membrane Filter Holder – 1 No.

Procedure

Record the set parameters for the sterilization cycle to be operated during the test for the loaded chamber heat penetration study in the Annexure-1 of Attachment-7.
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 supposed to most difficult points for steam penetration, also place biological indicator strip along with each temperature mapping probe. Record the position of the probes and the biological indicators in a representative schematic form the Annexure-2 of Attachment-7.
Connect the probes to suitable data logger, which can scan and print the actual temperature with respect to time.
Operate the High Pressure High Vacuum Steam Sterilizer as per SOP, and also start the data logger to record actual temperatures within the sterilization chamber with respect to time.

TEMPERATURE SENSOR PLACEMENT IN THE CRITICAL AREA FILTERS LOAD

(NOTE: The temperature sensors shall be placed in the pre determined locations with predetermined sensor numbers corresponding to the data logger channels).
SENSOR NUMBER
LOCATION IN THE CHAMBER
SENSOR NUMBER
LOCATION IN THE CHAMBER
1
Chamber drain
9
Inside Air Vent Filter -5
2
Aseptic door side middle
10
In Membrane filter holder
3
Non-aseptic door side middle
11
Near RTD-1
4
Inside 10” inch housing
12
Near RTD-2
5
Inside Air Vent Filter -1
13
Near RTD-3
6
Inside Air Vent Filter -2
14
Near RTD-4
7
Inside Air Vent Filter -3
15
Near RTD-5
8
Inside Air Vent Filter -4
16
Chamber middle top side
Justification for the temperature sensor location of choice
Sensor no.1-Control sensor of the equipment is located in the condensate drain. This is very important to know the temperature of the drain point to evaluate the cycle set parameters.
Sensor nos. 2, &3 - Any conduction of heat from the doors that may cause temperature drop at that particular point.
 Sensor no.4, 5, 6, 7, 8, 9 &10- to verify the heat penetration at inner most portion of Filtration assembly.
Sensor nos.11, 12, 13, 14, & 15- As these are temperature-recording probes at different points- it is necessary to verify/compare the temperatures obtained with strip chart of the equipment.
Sensor no.16- to verify the possible excessive heat during the cycle as the top portion of the chamber.
When the sterilization cycle completes;
1.  Collect thermograph from the multipoint temperature recorder of the sterilizer and preserve as Annexure-3 of Attachment-7
2.  Download the data from data logger into the computer for data-analysis and printing. Record the temperature observed at different locations in the Annexure-4 of Attachment-7
3.  Aseptically collect the exposed biological indicators and send the indicators after wrapping in a sterile enclosure to microbiology lab with necessary identification and the observation sheet as per Annexure-6 of Attachment-7
If the heat penetration study is acceptable perform three consecutive replicate runs to demonstrate cycle and sterilizer reproducibility.
Compile the data generated during the qualification test for complete evaluation of the system.

Acceptance Criteria

There should be uniform 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 121.1°C to 124°C during the complete sterilization hold period.

Observations and Results

Record the observations and results in formats enclosed as Attachement-7.

2.8  HEAT PENETRATION STUDY – OTHER ACCESSORIES

Objective
Objective of this test is to ensure that,
·  The steam is sufficiently penetrating into the innermost portions of accessories  load having gaskets , valves, clamps & sampling aids when subjected for sterilization to achieve desired temperature 121.1°C during the complete sterilization hold period with steam pressure of 1.2 Kg/cm2.
·  If sterilization temperature (121.1°C) is not achieved throughout the cycle, load configuration or size of the load has to be reviewed and cycles to be repeated.
·  Temperature spread within the range of 121.1°C to 124°C during sterilization hold period indicate that, uniform heating process which is achieved in the loaded chamber heat penetration study is not affected by load. There could be the possibility of lag period for attaining 121.1°C during heat penetration trials as the probes are placed deep into the load
·  Any Location(s) where the temperature indicator is placed, not achieving minimum sterilization temperature of 121.1°C during sterilization temperature hold period will be considered as cold spot.
·  Steam condensate collected during the sterilization cycle from the sample collection port should meet the requirements for Water for Injection USP.
Load details
·  For Filtered Disinfectant Solution Storage – 3 Nos
·  1” Teflon Gaskets – 100 Nos.
·  2” Teflon Gasket – 40 Nos
·  1” T.C. Valves – 20 Nos
·  2” T.C. Valves - 5 Nos
·  1” Clamp
·  2” Clamp

