Performance Qualification Protocol of Tunnel Sterilizer
Tunnel Sterilizer Description:
Sterilizing and depyrogenating tunnel enables the integration of the process of sterilization and depyrogenation of washed empty vials for sterile, continuous automatic filling in which all steps are performed under Grade A conditions.
The Tunnel is comprised of four zones, i.e. Drying zone, sterilizing zone, cooling zone, and Stabilizing zone.
Drying, cooling, and stabilizing zones are essential once-pass-through Laminar flow units comprising standard features, Pre-filters, motor blower assembly, and HEPA filters.
Extract and exhaust blowers are provided under the conveyor connected to the ambient using a common duct.
Vials are transferred by a stainless steel Wire Loop conveyor through drying, sterilizing, cooling, and stabilizing zones under non-turbulent class ISO 5 fresh air.
Container-controlled partition wall adjustment panels are provided at the entry and exit of the sterilizing zone.
In the feed area air taken from the room is prefiltered and aspirated by a blower through a HEPA filter. A laminar flow of air is sent vertically down onto the containers.
Containers passed nearer to the sterilizing zone are preheated up to 90-110°C by the hot air bleeding out of the sterilizing zone. the air along with water vapor is picked up underneath the conveyor belt by the extract blower and ejected outside.
Dried containers are moved over to the sterilizing zone. The sterilizing zone mainly comprises of resistance heating elements, HEPA filters, and temperature-regulating devices.
Hot filtered air is re-circulated in the sterilization zone, which sweeps the containers from top to bottom.
Differences exist in the hot air temperature and conveyor speed settings depending on the total mass of the glass required to be sterilized and cooled.
Equal pressure drop across the filter enables in creation of an extremely even distribution of air circulation.
This uniformly distributed hot air sterilizes and depyrogenates the containers. In the cooling and stabilization zones, containers are subjected to a Laminar flow of HEPA-filtered air taken from the room.
The airflow is profiled in such a way that the glass temperature as it excited the sterilizing zone would be transitioned at a nearly linear rate.
A stabilizing zone is used as a transition area separating the critical filling area and washing area.
A conveyor system is controlled by the container accumulation at the infeed and the set temperature for the conveyor starts.
SS wire Loop conveyor driven by a gear motor with AC frequency controlled drive and tension roller for maintaining the correct tension of the conveyor belt.
Control and recording systems for the parameters governing the sterilizing process such as air temperature, belt speed, and holding time are standard features.
The sterilizing and depyrogenating tunnel is equipped and controlled with an Allen Bradley PLC and MMI (Man Machine Interface) which in conjunction with RTDs, Infeed / Outfeed Proximity switches, Dot Matrix printers, and frequency converter (Variable speed drive) make the operations fully automatic.
Mode of operation enables one to opt a) Auto mode or b) Maintenance mode.
In the Auto mode, the operation of the tunnel is fully automatic. The tunnel starts and stops at a predetermined time. All blowers are running continuously except sterilizing zone heaters, which cut off when the temperature reaches the set temperature.
In the Maintenance mode, all operations are being done manually.
TABLE OF CONTENTS
| Description |
| Protocol Approval |
| Objective |
| Scope |
| System Description |
| Responsibilities And Identification Of Execution Team |
| Test Procedures |
| Recording Of Observations |
| Discrepancy And Corrective Action Report |
| Compilation, Review And Summary Report |
| Appendix |
| Requalification Criteria |
PROTOCOL APPROVAL
The signing of this approval page of the protocol indicates agreement with the qualification approach described in this document. If any modification in the equipment qualification approach becomes necessary, a revision through change control shall be prepared, checked, and approved. This protocol cannot be executed until approved by the personnel.
OBJECTIVE
The objective of this protocol is to provide a high degree of assurance in the sterilization and depyrogenation process, using a Tunnel sterilizer supplied by _______________________. The sterilizer is used to sterilize/depyrogenate glass vials (both molded and tubular) ranging from 5ml vials to 20ml vials.
Consecutive 3 runs for Heat penetration studies shall be carried out on each type of vial. These heat penetration studies shall include a Bacterial Endotoxin challenge study using Bacterial Endotoxin spiked vials (containing 104 EU/vial). The placement of the vials with the Endotoxin indicator in the vial load under test shall be in such a way that simulation of the worst-case consideration and normal load pattern can be established at a time.
