Performance Qualification of Isolator Systems

1.0 PROCEDURE

1.1 Validation Test Procedure:

The HVAC system will be considered qualified for consistent and reliable performance (Validated) on successful completion of the following tests.

1.1.1  Air velocity measurement studies
1.1.2  Integrity testing of HEPA filters 
1.1.3  Differential pressure monitoring
1.1.4  Non-viable particulate monitoring
1.1.5  Power failure recovery study
1.1.6  Airflow direction
1.1.7  Microbial monitoring
1.1.8  Lighting and sound level monitoring
1.1.9  Temperature and RH monitoring
1.1.10  Bio-decontamination study
1.1.11  Chamber leak test

To qualify the System, above tests 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 the ability of the isolator system to maintain the designed conditions.

1.2 Chamber and Glove Leak Test:

1.2.1 Objective:

1.2.1.1  To demonstrate that the isolator leak rate meets class-IV containment enclosure as per ISO 10648-2:1994(E)
1.2.1.2  To demonstrate that the there is no leakage in the gloves attached to the isolator.
1.2.2  Test Requirements:
1.2.2.1  Calibrated digital thermometer
1.2.2.2  Calibrated thermometer
1.2.2.3  Calibrated Differential pressure gauge
1.2.2.4  Compressed air

1.2.3 Test Procedure:

1.2.3.1  The room temperature and barometric pressure shall be measured during the test with the thermometer and barometer set up close to the isolator.

1.2.3.2  The isolator thermometer shall be suspended in the middle of the enclosure before the final sealing of the openings.

1.2.3.3  Before starting the leak test, the temperature and pressure in the containment enclosure to be tested and the test room shall be allowed to stabilize.

1.2.3.4  Set up the containment enclosure positive pressure to the required value of 250 Pa and then close the extract valve.

1.2.3.5  When the pressure and the temperature are stabilized, isolate the containment enclosure by shutting the valves, and measure the temperature and pressure in the containment enclosure for an hour at 15-minute intervals, together with the ambient pressure.

1.2.3.6  The first and last readings are used for the evaluation; the intermediate readings are used to control the test conditions.

1.2.3.7  During the test (duration 1 h) the following conditions should be fulfilled:

·  Internal enclosure relative pressure Variation shall be lower than 30 % of the initial value.

·  Internal temperature variations shall be lower than + 0.3 °C

·  Atmospheric pressure variations shall be lower than 100 Pa.

If these conditions are not entirely satisfied, the test shall be repeated.

1.2.3.8  The influence of temperature and pressure may be summarized as a Change of 1 °C in internal temperature corresponds to a Change in the internal pressure of 350 Pa.

1.2.3.9  The test report shall include the temperature and pressure measurements at the specified time intervals. The measured values, the calculated leak rate, and the assessment shall be entered in the following formula:

                                                  60            P_nT₁

      Hourly Leak Rate (T_f) = ------X {----------  - 1}

                                                   t              P₁T_n

Where,

        t is the duration of the test, in minutes

        P_l is the absolute pressure at the first reading, in Pascals

        Pn is the absolute pressure at the last reading, in Pascals;

        T_l is the temperature at the first reading, Kelvin

        T_n is the temperature at the last reading, Kelvin

1.2.4  Acceptance Criteria: The Leak tightness of the isolator should comply with the rate of leakage of a class 4 containment enclosure, in accordance with ISO 10648-2.

1.3 Air velocity measurement studies:

1.3.1 Objective:

To demonstrate that the air system is capable to balance and delivering sufficient air volumes to maintain a minimum cross-sectional velocity under the HEPA filter should be 0.45 m/s + 20% measured 6 inches downstream of the HEPA filter of Filling line isolator.

1.3.2 Test Requirements: Calibrated Thermo Anemometer  

      

1.3.3 Procedure:

1.3.3.1  Switch “ON” the Blowers of the isolators; let the blowers run for 15 minutes for stabilization.

1.3.3.2 Measure the Air Velocity of HEPA filter in Feet per Minute for each filter by placing the sensor of the instrument at approximately 6 inches below the grill and 6 inches away from the edge of filter grill. Take 5 readings as shown in the figure and average the 5 readings.