Procedure

Record the set parameters for the sterilization cycle to be operated drying the test for the loaded chamber heat penetration study in the Annexure-1 of Attachement-10.
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 supposed to most difficult points for steam penetration, also place biological indicator strip along with each temperature mapping probe. Record the position of the probes and the biological indicators in a representative schematic form the Annexure-2 of Attachment-10.
Connect the probes to suitable data logger, which can scan and print the actual temperature with respect to time.
Operate the High Pressure High Vacuum Steam Sterilizer as per SOP, and also start the data logger to record actual temperatures within the sterilization chamber with respect to time.

TEMPERATURE SENSOR PLACEMENT IN OTHER ACCESSORIES

(NOTE: The temperature sensors shall be placed in the pre determined locations with predetermined sensor numbers corresponding to the data logger channels).
SENSOR NUMBER
LOCATION IN THE CHAMBER
SENSOR NUMBER
LOCATION IN THE CHAMBER
1
Chamber drain
9
In bet. T.C. clamp 1 & 2
2
Aseptic door side middle
10
Inside the Disinfectant storage vessel
3
Non-aseptic door side middle
11
Near RTD-1
4
In between 20 mm Rubber stoppers pack
12
Near RTD-2
5
Inner side of Teflon coated flexible pipe
13
Near RTD-3
6
Between Teflon Gasket 1 & 2
14
Near RTD-4
7
In Between T.C. valves 1 & 2
15
Near RTD-5
8
In between P. vessel & stand
16
Chamber middle top side
Justification for the temperature sensor location of choice
Sensor no.1-Control sensor of the equipment is located in the condensate drain. This is very important to know the temperature of the drain point to evaluate the cycle set parameters.
Sensor nos. 2, &3 - Any conduction of heat from the doors that may cause temperature drop at that particular point.
Sensor no.4, 5, 6, 7, 8, 9 & 10- to verify the heat distribution & penetration at inner most portions of the Accessories.
Sensor nos.11, 12, 13, 14, & 15- As these are temperature-recording probes at different points- it is necessary to verify/compare the temperatures obtained with strip chart of the equipment.
Sensor no.16- to verify the possible excessive heat during the cycle as the top portion of the chamber.
When the sterilization cycle completes;
1.  Collect thermograph from the multipoint temperature recorder of the sterilizer and preserve as Annexure-3 of Attachment-10
2.  Download the data from data logger into the computer for data-analysis and printing. Record the temperature observed at different locations in the Annexure-4 of Attachment-10.
3.  Aseptically collect the exposed biological indicators and send the indicators after wrapping in a sterile enclosure to microbiology lab with necessary identification and the observation sheet as per Annexure-6 of Attachment-10.
If the heat penetration study is acceptable perform three consecutive replicate runs to demonstrate cycle and sterilizer reproducibility.
Compile the data generated during the qualification test for complete evaluation of the system.

Related: Computer System Validation

Acceptance Criteria

There should be uniform 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 121.1°C to 124°C during the complete sterilization hold period.

Observations and Results

Record the observations and results in formats enclosed as Attachement-10.

2.9 HEAT PENETRATION STUDY – CLEANING AIDS

Objective
Objective of this test is to ensure that,
·  The steam is sufficiently penetrating into the innermost portions of the Cleaning Aids load when subjected for sterilization to achieve desired temperature 121.1°C during the complete Sterilization hold period with steam pressure of 1.2 Kg/cm2.
·  If sterilization temperature (121.1°C) is not achieved throughout the cycle, load configuration or size of the load has to be reviewed and cycles to be repeated.
·  Temperature spread within the range of 121.1°C to 124°C during sterilization hold period indicate that, uniform heating process which is achieved in the Loaded chamber heat penetration study is not affected by load. There could be the possibility of lag period for attaining 121.1°C during heat penetration trials as the probes are placed deep into the load
·  Any Location (S) where the temperature indicator is placed, not achieving minimum sterilization temperature of 121.1°C during sterilization temperature hold period will be considered as cold spot.
·  Steam condensate collected during the sterilization cycle from the sample collection port should meet the requirements for Water for Injection USP.
Load details
*SS Buckets for Disinfectant Solution – 9 Nos.