Estimation of the FH value achieved during the sterilization hold period at each temperature-mapping probe. Biological indicators are not used as the intended cycle is for depyrogenation at a very high temperature and survival of the microorganisms is not possible after the depyrogenation cycle.
SCOPE
This protocol applies to the Tunnel sterilizer installed in the vial washing room of Production (sterile) area.
To be performed after the completion and authorization of Operational Qualification.
To be performed at the time of relocation or Requalification.
RESPONSIBILITIES AND IDENTIFICATION OF EXECUTION TEAM
Responsibilities: The group is comprised of representatives from each of the following departments and they shall be responsible for the overall compliance with this protocol.
| Department | Responsibility |
| Production | Execute the validation activity & provide the necessary support |
| Engineering & Utility | Participate & provide the necessary support for the validation activity |
| Quality Control | Testing of samples as per the test procedures |
| Quality Assurance | Monitoring, sampling & reviewing the validation activities. |
Identification of Executors: The personnel involved in the execution of this protocol shall be recorded Name, Designation, Signature, and Date.
TEST PROCEDURES
Test procedures are followed to qualify the equipment for its performance.
Measurement of Airflow Velocity
Tools required: Calibrated Anemometer.
Test procedure:
Ensure that the equipment is ON except for heating before the start of the observations before 30 minutes.
Calculate the number of location by using the following formula. Distribute all the locations equally for each filter outlet.
Number of locations = square root of 10 times of area in square meters but no less than 4, we have considered 4 sampling points for each filter.
The airflow velocity should be measured at approximately 150 mm to 300 mm from the filter face by using a calibrated Anemometer. The measuring time at each location should be also sufficient to ensure a repeatable reading.
Record a minimum of 3 readings in each location and calculate the average measured velocity. Record the calibration details of the instrument used for measurement and attach the calibration certificate.
Acceptance criteria:
For Drying zone: Airflow velocity across the HEPA filter should be within the range of 98 ± 20 feet / minute [0.5± 0.1 m/sec]
For Sterilising zone/Heat zone: Airflow velocity across the HEPA filter should be within the range of 157 ± 20 feet/minute [0.8± 0.1 m/sec].
For Cool zone: Airflow velocity across the HEPA filter should be within the range of 118 ± 20 feet / minute [0.6± 0.1 m/sec].
Differential Pressure Test
Tools required: Calibrated Magnehelic Gauge / Photohelic gauges.
Test procedure: Observe the differential pressure across the zones through the Magnehelic Gauge / Photohelic gauge and note down the reading before operation and during operation. Repeat the procedure for two more times.
Acceptance criteria:
Magnehelic gauge should show the reading at ‘0’ before operation.
During operation,
For Drying Zone: equal to or above2 MMWG.
For Sterilising zone: equal to or above 25 MMWG
For Cool zone: equal to or above 25 MMWG
Installed Filter System Integrity and Leakage Test (DOP/PAO Test)
Tools required: Aerosol generator, Aerosol Photometer (Calibrated), and Dioctyl pthalate or PAO Liquid (Poly Alpha Olefin).
Test procedure:
Operate the Tunnel steriliser as per the SOP.
Position the Aerosol generator and introduce Aerosol into the upstream air, ahead of the HEPA filters, at the concentration of 80-100 mg per litre of air at the filter’s designed airflow rating and set the instrument at 100% concentration.
Scan the downstream side of the filter with probe at scanning rate not to exceed 15cm2/Second. The probe should be held at a maximum distance of 5cm from the down stream filter face. Scanning should be performed over the entire downstream face of each filter, the perimeter of each filter, the seal between the filter frame and the grid structure, including its joints. Measurements of the aerosol upstream of the filters should be repeated at a reasonable time intervals between and after scanning for leaks, to confirm the stability of the challenge aerosol concentration.
Record the observations. Record the details of the instruments used including its calibration status, filter identification number and upstream and downstream concentration of Dioctyl pthalate/ PAO. Attach the calibration certificate of the photometer.
Acceptance criteria: Designated leaks are deemed to have occurred where a reading greater than 10−4 (0.01%) of the upstream challenge aerosol concentration. Alternative acceptance criteria may be agreed based on the filter efficiency.
Airflow Pattern Test
Tools required: Dry Ice.
Test procedure: Take Dry Ice and check for airflow direction in the zones. Observe the direction of the flow and the same shall be recorded.
Acceptance criteria:
The stream of air should be unidirectional and non-turbulent up to the working zone.
