ASEPTIC MEDIA FILL

APPROVAL SIGNATURES

Signing of this Approval page indicate the agreement with the validation approach
described in this document. Should modification be required an addendum shall be
prepared, checked and approved.

Name/designation Signature Date

Prepared by

Microbiology

Checked by

Manager, Production

Asst., Manager, QA

Approved by

DGM QA

CONTENTS
Sr No Index
A. Overview
1 Objective
2 Process Description of Vial Filling Operation
 3 Brief Plan of Media Fill
4 Equipment Description
5 Container closure Systems
6 Revalidation Frequency
7 Responsibility
B. Validation Methodology
1 Pre-validation Checks
2 Environmental Checks during Validation runs
3 Validation with Media
3.1 Sterilization of Lactose powder
3.2 Transfer of sterilized lactose container to critical area
3.3 Suitability test of lactose
3.4 Preparation of Media
3.5 Method of Growth Promotion Test
3.6 Component Preparation
3.7 Media Fill Operation
3.8 Growth Promotion Test of Lactose Filled Vials
3.9 Worst Case Consideration
3.10 Personnel Involvement
3.11 Incubation of Vials and observation
3.12 Destruction of Vials after incubation and observation
C Acceptance Criteria
D Action taken in case of System Simulation Test Failure
E Documentation
F Conclusion
 INTRODUCTION
The sterile powder vial filling process involves the following sub processes.
Washing and sterilization of rubber stopper direct sterilization of RFS rubber
stoppers
Steam sterilization of filling machine parts which directly comes in to contact
with sterile powder (a part from vial & stoppers)
Sterilization of garments, water filtration accessories, etc.
Storage of material intactly under LAF.
Transfer and handling of material in the aseptic area.
Continuous closure integrity.
Human handling and intervention during product processing.
Being a sterile product and each validated sub processes is having its own
limitations. Further the material handling and interventions during processing
cannot be validated directly. Similarly the product produced is only sampled
for microbial testing.
To ensure 100% testing at least on a limited quantity of vials aseptic media
fill in considered to be the best way to evaluate the freeness of the product
from microbial contamination.
A. OVERVIEW
1.0 OBJECTIVE
This protocol is designed to establish sufficient data to assure that the aseptic
powder filling process is adequate and drug product thus produced remains
sterile.
This protocol provides a standard procedure for the validation of aseptic dry
powder filling process environmental condition and practices to confirm its
acceptability in protecting the product from microbial contamination.
2.0 Responsibilities:
2.1 The pharmaceutical manufacturing group will be responsible for the
following:
2.1.1 Conducting the outlined procedure.
2.1.2 Ensuring that requirements outlined in this procedure are met
prior to ending a media fill run.
2.1.3 All events are completely and clearly documented on the
appropriate attachments of this SOP.
2.2 The validations department will be responsible for the following:
2.2.1 Reviewing requirements for all media fills. The requirements
will be documented in attachment 1.2. And 3.
2.2.2 Coordinating each media fill with the manufacturing group.
2.2.3 Submitting samples for growth promotion to microbiology.
2.2.4 Ensuring that the acceptance criteria per this procedure have
been met, and preparing a final report.
2.3 The packaging department is responsible; for the visual inspection of the
media fill vials.
2.3.1 The quality assurance department is responsible for approving
requirements for all media fill prior to fill. The requirements
will be documented in attachment 2.