Procedure

Record the set parameters for the sterilization cycle to be operated during the test for the loaded chamber heat penetration study in the Annexure-1 of Attachement-11.
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 supposed to most difficult points for steam penetration, also place biological indicator strip along with each temperature mapping probe. Record the position of the probes and the biological indicators in a representative schematic form the Annexure-2 of Attachment-11.
Connect the probes to suitable data logger, which can scan and print the actual temperature with respect to time.
Operate the High Pressure High Vacuum Steam Sterilizer as per SOP, and also start the data logger to record actual temperatures within the sterilization chamber with respect to time.

TEMPERATURE SENSOR PLACEMENT IN THE CLEANING AIDS

(NOTE: The temperature sensors shall be placed in the pre determined locations with predetermined sensor numbers corresponding to the data logger channels).
SENSOR NUMBER
LOCATION IN THE CHAMBER
SENSOR NUMBER
LOCATION IN THE CHAMBER
1
Chamber drain
9
In between SS bucket 5&6
2
Aseptic door side middle
10
In between SS bucket 8&9
3
Non-aseptic door side middle
11
Near RTD-1
4
Inside SS bucket  - 1
12
Near RTD-2
5
Inside SS bucket  - 3
13
Near RTD-3
6
Inside SS bucket  - 5
14
Near RTD-4
7
Inside SS bucket  - 7
15
Near RTD-5
8
In between SS bucket 2&3
16
Chamber middle top side
Justification for the temperature sensor location of choice
Sensor no.1-Control sensor of the equipment is located in the condensate drain. This is very important to know the temperature of the drain point to evaluate the cycle set parameters.
Sensor nos. 2, &3 - Any conduction of heat from the doors that may cause temperature drop at that particular point.
 Sensor no.4, 5, 6, & 7- to verify the heat distribution & penetration at innermost portion of stainless steel baskets.
Sensor no 8, 9 & 10- to verify the heat distribution & penetration at outside the baskets.
Sensor nos.11, 12, 13, 14, & 15- As these are temperature-recording probes at different points- it is necessary to verify/compare the temperatures obtained with strip chart of the equipment.
Sensor no.16- to verify the possible excessive heat during the cycle as the top portion of the chamber.
When the sterilization cycle completes;
1.  Collect thermograph from the multipoint temperature recorder of the sterilizer and preserve as Annexure-3 of Attachment-11
2.  Download the data from data logger into the computer for data-analysis and printing. Record the temperature observed at different locations in the Annexure-4 of Attachment-11.
3.  Aseptically collect the exposed biological indicators and send the indicators after wrapping in a sterile enclosure to microbiology lab with necessary identification and the observation sheet as per Annexure-6 of Attachment-11.
If the heat penetration study is acceptable perform three consecutive replicate runs to demonstrate cycle and sterilizer reproducibility.
Compile the data generated during the qualification test for complete evaluation of the system.

3.0  F0 CALCULATION

(a)  Numerical F0 VALUE

The actual observation obtained during the heat penetration studies at different temperature sensing location are compiled in the table and the observed temperature shall be subjected to fo calculation at that particular location the lethality factor calculation is done by using the formula
F0 = dt ∑10(T-121)/z
F0 = dt ∑(sum of lethality factor)
Where,
dt = time interval between successive temperature measurements.
T =observed temperature at that particular time (as actual temperature recorded).
Z  = change in the heat resistance of Geobacillus stearothermophilus
Spores as temperature is changed (10°C or mentioned in COA).

(b)  F0  value for biological indicators

The biological F0 value for biological indicator strip exposed during the sterilization can be calculated as follows.
               F0 = D121 (log A – log B)
               Where,
               D121 = D Value of the biological indicator at 121°C
               A =experimental biological indicator concentration or spore population
               B =desired level of sterility (SAL - 106)

(c)  Desired spore log reduction:

Calculate the desired reduction in spore log population by using the formula
SLR Desired = log A – log SAL desired
Where,
A = experimental population of biological indicator
SAL desired = desired level level of sterility (10-6)

(d)  Actual spore log reduction  :

Calculate the actual reduction in spore population by using the formula
SLR actual  = Fo/D121.
Where,
           Fo = Minimum calculated Fo value
              D121 = D value of Biological indicator
Acceptance Criteria
There should be uniform 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.
Observations and Results
Record the observations and results in formats enclosed as Attachement-11.

