SOP On Environment Monitoring (viable organisms) of Aseptic Processing areas
Objective: To lay down the procedure for Environment Monitoring (viable organisms) of Aseptic Processing areas.
Scope: This SOP is applicable for Environment Monitoring (viable organisms) of aseptic processing areas .
Responsibility:
Microbiologist or above of Microbiology Laboratory.
Head – Microbiology section.
Accountability:
Head – Quality Control.
Head – Quality Assurance.
Procedure:
Viable particle monitoring in sterile product manufacturing facility shall be performed by microbiologist, to ensure that the environmental conditions of aseptic area are in compliance with respect to the acceptable levels of total viable count.
Perform the viable particle monitoring in Sterile Filling Areas and other Critical areas by following methods.
Settle Plates Method (Passive air sampling).
Volumetric Air Sampling (Active air sampling by sieve impaction method).
Surface Monitoring by Swabs and Surface contact (RODAC) plates.
Personnel monitoring using Surface contact (RODAC) plates.
Perform the viable particle monitoring adjacent to Sterile Filling Areas by Settling Plates and Volumetric Air Sampling.
Preparation of plates and other accessories
Prepare and sterilize required number of SCDA (Soya bean Casein Digest agar Medium) plates / cassettes and pre-incubate as per SOP on Preparation of sterile media.
Sterilize required number of tubes containing 10 ml of normal saline solution (0.9 % W/V) and a swab and place in the test tube stand.
Sterilize required numbers of SS carriers required to carry plates / cassettes and other accessories to the facility to be monitored.
Place the Sterile pre-incubated media plates / cassettes under LAF working zone.
Disinfect the outer surfaces of the medium plates / cassettes and place them in the sterile SS carriers. Place the swab tubes stands in the carriers.
Carry the plates to the facility to be monitored by following the entry procedure of the sterile product manufacturing facility.
Precaution:
Before start the viable particle monitoring ensures that the HVAC system is in on condition.
To perform viable particle monitoring in Aseptic filling and other critical areas, disinfect outer surfaces of carriers and place them in the dynamic pass box of cooling zone for half an hour under the UV light.
Enter the aseptic processing area through change rooms following the Entry / Exit SOP.
After entering into the aseptic processing area, ensure that the air sampler is fully charged (Note: Air sampler is kept for charging in the aseptic area after every use).
Disinfect the outer surface of SS carrier placed in the dynamic pass box and take inside.
Take out the items from the SS bin and start the monitoring using the procedures mentioned below.
To perform viable particle monitoring in locations adjacent to Sterile Filling Areas, enter the areas as per the respective entry procedure and perform the monitoring.
Settle Plates exposure (Passive air sampling)
Expose the SCDA medium plates in the locations according to the pre-defined locations as per settle plate exposure schematic diagrams mentioned.
Exposure method.
Label each plate on bottom side with location code, sign (initial), date, exposure time and SET-I, SET-II for the first and second exposure of the day respectively and so on.
Bring each plate to the pre-defined locations
Open the lid of plate carefully.
Place the plate containing SCDA medium at the location and place the plate on the settle plate exposure stand.
Place the lid beside this plate internal side down.
Expose the plate for 4 hours.
After completion of exposure time, close the medium plate with lid carefully and mark the plate with closer time on the bottom side.
Place all the plates in SS carrier.
Alert and Action Level for settle plate
| Grade A | Grade B | Grade C | Grade D | |
| Alert level | 1 CFU | 4 CFU | 35 CFU | 0 CFU |
| Action Level | 0 CFU | 5 CFU | 50 CFU | 0 CFU |
Frequency:
The settle plate should be covered the entire filling activity. Make ensure aseptic connection activity shall also be covered during settle plate exposure. If the filling continues for more than four hours from the time of first exposure, the SET-II (second exposure of the same day) should be exposed at all predefined locations and so on.
During working days if there is no activity the plate should be exposed once in a day.
The plate shall be exposed on weekly off / holidays only if any operational activity is going on.
Settle plate exposure for adjacent to sterile filling areas shall be done on weekly basis.
Note: Switch off the UV light of pass box during settle plate exposure.
Volumetric Air Sampling (Active air sampling by sieve impaction method)
Perform the volumetric air sampling at the locations (1000 liters or M³/Location) according to the pre-defined locations of volumetric air sampling schematic diagrams mentioned.