2.4 The microbiology Department is responsible for:

 2.4.1 Conducting environmental monitoring (EM) per CP-
QC_MICRO-004.
2.4.2 Immediately reviewing the nonviable particle counts and notifying
management if alert and/ or action levels are exceeded
3.0 PROCESS DESCRIPTION FOR VIAL FILLING OPERATION:
3.1 Vial Washing
3.1.1 Approved vials are decartoned in the decartoning room. The
vials are placed into stainless steel boxes and transferred into
vial washing and sterilization room.
3.1.2 The vials are loaded onto the washing machine conveyor.
Washing of vials takes place in washing machine and the
washed vials are delivered into tunnel sterilizer with the aid of
conveyor.
3.1.3 The washed vials enter into the tunnel sterilizer under laminar
airflow protection.
3.1.4 The washed vials enter the tunnel sterilizer for sterilization &
Depyrogenation.
3.2 Vial sterilization & Depyrogenation
3.2.1 The tunnel sterilizer is a continuous belt type dry heat sterilizer
having drying, sterilization, cooling and stabilization zones to
facilitate sterilization and depyrogenation.
3.2.2 The washed vials first enter into the drying zone of the tunnel
sterilizer where the residual water in the washed vial gets
evaporated.
3.2.3 Then the vials enters into the sterilization zone where the vials
gets sterilized and depyrogenated by hot HEPA filtered air
(Class 100).
 3.2.4 Then the vials enter into cooling zone and get cooled by
HEPA filtered air (Class 100).
3.2.5 Finally the vial enters into the stabilization zone where the vials
are further cooled by HEPA filtered air (Class 100) and finally
delivered onto the vial filling machine turntable at a
temperature of 23 to 250C under unidirectional Air Flow zone.
3.3 Rubber stopper sterilization and transfer to filling room
3.3.1 Washed rubber stoppers sterilization.
3.3.1.1 The approved rubber stoppers are washed in rotary plug
washer and siliconised.
3.3.1.2 These rubber stoppers are loaded into the perforated
cassettes of the bung processor under LAF (class 100).
Each cassette is placed in the rotating carriage and the
rotating carriage is loaded into the bung processor and
subjected to steam sterilization and drying.
3.3.1.3 The rubber stoppers after sterilization and drying &
cooling are unloaded into previously sterilized non-
perforated rubber stopper holding canisters under
mobile laminar air flow work station (class 100)
3.3.1.4 These canisters are transferred into the sterile
quarantine area of the respective sterile powder filling
area and are stored under laminar airflow workstation.
3.3.1.5 The rubber stoppers stored in the canisters are
transferred into the filling room via hatch, kept under
LAF and are used at the time of filling.
3.3.2 Ready for sterilization rubber stoppers.
3.3.2.1 The rubber stoppers packed in Ready for sterilization
packs shall also be simulated.
3.4 Raw material transfer to filling area
3.4.1 Approved sterile RM is transferred from stores.
3.4.2 Kept the hatch & disinfected.
 3.4.3 Transfer canisters to API transfer room with LAF & dynamic
pass box.
3.4.4 Disinfect containers, keep in dynamic pass box.
3.4.5 Unload into aseptic area.
3.4.6 Store under LAF.
3.4.7 Transfer using mobile LAF to dynamic pass box opening into
filling area.
3.4.8 Unload into filling room through dynamic pass box and transfer
to filling machine.
3.5 Filling and stoppering
3.5.1 The filling machine is located in the class 100 zone. The
machine is provided with enclosure providing class 100 A
environment on the filling area and 100 for operating the
machine.
3.5.2 The filling machine will be fitted with suitable change parts
like indexing wheels with suitable pistons, powder hoppers and
rubber stopper hopper after steam sterilization and drying.
3.5.3 Filling machine is operated as per the standard operating
procedure.
3.5.4 In process sampling during filling operation is carried out as
per the standard operating procedure
3.6 Sealing
3.6.1 The filled and stoppered vials thus transferred from the filling
room are allowed onto the sealing machine track.
3.6.2 The sealing of the vials carried out as per the standard
operating procedure.
3.7 Filling area
3.7.1 The filling room is class 100 area. The filling & stoppering
machine is protected by an enclosure with doors suitable for
machine operation. The filling zone is class 100A and the other
surrounding area out side the filling zone is class 100B.
 3.7.2 The filling area is constructed with SS panels. The flooring is
painted with self-leveling epoxy painted.
3.7.3 Nitrogen/carbon dioxide and vacuum required for filling are
supplied into sterile area from a pendent through sterilizing
grade filters. A provision of compressed air instead of nitrogen
is provided for media fill operation
3.8 Environmental controls
3.8.1 Temperature of filling area is maintained not more than 250C
3.8.2 Relative Humidity of filling area is maintained not more then
30% 5%.
3.8.3 Pressure differential of the filling room is maintained at 0.5 mm
of water column higher when compared to sterile corridor and a
minimum of 1.5 mm of water column higher compared with
vial sealing and vial washing sterilization zone.
3.8.4 Microbiological monitoring of the filling room is carried out
daily for settling plate, centrifugal air sampler and swab testing.
Personnel working in the area are also monitored for bioburden.
3.8.5 Nonviable particulate counts are taken on daily basis to ensure
the cleanliness as per FED. STD. 209E
3.9 Gas purging if any
3.9.1 The vials are dosed with sterile powder with the aid of sterile
nitrogen. Where as in the case of sterile Ceftazidime and
sodium carbonate mixture filling, the empty vials is purged
with sterile CO2, dosed with sterile CO2 and before stoppering
once again purged with sterile CO2.
3.9.2 For media fill dosing of powder shall be done using
compressed air. Dont use other gases.
3.10 Optical testing of filled vials
 3.10.1 After filling and sealing the vials are subjected to optical testing
to detect for the presence of any bad seals (sealing defects),
surface defects, cracks on the vial surface, presence of any
distinguishable foreign particles in the powder etc., The
rejected vials are collected separately and destroyed. And the
good vials are allowed into the packing hall for labeling and
final packing.

4.0 BRIEF PLAN FOR MEDIA FILL

The vial filling process described above is simulated for media fill trials. Sterile
Lactose Powder is used for filling purpose, which is microbiologically inert.
Primary packaging material like vials, rubber stopper and Equipments for
washing, sterilization, filling and sealing of vials are same as used in routine
operations. At least 9000 Vials are filled with 0.5 gm Lactose powder followed by
5.0 ml sterile Soybean Casein Digest Media vials are stoppered and then sealed.
Different set, of primary packing material, used for vial filling, will be used for
the media fill trials. There will be one media fill trial with one set of primary
packaging material or theory of bracketing will be adopted. During filling, worst
case situations are simulated where possible. Media before and after filling is
checked for positive (Growth Promotion Test) and negative control (Sterility
Test). Filled vials are incubated at 22.5 2.5C for 7 days followed by 32.5 2.5
C for further 7 days.
4.1 Procedure:
4.1.1 To simulate the worst case conditions in the
filling room during media fills, the minimum
number of personnel to participate during the
media fill operations will be:
Filling room Production QA/QC Engineering Total

