The document outlines the media fill process and its validation, which simulates normal manufacturing operations for microbiological growth media to ensure aseptic conditions. It emphasizes the importance of validating aseptic processes as per pharmaceutical regulations to minimize contamination risks and details various protocol aspects, including frequency of runs, medium culture requirements, container considerations, and monitoring activities. Additionally, it specifies acceptance criteria for contamination rates and corrective actions in case of failures.

1. MEDIA FILL PROCESS AND
ITS VALIDATION
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2. What is Media Fill ?
 The Media fill or Broth fill technique is one in which a liquid
microbiological nutrient growth medium is prepared and filled
in a simulation of normal manufacturing operation.
 The microbiological growth medium such as Soybean Casein
Digest Medium (SCDM)is processed and handled in a manner
which simulates “normal” manufacturing process with same
exposure and possible contamination.
 The final container is then incubated and checked for turbidity
which indicate the microbial contamination.
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3. •Why the validation of aseptic process is required by
pharmaceutical regulations?
A “sterile product” is defined as “free of viable organisms”
As it is not practical examine every unit for confirmation of
sterility, all efforts are made to minimise the risk of contamination
(finishing, HVAC, pressure differentials, cleaning procedure,
monitoring programme)
Despite of many measures taken, contamination is an ever-
present danger because aseptic processing is a process being
operated in a controlled –but not sterile- environment and sample
numbers are too small; so that only gross contamination is likely to
be detected
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4. • Although media fills must duplicate aseptic manufacturing
conditions, it is not possible for them to be conducted in
exactly the same way as the manufacture of a production batch
of a pharmaceutical product.
• In aseptic processing, the greatest risk comes from the
personnel working in the clean room: the operators have to
participate in media fills.
• Environmental monitoring activities are required during
aseptic filling operations.
• It is usual to include the “worst case” conditions that can occur
in production runs.
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5. Aseptic process:
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6. washing machine
depirogenating tunnel
filling machine
stoppering machinecapping machine
Hands-on: Aseptic liquid filling line
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7. Media Fill Protocol
• Number and frequency of runs
• Medium culture (to replace the product)
• Number of units filled
• Container (vial) size
• Fill volume
• Line speed (or filling speed)
• Duration of fill
• Operators shifts
• Monitoring activities
• Interventions –both routine and non-routine
• Incubation method
• Acceptance criteria
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8. Number and frequency of runs
In start up simulation at least three consecutive separate
successful runs should be performed (it is recommended they are
performed in different days).
For on-going simulation, a routine semi-annual qualification is
recommended (one run)
Extraordinary media fill should be performed after all changes to
a product or line changes evaluated as a potential danger for the
aseptic process.
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9. Medium culture
The medium needs to support the growth of a wide variety of
microorganisms, including aerobic bacteria, yeasts and moulds
(non-selective medium).
For aerobic conditions: Soybean Casein Digest Medium (SCD)
also known as tryptone soya broth (TSB).
For anaerobic conditions: usually in a nitrogen environment fluid
thioglycollate medium (FTM).
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10. Medium culture
The media have to support the growth of microorganisms (growth
promotion test). The organisms to be tested are stated by
pharmacopoeia.
 Generally at the end of incubation period, some vials (taken from
the beginning, at half and at the end of the process) are inoculated
with < 100 CFU and incubated for 3 days (bacteria) and 5 days
(yeast and mould).
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11. Number of units filled
 Number of units filled should reflect the real batch size.
 It is allowed to fill a lowest number of units provided that the
number of units filled is sufficient to reflect the effect of potential
operator fatigue and adequately represents the maximum number of
interventions.
Some regulations suggest the number of units to be filled in
consideration of batch production size.
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12. Container size
The extremes of size containers should be considered.
The largest container (often filled at the lowest speed because of
its large fill volume) often has the large opening , so the potential
for microbial entry from the environment should be the greatest for
that size.
The smallest container (often filled at the highest speed for its
lower fill volume) represents the greatest handling difficulty; the
smaller container are more fragile and less stable and be more
subjected to breakage and jamming in the equipment.
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13. Container size
In the initial qualification two runs might be performed using the
largest container and the third run using the smallest container
In routine evaluation of the line, any container should be included
in the validation program
Clear containers should be used as a substitute for amber
containers to allow visual detection of microbial growth
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14. Fill volume
The volume of media filled into the containers need not the
routine fill volume.
It should be sufficient to contact the container-closure seal
surfaces (when the unit is inverted and swirled) and sufficient to
allow visual detection of microbial growth post incubation.
Smaller containers should not be over-filled as sufficient air
must be available in the container headspace to support the growth
of aerobic organisms (generally 25% of volume is not filled).
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15. Line (or filling) speed
The media fill should address the range of line speeds employed
during production. Sometimes more than one line speed should be
evaluated.