1.3.3.3 Multiply the result by 60 to determine the throughput per hour.

1.3.3.4 If there is more than one filter in the room, add the volume of air (throughput per hour) of each filter.

1.3.3.5 Divide the total air volume delivered (throughput per hour) into the room by room volume to get air changes per hour.

1.3.3.6  Multiply the average velocity by the calibration factor of the instrument and record the readings.

1.3.3.7  Measurement should be taken for a minimum of 15 seconds.

1.3.4 Acceptance Criteria: A minimum cross-sectional velocity under the HEPA filter should be 0.45 m/s + 20% or 90 FPM+ 20%.

1.4 HEPA Filters Integrity Test [DOP (Di-Octyl Phthalate) POA (Poly alfa olefin)]:

1.4.1 Objective: To establish the integrity of the HEPA filters installed in the isolators.

1.4.2 Test Requirements:

1.4.2.1  Calibrated Aerosol photometer
1.4.2.2  Calibrated DOP PAO generator
1.4.2.3  Compressed air/Nitrogen

1.4.3 Procedure:

1.4.3.1  Switch “ON” the Blowers of the isolators. Let the blowers run for 15 minutes for stabilization.
1.4.3.2  Directly test aerosol at the DOP PAO port by giving adequate pressure using nitrogen or compressed air at test pressure not less than 1.5 kg/cm².
1.4.3.3  Switch “ON” Aerosol Photometer and allow for stabilization for five minutes.
1.4.3.4  DOP upstream concentration should be 20 to 80 mg/liter of air. Adjust baseline concentration as 100 % and check for detection in the photometer.
1.4.3.5  Scan the HEPA Filter surfaces and periphery at a traverse rate of not more than approximately 10 ft/min by passing the receptor probe 1 inch from the filter surface, in overlapping strokes for any leakage.
1.4.3.6  Any leakage greater the acceptance criteria observed during scanning shall be identified and recorded.

1.4.4 Acceptance Criteria: During scanning percentage of the DOP PAO penetration shown by photometer should be less than 0.01% through the filter media and should be “zero” through mounting joints.

Also see: HVAC System Validation Tests

1.5 Differential Pressure Monitoring:

1.5.1 Objective: The objective of this test is to show that the differential pressure across the HEPA filters of the chamber, fresh air (BIBO), bleed air (BIBO) and return air (BIBO) of isolators are within designed values.

1.5.2 Test Requirements:

1.5.2.1  Calibrated Magnehelic gauges

1.5.3 Procedure:

1.5.3.1  Switch on the Filling line isolators blowers in process mode. Allow the isolators stabilize at this operating condition for approximately 30 minutes. Verify the set values and record for inlet air speed, the differential pressure of isolator chamber and differential pressure across HEPA filters of fresh air, bleed air and return air BIBO (Bag in bag out).
1.5.3.2  Use installed Magnehelic gauges or either external pressure-measuring device for recording pressures. Monitor the differential pressure across the HEPA filter of isolator chamber, fresh air, bleed air and return air for a minimum of 3 days.
1.5.3.3  Recording of differential pressure shall be done for a minimum of 3 days and observations shall be recorded.

1.5.4 Acceptance Criteria:

1.5.4.1  Filling, lyo and capping isolator pressures should be 70-80, 60-70 and 50-60 respectively.
1.5.4.2  Filtration isolator pressure should be 60-80 Pascal
1.5.4.3  Manufacturing and External washing isolator pressure should be not less than -40 to -60 pascals when compared with an adjacent room.
1.5.4.4  Pressure drop across BIBO filters should be in the range of 50 to 200 Pascals.

1.6 Temperature & Relative Humidity Monitoring:

1.6.1 Objective: The objective of this test is to verify that the isolators can maintain the required temperature and relative humidity as defined in operational instruction.

1.6.2 Test Requirements:

1.6.2.1  Calibrated Datalogger
1.6.2.2  Calibrated temperature and RH transmitters

1.6.3 Procedure:

1.6.3.1  Before starting the study ensure that Air conditioning system of isolators is in continuous operation for at least one hour prior to performing these tests.
1.6.3.2  Ensure that the measuring instrument is in the calibrated state with a valid calibration certificate. Place the temperature and RH transmitters as per the location mentioned in the Appendix-1. 
1.6.3.3  Distribution studies shall be performed at working height level. Monitor the temperature and relative humidity inside the isolators for a minimum of 24 hours with an interval of 5 minutes.
1.6.3.4  Temperature and relative humidity study shall be conducted with extended hour’s production run to establish the maximum heat load during operation.