Class 100 Powder Filling Room 2 1 1 4

4.1.2 Filling and QA/QC operators must remain in the room during
the fill except during changing gloves, goggles, etc.
4.1.3 Operator requirements.
4.1.3.1 Each operator working in a class 100 area will require
to be certified for level I certification. The following are
the minimum requirements for operators to maintain
class 100 Certification.
4.1.3.2 Gowning training per CP-QC-MICRO-016.
4.1.3.3 Gowning certification per CP-QC-MICRO-008.
4.1.3.4 Operators are required to participate in at least one
media fill per year for annual certification (except initial
media fill).
4.1.3.5 Operators trained to perform setup of fill line must also
p0erform setup of fill line for media fill.
4.1.3.6 The amount of time required for everyone to be in the
class 100 rooms is 2 hours minimum.
4.1.3.7 All the employees entering the filling room will require
to be monitored per CP-QC-MICRO-004.
4.1.3.8 Contrary to the regular fills, vials with high and low
fills, particles, cosmetic defects, etc., will be included as
part of media fills. Vials will not be discarded unless
they are cracked, broken, have no cap or no stopper, or
not properly filled (no media/no water). The vials are
also rejected if the stoppers and caps are improperly
applied and the contents of the vials have exposed to the
non-sterile environment.
4.1.3.9 The duration of routine media fill runs will be the time
required to fill minimum of 5000 vials at the slowest
speed (20-25units per minute for all sizes of vials).
4.1.3.10 The inspection log sheet (Attachment 10) in the
batch record will list the vial and box number of media
filled vials.
4.1.3.11 Sanitize the fill line surfaces such as conveyor
belt, and the other machine parts that are not subject to
autoclave, by wiping with 70% Sterile Isopropyl
Alcohol.
4.1.3.12 Growth promotion samples will be pulled
randomly during the process simulation. After
inoculation, the samples will be incubated in parallel
with the process simulation units.
 4.1.3.13 Following are the reasons to invalidate the
media fill:
4.1.3.14 Failure of growth promotion test of media.
4.1.3.15 Power outage for more than 3 hours.
4.1.3.16 After the initial qualification, one (1) media fill
will performed twice a year. This stipulates that there
were no changes in normal production procedures and
no action limits were exceeded.
4.1.3.17 FILLING LINE (Refer to Attachment 1,2 or 3
for media fill requirements.
4.1.3.18 Setup the filling equipment and components per
Aurobindo Pharma standard operating procedures for
filling operations.
4.1.3.19 The sterilized filling equipments and
components can be held in the class 100 area for no
more than three (3) days prior to fill date.
4.1.3.20 The media fill start time, break period start and
end time, and media fill completion time will be logged
in Attachment2.
Justification for using sterile Lactose to simulate the vial filling process
1. Flow ability of Lactose through hopper and dosing wheel is good.
2. It does not inhibit the growth of microorganisms at the selected concentration
(approximately 10% w/v).
3. Lactose is soluble in media and water.
4. Could be sterilized by gamma radiation.
Reference for usage of Lactose as placebo materials are also available with process
simulation testing for aseptically filled products Vol. 50 No 6, Nov-Dec 1996,
supplement Appendix 1 Page 5.13 which is based on PDA guideline Technical
monograph no 2 validation of aseptic filling for solution drug products 1980 and
Technical Report no. 6, validation of aseptic drug powder filling process 1984
5.0 EQUIPMENT
Following equipment are utilized during media fill trials
Name of the equipment Tag Nos.
Linear vial washer VWD001
Bung processor BWD001
Tunnel sterilizer TSD001
Sterile powder Filling machine VFD001
Vial sealing machine SMD001
Optical testing machine OTD001
6.0 CONTAINER CLOSURE SYSTEMS
Vial Stopper Seal
5 ml Molded 20mm Grey butyl 20mm Flip off
20mm Bromo butyl 20mm Tear of
7.5 ml Molded 20mm Grey butyl 20mm Flip off
20mm Bromo butyl 20mm Tear of
10 ml molded 20mm Grey butyl 20mm Flip off
20mm Grey Bromo butyl ready for 20mm Tear off
sterilization
10 ml tubular 20mm Bromo butyl
20mm Grey Bromo butyl ready for 20mm Flip off
sterilization
15 ml molded 20mm Grey butyl 20 mm Flip off
20mm Bromo butyl
15ml Tubular 20mm Bromo butyl
20mm Grey Bromo butyl ready for 20mm Flip off
sterilization
20 ml molded 20mm Grey butyl 20mm Flip off
20mm Bromo butyl
20 ml tubular 20mm Bromo butyl
20mm Grey Bromo butyl ready for 20mm Flip off
sterilization
30 ml tubular 20mm Bromo butyl
20mm Grey Bromo butyl ready for 20mm Flip off
sterilization
50 ml Molded 32mm Bromo butyl
32mm Grey Bromo butyl ready for 32mm Flip off
sterilization
100 ml Molded 32mm Bromo butyl
32mm Grey Bromo butyl ready fir 32mm Flip off
sterilization bags
For Media fill purpose-bracketing approach shall be adopted. Trials shall be taken
with different packing system and all packing system shall be simulated over a year.
In case if any new container closure system / type in included (a part from those
described above) shall be simulated before commercial production of that particular
size.
1st half Year :