Drying zone: airflow towards the vial washing side.
Sterilising zone: airflow towards the drying zone.
Cool zone: airflow towards Cooling zone and airflow from Vial filling side.
Non-Viable Particle Count Test
Tools required: Calibrated Air borne particulate counter.
Test procedure:
Calculation of Measuring Points (Sampling Locations)
Calculate the number of location by using the following formula. Distribute all the locations equally in the clean zone.
Number of locations = Square route of the area in meters
Sampling:
Tunnel sterilizer blowers shall be in continuous operation for at least 30 minutes prior to performing these tests. AHU of the particular room should also be in operation.
Sampling shall be done at rest conditions at pre-determined locations.
The non-viable particulate counts shall be done before and after the de-pyrogenation cycle. If the trails are conducted continuously on the same day, then the particulate count are taken before starting the first trial and after the completion of last trial.
Set the particle counter in ISO mode. Position the sampling probe towards airflow at work level and Sample at least 3 cycles for each location. Each cycle should be Approx. 28 litres/minute.
Paste the printouts generated by the particle counter on A4 page and shall be photocopied.
Verify the printouts for all calculations and acceptance criteria as mentioned below. Attach the photocopies of the same to the annexure –1. Attach the calibration certificate of Particle Counter.
Acceptance criteria:
All the readings of the test should pass the below-mentioned criteria.
The maximum permitted number of particles/m3 equal to or above 0.5μm is 3500 and 5μm is 20.
Heat Distribution Study For Empty Chamber
Tools required: Calibrated Data logger
Test procedure:
Operate the tunnel steriliser as per the SOP.
10 number of temperature sensors shall be passed randomly along with the conveyor from the drying zone. Ensure that the entire conveyor area to be covered during mapping. The placement temperature sensors in empty chamber. The natural progression of sensor on the conveyor that occurs during actual running of the tunnel shall be simulated.
Record the set parameters for the sterilization/Depyrogenation cycle to be operated during the test.
Record the position of temperature sensors in representative schematic form.
Connect the probes to suitable data logger, which can scan and print the actual temperatures with respect to time.
Start the data logger to record the actual temperatures within the sterilization zone with respect to time.
When the sterilization / Depyrogenation cycle completes:
When all the temperature sensors cross the cooling zone, Stop the conveyor belt of sterilizing tunnel, switch off the data logger and pull out the probes.
Collect print out from the printer of the tunnel control panel and attach to the annexure-1 with attachment No.
Download the data from the data logger into the computer for data analysis and printing. Review and attach the printouts with attachment No.
Repeat two more consecutive runs to demonstrate sterilization cycle reproducibility.
Acceptance criteria
There should be uniform temperature distribution across the conveyor belt during the hold period.
The temperature at each temperature sensor should be 325°C ± 250C for NLT 1 Minute during the depyrogenation cycle.
Note: The temperature sensors shall be placed in a straight line across the belt, tied to the SS wire, during heat distribution studies.
Heat Penetration Study For Load Chamber
Tools required: Calibrated Data logger
Test procedure:
Operate the tunnel steriliser as per the SOP.
Empty glass vials (as required) shall be fed to the tunnel sterilizer from the vial washing machine.
10 numbers of Vials spiked with a known amount of Endotoxin (Endotoxin indicator vials) as per SOP and 10 numbers of empty vials with temperature sensors shall be placed in the load as depicted below, to simulate normal sterilization and simulated worst condition in a single load. Both the vials with endotoxin and temperature sensors must be placed side by side.
The placement of Endotoxin indicator vials and temperature sensors in empty vials. The vials shall not be tied with wire to keep them together. The natural progression of the vial on the conveyor that occurs during the actual running of the tunnel shall be simulated.
Record the set parameters for the sterilization cycle to be operated during the test.
Record the position of the vials with temperature sensors and vials with Endotoxin in representative schematic form.
Connect the probes to a suitable data logger, which can scan and print the actual temperatures concerning time.
Start the data logger to record the actual temperatures within the sterilization zone concerning time.
During the validation run inspect the vials in the tunnel sterilizer for the following parameters:
Look through the glass window of the tunnel to the following shall be verified
Position of the vials on the conveyor.
Movement of the vial on the conveyor belt.
To inspect the incoming vials to the in-feed turntable of the powder filling machine.
Breakage / Cracks in vials on sterilization.
Presence of any spot on the sterilized vial surface.
