MEDIA FILL VALIDATION PROTOCOL Logo

ASEPTIC PROCESS Protocol No:

Company Name and Address
SIMULATION STUDY

ASEPTIC PROCESS SIMULATION STUDY

Product Media Fill Validation

Generic Name Soyabean Casein Digestive Medium
Batch Size 10, 000 Vial/batch
Type of Validation Aseptic Process Simulation Study
Reason For Validation Scheduled Revalidation
Effective Date

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ASEPTIC PROCESS Protocol No:

Company Name and Address
SIMULATION STUDY

Sr. No. Subject Page No.

1.0 Preapproval 3

2.0 Objectives 4

3.0 Scope 4

4.0 Responsibilities 5

5.0 Definition 6

6.0 Reference Documents 6

7.0 Validation Matrix 7

8.0 Verification Of Primary Packaging Materials(Container & Closures) 7

9.0 Description of Equipment and Facility involved in Process Simulation: 8

10.0 Verification of Standard Operating Procedure 9

11.0 Acceptance Criteria 10

12.0 Methodology 11 - 12

13.0 Elements of Process Simulation Test 12 - 16

14.0 Rationale For The “Worst Case” Parameter Chosen 17

15.0 Process Steps 18 - 21

16.0 Process Flow Chart 22

17.0 Monitoring Parameters 23 - 25

18.0 Training Details 26

19.0 Sampling Plan 27

20.0 Execution 28

21.0 Failure Investigation and Corrective Action 28 - 30

22.0 Revalidation 30

23.0 Methods For Recording and Evaluating Results 31

24.0 Annexures 32

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ASEPTIC PROCESS Protocol No:

Company Name and Address
SIMULATION STUDY

1.0 PREAPPROVAL

Signing of this Aseptic Process Simulation study Protocol indicates agreement with the Validation
Master Plan approach of the equipment. Further if any changes in this protocol are required, protocol
will be revised and duly approved.

PREPARED BY

Functional area Name Signature Date

Quality Assurance

CHECKED BY:

Functional area Name Signature Date

Engineering

Production

Quality Control

Quality assurance

APPROVED BY:

Functional area Name Signature Date

Head Engineering

Head Manufacturing

AUTHORISED BY:

Functional area Name Signature Date

Head – QA

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ASEPTIC PROCESS Protocol No:

Company Name and Address
SIMULATION STUDY

2.0 OBJECTIVE
Objective of This protocol is to provide a written guideline for the validation of the aseptic
process and practices to confirm its acceptability in protecting the aseptically filled product from
microbial contamination. The results can be useful to detect and identify process or procedural
weakness that can lead to microbiological contamination of product.
➢ To demonstrate the capability of the aseptic process to produce sterile drug products.
➢ To evaluate the processing steps used to manufacture a sterile product, by aseptic
processing.
➢ To qualify or certify aseptic processing personnel

3.0 SCOPE
The process simulation test shall follow as closely as possible the routine aseptic manufacturing
process and include all critical subsequent manufacturing steps. All equipment shall remain the
same wherever practicable for the routine process. The media fill shall emulate the regular
product fill situation in terms of equipment, processes, personnel involved and time taken for
filling as well as for holding. Appropriate combinations of container size and opening as well as
speed of the processing line shall be used (preferably at the extremes).

This study covers Definition, Reference documents, Verification of primary packing material,
Critical instruments involved, Standard operation procedure, Methodology, Rational, process
flow diagram, Monitoring parameters, process details, Training details, Execution, Failure
investigation and corrective action and revalidation for process simulation carried out in
XXXXXXXXXX (Company Name).

The process simulation shall represent the “worst case situation” and shall include all
manipulations and interventions likely to happen during actual manufacturing process.

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ASEPTIC PROCESS Protocol No:

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4.0 RESPONSIBILITIES
The validation group comprising of representatives from each of the following departments
should be responsible for the overall compliance with this protocol:

Department Responsibility
Preparation and Final review of Validation Protocol and reports and its
compliance to meet the acceptance criteria of the Validation protocol.
Approval of final report. Review and Approval of protocol and final
report.
Review Media fill batch specific documents prior to the schedule of
Media Fill.
Quality Assurance
Provide oversight to ensure all planned activities are properly executed.
Ensure that Media fill Trials are conducted according to routine
procedure with the adherence to Quality.
Ensure all relevant documentation is audited for current Good
Documentation Practices.
Ensure appropriate personnel participate in the media fill trials.
Providing the utility services.
Engineering Ensure environmental conditions are maintained.
Approval of the final report.
Scheduling and conducting the validation run, checking the operations
and recording data as per the procedures outlined in the Protocol.
Production Review of Validation protocol and final report.
To ensure that the units are available for incubation. Execution of
protocol.
Carrying out the environmental monitoring (settle plate, swab testing and
personnel monitoring, etc) in critical areas during media fill trials.
Reporting the results.
Review of Validation protocol and final report.
Sampling of media as defined in QC SOP.
Quality Control To count the units given by manufacturing, before incubation. Visually
inspect the presence or absence of microbial growth, count and reconcile
the number of units after 7 & 14days of incubation.
Routine monitoring of media filled units during incubation.
Review of protocol, testing of samples, recording of results and final
result submission.
Head Engineering Approval of Protocol
Head Manufacturing Approval of Protocol
Head – QA Final authorization of protocol

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ASEPTIC PROCESS Protocol No:

Company Name and Address
SIMULATION STUDY

5.0 DEFINITIONS
5.1 Media Fill (Process simulation): Method of evaluating an aseptic process using a
microbial growth medium.
5.2 Aseptic Filling: Operation where the pre-sterilized product is filled and packed in
sterilized containers and closures in aseptic processing zones.
5.3 Environmental Monitoring Programme: Defined documented Programme, which
describes the routine particulate and microbiological monitoring of processing and
manufacturing areas, and includes a corrective action plan when action levels are
exceeded.
5.4 Growth Promotion Test: Test performed to demonstrate that media would support
microbial growth.
5.5 Sampling Frequency: Established period for collecting samples.
5.6 Sterilization: A process, by which all viable microorganisms are removed or destroyed,
based on probability.
5.7 Shift: Scheduled periods of work or production, usually less than 12 hours in length,
staffed by alternating groups of workers.