Trial Vial Stopper Seal Minimum No

of vials for fill
simulation
1st 5 ml Molded 20mm Grey butyl 20mm Flip off 4500
20mm Bromo butyl 20mm Tear off 4500
2nd 10 ml Molded 20mm Grey butyl 4500
20mm Grey Bromo butyl 20mm Flip off 4500
ready for sterilization
3rd 15 ml Molded 20mm Grey butyl 20mm Flip off 4500
20mm Bromo butyl 4500
4th 15 ml Tubular 20mm Bromo butyl 4500
20mm Grey Bromo butyl 20mm Flip off 4500
ready for sterilization
5th 30ml Tubular 20mm Bromo butyl 4500
20mm Grey Bromo butyl 20mm Flip off 4500
ready for sterilization
2nd half of Year:
Trial Vial Stopper Seal Minimum No
of vials for fill
simulation
1st 7.5 ml Molded 20mm Grey butyl 20mm Flip off 4500
20mm Bromo butyl 20mm Tear off 4500
10 ml Tubular 20mm Bromo butyl 4500
2nd 20mm Grey Bromo butyl 20mm Flip off 4500
ready for sterilization
3rd 20 ml Molded 20mm Grey butyl 20mm Flip off 4500
20mm Bromo butyl 4500
20 ml Tubular 20mm Bromo butyl 4500
th
4 20mm Grey Bromo butyl 20mm Flip off 4500
ready for sterilization
5th 50 ml Molded 32mm Bromo butyl 4500
32mm Grey Bromo butyl 32mm Flip off 4500
ready for sterilization
7.0 REVALIDATION FREQUENCY
Scheduled Revalidation:

Three Media fill trials to be carried out once in six month with one trial with each
type of container closure system.
Unscheduled Revalidation:
Revalidation are also carried out in some special cases:
a. After maintenance of major breakdown in Filling Machine, Sealing Machine and
HVAC system
b. Change in the Vial Filling Machine and Sealing machine itself or their critical
parts
8.0 RESPONSIBILITIES
Validation team
Validation team consist of the key personnel from the following department
Production
QC
QA
Engineering
General responsibly of validation team
Monitoring of protocol completeness, accuracy, technical excellence and
applicability.
Scheduling of validation.
Conducting of validation runs.
Data compilation and review.
Validation reports preparation and recommendation thereafter (if required).
Schedule of revalidation.
The Engineering Department shall be responsible for the following.
Carrying out the repairs/modifications (if required) prior to validation runs.
The responsibility of QC - Microbiology Lab shall be as follows:
Preparation and qualification of the microbiological growth support medium.
 Carrying out the monitoring (settling plate, air sampling, swab testing and
personnel monitoring) in critical area during media fill trials.
Inoculation and incubation of empty vials and stoppers, sterility test of lactose
used for media fill trials.
Incubation of the remaining media to confirm its sterility.
Observation of incubated filled vials after 7 and 14 days.
To carry out investigations in case the media fill trial fails.
Detecting the organism in the vials showing microbial growth.
Responsibilities of Production Department are:
Procuring sterile Lactose powder (gamma radiated).
Providing the machine when agreed upon with the validation group.
Providing machine operators and supervisory staff.
Sterilization of media, empty vials, rubber stoppers and drying of rubber stoppers.
Providing samples wherever necessary.
Carrying out the aseptic filling process.
Checking the damaged vials after filling and sealing.
Incubation and ensuring security of media filled vials.
Cleaning and sanitization of area and equipments after media fill.
Destruction of media filled vials after evaluation and authorizations from Q.A.
9.0 B. VALIDATION METHODOLOGY
9.1 PRE-VALIDATION CHECKS
Prior to taking up the validation of aseptic filling, it should be ensured that all
equipments, utilities and processes are validated and all instruments are
calibrated.
It should be ensured that the aseptic filling room is at a positive pressure with
respect to the surrounding areas.
Particulate count for non-viable particles (static conditions) should be within
acceptable limits of environment control sop.
 9.2 CHECKS DURING THE VALIDATION RUNS
9.2.1 Personnel Monitoring:
Ensure that personnel entering the sterile area have followed the gowning and
entry procedures as per standard Operating Procedure and have been qualified
as per standard Operating Procedure. Personnel should undergo, monitoring
by RODAC plate method after completion of the trial and before leaving the
sterile area for monitoring bio-burdens levels.
9.2.2 Environmental Monitoring
Environmental Monitoring of the sterile area should be done as per standard
Operating Procedure
Swab test for surfaces like walls/floor etc. of the filling room and surfaces of
the vial-filling machine.
Monitoring of microbial counts in air shall be done by air sampling by settling plate
method.