When the sterilization cycle completes:
When all the vials, are attached with temperature indicating probe and endotoxin indicator vials, and vials cross the cooling zone, Stop the conveyor belt of the sterilizing tunnel, switch off the data logger, and pull out the probes.
Collect print out from the printer of the tunnel control panel, review, and attach to annexure-1 with attachment No.
Download the data from the data logger into the computer for data analysis and printing. Review and attach the printouts to annexure-1 with attachment No.
Wrap the exposed endotoxin indicator vials with sterile aluminum foil in the filling room and identify them suitably. Send the exposed vials to the Microbiology lab to estimate the quantity of the residual endotoxin as per the SOP. Record the results.
Repeat two more consecutive runs to demonstrate sterilization cycle reproducibility.
Acceptance criteria
There should be uniform temperature distribution across the conveyor belt during the hold period.
The temperature at each temperature sensor should be within ± 250C during the depyrogenation cycle.
The FH value should be more than the value calculated in the FH value calculation sheet proving the sterility assurance by 12-log reduction. (Using assumed D-value and worst case heat resistant spore population)
The temperature distribution is uniform throughout the sterilization period i.e. 325°C ± 250C for NLT 1.0 min.
Endotoxin-spiked vials exposed to the depyrogenation cycle should show more than a 3-log reduction in the endotoxin content when tested.
The conveyor should be packed with vials.
The vial should move on the conveyor belt in an erect condition. No vial should be tilted.
No breakage of the vial should be observed.
No charring spot should be observed on the surface of the vials.
RECORDING OF OBSERVATIONS
Record the observations after the execution of each test procedure, in the annexure (Recording of Observations For Performance Qualification).
DISCREPANCY AND CORRECTIVE ACTION REPORT
Document any discrepancies observed during the Performance qualification of the equipment in Annexure. Include the corrective actions of the same. When all the discrepancies are satisfactorily resolved or an approved action plan is developed it ensures that the discrepancy will be resolved.
COMPILATION, REVIEW, AND SUMMARY REPORT
Compile and review that all test functions have been completed, reconciled, and attached to this protocol. Verify that the approvals for deviations have been taken and are resolved appropriately to the satisfaction.
Performance Qualification shall be considered acceptable when all the conditions specified in the test procedures have been met.
Prepare the summary report in the annexure (Performance Qualification Report) and submit this for review, approval, and authorization to the Validation Core Team.
APPENDIX
Abbreviations and definitions
| Abbreviation | Definitions |
| PQP | Performance Qualification Protocol |
| SOP | Standard Operating Procedure |
| QA | Quality Assurance |
| QC | Quality Control |
| NA | Not Applicable |
| No. | Number |
| PLC | Programmable Logic Controller |
References
SOP for Qualification of Equipment and Utilities
Validation Master Plan
SOP No. QC224 (Recovery Of Endotoxin From Rubber Stoppers, Vials, Aluminium Containers And Rubber Bungs)
Enclosures
Annexure (Recording Of Observations For Performance Qualification)
Annexure (Performance Qualification Report)
REQUALIFICATION CRITERIA
Requalification criteria are as follows
If any major changes or modifications in the equipment.
Change of cycle program
Inclusion of new load
Placement Of Endotoxin Indicator Vials And Vials With Temperature Sensors On The Conveyor
| Sensor No. | Location | Justification for location selection |
| 1 and 6 | The left side of the conveyor belt. | To verify uniform heat penetration in the vials on the left side of the conveyor belt in the tunnel, which is considered to be the critical area where there may be a possibility of a cold spot because the hot air re-circulation ducts are below the conveyor belt. |
| 4 and 9 | Towards the left side from the middle of the conveyor belt. | To verify uniform heat penetration in the vials across the conveyor belt towards the left side from the middle of the conveyor belt. |
| 2 and 7 | In the middle portion of the conveyor belt. | To verify uniform heat penetration in the vials across the conveyor belt in the middle portion of the conveyor belt. |
| 5 and 10 | Towards the right side from the middle of the conveyor belt. | To verify uniform heat penetration in the vials across the conveyor belt towards the right side from the middle of the conveyor belt. |
| 3 and 8 | The right side of the conveyor | To verify uniform heat penetration in the vials on the right side of the conveyor belt in the tunnel, which is considered to be the critical area where there may be a possibility of a cold spot because the hot air re-circulation ducts are below the conveyor belt. |
Note: For Heat distribution study, only temperature sensors shall be considered.