6.0 REFERENCE DOCUMENTS

➢ PIC/S Guideline –Recommendation on the Validation of Aseptic Process.
➢ ISO14698-1, 2003-09-01 Clean rooms and associated controlled environments -
biocontamination control — Part 1: General principles and methods
➢ USFDA Guidance for industry – Sterile drug products produced by Aseptic processing
cGMP - September – 2004
➢ Volume 4 EU Guidelines to Good Manufacturing Practice Medicinal Products for Human
and Veterinary Use

Following Documents shall be verified prior to commencement of the Media fill

processing.
➢ Media fill Batch record.
➢ Packaging Material /Specifications of, container/closure to be checked before Media filling.

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ASEPTIC PROCESS Protocol No:

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7.0 VALIDATION MATRIX

7.1 Frequency of periodic monitoring is 6month ± 15Days
7.2 Following test matrix is prepared for the Media fill validation
7.3 All the Vial size should be covered with in the 2year.
7.4 After 2year matrix should be repeated till the revision of the protocol.
1st 2nd 3rd 4th
Vial No of No of No of
Applic Applicab Applica No of runs Applica
Size runs runs runs
ability ility bility required bility
required required required
7.5ml  NA  1  NA  NA

10ml  NA  NA  NA  1

15ml  1  1  1  NA

20ml  1  NA  NA  NA

30ml  NA  NA  1  1

8.0 VERIFICATION OF PRIMARY PACKAGING MATERIALS (CONTAINER &

CLOSURES)
Verify the type of containers and closure used in process simulation test and record in the
validation report.

S. No. Material Name

01 Glass vials

Gray butyl rubber Bungs, Bromo Butyl rubber bungs and RFUs rubber
02
bungs

03 Aluminum Flip – off Seal

9.0 DESCRIPTION OF EQUIPMENT, FACILITY AND UTILITIES INVOLVED IN

PROCESS SIMULATION

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ASEPTIC PROCESS Protocol No:

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SIMULATION STUDY

All the major equipments used for process, facility and utility as listed below shall be verified
for Installation, Operational and Performance Qualification and recorded in the validation
report.
Integrity testing of HEPA filters to be verified and should be recorded in the validation report.
Standard operating procedure for Filter Cleaning, Maintenance, Integrity testing of HEPA filters
and sanitization of water system shall be included and verified in its report. The reference
protocol of the equipments shall be verified.
The following equipments are used during the Media Fill.

Sr. No. Name of Equipment Equipment I.D. No. Qualification Status

1 Laminar Air Flow Should be Qualified
2 Analytical Balance (Capacity: 220gm) Should be Qualified
3 Vial Decartoning Machine Should be Qualified
Automatic High speed linear vial
4 Should be Qualified
washing machine-300
Sterilization & Depyrogenation vial
5 Should be Qualified
tunnel
6 Autoclave cum Bung Processor Should be Qualified
Automatic Injectable Dry Powder
7 Should be Qualified
Filling with Rubber Stoppering
Automatic high speed eight head vial
8 Should be Qualified
sealing machine

Facility Involved: (HVAC System)

S. No Name of Area Qualification/Validation Status

1 Vial Filling/Sterile Area Should be Qualified

2 Change Rooms Should be Qualified

3 Washing/Sterilization Area Should be Qualified

10.0 VERIFICATION OF STANDARD OPERATING PROCEDURE

Standard operating procedure for the following operation shall be verified and recorded in the
validation Report.

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ASEPTIC PROCESS Protocol No:

Company Name and Address
SIMULATION STUDY

PROCEDURE TITLE Procedure No.

Entry & Exit in Aseptic area

Operation and cleaning of Autoclave cum Bung processor

Operation and cleaning of the Vial Washing Machine

Operation and cleaning of Sterilizing Cum Depyrogenating Tunnel

Operation and cleaning of Vials filling and Bunging machine

Operation and cleaning of Automatic High Speed Eight Head vial sealing machine

Operation and cleaning of mobile Laminar Air Flow

Operation and cleaning of Laminar Air Flow Station

Cleaning and sanitization of Aseptic area

Washing, Sterilization, and Storage of Aseptic Area Garments

Washing, Sterilization and Storage of Aluminium seal

Washing, Sterilization, Unloading and Storage of Rubber Bungs

Washing, Sterilization, Unloading and Storage of machine parts

Preparation, usage and Destruction of Disinfectant solution in Injectable area

Environmental and Personnel Monitoring of production Area

Procedure for Performing Sterility Test

Bacterial Endotoxin Test

Growth Promotion Test

Procedure for Bioburden Testing

11.0 ACCEPTANCE CRITERIA

The aim of the process simulation shall be zero contaminated units.
11.1 When Filling < 5000 units, then NO contaminated units should be detected

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ASEPTIC PROCESS Protocol No:

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SIMULATION STUDY

➢ One (1) contaminated unit is considered cause for revalidation, following

investigation.
11.2 When Filling between > 5000 units to < 10000 units, the
➢ One (1) contaminated unit should result in an investigation, including
consideration of a repeat media fill
➢ Two (2) contaminated units are considered cause for revalidation, following
investigation.
11.3 When Filling >10000 units, then
➢ One (1) contaminated unit should result in an investigation.
➢ Two (2) contaminated units are considered cause for revalidation following
investigation.