Monitor the environmental conditions for temperature, differential pressure,

relative humidity and Non-viable airborne particle counts.
The critical area should meet the specifications of class 10,000 clean room and the
area under vertical laminar air flow unit should meet the specifications of class 100
clean room as per the Federal Standards 209E.
Environmental and personnel monitoring result should comply the acceptance limit as
written in Standard Operating Procedure for Environmental Monitoring
9.3 VALIDATION WITH MEDIA
At least 9000 vials shall be filled for each trial run in duration of 8 hours (an
additional step of filling media in the empty vials reduces the normal operating speed)
to simulate the conditions.
9.4 Sterilization of Lactose powder:
Lactose powder (endotoxin free) shall be packed in Aluminum foil pouches (5 Kg per
pack) wrap these packs in double polybags. These packs shall be packed in the
shippers as specified by ISOMED. Each shipper is filled with two packs of 5kg
Lactose and a 50 gm sample pouch is placed below the pouches for sterility testing
and growth promotion test of the Lactose powder after sterilization. These shippers
are then sent for sterilization by gamma radiation at ISOMED (BARC), Mumbai.
9.5 Transfer of Sterilized Lactose packs into critical area
On receipt of lactose packs after sterilization, the pack shall be vacuum cleaned. The
outer carton and polybag shall be removed in the decartoning room. The shippers are
opened and sample packs of the Lactose are collected and tested for sterility and
growth promotion. The container shall be sanitized as per standard Operating
Procedure and shall be transferred to sterile quarantine. The packs shall be stored in
sterile quarantine under LAF unit. Disinfect the packs as per SOP and transfer to
filling room prior to vial filling operation.
9.6 Suitability Test of lactose
a. Dissolve 500 mg sterile Lactose in 5 ml of Soybean Casein Digest
media in a test tube to check solubility of Lactose in media.
b. Establish the concentration lactose, which should be non-inhibitory.
Microbiology Department shall maintain the record of suitability study
9.7 Preparation of Media :
Dissolve 600.0 gm of SCDM powder in 20 L. of WFI (DW) in SS carboys and
distribute in three carboy i.e. approximately 6.5L liters/carboy (Concentration of
media 30 gm SCDM/lit). Close the Carboys. Sterilize the carboys at validated
sterilization parameter. Unload the media in critical area. Transfer the Media Carboy
to Powder Filling Room as per SOP. Remove 800 ml media aseptically from each
trial for Growth Promotion Test.
Note :
A) Store the sterilized carboys filled with media under LAF till it is used. The media
preparation and sterilization will be done in presence of Microbiologist.
B) Sterilized media should be used for media fill trial only after it passes in GPT and
shows no contamination when stored for 7 days at room temperature.
9.8 Method of Growth Promotion test
Objective of the test: To ensure that media used for the sterility test is capable
of microbial growth promotion
 a. Aseptically withdraw 800 ml of autoclaved media and aseptically
transfer 100 ml each in 8 sterilized tubes.
b. Out of the 8 tubes, 2 tubes are inoculated with about 10 100 cells of
Candida albicans ATCC 10231, 2 tubes with about 10 100 cells of
Bacillus subtillis ATCC 6633 and 2 tubes with about 10 100 cells of
Aspergillus niger ATCC 16404. The Inoculum for growth promotion
test are prepared as per the SOP.
c. Incubate the tubes inoculated with Bacillus subtillis at 32.5 2.5C for
maximum 3 days and incubate the tubes inoculated with Candida
albicans and Aspergillus Niger at 22.5 2.5C for maximum 5 days.
d. Incubate 2 tubes (Un-inoculated) of SCDM at 22.5 2.5C for 7 days
followed by 7 days at 32.5 2.5C as Negative Control.
Acceptance Criteria: Media inoculated with microorganism should promote
growth of microbes and un-inoculated media should remain sterile.
9.9 Component Preparation
Components i.e. Vials, machine parts are washed. Rubber Stoppers are sterilized,
dried and transferred to vial filling room. Following SOPs are applicable.

Operation SOP No
Washing of Rubber stopper
Steam Sterilization and Drying of Rubber stopper
Cleaning of vial filling machine parts
Steam Sterilization of Filling Machine Parts
Washing of Vials
Tunnel Sterilization of Vials