ANNEXURE -1
Details Of Operational Qualification Report
Report No. /Date Of Authorisation
Identification Of The Executors
| S. No. | Name | Designation & Department | Sign & Date | Training Details |
Comments:
Measurement of Airflow Velocity
Drying Zone:
Name of the Instrument: Anemometer
Calibration certificate No:______________________ Validity: From:__________To:__________
Calculations:
Minimum Number of measuring locations = 4 locations are decided for measuring velocity.
Acceptance Criteria: Airflow velocity across the HEPA filter should be within the range of 98 ± 20% Feet/Minute.
| Filter No. | Filter/ Grill Area (A): | ||||||
| Location | Air velocity in Feet/Minute | Air Flow
F = V x A CFM |
|||||
| Run 1 | Run 2 | Run 3 | Average | Average of all locations | |||
| Location 1 | |||||||
| Location 2 | |||||||
| Location 3 | |||||||
| Location 4 | |||||||
Hot Zone:
Name of the Instrument: Anemometer
Calibration certificate No:______________________ Validity: From:__________To:__________
Calculations:
Minimum Number of measuring locations = 4 locations are decided for measuring velocity.
Acceptance Criteria: Airflow velocity across the HEPA filter should be within the range of 138 ± 20% Feet/Minute.
| Filter No. | Filter/ Grill Area (A): | |||||||
| Location | Air velocity in Feet/Minute | Air Flow
F = V x A CFM |
Checked by Sign / Date | |||||
| Run 1 | Run 2 | Run 3 | Average | Average of all locations | ||||
| Location 1 | ||||||||
| Location 2 | ||||||||
| Location 3 | ||||||||
| Location 4 | ||||||||
| Filter No. | Filter/ Grill Area (A): | |||||||
| Location | Air velocity in Feet/Minute | Air Flow
F = V x A CFM |
Checked by Sign / Date | |||||
| Run 1 | Run 2 | Run 3 | Average | Average of all locations | ||||
| Location 1 | ||||||||
| Location 2 | ||||||||
| Location 3 | ||||||||
| Location 4 | ||||||||
Cool Zone:
Name of the Instrument: Anemometer
Calibration certificate No:______________________ Validity: From:__________To:__________
Calculations:
Minimum Number of measuring locations = 4 locations are decided for measuring velocity.
Acceptance Criteria: Airflow velocity across the HEPA filter should be within the range of 118 ± 20% Feet/Minute.
| Filter No. | Filter/ Grill Area (A): | |||||||
| Location | Air velocity in Feet/Minute | Air Flow
F = V x A CFM |
Checked by Sign / Date | |||||
| Run 1 | Run 2 | Run 3 | Average | Average of all locations | ||||
| Location 1 | ||||||||
| Location 2 | ||||||||
| Location 3 | ||||||||
| Location 4 | ||||||||
| Filter No. | Filter/ Grill Area (A): | |||||||
| Location | Air velocity in Feet/Minute | Air Flow
F = V x A CFM |
Checked by Sign / Date | |||||
| Run 1 | Run 2 | Run 3 | Average | Average of all locations | ||||
| Location 1 | ||||||||
| Location 2 | ||||||||
| Location 3 | ||||||||
| Location 4 | ||||||||
| Filter No. | Filter/ Grill Area (A): | |||||||
| Location | Air velocity in Feet/Minute | Air Flow
F = V x A CFM |
Checked by Sign / Date | |||||
| Run 1 | Run 2 | Run 3 | Average | Average of all locations | ||||
| Location 1 | ||||||||
| Location 2 | ||||||||
| Location 3 | ||||||||
| Location 4 | ||||||||
Comments:
| Differential Pressure Test |
| Photohelic Gauge ID | Calibration Details | From | With respect to | Differential pressure in MMWG | Limit on display
in mmwc |
||||
| Certificate No. | From | To | Run – 1 | Run – 2 | Run – 3 | ||||
| Drying zone | Vial washing side | Equal to or above 21.2 | |||||||
| Hot zone to | Drying zone | Equal to or above 25 | |||||||
| Sterility testing room | Change room 2 | Equal to or above 25 | |||||||
Installed Filter System Integrity and Leakage Test (DOP /PAO Test)
Name of the Instrument: Aerosol Photometer
Calibration Certificate No:______________________Validity: From:__________To:__________
Aerosol used:________________________________
Acceptance Criteria: Designated leaks are deemed to have occurred where a reading greater than 10−4 (0.01%) of the upstream challenge aerosol concentration and should be ‘zero’ through mounting joints. Alternative acceptance criteria may be agreed based on the filter efficiency.