1.6.4 Acceptance Criteria:

1.6.4.1  Temperature inside the isolators should be not more than 23+ 2 °C.
1.6.4.2  Relative Humidity inside the isolators should be not more than 55 + 5 %.

1.7 Non Viable Particulate Monitoring:

1.7.1 Objective: The objective of this test is to verify that the concentration of airborne particles inside isolators meets the ISO class requirements.

1.7.2 Test requirements: Calibrated non viable particle counter

1.7.3 Procedure:

1.7.3.1  Switch on the isolators in process mode. Verify the set values for inlet air speeds and differential pressure across HEPA filters of all isolators.
1.7.3.2  Allow the isolators stabilize at this operating condition for approximately 30 minutes. Record the actual values of inlet air speeds and differential pressure across HEPA filters of all isolators.
1.7.3.3  Connect the measuring probe with silicon hose to the particle counter. Set the particle counter according to appropriate test equipment operating procedures at different sampling locations mentioned in the attached drawing of Location mentioned in Appendix-2 of Non-Viable Particle Count Monitoring in the isolators at the working height.
1.7.3.4  The air sample volume from each location shall be 1 m³. Run the counter using 0.2-micron purge filter to purge the sampling port. Perform this test during start-up of the test.
1.7.3.5  Connect the probe to the sampling port. Position the sampling probe vertically upward. For operational condition, all the machine should run at optimum speed.

1.7.4 Acceptance criteria:

1.7.4.1  > 0.5 micron particles       - NMT     3520
1.7.4.2  > 5.0 micron particles       - NMT        29 

1.8 Power failure Recovery Study:

1.8.1 Objective: The objective of this test is to verify that the effect of the power failure on the concentration of airborne particles inside isolators.

1.8.2 Test Requirements: Calibrated Non - viable particle counter

1.8.3 Procedure:

1.8.3.1  Ensure that the entire isolators are clean.
1.8.3.2  Ensure that the Non - viable particle counter is in a valid state with valid calibration certificate.
1.8.3.3  Set up the particle counter at the center of the isolator.
1.8.3.4  The sampling rate should be one minute and set the delay time of the particle counter to not more than 10 seconds.
1.8.3.5  Switch ON the isolators.
1.8.3.6  After about 30 minutes start the airborne particle counter in auto mode at rest state.
1.8.3.7  Switch OFF the isolators after about one minute sample to simulate the power failure condition and check the time at which the cumulative count of either 0.5 or 5m particle exceeds the limit.
1.8.3.8  Switch ON the isolators and check the time at which the cumulative count of both 0.5m or 5m particles are almost equal to the initial counts.

1.8.4 Acceptance Criteria:

1.8.4.1  Isolator should maintain the class limits minimum two minutes during the power failure.
1.8.4.2  Time taken to recover almost all equal to initial count should not be more than 5 minutes for all filling line isolators.

1.9 Lighting and Sound level monitoring:

1.9.1 Objective: The objective of this test is to verify that the lighting inside isolators and sound levels from isolators.

1.9.2 Test Requirements:

1.9.2.1  Calibrated Light intensity meter
1.9.2.2  Calibrated sound level meter

1.9.3 Procedure:

1.9.3.1  Ensure that the entire isolators are clean.
1.9.3.2  Switch “ON” the isolators. Let the blowers run for 30 minutes for stabilization.
1.9.3.3  Set the light intensity meter in the range of 0-1999 LUX and take the readings from the workplace.
1.9.3.4  Set the sound level meter weighting in A-level, range in 50~80 and response in Full scale.
1.9.3.5  Take the sound level readings from the distance of one meter and record the details.

1.9.4 Acceptance Criteria:

1.9.4.1  Lighting levels inside the isolators should not less than 500 LUX.
1.9.4.2  Sound levels from the distance of one meter should not more than 90 dB.

1.10 Air Flow Direction (Smoke Test):

1.10.1 Objective:

1.10.1.1  To verify the unidirectional parallel airflow throughout the positive isolators and the capability of the HEPA filter unit to limit the dispersion and turbulence of air so as to maintain ISO - 5 environmental conditions.
1.10.1.2  To verify the negative air flow through the negative isolators so as to maintain the negative pressure inside the isolator.