9.10 Media Fill Operations

a. Obtain the line clearance before starting filling operation of the batch
as per Standard Operating Procedure for any left over Raw Material,
Vials, Rubber stoppers of previous batch.
b. Check the area and machine for cleanliness.
c. Check the temperature, Differential Pressure and Relative Humidity of
Powder filling Room.
d. Transfer carboy-containing media to filling room. Connect volumetric
liquid filling assembly with the carboy
e. Transfer the sterilized rubber stopper, Lactose packs and machine parts
to powder filling room and assemble the machine parts
f. Check and ensure that all media filled carboy containers are free from
microbial growth / turbidity.
g. Take 10 vials in a sterile conical flask containing 500 ml SCD media.
Incubate the media at 22.5 2.5C for 7 days followed by further 7
days at 32.5 2.5C. Check for any microbial growth in the flasks.
h. Adjust the powder dosing to deliver 0.5 g of Lactose /vial.
i. Adjust the liquid filling machine to deliver 5.0 ml media in each vial.
j. Run the machine to fill only media for checking of volume variation.
Approximately 50 vials may require for volume adjustment and
checking. These vials will be sealed and kept separately for
investigation purpose if required in case of failure.
k. Run the machine to fill only Lactose for checking of weight variation.
Approximately 200 vials may require for weight adjustment and
checking. These vials will be sealed and kept separately for
investigation purpose if required in case of failure.
l. Record three consecutive set of observations of weight and volume
checking from all filling station
m. Operate the Filling Machine and Turn Tables to fill Lactose followed
by liquid media in Vials
n. Seal filled and stoppered vials
o. Following sequence will be followed for normal operation:
p. Vials from Tunnel Outlet TurnTable sterilized Lactose powder
Media fill Stoppering Sealing Visual inspection Invert the
vials Incubation.
q. Collect these media and Lactose filled, stoppered and sealed vials in SS
trays identify the trays with number and time of filling. Invert the vial
 so that the media comes in contact with all the inner surfaces of vial
and stoppers.
r. Sample 30 vials for growth promotion test.
s. Fill at least 5000 vials excluding weight/volume variation check vials
with one set of container closure system (10000 vials per trial). After
completion of filling obtain line clearance from In Process QA
chemist.
t. Operate the machine at minimum, maximum and normal production
speed.
u. Requirement of Raw material and packing material:
i. Sterile lactose: 5.1 Kg
ii. Vials: 10200
iii. Stopper: 10200
iv. Seal: 10200
9.11 Growth Promotion Test of Lactose Filled Vials
Objective of the Test: To ensure that lactose filled in the sterile glass vials
and sealed by rubber stopper and aluminum seal, does not inhibit the growth
of microorganism
a. Collect 30 filled and sealed vials
b. 5 vials are inoculated with about 10 - 100 cells of Candida albicans
ATCC 10231, 5 vials are inoculated with about 10 - 100 cells of
Bacillus subtillis ATCC 6633, 5 vials are inoculated with about 10 -
100 cells of Aspergillus niger ATCC 16404 and five vials each of 3
environmental isolates are inoculated with about 10-100 cells. The
innoculum for growth promotion test are prepared
c. Incubate the vials inoculated with Bacillus subtillis at 32.5 2.5C for
maximum 3 days incubate the vials inoculated with Candida albicans
and Aspergillus niger at 22.5 2.5C for maximum 5 days and
incubate the environmental isolates as per their respective growth
conditions.
 Acceptance Criteria: Media should promote the growth within the stipulated
period.
9.12 Worst case consideration:
Following conditions will be simulated to have worst case condition during the
trials (indicated against the step):
1 To conduct media fill trial simulating change of personnel, tea break, lunch
break etc.
2 Deliberately switch off the HVAC system and LAFs in critical area ( Cooling
& Filling Zone) for 5 minutes in any one of the trials
3 Simulate the interlocking failure by switch off the interlocking system for 30
minutes during filling operation (in any one of trials).
4 Ask one maintenance person (qualified to enter in sterile area) to enter into
filling zone with his toolbox and to stay for 4 Hrs.
5 One microbiologist to enter the area for monitoring purpose during media fill
trials.
6 To extend the filling time from 8 Hrs to 16 Hrs in any one of the three trials.
7 Intervene the filling operation by simulating the following operation
a) Adjustment of the fill weight
b) Charging and recharging of the Lactose in hopper
c) Charging and recharging of rubber stopper in hopper
d) Adjustment of the sensors if required or touch the sensors
e) Set the rubber stopper chute
f) Setting of Nitrogen dosing and setting or Carbon Dioxide Purging (if
applicable)
g) Adjustment of roller for pressing of stopper
h) Removal of choked piston
i) Removal of dropper filled and unfilled vials from turntable
j) Cleaning of conveyor on dropping of filled vials on conveyor
(deliberately simulate the operation by stopping the filling operation
and pouring the content of vial on the conveyor
 k) Open the entry door of powder filling room so that, differential
pressure of the room goes below standard differential pressure
l) Stop the sealing machine and sterilizing tunnel when the turntable of
filled and unfilled vials is full. Stop the filling operation. Let the
turntable rotate for 30 minutes
m) Ask maintenance person to simulate minor maintenance job.
9.13 INTERVENTIONS
9.14 The following interventions will be performed throughout the process
of the fill. All interventions must be completed prior to the end of the fill.
9.14.1 Change the count on the dosing wheel to adjust the weight of
the powder.
9.14.2 Adjust sensors.
9.14.3 Adjust the stopper arm for height.
9.14.4 Remove the broken glass vials from the fill line.
9.14.5 Perform a clean up of a powder spill using a vacuum cleaner.
9.14.6 Open safety panels on the filling machine. Keep the panels
open for 10 minutes during the machine stoppage.
9.14.7 Hold the filling room door open for 60 seconds.
9.14.8 Shut the filling line down for a minimum of 1 hour, but do not
clear the in-feed table. This operation can be coordinated at the
break time.
9.14.9 Open the swing-open conveyor to get behind the machine and
spend 5 minutes simulating checking the pressures on the
Magnehelic Gauges.
9.14.10 Cause the conveyor jam at in feed and out feed and
unjam the conveyor.
9.14.11Cause the vial jam and unjam at the star wheel.
9.14.12 Record actual or unexpected interventions(s) on
9.14.13 Attachment 2,3 or 4.
9.15 BREAK PERIOD
9.15.1 The machines will be stopped during the break period will be
of 1 hour duration. This time includes time needed for
gowning, degowning and actual break time. The interventions
outlined in section 5.13.3 may be timed properly to coincide
with the scheduled break. Any operational malfunctions
experienced before the simulation can be considered to satisfy
the simulation requirements. When logging the events, indicate
if the intervention was actual or simulated.
9.15.2 Run the machine in the slowest possible production speed to
simulate the worst-case condition. Maintain the same speed
(20-25 vials per minute) through out the fill period. Log the
actual line speed setting used at this time.
9.15.3 Vials will be removed from the fill line if the following occur:
9.15.3.1 Vials with no stoppers or defective stopper.
9.15.3.2 Vials with no powder.
9.15.3.3 Vials with no water.
9.15.3.4 Tipped vials.
9.15.3.5 Vials with no seals or defective seals.
9.15.3.6 The vials removed from the fill line inside the
clean rooms will be discarded.
 9.15.4 After the sealing operation, shake the vials to facilitate the
dissolu8tion of the media in water. Shake or vortex till a clear
solution is obtained. Invert each vial to let the media solution
come in contact with all sides of the vial including the stopper.
Examine each vial for clarity, glass defects, proper placements
of stopper and seal. Reject the vials found defective. Number
the inspected vials with marking pen with sequential numbers
starting from one (1) after inspection and arrange the vials in
boxes. Label the boxes with lot number, number of vials inside
the box, fill date and sequential vial numbers from beginning to
end contained in the box. Deliver the boxes to Microbiology
laboratory for incubation.
9.15.5 After each fill, if necessary. Remove dosing wheel, water
pump, and water carboy and stopper bowl and sterilize for the
next usage.
9.15.6 The media filled containers will be incubated for two 7-day
periods at 20-25oC.
9.15.7 Visual inspection will be performed during incubation period
on the last day of each 7-day period. Inspection will be
performed by QA/QC personnel and will be recorded at
Attachment 1 on CP-QC-MICRO-021.
9.15.8 For all media fills, refer to Attachment 1,2 or 3 for
validation/QA requirements.
9.16 Personnel Involvement:
During Media filling operation minimum 04 and maximum 06 number of
persons will be present in side the filling room. All persons involved in routine
aseptic operation should be included in any one of the following critical aseptic
operations in the trial.
a. Unloading of stopper from steam Sterilizer
b. Unloading of machine parts from steam Sterilizer
c. Charging of RM and rubber stoppers into hopper
 d. Vial filling machine assembling and setting
e. Vial filling operation.
All person involved in routine filling operation should involve in media filling
operation over a period of 1 year
One Microbiologist (QC), qualified to enter into critical area, will enter into
critical area for area monitoring, personnel monitoring and sampling. All
microbiologists involved in sampling and environmental monitoring during
normal manufacturing periods will take part in system simulation run over a
period of one year.
The maintenance personnel, qualified to enter into critical area, shall be required to
enter the critical area and to remain in the area for at least 4 hrs. Duration with the tool
box and will be monitored for personnel counts in all the trial runs. (The maintenance
person will follow the entry & exit procedures as per normal production runs, but will
not interfere in the proceedings during the system simulation trials).
Area of No. of Duration in Remarks
Operation Persons Aseptic Area

