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STANDARD OPERATING PROCEDURE

Title: Aseptic Fill Environmental Monitoring


Effective Date: _____________

Approvals (Signature and Date):

_______________________________ ____________________________ __________________________


Responsible Department Head Technical Authority QA/QC

1. PURPOSE

1.1 To specify the procedures for environmental monitoring during aseptic filling operations.

1.2 To specify the environmental and personnel monitoring alert and action level specifications for aseptic
filling events and contamination investigation procedures when these levels are exceeded.

2. SCOPE

2.1 This procedure applies to the Aseptic Fill Room 114K during aseptic filling operations.

3. RESPONSIBILITY

3.1 It is the responsibility of the Quality Control and Manufacturing departments to perform environmental
monitoring.

3.2 It is the responsibility of the QC and Manufacturing supervisors to ensure that QC and Manufacturing
personnel performing environmental monitoring have been properly trained in aseptic technique and the use
of all monitoring equipment and procedures.

4. REFERENCES AND APPLICABLE DOCUMENTS

4.1 09-0035-SOP-1.0, Environmental Monitoring Using The Biotest Centrifugal Air Sampler

4.2 09-0036-SOP-1.0, Environmental Monitoring Using RODAC Plates

4.3 09-0037-SOP-1.0, Routine Environmental Monitoring Program in Manufacturing Facility

4.4 09-0038-SOP-1.0, Aerosol Particle Counting Using the MetOne 200L

4.5 Documents used to develop the aseptic fill environmental program include:

4.5.1 Pharmacopial Forum. Page 440. Mar-Apr 1995. Volume 21, Number 2. 1116 Microbiological
Evaluation and Classification of Clean Rooms and Clean Zones.

4.5.2 Federal Standard 209E

4.5.3 02-0031-PVP-1.0, Process Validation of Aseptic Filling of Biotin Conjugated Murine Anti-Human
CD34 Monoclonal (12.8 Biotin) Antibody

4.5.4 Food and Drug Administration “Guideline on Sterile Drug Products Produced by Aseptic
Processing” June 1987

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4.5.5 Barber, Thomas A. 1993. “Pharmaceutical Particulate Matter Analysis and Control”

5. MATERIALS AND EQUIPMENT

5.1 Calibrated Biotest RCS Centrifugal Air Sampler and tripod stand, Biotest Diagnostics

5.2 Tryptic Soy Agar air strips for the Biotest RCS Air Sampler, Biotest Diagnostics

5.3 Rose Bengal Agar air strips for the Biotest RCS Air Sampler, Biotest Diagnostics

5.4 Tryptic Soy Agar + Lecithin + Polysorbate 80 Contact (RODAC) 30cm2 plates, PML Microbiologicals

5.5 Tryptic Soy Agar 15 x 150mm plates, PML Microbiologicals

5.6 Sabouraud Dextrose 15 x 150mm plates, PML Microbiologicals

5.7 Laser Particle Counter capable of detection down to 0.5 micron with tubing and isokinetic probe

5.8 30-35° C Incubator

5.9 28-30° C Incubator

6. HEALTH AND SAFETY CONSIDERATIONS

6.1 Use safety glasses when working with 70% IPA.

6.2 Follow proper gowning procedures when entering aseptic fill room to minimize contamination.

7. DOCUMENTATION REQUIREMENTS

7.1 Aseptic Fill Environmental Checklist: Attachment A

7.2 Aseptic Fill Environmental Monitoring Test Report: Attachments B-H

7.3 Completed Checklists and Reports are to be kept in notebooks in the QC Laboratory. Copies of completed
reports shall be placed into the product lot file.

8. PROCEDURE

8.1 ENVIRONMENTAL MONITORING

8.1.1 Environmental monitoring shall be performed during every aseptic filling event by properly trained
personnel. All monitoring activities and equipment involved shall not disrupt the laminar air flow
nor violate the sterile field in any way. The sterile field consists of the area between exposed
product or containers and the clean air source under laminar flow.

8.1.2 Use of proper aseptic technique is critical to this test. Initial environmental monitoring shall be
performed after the last sanitization of the filling room before operations begin as baseline data of
the filling room conditions prior to commencing operations.