Perform Air sampling with sieve impaction Air Sampler, as per SOP entitled (SOP on Operation, Calibration, Cleaning and Maintenance of Air T Air Sampler).
Disinfect the sieve before and after sampling of every location with 70% IPA.
Perform the volumetric air sampling starting from high clean area location to low clean area location sequentially.
After completion of test label each plate on bottom with location code, sign (initial), date and SET-I and SET-II for the first and second sample of the day respectively and so on.
Place all the plates in SS carriers.
Alert and Action Level for Volumetric Air Sampling
| Grade A | Grade B | Grade C | Grade D | |
| Alert level | 1 CFU | 8 CFU | 70 CFU | 140 CFU |
| Action Level | 1 CFU | 10 CFU | 100 CFU | 200 CFU |
Frequency:
During Filling activities volumetric air sampling should cover all the predefined locations of volumetric air sampling in the SET-I (first Sample of the day). If the filling activity continues for more than four hours from the time of SET-I sampling the SET-II (Second sample of the day) should cover all the Grade ‘A’ and ‘B’ area for the same and so on.
If there is no activity all the locations should be sampled at least once in a week.
Volumetric air sampling for adjacent to aseptic filling areas shall be done on weekly basis.
Surface Monitoring by Swabs and 55 mm Surface contact (RODAC) plates
Perform the surface monitoring in the locations according to the pre-defined locations and surface monitoring schematic diagrams mentioned.
Perform the surface monitoring by following two methods based on the location detailed to be monitored.
Swab method:
- Aseptically open the test tube containing swab and take out the sterile swab partially, press the swab to the internal walls of the tube gently to remove excess saline and take out completely.
- Take Swab 25 cm2 (5 cm x 5cm) area in the direction specified 1.Horizontal Strokes 2. Vertical Strokes using second side of swab after horizontal stroke (5 cmx 5cm)
- Place the swab in the saline containing tube and close.
- After completion of test label each tube with location code, time, sign (initial) and date.
- Perform the sampling in all the locations as per the sampling plan using same procedure.
- Place all the swab tubes and the stands in SS carrier bin.
5.12Surface contact (RODAC) plates method :
- Perform the surface monitoring in flat surfaces using surface contact (RODAC) plates.
- Carefully open the RODAC plate, invert and contact the surface of agar to the surface to be monitored. Press the plate gently to expose the whole surface of agar to the sample surface.
- Slowly take back the plate and close the plate with lid. Take care to not to leave any traces of agar medium to the surface monitored.
- Disinfect the sampled area with 70%IPA and clean with sterile lint free duster.
- After completion of test label each plate on bottom side with location code, time, sign (initial) and date.
- Perform the sampling in all the locations as per the sampling plan using same procedure.
- Place all the plates in SS carriers bin.
Alert and Action Level for Surface swab /Surface contact (RODAC) Sampling
| Grade A | Grade B | Grade C | Grade D | |
| Alert level | 1 CFU | 4 CFU20 | CFU | 40 CFU |
| Action Level | 1 CFU | 5 CFU | 25 CFU | 50 CFU |
Frequency :Perform the contact plate and surface swab sampling after completion of filling Activity.
Personnel Monitoring by Surface contact (RODAC) plates
Perform the personnel monitoring for all the personnel working in the Aseptic filling area (including microbiologist) using 55 mm Surface contact (RODAC) plates.
Perform the personnel monitoring for four locations and it should include both hands. Perform the sampling in any two other locations by rotation for persons regularly working in Aseptic areas.
Sampling:
- Perform the personnel monitoring using Surface contact (RODAC) plate.
- Carefully open the RODAC plate, invert and contact the surface of agar to the surface to be monitored. Press the plate gently to expose the whole surface of agar to the sample surface.
- Slowly take back the plate and close the plate with lid. Take care to not to leave any traces of agar medium to the surface monitored.
- After completion of test, label each plate on bottom side with name of the person location code, time, sign (initial) and date.
- After complete sample collection, place all the plates in SS carriers.
Alert and Action Level for Personnel monitoring Sampling
| Gloves | Garment | ||
| Upper Garments | Booties | ||
| Alert level | 1 CFU | 3 CFU20 | 4 CFU |
| Action Level | 1 CFU | 4 CFU | 5 CFU |
Frequency
Perform the monitoring of the persons before they leave the manufacturing area.
After complete sampling of environment and personnel keep the SS carriers in the dynamic pass box and come out through exit change rooms as per SOP.
