STANDARD OPERATING PROCEDURE

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
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
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 samplers
initials for each
phase of monitoring to be done.
8.1.8 Perform Air-Viable sampling using the Biotest RCS air sampler per 09-0035SOP-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 090038-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.
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.

Receiving Vessel
(Filtrate)
Watson-Marlow 505Di Pump
with 505LA Pumphead
Bulk Product
Vessel
Filtration Tubing Millipak Filter
Assembly
Room 114K
Class 100 Area
Front
Particle
Counter
Settling Plates
Figure 1. Standard Configuration During Product Filtering (Clinical Antibody, Media
Fill)
Watson-Marlow 505Di Pump
with 505L Pumphead
Filtered
Product
Filling Tubing
Assembly
Filling and Stopper Seating Crimping
Settling Plates

Particle
Counter
Stand
Vial Tray Front
Room 114K
Class 100 Area
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,
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-togown 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 operators 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 30-35C
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 2830C 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 09-0036SOP-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
(09-0065SOP-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 2-8C refrigerator
as retain samples
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
Method Clean Zone
Class Alert Level Action Level
Non-Viable Particulates
(Baseline and Set-up)
1000 None Avg. >1000 Particles 0.5mm/ft3
Avg. >7 Particles 5.0 mm/ft3
Non-Viable Particulates (Setup)
100 None Avg. >100 Particles 0.5 mm/ft3
Non-Viable Particulates
(During Aseptic Processing)
100 >5% samples or two
consecutive samples with
100 Particles 0.5mm/ft3
100 Particles 0.5mm/ft3
for 3 consecutive samples
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.