| S. No. | Filter ID No. | Efficiency
% |
Upstream Concentration % | Downstream Concentration % | Mounting joints
% |
| Drying zone | |||||
| 1 | |||||
| Hot zone | |||||
| 1 | |||||
| 2 | |||||
| Cool zone/Stabilizing zone | |||||
| 1 | |||||
| 2 | |||||
| 3 | |||||
Airflow Pattern Test
Chemical Used: Titanium Tetra Chloride or Dry Ice
Acceptance criteria:
Air should flow from the higher-pressure zone to the low-pressure zone.
| Airflow Pattern/Direction | Airflow toward Pre-filter
Pass/Fail |
||
| From (High-pressure zone) | To (Low pressure zone) | Pass / Fail | |
| Drying zone | Vial washing side | ||
| Hot / Sterilising zone | Drying zone | ||
| Cool zone | |||
| Vial filling side | Cool zone | ||
Non-Viable Particle Count Test
Stage: Before / After Depyrogenation cycle
Vial Size:
Zone Name: Drying Zone
Name of the Instrument: Laser-Based Air Borne Particulate Counter.
Calibration Certificate No:___________Validity: From: _________To:_________
Test Conditions: At Rest; Specified Class: ‘A’
Calculations:
Number of locations = 5 locations are decided for sampling
Sample Volume =3 cycles per location
Acceptance limit: Maximum permitted number of particles/m3 equal to or above 0.5μm: 3500; 5μm: 20
Observations from raw data
| S. No. | Checks |
| 1 | Date of testing |
| 2 | Date of completion |
| Checks | Observation (Yes / No) |
| Number of cycles per location is met with the above calculations. | |
| Number of sampling locations is met with the above calculations. | |
| Each cycle of each location met the acceptance criteria | |
| The average of each location is met with the acceptance criteria | |
| The mean of all locations is met with the acceptance criteria | |
| 95% of UCL complies with the acceptance criteria. |
Zone Name: Hot/Sterilising Zone
Name of the Instrument: Laser-Based Air Borne Particulate Counter.
Calibration Certificate No:___________Validity: From: _________To:_________
Test Conditions: At Rest; Specified Class: ‘A’
Calculations:
Number of locations = 7 locations are decided for sampling
Sample Volume = 3 cycles per location
Acceptance limit: Maximum permitted number of particles/m3 equal to or above 0.5μm: 3500; 5μm: 20
Observations from raw data
| S. No. | Checks |
| 1 | Date of testing |
| 2 | Date of completion |
| Checks | Observation (Yes / No) |
| Number of cycles per location is met with the above calculations. | |
| Number of sampling locations is met with the above calculations. | |
| Each cycle of each location met the acceptance criteria | |
| The average of each location is met with the acceptance criteria | |
| The mean of all locations is met with the acceptance criteria | |
| 95% of UCL complies with the acceptance criteria. |
Zone Name: Cool Zone
Name of the Instrument: Laser-Based Air Borne Particulate Counter.