Manufacturing 04 04 Hrs x 2 Times Come out for Lunch after 4 hrs and will
go in aseptic area again after changing
garments
Microbiologist 01 04 Hrs x 2 Times Wear fresh garments for each entry. To
[QC] be present during filling operation.
Supervisor 01 02 Hrs x 2 Times Wear fresh garments for each entry
OR
Maintenance 01 04 Hrs x 1 Time Will enter with toolbox. To be present
during filling operation.
9.17 Incubation of Vials
9.17.1 Incubation at 22.5 2.5C
Transfer the properly identified SS trays containing media
filled vials into the Incubation room.
Maintain the room temperature at 22.5 2.5C for 7 days.
After 7 days of incubation at 22.5 2.5C observe each
vial visually for any type of growth/Turbidity.
Identify the vials with growth/turbidity (if any) and send
these to Microbiology laboratory for further investigations.
 9.17.2 Incubation at 32.5 2.5C
Maintain the temperature of incubation room at 32.5
2.5C for next 7 days.
After 7 days of incubation at 32.5 2.5C observe each
vial visually for any type of growth/Turbidity
Identify the vials with growth/turbidity (if any) and send
these to Microbiology laboratory for further investigations.
9.18 Growth Promotion Test of Lactose Filled Vials after 14 days
Incubation
Objective of the Test: To ensure that Lactose filled in the sterile glass vials
and sealed by rubber stopper and aluminium seal, does not inhibit the growth
of microorganism after incubation of 14 days.
a. Collect 30 filled and sealed vials
b. 5 vials are inoculated with about 10 - 100 cells of Candida albicans
ATCC 10231, 5 vials are inoculated with about 10 - 100 cells of
Bacillus subtillis ATCC 6633, 5 vials are inoculated with about 10 -
100 cells of Aspergillus niger ATCC 16404 and five vials each of 3
environmental isolates are inoculated with about 10-100 cells. The
inoculums for growth promotion test are prepared.
d. Incubate the vials inoculated with Bacillus subtillis at 32.5 2.5C for
maximum 3 days and incubate the vials inoculated with Candida
albicans and Aspergillus niger at 22.5 2.5C for maximum 5 days
and incubate the environmental isolates as per their respective growth
conditions.
Acceptance Criteria: Media should promote the growth within stipulated
period.
9.19 Destruction of Media filled Vials after 14 days Incubation
Media filled Vials are steam sterilized and destroyed by incineration after
the 14 days incubation and observation.
 C. ACCEPTANCE CRITERIA:
9.20 ACCEPTANCE CRITERIA:

9.20.1 An acceptable SCDM (or TSB) media fill run will consist of a
minimum of 5000 units filled with sterile SCDM (or TSB)
powder followed by sterile Water For Injection.

9.20.2 The duration of each media fill run will be the time required to
fill minimum of 5000 vials of each size at the lowest speed of
the machine (20-25 vials per minute).