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ASEPTIC PROCESS Protocol No:

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SIMULATION STUDY

12.0 METHODOLOGY
12.1 Process definition
The process is defined as all steps from the washing and sterilization of the container and
closure, to the point the drug is sealed. All equipment remains the same as for the routine
process with the addition of a device to add WFI to the media powder. All the parameters
related to different equipment and environment shall be maintained the way it is
maintained during normal production run. No extra cleaning and sanitization shall be
done prior to and during filling operation. The maximum time frames for storage of the
sterile drug container and closures prior to aseptic assembly shall be factored into the
simulation.
12.2 Methods selected for the process simulation tests
12.2.1 All the vials shall be filled by filling the powder into the vials online followed by
online liquid (SWFI) filling. Accordingly, a liquid filling machine is added to the
filling line after to the powder filler.
12.2.2 Filtered compressed air shall be used as the substitute for the sterile inert gases
used during normal production.
12.2.3 The containers and closures, and equipment and filling parts shall be cleaned and
sterilized using the standard operating procedures (SOPs) listed in Section 9.0.
12.2.4 The speed of the filling machine shall be the determined fill rate for the container
size being utilized in Section 11.3.5.
12.2.5 The vials with the WFI and media shall be bunged and sealed, inspected on the
on-line inspection belt, and shall be collected in sequentially numbered trays or
boxes for incubation.
12.2.6 The vial shall be rejected at inspection only for leakage or distortion of the seal or
cap, or cracked glass. All vials that would otherwise be rejected for minor
cosmetic defects shall be passed and incubated.
12.2.7 The time of collection shall be noted. The filled units shall be briefly inverted and
swirled manually by inverting the box 2 or 3 times, after filling to assure closure
contact with the medium.
12.2.8 There shall be a reconciliation of the number of vials after incubation with the
number of filled vials that were passed after inspection. The acceptance criteria

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ASEPTIC PROCESS Protocol No:

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SIMULATION STUDY

are 100%. A missing vial shall be grounds to invalidate the test unless the test has
failed because of contaminated vials exceeding the acceptance criteria.

13.0 ELEMENTS OF PROCESS SIMULATION TEST

The following parameters must be considered during media fill validation
13.1 Filling Line set-up:
13.1.1 Since the filling line set-up entails manual assembly of the equipment and requires
more manipulation of critical surfaces than subsequent filling operations.
13.1.2 The set-up activities have been included in the media fill operation to detect the
potential contamination from machine set-up activities.
13.2 Number and Type of Interventions and stoppages to be included
13.2.1 The Media fill operation will include all the normal activities, which occur during
an aseptic filling operation (i.e. weight adjustments, container closure re-supply
etc.) in order to substantiate the acceptability of those practices in routine
operation.
13.2.2 Initial settings such as machine setting, weight setting and volume setting shall
not be considered as intervention but the vials shall be collected in the first tray
and incubated.
13.2.3 During the media filling normal weight checks on the quantity of media shall be
performed every half hour. The vial once removed from the conveyor for weigh
checks shall not be returned to the line or counted towards the number to be
incubated. However, the weight taken will be recorded for the total reconciliation.
13.2.4 During filling and sealing operations all the interventions listed in Section 13.3.3
shall be performed in such a way that one tray contains one intervention.
13.2.5 It is possible that non-planned interventions may be necessary to correct for
container breakage, fluid leakage, closure jams, which may occur during process
simulation. To the extent that these types of problems occur independently, and
are rectified during a successful process simulation test, but they will not count to
the number of interventions required below.
13.2.6 Details of the planned required interventions to be carried out during media filling
are given below.

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ASEPTIC PROCESS Protocol No:

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13.3 List of interventions to be deliberately performed

Sr. Minimum No.
Planned permitted Interventions
No. of Times

01 Adjustment of weight in the dosing wheel 02

02 Adjustment of stopper holding spring 02

03 Transfer of powder from Blender to Canister 02

04 Transfer of stoppers from bag to hopper 05

05 Picking unstoppered vials from the out feed to reject bin 08

06 Adjustment of separator 02

07 Picking up of fallen vials from turn table In-feed turn table for filling 03

08 Cleaning of spilled powder with lint-free mop 02

09 Minimum speed 02

10 Maximum speed 02

11 Optimum speed 02

12 Adjustment of turn table over load sensor 03

13 replacing of piston from wheel 01

14 Movement of person to backside of filling machine through swing conveyor 04

15 Checking the weight of the filled vial on balance 07

16 Adjustment of dosing air 02

17 Pushing stopper in the channel by forcep. 05

18 Entry of maintenance person for repairing electric panel and door setting 01

19 Power failure for 3 minute 01

20 AHU of Filling Area switched off for 5 minutes 01

21 Change of hopper, Port wheel and other machine Parts 01

13.4 Container Size

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ASEPTIC PROCESS Protocol No:

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SIMULATION STUDY

Process simulation trials shall entail the filling of the containers (as per matrix) on a
given filling line as per the schedule.
13.5 Filling speed/line speed
The fill speed to be used for any container shall be set at the low end of the Filling speed
since his represents the worst case for exposure to the Class A LAF air.
Simulated media fill speed Routine speed
Vial size/ML
(Not more than or equal to) (Vials/Hour)
7.5ML(M) 4000 Vials/Hour NLT 5000 Vials/Hour
10ML(M) 4000 Vials/Hour NLT 5000 Vials/Hour
15ML(M) 4000 Vials/Hour NLT 5000 Vials/Hour
20ML(M) 4000 Vials/Hour NLT 3000 Vials/Hour
30ML(M) 4000 Vials/Hour NLT 2000 Vials/Hour

13.6 Filled weight of medium and amount of sterile WFI to be filled into the containers
13.6.1 The volume of medium filled in the vial shall be NLT 50% ±0.5ML of the labeled
vial size. This volume is sufficient to enable contact of all the container-closure
seal surfaces when the container is inverted and to allow the detection of growth,
and it allows sufficient space for oxygen necessary for the growth of aerobes.
13.6.2 The filled weight of the sterile dehydrated medium powder shall be as per the
recommendations of the manufacturer depending upon the volume of sterile water
for injection filled in each size of vial.

Vial size/ML Dry Media fill weight Sterile WFI Volume

7.5ML(M) 120± 5% mg 4.0 ± 0.5ML

10ML(M) 150± 5% mg 5.0 ± 0.5ML

15ML(M) 240± 5% mg 8.0 ± 0.5ML

20ML(M) 300± 5% mg 10.0 ± 0.5ML

30ML(M) 450 ± 5% mg 15.0 ± 0.5ML

13.7 Duration of fill

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13.7.1 The media fill simulation test shall cover all the shifts as to qualify the
production run.
13.7.2 The duration of media fill shall be sufficient to fill the enough containers to
determine the contamination rate and must be more than the duration of
commercial batch.
13.7.3 Normal aseptic manipulations such as initial set-up activities, adjusting fill
weights or volumes, changing equipment and manual maintenance operations
shall be included in process simulation tests.
13.7.4 Process simulation tests also shall be of sufficient duration to include a
representative number of typical interventions, which might occur during an
actual production filling operation Where they are part of normal operations,
gown changes, breaks and shift changes shall be simulated.
13.7.5 The minimum time starting from initial machine set-up till completion of filling
operation and the minimum duration shall be of 3 shifts.
13.8 Number of units to be filled and number of runs
Standard batch size of vials for media filling shall be 10,000 and NLT 9,000 vial shall be
collected for the incubation.
13.9 Selection of Media
The medium selected for the Aseptic Process Simulation test shall be sterile Soya bean
Casein Digest Medium (SCDM) as it is capable of supporting a wide range of
microorganisms and is clear to allow observation for turbidity with ease.
13.10 Incubation Conditions-Incubation time and temperature for the filled units
13.10.1 All vials collected will be subject to incubation unless they are rejected at the
inspection stage for reasons mentioned above. Minor cosmetic blemishes do not
disqualify the vial and they must be incubated with all other vials. The
temperature chosen is based upon its ability to recover microorganisms normally
found.
13.10.2 The vials collected from the Process simulation shall be incubated at 20-25°C for
7 days followed by incubation at 30-35°C for 7 days for a total incubation
period of 14 days
13.10.3 Steps shall be taken to ensure wetting of all the inner surface of the container,
and closure, by the medium, e.g. by shaking or inversion during initial

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inspection, and by inverting the containers part-way through the incubation

period after 7 days during the change of incubators.
13.11 Personal Involvement
To qualify personnel working in the aseptic area from all related department by ensuring
that hey are present during media fill.
13.11.1 During filling operation maximum four person from the production dept. will be
present.
13.11.2 Minimum One microbiologist/Environmental monitoring personnel will perform
area monitoring, personnel monitoring and sampling
13.11.3 Minimum one maintenance person will enter aseptic area with his toolbox and
will do simulation of maintenance activity using sterile tools already available in
the area. (Grade –B)
13.11.4 The In-process QA and validation persons will also enter the aseptic area during
the media fill and will remain in the area (Grade B).
Note: A maximum of six people at a time should remain present during filling
that include person from production, microbiologist, maintenance, IPQA and
validation department. if it is required for a person to enter the filling room for
any activity or simulation when there are already six persons present then one of
the person from filling room should come out such that there is no impact on the
filling activity.

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14.0 RATIONALE FOR THE “WORST CASE” PARAMETER CHOSEN

14.1 Using sterilized materials, Components and closures, which have remained in the aseptic
processing area for extended period.
14.2 Increasing the size of the fill crew to more than the number necessary to fill the batch.
14.3 Using a growth-promoting medium in the process simulation test rather than an
inhibitory and preserved formulation.
14.4 Double dose in largest size container for more exposure during filling.
14.5 Increase retention time of aseptic area workmen.

15.0 PROCESS STEPS

15.1 Preparation of Sterile water for Injection
15.1.1 Take required quantity of water for injection in a clean stainless steel (S.S)
pressure vessel from the pendent of unit preparation area and cool it.
15.1.2 Transfer the stainless steel (S.S) pressure vessel containing water for injection into
the Interim Raw Material room (IRM) for filtration through static pass box.
15.1.3 Connect the following by using silicone tube;
➢ Outlet of compressed air with inlet of pressure vessel
➢ Outlet of pressure vessel (in IRM) with inlet of filter holder through the
S.S. pipe
➢ Outlet of filter holder with S.S pressure vessel inside the blending room
(aseptic area)
15.1.4 Open the compressed air valve from the pendent to get the pressure in between
1.0 to 1.5 kg/cm2
15.1.5 Start the filtration process using 0.22µ membrane filter.
Note: Perform in-process checks for filters before and after filtration as per the
Standard Operating Procedure, “Procedure for pre filtration in-process checks and
post filtration Bubble point test for membrane holder.
15.2 Decartoning of vials.
Dedust the cartons, decartonate and remove the vials. Arrange in the S.S. trays and stack
for feeding to the vial washing machine.