Bring all the medium swab tubes / plates / cassette carriers to microbiology for incubation.
Perform the testing of swabs collected in the sterile filling area, in MLT testing room.
First, vortex the solution (saline) contained in each tube along with swab and filters through a sterile 0.45 µm membrane filter and rinses the membrane filter with 100 ml X 3 of 0.1% peptone water and place the membrane filter on pre-incubated SCDA medium plate. Use different filter holder for each swab.
Incubation :Incubate all plates / cassettes of environmental monitoring and personnel monitoring at 20-25 0C for 72 hours and at 30-35 0C for further 48 hours. Observe the plates / cassettes after 72 hours followed by further 48 hours and record the results in respective report given as per Annexure.
Sampling for Anaerobes :Sampling for anaerobes should be done by the microbiologist as predefine location given in table-1 by using the same procedure as mention in different section of this SOP.
| S No. | Type of monitoring | Alert level | Action Level | Frequency |
| 1 | Settle Plate | <1 CFU | 1 CFU | Monthly |
| 2 | Volumetric sampling | <1 CFU | 1 CFU | |
| 3 | Surface monitoring | <1 CFU | 1 CFU | |
| 4 | Personnel monitoring finger dab(Gloves) | <1 CFU | 1 CFU | |
| 5 | Personnel monitoring Upper Garments | <1 CFU | 1 CFU |
Incubation : Incubate all plates / cassettes of environmental monitoring and personnel monitoring at 30-35 0C for 72 hours. Observe the plates / cassettes after 72 hours and record the results in respective report.
Action to be taken when Alert or action level exceed
1.Alert Level (If exceeded) :
The organisms should be identified up to genus level by using Mini API SOP
Check whether the organisms identified are objectionable or not ; if yes than identify the source of the organisms;
The other sample location should be checked for the same;
Check whether the subsequent samples from the same location and other sample locations also exceed Alert level;
The observation should be recorded for future reference;
2.Acton Level (If exceeded) :
The microbiologist should report the deviation to QC Manager, QA Manager, Production Manager and Engineering Manager regarding the excursion;
The product batches should be identified by the microbiologist that is filled during environment sampling period;
The microbiologist should check whether the action level is exceeded in only one sampling location or also in other locations;
The microbiologist should check whether the action level is exceeded in only one sampling location or also in other locations upon subsequent sampling intervals;
The organisms should be identified up to genus level by using Mini API SOP.
Check whether the organisms identified are objectionable or not ; if yes than identify the source of the organisms;
Investigation should be done regarding the source and cause of such high counts;
Check sterilization, handling and use of garments;
Historical trend of the sampling locations under question must be checked;
Check for any change made in sanitation, Sanitization or sterilization procedures;
Check for any untoward incident that may have occurred in the recent past;
Identify corrective action and preventive actions (CAPA) as per SOP.
Implement CAPA and do the follow-up;
Record the investigation and conclude;
Documentation
Record the results details of settle plate exposure for Sterile Filling Areas and other Critical areas in the report..
Record the results details of settle plate exposure for locations adjacent to Sterile Filling Areas in the report given.
Record the results details of Volumetric Air sampling for Sterile Filling Areas and other Critical areas in the report..
Record the results details of Volumetric Air sampling for Adjacent to Sterile Filling Areas in the report.
Record the results details of Surface Monitoring by Swabs and Surface contact (RODAC) plates in Sterile Filling Areas and other Critical areas in the report..
.Record the results details of Personnel Monitoring by Surface contact (RODAC) plates in Sterile Filling Areas and other Critical areas in the report..
Record the results of sampling for anaerobes in the report.
Note: If there is no count observed, the results should be recorded as nil.
Acceptable levels : .The results of monitoring should be within the levels mentioned in the respective annexure.
Rationale of Sampling Location
Settle plate and volumetric air sampling the locations are selected on the following basis:
1) Based on air traffic pattern i.e. Area near the return air raiser.
2) Area near those areas where product is exposed to the environment.
3) Area near the primary packing material.
4) The area which is difficult to clean.
3) Other area which may be hidden or assumed as critical with respect to the microbial contamination.
Surface monitoring locations are selected on the following basis:
1) Product contact surfaces.
2) Frequently in touch with operators.
3) Peripheral surfaces.
4) Difficult to clean area.
5) Aseptic connections.
6) Aseptic area equipment surfaces.
7) Working location which comes to closed to operators.