8.1.3 After baseline, aseptic fill monitoring shall be done at set-up, during filtering and filling
operations, and at the end of the event. Set-up is defined as the time when all materials and
supplies involved in aseptic operations (filtering or filling) have been introduced into the class 100
zone, and set-up procedures are being completed before actual operations where product is
exposed. The end of the event is defined as the time when all aseptic operations have been
completed and all vials have been filled and capped prior to clean-up.

8.1.4 For media fills and clinical antibody fills, both filtering and filling operations should be considered
separate aseptic processing events based on 02-0031-PVP-1.0, therefore monitoring should be
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performed according to the Table 1 schedule below. Filtering and filling for other non-clinical
products may be considered a singular event provided all operations occur during a single shift and
do not involve re-introducing materials and supplies into the class 100 zone.

8.1.5 Perform aseptic fill monitoring according to the following schedules in Tables 1 and 2:

Table 1. Aseptic Fill Environmental Monitoring Schedule (Media Fills, Clinical Antibody)
Monitoring Phase Air Site Numbers Surface Site Personnel Responsibility
Viable Non-Viable Numbers Sites
Baseline 1,2 1,2 1-7 QC/Mfg.
Filter Set-up 1,2 1,2,3,5 1-7, 79-81 82-86 QC/Mfg.
During Filter 3,4 4* ** QC/Mfg.
Filter End 87-91 QC/Mfg.
Fill Set-up 1,2 1-7, 79-81 82-86 QC/Mfg.
During Fill 3,4 4* ** QC/Mfg.
Fill End 1,2 1-7 87-91 Mfg.

Table 2. Aseptic Fill Environmental Monitoring Schedule (Non-clinical product)


Monitoring Phase Air Site Numbers Surface Sites Personnel Responsibility
Viable Non-Viable Numbers Sites
Baseline 1,2 1,2 1-7 QC/Mfg.
Set-up 1,2 1,2,3,5 1-7, 79-81 82-86 QC/Mfg.
During 4,5 4* ** QC/Mfg.
End 1,2 1-7 87-91 Mfg.
* Non-Viable particle counting no more than one foot from where vial filling occurs.
** Personnel monitoring only for operator changes, regowning, additional personnel or shift changes.

8.1.6 NOTE: To aid in ensuring that all monitoring is performed for each phase at each site, complete
the Aseptic Fill Monitoring checklist (Attachment A) upon completion of each task.

8.1.7 Label RODAC plates, 15 x 150mm TSA and Sabouraud Dextrose plates, TSA and Rose Bengal air
strips, with date, site number or site identification, sample time, and sampler’s initials for each
phase of monitoring to be done.

8.1.8 Perform Air-Viable sampling using the Biotest RCS air sampler per 09-0035-SOP-1.0 for site
locations 1 and 2 in the aseptic fill room. Additionally, use settling plates (15 x 150 mm Tryptic
Soy Agar and Sabouraud Dextrose Agar for yeasts and molds) as a continuous air viable test in the
class 100 zone during operations. Place one plate on the left and one on the right side of
operations on the bench surface with the exposed media facing up being cautious not to interfere in
the sterile field. Place the first set of plates just prior product exposure processing.

8.1.9 Replace settling plates every hour alternating between Tryptic Soy Agar and Sabouraud Dextrose
Agar until aseptic processing is completed. Record on the settling plate data sheet the site location
where monitoring occurred. (Refer to Aseptic Filling Area Monitoring Site Map)

8.1.10 Perform RODAC surface sampling per 09-0036-SOP-1.0 for site locations 1-7 in the aseptic fill
room. In addition, during event set-up, use RODAC plates to sample the surfaces of the product
container, the filling pump, or other equipment if used. Also collect a sample from packaged
sterilized supplies (i.e. vial trays, caps or stopper containers etc.) that are brought into the class 100
zone. Record on the RODAC data sheet the time and description of sampling locations.