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15.3 Inspection of Vials.

Inspect the vials visually for absence of black particles, broken glasses, fibers and other
damages. Segregate the rejected vials, and destroy. Account for the rejection.
15.4 Vial Washing
15.4.1 Approved vials are decartoned in the decartoning room and visually inspected
for any physical deformities and placed into plastic crates and transferred into
Vial washing and sterilization room
15.4.2 The vials are loaded onto the washing machine conveyor. Washing of vials takes
place washing machine and delivers the washed vials into tunnel sterilizer for
sterilization and depyrogenation.
15.4.3 The washed vials then enter the tunnel sterilizer under laminar airflow protection.
15.5 Vial sterilization
15.5.1 The tunnel sterilizer is a continuous belt type dry heat sterilizer having drying,
sterilization, cooling and stabilization zones to facilitate sterilization and
depyrogenation.
15.5.2 The washed vials first enter into the drying zone of the tunnel sterilizer and where
the residual water in the washed vials gets evaporated.
15.5.3 Then the vials enter into the sterilization zone where the vials get sterilized and
depyrogenated by hot HEPA filtered air (Class 100).
15.5.4 Then the vials enter into cooling zone and get cooled by HEPA filtered air (Class
100).
15.5.5 Finally the vials 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.
15.6 Rubber stopper sterilization and transfer to filling room
15.6.1 The approved rubber stoppers are washed, siliconised in bung processor and
subjected to steam sterilization and drying.
15.6.2 The rubber stoppers after sterilization and drying & cooling are unloaded into
previously sterilized non-perforated rubber stopper holding canisters.

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15.6.3 These canisters are transferred into the sterile cool zone area of the respective
sterile powder filling area by placing in the mobile Laminar air flow work station
and are stored under laminar airflow workstation.
15.6.4 The rubber stoppers stored in the canisters are transferred into the filling room,
kept under LAF and are used at the time of filling.
15.6.5 The rubber stoppers packed in Ready for sterilization packs shall also be
simulated.
Note: In case of using ready for use rubber bung the rubber bung shall be
transferred through the dynamic pass box, before transferring, mop the external
area of the bag containing RFU rubber bung with 70% IPA and hold the sample
for NLT 15 min and then pass in production filling area with again mopping
with 70% IPA.
15.7 Sterilization of Flip-off aluminium seals
15.7.1 Sterilize the flip off Aluminium seals in Autoclave as per current SOP No. BP/064
“Operation and cleaning of Autoclave cum Bung processor”
15.7.2 A copy of temperature log should be attached with the media fill record.
15.8 Sterilization of machine parts.
15.8.1 Sterilize the machine parts in autoclave as per current SOP No. BP/064
“Operation and cleaning of Autoclave cum Bung processor”.
15.8.2 A copy of autoclave temperature log shall be attached with the media fill record.
15.9 Filling & Stoppering:
15.9.1 Take up the sterile medium for filling in previously sterilized vials under
compressed air and vacuum using vial filling and adjusted to fill weight.
15.9.2 The filling machine is covered by laminar flow workstation providing class 100
backgrounds for filling machine. The filling machine was provided with an
enclosure with doors.
15.9.3 Ensure that the filling line should be assembled prior to the filling operation.
15.9.4 Load the sterile medium into the hopper of the filling machine after ensuring that
the hopper, fiber disc, port hole and stoppering unit assembly is sterilized.
15.9.5 SWFI filling, numeric injector needle has to be fitted before powder filling
station.

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15.9.6 Adjust the required volume of SWFI and weight of the powder delivered into the
sterile Vials.
15.9.7 Carry out the filling operation by consuming media
15.9.8 Check vials from each port during filling for fill weight and volume at
predetermined intervals.
15.10 Sealing
15.10.1 The filled and stoppered vials thus transferred from the filling room are allowed
onto the sealing machine track.
15.10.2 The sealing of the vials carried out as per the standard operating procedure.
15.11 Inspection of vials.
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 liquid etc., The rejected Vials are
collected separately and destroyed. And the good Vials are transferred for incubation.
15.12 Incubation
Process simulation test shall be incubated at 20-250c for 7 days followed by 7 days of
incubation at 30-350c with vials inverted to make a total incubation period of 14 days.
15.13 Container Inspection
15.13.1 Only personnel who have had specific training in the visual inspection of media-
filled units shall perform inspection of vials.
15.13.2 Inspection of vials shall be done by visual examination by the microbiologist on
3rd, 7th, 11th and 14th day of incubation.
15.13.3 The units shall be examined visually for evidence of growth in the incubation
room using the inspection hood with illuminated background.
15.13.4 Containers shall be manipulated during the inspection process to ensure
detection of turbidity in the vials and closure surfaces. Any positive(s) noted
during routine inspection should be recorded and shall be removed from
incubation room for further investigation/identification.
15.14 Media growth promotion Test
Perform the growth promotion test on the samples (media fill vials) collected during
media fill. The samples shall be tested for GPT for initially and also after 14 days of
incubation. The growth promotion test will be conducted according to the GPT SOP

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15.15 Media Destruction record

Media filled Vials without any contamination after final inspection, incubation and
growth promotion tests will be destroyed by crushing and vials with growth after
isolating the organisms will be destroyed after autoclaving at 121 .1 ºC for 30 minutes.
Records for the same will be maintained.