1.10.2 Test Requirements: Fog generator (Glycerin) and Digital camera

1.10.3 Procedure:

1.10.3.1  Switch “ON” the isolators. Let the blowers run for 30 minutes for stabilization.
1.10.3.2  Hold the smoke generator pointing in the direction of airflow below the HEPA filter.
1.10.3.3  Verify that the path of smoke is parallel to the airflow direction.
1.10.3.4  Take video at each point. Ensure that the installed equipment is cleaned and sanitized immediately after performing the test.

1.10.4 Acceptance Criteria: The Smoke Flow Pattern should coincide with the designed specifications laid down in the isolators manual and should maintain

1.10.4.1  The unidirectional air flow up to the working height
1.10.4.2  The flow from the HEPA to the Exhaust Cut out

1.11 Microbiological Environmental Monitoring:

1.11.1 Objective: The objective of this test is to verify that the isolators can maintain the required cleanliness with respect to microbiological contamination as defined in international Standards.

1.11.2 Test Method: Three types of tests shall be carried out under microbial monitoring i.e. Active Sampling, Passive Sampling and surface monitoring.

1.11.3 Passive Air Sampling (Settle plate method):

1.11.3.1  Switch “ON” the isolators. Let the blowers run for 30 minutes for stabilization. Expose the labeled plates at the designated locations inside the Isolators listed in Appendix-3.
1.11.3.2  Ensure that the surface of the agar is open to the environment by removing the lid of the Petri plate and placing it in the adjacent position.
1.11.3.3  After completion of the exposure period of for 4 hrs and note the time for exposure, close the lid and transfer the closed plate to the stainless steel container. Bring the container back to the Laboratory.
1.11.3.4  Keep one un-exposed plate from a lot of plates carried into the manufacturing areas as negative control and label it accordingly.
1.11.3.5  Incubate all the exposed SCDA plates along with the negative control at 20 -25°C for 3 days, followed by 30-35°C for 2 days.
1.11.3.6  Perform passive sampling under the Filling line isolators daily for a period of 03 days.

1.11.4 Active Air Sampling:

1.11.4.1  Switch “ON” the isolators. Let the blowers run for 30 minutes for stabilization.
1.11.4.2  Keep the air sampler at the designated location, place the labeled plate in the aspirating head and open the Petri plate cover.
1.11.4.3  Take care not to touch the inside of the Petri plate cover and the sieve during this operation.
1.11.4.4   Close the aspirating head with the sieve and start the air sampler to sample 1000 liters (1 m3) of air.
1.11.4.5  Expose the labeled plates at the designated locations inside the Isolators listed.
1.11.4.6  Incubate all the SCDA plates at 20–25°C for 3 days, followed by 30-35°C for 2 days.
1.11.4.7  Perform Active air sampling under the isolators daily for a period of 03 days.

1.11.5 Surface monitoring (Swab method):

1.11.5.1  Switch “ON” the isolators. Let the blowers run for 30 minutes for stabilization.
1.11.5.2  Identify the locations which are to be sampled by swab. 
1.11.5.3  Wet swab by dipping in sterile saline and swab the surface of 25 cm².
1.11.5.4  Transfer the swab into the sterilized saline tubes and transfer to laboratory but do not store exceeding the swab hold time period.
1.11.5.5  Incubate the media Petri plates contains filter after completion of filtration at 20–25°C for 3 days, followed by 30-35°C for 2 days.
1.11.5.6 Perform surface monitoring under the isolators daily for a period of 03 days.

1.11.6  Acceptance Criteria:

Grade	Air Sample (Cfu/m3)	Settle plate (cfu/ Plate)	Surface monitoring (cfu/swab)
A	<1	<1	<1
C	100	50	25

1.12. Bio-Decontamination Study:

1.12.1 Objective:

To demonstrate that sterilization process through vaporized Hydrogen Peroxide to the positive isolators.