3.1 ANALYSIS OF THE STEAM CONDENSATE

Objective

Objective of this test is to ensure that,
The pure steam used for sterilization process should be of the desired quality and the condensate collected at the sterilization chamber drain point should meet the specification of water for injection as per USP.

Procedure

The steam condensate collected during the sterilization cycles performed during Empty Chamber Heat Distribution Study and heat penetration study of different loads should be tested as per water for injection specifications USP.
Record the observations in the Annexure-12 of relevant Attachments.
Compile the data generated during the qualification test for complete evaluation of the system.

Acceptance Criteria

The steam condensate sample collected at the drain point of the sterilization chamber should comply with water for injection specifications as mentioned in USP.

Observations and Results

Record the observations and results in formats enclosed as Annexure-12 of relevant Attachments.

3.2 VACUUM BREAK FILTER INTEGRITY TEST

Objective: 

The objective of the test procedure is to ensure that.
·  The 0.2-micron filter being used as vacuum break filter after completion of sterilization and vacuum cycle to provide sterile air to protect the sterilized load from possibility of contamination which may compromise the sterility of the load.
·  This test confirms the consistent integrity of the 0.2 micron filter which provides sterile air into the sterilizer by determining the Bubble point of the of the filter using Integritest Exacta instrument.

Procedure:

·  Wet the filter to be tested by wetting fluid based on the type of the filter used (hydrophilic/ hydrophobic). In this case wet the filter with 60% Iso Propyl Alcohol Solution
·  Connect a regulated supply of dry clean air or nitrogen at 35-120 psi (2.4-8.5 bar) to the inlet tubing assembly of the instrument with ¼” NPTF side of the filter.
·  Connections to the filter housing are made with the outlet tubing assembly. Two connections are made to instrument.
·  Plug the power cord and switch on to go to editor screen of the instrument (Integritest Exacta)
·  Enter the filter catalog number, appropriate wetting fluid i.e. 60% IPA solution, filter description and the type of the filter.
·  Following the important steps for the bubble point testing.
a)  System check
b)  Clearing the housing from excess fluid.
c)  Determination of the upstream volume.
d)  Pressurizing the system to the diffusion test pressure (80% of the bubble point specification if the diffusion test pressure was not entered into the filter editor)
e)  Allow the pressure to stabilize.
f)  Increment pressure until bubble point is determined.
g)  Increase the pressure to 1 psi below the bubble point specification (Bubble point for the filter Optiseal filter code no- **** is NLT 18 psig).
h)  Increase pressure in various increments above the bubble point values, until the test is complete.
·  Calculate pressure decay 
·  The machine determines the changes in flow by interception of the tangents method. The intersection between the diffusion curve and a linear fit of the flow rates above the bubble point is defined as the bubble point.

Acceptance criteria

The bubble point determined by the above method should be not less than-18 psig.

Observation and results

Record the observation and results as print out in annexure-1 of attachment-12.
REVALIDATION SCHEDULE

3.2 SCHEDULED REVALIDATION

Study
Frequency
Validation
Routine
Calibration of   all instruments e.g. Pressure Gauge, Compound   Gauges, Timer, DTI, Data logger, Probes.
Every Six Month
Not required
Vacuum Hold Test
Every six month three trials
Every day one cycle
Test for the presence of non-condensable gases in pure steam used for steam sterilization
Every Six month three trials
Once in a Month one trial
Bowie Dick Test
Every six month three trials
Once in a week one trial.
Empty-chamber heat distribution studies (3 cycles)
Every six month three trials
Not required
Loaded chamber heat distribution and heat penetration studies.  (3 cycle each with specific loading pattern).
Every six month three trials for each load
Not required

3.3  UN-SCHEDULED REVALIDATION

Revalidation shall be carried out in case of 
·  Major maintenance of critical parts
·  Change of cycle program
·  Inclusion of new load
For unscheduled revalidation Empty chamber and loaded chamber revalidation shall be carried out. The pattern of the study shall be based on case to case basis and properly documented.
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  1. thanks for the explanation.
    shall I know is there any guideline or requirement for the acceptance criteria mentioned here?

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