Calibration Certificate No:__________Validity: From: _________To:_________
Test Conditions: At Rest; Specified Class: ‘A’
Calculations:
The number of locations =15 locations are decided for sampling
Sample Volume = 3 cycles per location
Acceptance limit: Maximum permitted number of particles/m3 equal to or above 0.5μm: 3500; 5μm: 20
Observations from raw data
| S. No. | Checks |
| 1 | Date of testing |
| 2 | Date of completion |
| Checks | Observation (Yes / No) |
| Number of cycles per location is met with the above calculations. | |
| Number of sampling locations is met with the above calculations. | |
| Each cycle of each location met with the acceptance criteria | |
| Average of each location is met with the acceptance criteria | |
| Mean of all locations is met with the acceptance criteria |
Heat Distribution Study For Empty Chamber
| DEPYROGENATION CYCLE PARAMETERS | |||
Parameter |
PLC set point |
Parameter |
PLC set point |
| Set point (deg C) | HZ over shoot (deg C) | ||
| Conveyor start temperature (deg C) | Conveyor stop temperature (deg C) | ||
| Conveyor speed (mm/min) | CZ over shoot (deg C) | ||
| Data logging time (min) | Night mode HZ temp (deg C) | ||
| Recipe type | — | ||
Observations from Printouts:
| Checks | Run – 1 | Run – 2 | Run – 3 |
| Date of testing | |||
| Date of completion | |||
| Done by |
| Checks | Observation Yes / No | ||
| Run – 1 | Run – 2 | Run – 3 | |
| The temperature at each temperature sensor should be within ± 250C during the depyrogenation cycle. | |||
| The temperature distribution is uniform throughout the sterilization period i.e. ± 250C to set temperature for at least 3.0 min. | |||
| Conveyor speed (mm/Min) is observed as per the set point throughout the cycle. | |||
| A nonviable particle count has been taken after validation runs and complies with the acceptance criteria. | |||
Heat Penetration Study
| DEPYROGENATION CYCLE PARAMETERS | |||
Parameter |
PLC set point |
Parameter |
PLC set point |
| Set point (deg C) | HZ over shoot (deg C) | ||
| Conveyor start temperature (deg C) | Conveyor stop temperature (deg C) | ||
| Conveyor speed (mm/min) | CZ over shoot (deg C) | ||
| Data logging time (min) | Night mode HZ temp (deg C) | ||
| Recipe type | — | ||
Observations from Printouts:
| Checks | Run – 1 | Run – 2 | Run – 3 |
| Vial type/size | |||
| Date of testing | |||
| Date of completion | |||
| Done by |
| Checks | Observation Yes / No | ||
| Run – 1 | Run – 2 | Run – 3 | |
| The conveyor is packed with vials during the run. | |||
| Is the vial moved on the conveyor belt in erect condition? No vial should be tilted. | |||
| Any breakage of the vial was observed during the run. | |||
| The temperature at each temperature sensor is within ± 250C during the depyrogenation cycle. | |||
| The temperature distribution is uniform throughout the sterilization period i.e. ± 250C to set temperature for at least 3.0 min. | |||
| Conveyor speed (mm/min) is observed as per the set point throughout the cycle. | |||
| Exposed endotoxin indicators are meeting the minimum 3-log reduction | |||
| Non viable particle count has taken after validation runs and complies the acceptance criteria. |
Discrepancy And Corrective Action Report
ANNEXURE -2
Heat Penetration Study For Load Chamber
Spiking of Endotoxin Vials
Vial Type/ Size:
| Endotoxin Indicators | ||||||
| Lot No. | Amount | |||||
| Expiry Date | Reconstitution on | |||||
| LRW Details | ||||||
| LRW Lot No.: | Expiry Date: | |||||
| Date of Opening: | ||||||
| Spiking details | ||||||
| Micropipette ID: | LAF ID: | |||||
| Validation CSE Potency: | Reconstitution: | |||||
| Spiked volume: | Number of Spiked vials: | |||||
| Done By: | ||||||
Recovery of Spiked Endotoxin
Confirmation Of Labelled Claim Sensitivity Of The Lysate
| LRW Details | LAL Details | Control Standard Endotoxin Details | ||||
| LRW Lot No. | LAL Lot No. | Lot No. | ||||
| Expiry Date | Sensitivity | Expiry Date | ||||
| Date of Opening | Date of Reconstitution | Conc. | ||||
| Expiry Date | Reconstituted on | |||||
| Endotoxin Indicators | ||||||
| Lot No. | Amount | |||||
| Expiry Date | Reconstitution on | |||||
Control Standard Series
| Tube No. | LRW ( ml) | Vol of CSE added from Tube | Final CSE Concentration |
| 01 | |||
| 02 | |||
| 03 | |||
| 04 | |||
| 05 | |||
| 06 |
Observation:
Time of Incubation: Time of Observation :
Temp. at Incubation: Temp. at Observation :
| Replicate No. | Observation at different concentrations | End Point | Log 10
End Point |
– ve Control
(0.1 ml LRW + 0.1 ml lysate) |
|||
| 2 λ | λ | λ/2 | λ/4 | ||||
| I | |||||||
| II | |||||||
| Mean of log 10 End Point | |||||||
| Geometric Mean = Antilog 10 of Mean End Point | |||||||
Remarks: The geometric Mean End Point concentration is within the / Not within the ± two-fold of the labeled claim sensitivity of the lysate.