8.1.11 Perform non-viable particulate counting for site locations 1 and 2 per 09-0038-SOP-1.0 during the
baseline and set-up phases. Perform additional counting at set-up for sites 3 and 5 after all
processing equipment has been appropriately disinfected and introduced into the class 100 zone.

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8.1.12 To monitor non-viable particles during aseptic processing, place the sensor on a suitable tripod or
stand. Place the sensor no more than one foot away from the bench top and within 12 inches of
where filtering or filling occurs. This monitoring is represented as non-viable site location 4. Set
the counter to sample continuously by taking at least one sample every five minutes.

8.1.13 Specific placement of airborne viable settling plates and the isokinetic particle counting probe with
tripod stand for media fills and clinical vial filled antibody is given below in figures 1 and 2.
These placements reflect the class 100 zone monitoring procedures that should be employed during
aseptic processing per 02-0031-PVP-1.0, and as specified in MBR 850005-02 (ascites), MBR
850016-02 (TC), and MBR 850022-01 (Tryptic Soy Broth Filling). Ensure that the particle count
sensor is not less than 12 inches from the pump equipment.

Watson-Marlow 505Di Pump Receiving Vessel


with 505LA Pumphead Settling Plates (Filtrate)

Room 114K
Class 100 Area
Front
Particle Bulk Product Filtration Tubing Millipak Filter
Counter Vessel Assembly
Figure 1. Standard Configuration During Product Filtering (Clinical Antibody, Media Fill)

Watson-Marlow 505Di Pump Room 114K


with 505L Pumphead Stand Settling Plates Class 100 Area

Filling and Stopper Seating Crimping

Particle Filtered Filling Tubing Vial Tray Front


Counter Product Assembly
Figure 2, Standard Configuration During Product Filling (Clinical Antibody, Media Fill)

8.2 PERSONNEL MONITORING

8.2.1 Personnel monitoring shall be performed on all manufacturing personnel after entering the filling
room at the set-up phase immediately prior to beginning actual aseptic processing operations in the
class 100 zone. Personnel shall be monitored again just after finishing aseptic operations before
clean-up and exiting. Personnel shall always be monitored in the event of operator changes,
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regowning, or shift changes, after entering and before exiting the aseptic filling room.

8.2.2 Use RODAC plates to sample the chest/collar area preferably at the hood-to-gown joint. The
hood-to-gown seam is the area of the gown that may be touched most by the first set of gloves
during the gowning procedure and therefore may be a general indicator of gowning effectiveness
and technique.

8.2.3 Use RODAC plates to sample operators’ wrist area joint between the glove and gown anterior side
which is the location that is generally in closest proximity to product materials and vials during
processing and pose a greater exposure risk to open containers.

8.2.4 Use RODAC plates for monitoring the operator’s hands. Sample the gloves across the fingers and
finger tips where filling materials and containers are predominantly handled.

8.2.5 Technicians performing monitoring shall also be monitored initially as described above at set-up,
and once more prior to exiting the filling room.

8.2.6 All sampled areas should be thoroughly and carefully wiped clean using 70% IPA and sterile wipes
immediately after sampling before resuming manufacturing or monitoring duties. Avoid excessive
wetting of the Tyvek gown with the disinfectant.

8.3 SAMPLE HANDLING, RECORDING, AND STORAGE

8.3.1 Immediately after all aseptic fill monitoring has been completed, all Tryptic Soy Agar air strips,
RODAC plates, and Tryptic Soy Agar settling plates should be placed in a 30-35°C incubator in
the QC laboratory for 48-120 hours.

8.3.2 Place all Rose Bengal air strips and Sabouraud Dextrose settling plates in a 28-30°C incubator in
the QC Laboratory for 5-7 days.

8.3.3 Manufacturing or QC personnel should record on the environmental monitoring data sheets the
name, part number and lot number of product being filled. Also record the test date, time of
sample, initials of technician performing sampling, and all the test media information including the
name of the manufacturer, lot numbers, expiration dates,

8.3.4 Record the results of non-viable particle counting on the test report attachment and fill out the
Particle Counting Data Sheet Attachment. Photocopy the particle count printouts and paste on the
data sheet.