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ASEPTIC PROCESS Protocol No:

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16.0 PROCESS FLOW CHART

Washing of R/S Washing of m/c parts Decartoning of Vials Gamma Irradiated Collection
SCDM media of WFI
Sterilization
of Aluminum
seals

Washing of Vials Sanitization of Aseptic

Sterilization of R/S Sterilization of m/c parts
media container Filtration of
WFI

De-pyrogenation of
Vials Transfer of media
container to
Aseptic area

Unplanned Temp./ RH/ DP

Interventions

Aseptic
Environmental Settle Plate
Interventions Filling/Plugging/Sealing of vials Monitoring

Personnel
Monitoring
Planned
Interventions
Collection/Inspection of Vials
Surface
Monitoring

NVPC
Transfer of Vials for Incubation

Incubation of Vials

Visual Inspection of GPT

Incubated Vials

Destruction of Media Filled Vials

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17.0 MONITORING PARAMETERS

17.1 Environmental Monitoring Parameters
Sr. ACCEPTANCE
ENVIRONMENTAL PARAMETERS FREQUENCY
No. CRITERIA
1.0 WASHING AND STERILIZATION AREA
Initial and every
01 Temperature NMT 25°C
4hours
Initial and every
02 Relative Humidity NMT 45%
4hours
2.0 FILLING AND SEALING AREA
Initial and every
01 Temperature NMT 20°C
2 hours
Initial and every
02 Relative Humidity NMT 45%
2 hours
Non-Viable Particle Count before filling
03
(Preparatory Phase)
04 Non-Viable Particle Count: End Phase
05 Non-Viable Particle Count after filling
Viable Particle Count: Active air sampling
06
(Preparatory Phase)
07 Active air sampling (filling phase) As per Monitoring Should comply as per
Viable Particle Count: Passive air sampling Plan Monitoring Plan
08
(Preparatory Phase)
09 Personnel Monitoring (Preparatory Phase)
10 Personnel Monitoring (filling phase)
11 Swab Testing (Preparatory Phase)
12 Swab Testing (filling phase)
13 Pressure Differential of Sterile Area
Filling Vs Corridor NLT 1.0mm of WC
Sealing Vs Corridor NLT 1.0mm of WC
Blending Vs Corridor Initial and every 2 NLT 1.0mm of WC
hours
Cooling Vs Corridor NLT 1.0mm of WC
Change Room - III Vs Corridor NLT 1.0mm of WC

17.2 Process Monitoring Parameters of Autoclave Processing

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Sr. MONITORING ACCEPTANCE

PROCESS PARAMETERS FREQUENCY
No. PARAMETERS CRITERIA
1.0 BUNG WASHING
Rinsing with Purified Water:
1.1 NLT 5Mins.
NLT 5Mins.
Rinsing with Water for
1.2 NA NA NLT 5Mins.
Injection NLT 5Mins
Siliconization Time:
1.3 NLT 15Mins.
NLT 15Mins.
2.0 BUNG STERILIZATION

2.1 Sterilization Time: 30 Mins Fo Value NA NLT 30 5Mins.

Sterilization temp. :
2.2
121.1° C to 124.0° C Complies sterility
Sterility Once
Test
2.3 Vacuum drying: 45 Mins.

3.0 STERILIZATION OF ALUMINIUM SEAL

3.1 Sterilization Time: 30 Mins. Fo Value NA NLT 30Mins.

Sterilization temp. :
3.2
121.1° C to 124.0° C Complies sterility
Sterility Once
Test
3.3 Vacuum drying: 30 Mins.

4.0 STERILIZATION OF MACHINE PARTS

4.1 Sterilization Time: 30 Mins. Fo Value NA NLT 30Mins.

Sterilization temp. :
4.2
121.1° C to 124.0° C Complies sterility
Sterility Once
Test
4.3 Vacuum drying: 30 Mins.

5. 0 STERILIZATION OF FILTRATION ASSEMBLY

5.1 Sterilization Time: 30 Mins. Fo Value NA NLT 30 Mins.

Sterilization temp. :
5.2
121.1° C to 124.0° C Complies sterility
Sterility Once
Test
5.3 Vacuum drying: 30 Mins.