1.12.2 Test Requirements:

1.12.2.1  VHP Generator (Fogstar Power Jet)

1.12.2.2  Hydrogen Peroxide (15%) + Peroxyacid (0.2%)

1.12.3 Test Method:

1.12.3.1  The bio-decontamination cycle for Positive Isolators requires using hydrogen peroxide vapor generator. The generator is placed in the fresh air AHU, which is supply to all positive isolators of filtration, filling, lyo and capping.

1.12.3.2  Recirculation of the Hydrogen peroxide vapor through each Isolator module including associated BIBO HEPA filters is achieving by isolator blowers. This control state will continue through gassing dwell phase of the cycle.

1.12.3.3  Ammonia integrity study shall be performed to ensure that the filling line isolators and gloves are leak tight.

1.12.3.4  Prepare the 2% hydrogen peroxide solution by diluting the 15% hydrogen peroxide with cold water for injection.

1.12.3.5  The generator shall be set by adjusting the vapor control knob to maintain the gas concentration at above 5 ppm inside the isolator.

1.12.3.6  Keep the five biological indicators in each location inside the chamber as per mentioned in Appendix-4.

1.12.3.7  Start the generator and record the H₂O₂ concentrations inside the chamber. Monitor and record the physical parameters and isolator component status.

1.12.3.8  BI’s should be removed from the Isolator by hourly basis from each location till completion of five hours.

1.12.3.9  Pass the collected BI’s to Microbiology Laboratory for Incubation at 55°C to 60°C for seven days and observe daily for evidence of growth. Growth is indicated by turbidity (a cloudy appearance); no growth is indicated by the broth remaining clear.

1.12.3.10  BI’s should be collected in a manner that minimizes any risk of cross-contamination between BI’s.

1.12.3.11  After completion of five hours, stop the generator and observe the H₂O₂ concentration inside the chamber and continue the monitoring up to the concentration level below 1 ppm.

1.12.3.12  Perform post microbial monitoring with following techniques during performance qualification studies;

·  Active air sampling (Volumetric air sampling)

·  Passive air sampling (Settle plate method)

·  Surface monitoring (Swab method)

1.12.4 Acceptance Criteria:

1.12.4.1  All Geobacillus stearothermophillus biological indicators having the population of 10⁶ should show no growth during 7 days of incubation.

1.12.4.2  Concentration of hydrogen peroxide should be less than 1 ppm at the end of the cycle.

1.12.4.3  Surface Temperature should be 15 to 30°C.

1.12.4.4  Relative humidity should be 30 to 70%.

Related: Types of Biological Indicators

2.0 RE-QUALIFICATION:

2.1 Un Scheduled Revalidation:

Revalidate the equipment in the following cases:

2.1.1  Change of location.

2.1.2  Break down of Critical parts.

2.2 Scheduled Revalidation:

S.NO.	TESTS TO BE CARRIED OUT	FREQUENCY
1.	Air velocity measurement studies	6 months + 2 weeks
2.	Integrity testing of HEPA filters	6 months + 2 weeks
3.	Differential pressures monitoring	During operation
4.	Temperature and Relative humidity monitoring	During operation
5.	Non-viable particulate monitoring	During operation
6.	Power failure recovery study	Annually + month
7.	Airflow direction	Annually + month
8.	Microbial monitoring	During operation
9.	Bio-Decontamination	6 months + 2 weeks
10.	Chamber and gloves Leak test	6 months + 2 weeks
11.	Lighting and sound levels	Annually + month

3.0 RECORDING:

3.1  Record the observation after the execution of each test procedures in the performance qualification record.

3.2  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.

3.3  Prepare the summary report and submit this for approval and authorization to Validation Core Team.

3.4  Document the discrepancies observed during the Performance qualification of the equipment in performance qualification record.

3.5  When all the discrepancies are satisfactorily resolved or an approved action plan is developed which ensures that the discrepancy will be resolved.

3.6  Training shall be conducted before commencing the validation studies and record the same in training format.

4.0 REFERENCES:

4.1  ISO 10648-2, Classification according to leak tightness and associated checking methods.

4.2  ISO 13408-6, Aseptic processing of health care products-Isolators.

4.3  Area fumigation with Hydrogen peroxide vapor, Steris Corporation, Mentor, Ohio

4.4  VHP cycle development report for a 6 glove isolator, Walker barrier system

Submitted By:

Alluri Sitaramaraju

Estima Pharma Solutions

E-mail - alluri2025@estimapharma.com