8.3.5 After the appropriate incubation period, QC microbiology personnel shall remove the media plates
and airstrips and count the number of colonies per 09-0035-SOP-1.0 and 09-0036-SOP-1.0.
Record the count numbers and calculations in the corresponding place on the data sheets.

8.4 CHARACTERIZATION OF MICROORGANISMS

8.4.1 Isolates recovered from from each site and operator during aseptic filling events, including media
fills, require identification of all different types present in terms of Gram stain and colony
morphology (09-0040-SOP-1.0); as well as genus or species identification if possible (09-0065-
SOP-1.0).

8.4.2 In cases of multiple CFUs on a single plate or airstrip where colony type is visually the same, pick
an isolated colony for Gram stain from each quadrant. Identify to the genus and species level if
possible all different types obtained.

8.4.3 If plate results show a lawn of colony growth, pick a representative section and streak for colony
isolation. Perform Gram stain and identification on different organism types.

8.4.4 All original sample isolates shall be labeled and stored in a 2-8°C refrigerator as retain samples
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until: positive identifications are completed; product sterility testing is completed; and, if
applicable, environmental monitoring or sterility test investigations are completed.

8.5 INVESTIGATION PROCEDURES FOR EXCEEDED MONITORING LEVELS

8.5.1 Alert levels are established as recommended in 02-0031-PVR-1.0, and Action levels are referenced
from the Pharmacopial Forum Section 1116 Microbiological Evaluation and Classification of
Clean Rooms and Clean Zones. Alert and Action levels are given in Table 3 below:

Table 3. Aseptic Fill Room 114K Alert and Action Levels by Method and Clean Zone Class
Clean Zone
Method Alert Level Action Level
Class
Non-Viable Particulates 1000 None Avg. >1000 Particles ≥ 0.5µm/ft3
(Baseline and Set-up) Avg. >7 Particles ≥5.0 µm/ft3
Non-Viable Particulates (Set- 100 None Avg. >100 Particles ≥ 0.5 µm/ft3
up)
Non-Viable Particulates 100 >5% samples or two 100 Particles ≥ 0.5µm/ft3
(During Aseptic Processing) consecutive samples with for 3 consecutive samples
100 Particles ≥ 0.5µm/ft3
Surface Viables 1000 >0.5 CFU Average/Plate >10 CFU/Plate
100 >0.1 CFU Average/Plate >3 CFU/Plate
Air Viables - RCS 1000 >0.5 CFU/ ft3 >0.5 CFU/ ft3
Air Viables - Settling Plate 100 >1 CFU/fill >1 CFU/Plate/hour
Personnel 1000 >0.5 CFU Average/Plate >10 CFU/Plate (chest/collar)
100 (All samples) >3 CFU/Plate (hand gloves)
100 >5 CFU/Plate (wrist gown)

8.5.2 If any alert and action level has been exceeded, as soon as possible notify:

• Head of Manufacturing
• Head of QA/QC
• QC Supervisor

8.5.3 For airborne viables, the alert or action level is exceeded if either the TSA or Rose Bengal air
strips yield results that exceed the level. For airborne particulates during baseline and set-up, the
action level is exceeded only if the average count of three exceeds the level, i.e. individual counts
may exceed the action level as long as the average of all counts is below the action level.

8.5.4 If particle count action levels are exceeded during baseline or set-up phases and there are known
assignable causes (i.e. equipment manipulation, disinfectants, or aerosols), then resampling should
be continued at that site for at least three more samples to determine if the environment is still
under controlled conditions. If the resampling results are still out of these specifications with no
assignable causes, then this constitutes an action level failure. The technician performing particle
counting should immediately notify those listed in 8.5.2 and inform manufacturing operators to
suspend duties prior to product exposure pending investigation and retesting as outlined below.

8.5.5 When non-viable particle count alert levels are exceeded in the class 100 zone during aseptic
processing, the particle count instrument will sound an alarm notifying operators that levels have
been exceeded. If possible, operators shall note by description any activities occurring before and
during the time of exceeded levels that may have contributed to the particle counts. This notation
may be referenced in an investigation if applicable.

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