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17.3 Process Monitoring Parameters of Vial Washing, Sterilizing Tunnel & Vial Filling
Machine
1.0 WASHING OF VIALS
1.1 Machine Pressure Pressure Pressure
Vial Pressure
Speed Frequency Recycled Compresse Purified
Size WFI
(Vials /Min. water d Air Water
Every NLT NLT NLT NLT
7.5ml 230 - 300 2 2 2
1Hour 0.5kg/cm 1.0kg/cm 0.5kg/cm 0.5kg/cm2
Every NLT NLT NLT NLT
10ml 230 - 300 2 2 2
1Hour 0.5kg/cm 1.0kg/cm 0.5kg/cm 0.5kg/cm2
Every NLT NLT NLT NLT
15ml 230 - 300 2 2 2
1Hour 0.75kg/cm 1.2kg/cm 0.75kg/cm 0.75kg/cm2
Every NLT NLT NLT NLT
20ml 230 - 300 2 2 2
1Hour 1.0kg/cm 1.2kg/cm 1.0kg/cm 1.0kg/cm2
Every NLT NLT NLT NLT
30ml 230 - 300 2 2 2
1Hour 1.2kg/cm 1.5kg/cm 1.2kg/cm 1.2kg/cm2
2.0 STERILIZATION OF VIALS
2.1 Temp. of Hot Pressure Differential Reading of Tunnel
Conveyer
Vial Frequency Zone( in 0C) (In mm of wc)
Speed
Size Drying Hot Cool Stabilizing
(mm/min) In Out
Zone Zone Zone Zone
140 Every NLT NLT
7.5ml 8 - 15 18 - 30 8 - 15 8 - 15
1Hour 300 300
Every NLT NLT
10ml 140 8 - 15 18 - 30 8 - 15 8 - 15
1Hour 300 300
Every NLT NLT
15ml 120 8 - 15 18 - 30 8 - 15 8 - 15
1Hour 300 300
Every NLT NLT
20ml 120 8 - 15 18 - 30 8 - 15 8 - 15
1Hour 300 300
Every NLT NLT
30ml 100 8 - 15 18 - 30 8 - 15 8 - 15
1Hour 300 300
3.0 FILLING OF VIALS
3.1 Vial Speed of Filling Frequency Target Fill weight Target Fill Volume
Size Machine (In mg) (In ML)
7.5ml NLT 2000 Vials/Hrs Every 1Hour 120 ± 5% 4.0 ± 0.5
10ml NLT 2000 Vials/Hrs Every 1Hour 150 ± 5% 5.0 ± 0.5
15ml NLT 2000 Vials/Hrs Every 1Hour 240 ± 5% 8.0 ± 0.5
20ml NLT 2000 Vials/Hrs Every 1Hour 300 ± 5% 10 ± 0.5
30ml NLT 2000 Vials/Hrs Every 1Hour 450 ± 5% 15 ± 0.5
18.0 TRAINING DETAILS

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Each person who works in an aseptic suite shall participate in a simulation test on a periodic
basis. All selected new persons, who are considered for sterile area entry and aseptic operations
should undergo training of SOPs. Logs of personnel who have participated in each fill shall be
maintained as part of the documentation. All the personnel who enter the aseptic processing
area, including technicians and Micro laboratory personnel shall be covered in a media fill at
least once in a year for re authorization.

Name of Person Department

CO-ORDINATOR (Head-Q.A.) Q.A.

SUPERVISORY STAFFS

Head - Production Production

Manager Production

Executive(s) Production

Officer(s) Production

Executive(s) Q.A./IPQA

Officer(s) Q.A./IPQA

Executive(s) Microbiology

Officer(s) Microbiology

Manager Engineering

Executive(s) Engineering

Officer(s) Engineering

OPERATORS

Technician Engineering

Operators Production Operator

Helper (Material transfer) Production Operator

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19.0 SAMPLING PLAN

Sampling Items Stages Quantity of Sample Test Performed

100ml+10ml+200ml+500ml LPC + BET + MLT +

Water for injection Vial washing
Chemical

Viable particle count Before media fill, during media fill and after media fill

Non viable particle

Before media fill
count
Sterile Soyabean
casein digest media After Blending 6g Sterility
(SCDM)
Sterile Soyabean
Before loading into the
casein digest media 30 g GPT, Sterility, pH
hopper
(SCDM)
BET + MLT +
Water for injection Before filtration 10ml+200ml+500ml
Chemical

Filter sterile water

After filtration 100ml+10ml+100ml Sterility + BET + LPC
for injection

Liquid SCDM After filling 50 ml pH

After sterilization
(Initial, Middle & End 21 Rubber Plugs from each
Rubber Plugs Sterility and BET
of the filling stage
operation)
After sterilization
Flip-off Aluminium (Initial, Middle & End 21 Flip-off Aluminium seal
Sterility and BET
seal of the filling Plugs from each stage
operation)
Initial, Middle & End
Depyrogenated
of the filling 21 vials from each stage. Sterility and BET
Vials
Operation.
After 14 days of
Media fill vial 20 vials GPT
incubation

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20.0 EXECUTION
20.1 Execution of the protocol is performed through the batch record.
20.2 The batch record shall give detailed instructions on how to perform the process
simulation test. It shall be written in the same format as a normal batch record and
contain all the normal data and sign of elements.
20.3 All information and records such as the cleaning records for pieces of equipment used,
release stickers for the containers and closures etc., shall be attached to the batch
manufacturing record.
20.4 All interventions, planned or unplanned, and stoppages must be documented in the record
as to the type of intervention, time the intervention occurred, duration of the intervention
or stoppage and number of the box or tray being filled.
20.5 Document (but not limited) to the following:
➢ Number of units filled
➢ Number of units incubated
➢ Number of units positive
➢ Number of units rejected for cause (damaged container, defective seal)
➢ Growth promotion of medium (“Before Filling, During Filling and After” incubation)

21.0 FAILURE INVESTIGATION AND CORRECTIVE ACTION

21.1 A contaminated container should be examined carefully for any breach in the integrity of
the container system. A damaged container should not be considered in the evaluation
(Acceptance) of the aseptic processing capability of the process. All positives from
integral containers should be checked to determine of the turbidity is microbiologically
related, if it is microbial growth, the organism should be identified to genus, and
whenever possible to species.
21.2 A comprehensive consistent sampling and identification scheme shall be part of the
investigation and determination of the contaminant source. In the instance when the
process simulation test does not meet the established acceptance criteria all possible
sources of contamination should be investigated. A detailed history of the investigation is
to be maintained.
21.3 The identity of the microorganism from the contaminated units should be determined.
The identification of the contaminant should be compared to the database of the
organisms recently identified. The biochemical (genus / species) profile of the
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contaminating microorganisms can then be compared to that of microorganisms obtained