9.20.3 Requirements listed in Attachment 1,2 or 3 have been met as

appropriate.
9.20.4 Positive-control units filled with SCDM (or TSB) must exhibit
growth of the specified growth promotion organisms at the
specified incubation parameters per CP-QC-MICRO-015.
9.20.5 Ideally the contamination rate should be zero. However, the
FDA guidelines suggest the contamination rate of less than
0.1% with a 95% confidence level. Initial qualification requires
3 successful consecutive media fill tests. During the initial
qualification, if ther are two (2) contaminated units in a single
run, or one (1) each in 2 run, the qualification is stopped, cause
is investigated and 3 media fill batches are repeated.

If the level of contamination exceeds 01 vial per 9000 vials, then cause of
contamination should be investigated and one more media fill run should be
conducted. If the number of contaminated units exceeds 03 per 9000 vials,
investigate the reasons of failure, rectify and then repeat the trials.
The aseptic drug powder filling process shall be considered validated if all media fill runs
are completed successfully.
D. ACTION TAKEN IN CASE OF SYSTEM SIMULATION FAILURES
In the event of any one or more trial results out of three trials fails in the test for
sterility, before starting fresh product campaign, following actions will be taken.
Characterisation of microorganisms up to species level, shall be carried out to find
out the source of contamination.
Review of following records for 60 days prior to system simulation trial failure after
the failing batch (Lactose trial) processing is carried out:
In addition to this following observations will be closely controlled and monitored
for subsequent 60 days.
a. Environmental records for manufacturing and testing area for temperature,
relative humidity, differential pressure and non-viable particulate counts.
b. Microbiological monitoring, reports of manufacturing and testing area for
settling plate, air sampling, surface monitoring and personnel monitoring.
c. Sterilization records for garments, filters, vials, stopper, machine parts, media
etc.
d. Filter integrity record of nitrogen, Carbon Dioxide and air supply filters.
e. WFI monitoring records.
f. Cleaning and sanitizing records.
g. Batch records of system simulation trials to find out the sign of any failures or
anomalies.
h. All other points mentioned in SOP for investigation will be included for
investigation.
Based on the above review, the most likely cause of failure will be identified and an
action plan will be made to avoid such occurrence in future.
The area will be monitored for bio-burden and after getting satisfactory area
monitoring reports for 3 consecutive days; three trials with Lactose will be taken.
All three such trials should pass as per the acceptance criteria for sterility.
Note: No commercial production batches will be produced till media fill runs pass as
per acceptance criteria.
1.1.1 CORRECXTIVE ACTIONS:

1.1.1.1 If any positive unit (s) is/are identified such that an alert or action
level is reached, an investigation will be performed and
documented per CP-QC-MICRO-009.

1.1.1.2 The investigation will include but not be limited to the following:
 1.1.1.2.1 Microbial environmental monitoring data.

1.1.1.2.2 Particulate monitoring data.

1.1.1.2.3 Bio burden data (Prior to pre-filtration and prior to

final filtration).

1.1.1.2.4 Personnel monitoring data (finger impressions, etc.,).

1.1.1.2.5 Sterilization for media, equipment and commodities.

1.1.1.2.6 HEPA filter evaluation (airborne particle levels,

smoke challenge testing, velocity measurements, etc).

1.1.1.2.7 Room airflow patterns and pressures.

1.1.1.2.8 Operator techniques and training.

1.1.1.2.9 Unusual events that occurred during the media-fill.

1.1.1.2.10 Storage conditions of sterile commodities.

1.1.1.2.11 Identification of contaminants.

1.1.1.2.12 House keeping procedures and training.

1.1.1.2.13 Calibration of sterilization equipment.

1.1.1.2.14 Pre- post- filter integrity test data.

1.1.1.2.15 Product and/or process defects, and/or limitation of

inspection process.

1.1.1.2.16 Documented disqualification of samples for obvious

reasons prior to final reading.

1.1.1.3 When action levels are exceeded: The investigation will include a
prompt review of all appropriate records relating to the aseptic
production lots between the current media fill and the last
successful media fill. The filling room will immediately placed out
of service and an Out of service tag will be placed by the
entrance of the filling room. A written notification will be
distributed by quality assurance to management.

1.1.1.4 Alert and action level: The number of verified positives should be
less than 3 contaminated vials per fill (5000 vials, with
contamination rate of less than 0.1% with a 95% confidence level).
1.1.2
When action and alert levels are exceeded, routine production will not be
resumed until acceptable media test run results are achieved.

E. DOCUMENTATION
The following documents shall be maintained in one binder.
 Summary report of Media fill trial
Approved Validation protocol.
Executed Batch Production Record for System Simulation Test ( Media Fill
Trials).
Environmental and personnel monitoring report of the critical area during the
trials.
Analysis of experimental results of the trials.
Investigation report and discussion for the cause in case of failure trial (i.e. not
meeting the acceptance criteria).
Investigation report for microbiological identification if growth is observed.
Recommendation for the corrective action [if any] required.
Summary and Conclusion duly approved by Head of the Quality Assurance
Department.

The observations during the system simulation test (Media fill trials) should be
recorded in the System Simulation Test record and its attachments .

F. Conclusion:

Acceptable result of all media run will establish the suitability of Standard Operating
Procedure for area cleaning and disinfection, cleaning and sterilization of
component, aseptic transfer of component, raw material and primary packaging
material, Environmental control system for temperature, Relative Humidity,
Differential pressure and viable / nonviable particulate load and finally the training
and practices of personnel.