Done By:
RECOVERY OF ENDOTOXIN IN UNEXPOSED SPIKED VIALS
No. of Unexposed vial used for determining the recovery = 01
Endotoxin Concentration Spiked on each vial= 10000 EU /vial
The volume of LRW required for dilution = 4.0 ml
The total concentration in the vial will be 10000/4 =2500 EU/ ml
Dilutions:
| Tube No. | The volume of LRW added | Volume of Endotoxin Concentrations | Final Concentration | Dilution |
| 0.9 ml | 0.1 ml of 2500 EU/ml | 250 EU /ml | 1: 40 | |
| 0.9 ml | 0.1 ml of Tube No.1 | 25 EU / ml | 1: 400 | |
| 0.9 ml | 0.1 ml of Tube No.2 | 2.5 EU / ml | 1: 4000 | |
| 1.8 ml | 0.2 ml of Tube No.3 | 0.25 EU / ml | 1: 40000 | |
| 1.0 ml | 1.0 ml of Tube No.4 | 0.125 EU / ml | 1: 80000 | |
| 1.0 ml | 1.0 ml of Tube No.5 | 0.06 EU / ml | 1: 160000 |
Observation:
Time of Incubation: Time of Observation:
Temp. at Incubation: Temp. at Observation:
| Dilution | Sample | Vol. of Sample | Vol. of Lysate | Result |
| · – ve Control | Test | 100 µl of LRW | 100 µl | |
| Duplicate | 100 µl of LRW | 100 µl | ||
| · 1 : 40 000
|
Test | 100 µl | 100 µl | |
| Duplicate | 100 µl | 100 µl | ||
| · 1 : 80 000
|
Test | 100 µl | 100 µl | |
| Duplicate | 100 µl | 100 µl | ||
| · 1 : 160000
|
Test | 100 µl | 100 µl | |
| Duplicate | 100 µl | 100 µl |
ENDOTOXIN CONTENT IN EXPOSED VIALS AFTER DEPYROGENATION
Concentration of Endotoxin Spiked on each vial = 10000 EU / vial.
Assuming that after depyrogenation a three-log reduction must have taken place, the total concentration on each vial will be = 10 EU / vial.
The volume of LRW used for Dilution = 4.0 ml/vial
Therefore the total concentration in the vial will be = 2.5 EU / ml
Dilutions:
| Tube No. | The volume of LRW added | Volume of Endotoxin Concentrations | Final Concentration | Dilution |
| 1. | 1.8 ml of LRW | 0.2 ml of 2.5 EU / ml | 0.25 EU /ml | 1: 40 |
| 2. | 1.0 ml of LRW | 1.0 ml of 0.25 EU / ml | 0.125 EU /ml | 1: 80 |
| Dilution | Sample | Vol. of Sample | Vol. of Lysate |
| · 1: 40 | Test | 100 µl | 100 µl |
| · 1: 40 | Duplicate | 100 µl | 100 µl |
| · 1: 80 | Test | 100 µl | 100 µl |
| · 1: 80 | Duplicate | 100 µl | 100 µl |
Observation:
Time of Incubation: Time of Observation :
Temp. at Incubation: Temp. at Observation :
| Vial No. | Dilution 1: 40 | Dilution 1: 80 | Vial No. | Dilution 1: 40 | Dilution 1: 80 | ||||
| Test | Duplicate | Test | Duplicate | Test | Duplicate | Test | Duplicate | ||
ESTIMATION OF ENDOTOXIN REDUCTION FOR RUN NO._______
Recovered endotoxin per = Reciprocal of the last dilution which Exposed vials were positive X lambda (Sensitivity of the Lysate).
Last positive Dilution =…………….
Therefore the reciprocal will be =…………….
Sensitivity of the Lysate = 0.125 EU / ml
= ……………. X 0.125 EU/ ml
= ……………….. EU / vial
Recovered endotoxin per = Reciprocal of the last dilution which
The unexposed vial was positive X lambda (Sensitivity of the Lysate).
- Last positive Dilution =…………….
- Therefore the reciprocal will be =…………….
- Sensitivity of the Lysate = 0.125 EU / ml
= ………………. X 0.125 EU/ ml
= ……………….. EU / vial
therefore the amount of endotoxin reduced after depyrogenation
= log (b) – log (a)
= log …………- log ……. ….
= ……………….log reduction
Anti log of (log reduction) = ……………….fold reduction
INTERPRETATION:
There is a greater / less than 3 log reduction in endotoxin in the Depyrogenated vial. Hence depyrogenation cycle was successful / not successful.