from the sterility tests, bio-burden and environmental monitoring programmes in order to
help identify the potential source of the contaminant.
21.4 These isolates should be checked for possible identification matches, as should isolates
for any areas, which exceed their count limits or are trending upward.
21.5 Processing records should be reviewed. Any deviations, downtimes and repairs before or
during filling should be noted. Filter integrity testing results and all Sterilization records
associated with the product components and equipment should be examined. Cleaning
and sanitization records should be reviewed. Critical systems (HVAC, Compressed Air
/Water, Steam) should be reviewed for documented changes and re-qualification or
acceptance criteria for those changes.
21.6 Calibration records should be checked. All HEPA filters in the filling area should be
checked and rectified if warranted. Training records for all individuals (Production,
Maintenance, Cleaning) involved in the fill should be reviewed to assure proper training
was provided.
21.7 Validation records can be reviewed for any procedure or process changes. All deviations
from the original validation should have an associated justification for not performing a
new validation.
21.8 Based upon the outcome of investigation, the cause of the failure is either assignable or
not assignable. It may be clearly assignable to a single source or vaguely associated with
multiple system or process, which required re-defining. If the cause is assignable
corrective action needs to be taken and documented. The root cause and the corrective
action shall detect the no. of process simulation test required to demonstrate that the
process is operating within the expected parameters.
21.9 If no cause can be found the process should be validated as though it were a new process.
Multiple consecutive process simulation tests should be performed to demonstrate the
ability to consistently produce acceptable product.
21.10 The failure investigation report should contain
21.10.1 A summary of occurrence
21.10.2 All systems investigated, not just the system tied to the failure.
21.10.3 A conclusion as to causes and supporting documentation.
21.10.4 Potential effect on previous batches produced.

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21.10.5 Corrective action taken.

21.11 Outcome of additional process simulation tests, if performed.
21.12 Appropriate signatures. In addition to the signatures of the investigators of the individual
systems, the head of Production and head of Quality shall sign the overall reports.
21.13 The investigation needs to be completed in a timely fashion. It may be necessary to issue
an interim report, with anticipated time lines, if the investigation or corrective actions
require excessive time.
22.0 REVALIDATION
22.1 There shall be a routine process simulation test program for the aseptic filling line, which
should be performed at least twice per year. All container/closure systems should be
covered in media fill at least once in 2 years. Performance of process simulation tests
prior to the scheduled reassessment may be necessary following a process change of such
scope that previous qualification studies would be initiated. In such cases, the number of
process simulation tests may vary, depending upon the extent of the change. Examples of
such changes include:
22.2 Major Modifications to the equipment or immediate product containers / closures
(Interchanging standard parts does not constitute a major equipment modification)
22.3 Modification to equipment of facilities, which potentially affects the air quality of
airflow in the aseptic environment. Major changes in the number of production personnel
or initiation of second (or third) shift production when the facility has been qualified only
for single shift operations.
22.4 Major changes to the aseptic production process and / or procedures.
22.5 It also may be necessary to re-qualify with acceptable process simulation test in response
to adverse trends or failures in the on going monitoring of the facility or process, such as:
22.6 Continued critical area environmental monitoring results above the alert / action levels.
22.7 An increased incidence of product sterility test failures.
22.8 Breach of asepsis in the aseptic processing area.
22.9 When such incidents occur, the process and any changes, which may have occurred since
the previous qualification, should be evaluated. Appropriate action can then be taken to
restore the facility or process to its “qualified state” has been re-established testing may
be appropriate to assure that the “qualified state” has been re-established.
23.0 METHODS FOR RECORDING AND EVALUATING RESULTS

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23.1 The Protocol should be pre-approved prior to its execution and the protocol should have
detailed sampling plan and acceptance criteria for Process Simulation test parameters.
23.2 Wherever applicable the graph and data print outs of critical process parameters shall be
obtained and attached.
23.3 Data shall be recorded using pen in the report and the recorded data shall be identified by
date and signature of responsible person.
23.4 All parameters shall be recorded in relevant records (e.g. Forms, Media Fill Batch
record.)
23.5 For environmental parameters like viable and non-viable particles, count shall be done as
per the current environmental monitoring plans. However, all personnel entering the
aseptic area during media fill shall be examined for personnel hygiene
23.6 For parameters, which are recorded more frequently, minimum and maximum values
shall be presented.
23.7 For all results reference to source document shall be given.
23.8 The final report is a summation of the data from the batch record and environmental
monitoring samples. Based upon this information, a conclusion is drawn regarding the
acceptability of the manufacturing process and facility.

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24.0 ANNEXURES
The following documents to be attached with the validation report.
Annexure No Title of Document
1.0 Test Report for Sterile Soyabean Casein Digest Medium (SCDM)
2.0 Manufacturer’s certificate of analysis (COA) for sterile SCDM
3.0 Media Growth Promotion Test Report
3.1 Before Media Fill
3.2 During Media Fill
3.3 After Incubation
4.0 Microbial Monitoring Report
4.1 Passive Air Sampling
4.2 Active Air Sampling
4.3 Surface Monitoring – Swab Report
4.4 Personnel Monitoring, Glove and Garment monitoring
5.0 Sterility Report for
5.1 Water for Injection
5.2 Vials
5.3 Rubber Bungs
5.4 Aluminium Seals
5.5 SCDM
6.0 Dynamic Pass Box Entry Record
7.0 Aseptic Area Entry and Exit Log
8.0 Temperature Record of Incubation Room
9.0 Media Fill Vial Observation Record
10.0 Material Destruction Note (Post Incubated Vials)
11.0 Personnel Participate in Media Fill

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