The speed chosen for each batch during simulation should be
justified.
Use of high line speed is justified for manufacturing processes
characterized by frequent interventions or a significant degree of
manual manipulation.
Use of low speed is justified for manufacturing processes
characterized by prolonged exposure of sterile components in the
aseptic area.
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16. Duration of fill
In general media fills should be long enough to include all of the
required interventions and stoppage and should reflect the potential
operator fatigue: a typical media fill might be at least 3-4 hours
long. Ideally a media fill should use more units than are in the
product being simulated (for all batches up to 5000 units).
 For very large batches or long campaigns, some blank units
(either empty or water filled) are used to maintain operating
conditions during the simulation: this technique can be used to
validate processes that may run for several days in order to validate
the full length of the longest approved campaign
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17. Operators shifts
Each operator performing aseptic processes are requested to
participate in media fill.
Set-up and line operators should be part of not less than one
process simulation per year. Operators such as line mechanics and
environmental samplers should be managed in a similar manner.
A maximum number of personnel present in the aseptic
processing room should be established.
When a firm operates on multiple shifts, the second and third shift
should be included in the media fill programme.
In case of manual operations (filling), each line operator should
participate into all three initial validation runs and at least one run
in re-validation (every six months)
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18. Environmental monitoring activities
There are regulatory and pharmacopoeia references that states the
microbial conditions.
Air sampling using either active and passive sampling methods
should be performed during the execution of the process. Surface
sampling is best performed at the end of aseptic process. Also
personnel should be monitored
Microbiological monitoring (air, surfaces, personnel) and particle
monitoring should be performed during media fill employing the
same procedure in force
Sometimes the number of sampling locations might be increased
respect the routine procedure
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19. Interventions –both routine and non-routine
Media fill records should document all interventions performed
and the number of units removed.
Routine interventions: aseptic line set-up in which sterilised parts
are removed from protective materials and installed is a potential
danger; it is common to identify the first containers filled as they
may be more indicative of potential problem with aseptic assembly.
Other routine interventions: stoppers bowl feeding, remove fallen
vials, remove jam stoppers, operators breaks, gloves change,
environmental monitoring.
Non routine interventions (occur randomly): glass breakage,
change / reset of filling needles, interventions on weight
adjustments, sensor failure, rail adjustments.
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20. Incubation methods
Any filled units should be inspected prior to incubation; any
defects that compromise the container closure or non-integral units
are rejected and documented.
Divergence in industry practice: incubation is performed for 14
days at 20-35°C (+/- 2,5°C): it is performed for 7 days at 20-25°C
and further 7 days at 30-35°C; it is performed for 7 days at 30-35°C
and then move the filled containers to 20-25°C
The lack of agreement suggest that the selection of incubation
conditions employed.
Units are incubated in an inverted position for the first half of the
incubation period and then returned to an upright position for the
remainder.
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21. Acceptance criteria
The target should be zero growth but a contamination rate less
than 0,10% with 95% confidence level is acceptable (approx. 1
contaminated unit in 5000 filled units).
FDA and PDA agree that the target should be zero contaminated
units regardless of size of run
It is important to note that “invalidation” of a media fill run
should be a rare occurrence
Each failure should be investigated
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22. Filled units per
run
Contaminated units
permitted (action level)
3000 0
4750 1
6300 2
7750 3
9150 4
10510 5
11840 6
13150 7
14430 8
15710 9
16960 10
The table indicates the
maximum permitted
number of contaminated
units per various Media
Fill “run sizes” to
indicate a 0,10%
contamination limit with
a 95% confidence level
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23. Contamination
• The root cause of a failure (contamination), or at least the
most probable one, must be identified
• It is important to be able to isolate and identify (to species
level) the microorganisms
• An appropriate corrective action / preventive action plan
must be implemented
• The impact of the failure on product lots already released (if
any) must be evaluated
• After the corrective actions have been implemented, a new
media fill study is performed to confirm their efficacy
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24. 08/10/15 25
CR decreases during filling
Con. spike during filling
Con. spike followed by an
increase
Isolated events (few
contaminated units per
run)
CR increasing during
filling

25. Validation

26.  OBJECTIVE
The Objective of validation protocol is to establish
documented evidence that the process employed for aseptic
processing of Parenterals liquid/Ophthalmic solution will
produce the desired results consistently, within the specified
acceptance limits, when performed as per the latest Standard
Operating Procedures.
 SCOPE
The Validation protocol describes the procedure for the
total Process Simulation (Media Fill).
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27. RESPONSIBILITIES
Sr. no . Responsibility Name of the
department
1 Preparation of Protocol QC
2 Provision of qualified personnel to assist
in the protocol preparation and
execution
QC, QA,
Production and
Maintenance
3 Verification of Protocol QC and
Production
4 Approval of protocol QA
5 Final determination of System
Acceptability
QA
6 Review and assembling of data into a
final report
QA
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28. PRE-REQUISITES
• Approved Soybean casein digest broth
• Environmental Monitoring of manufacturing areas by Plate
Exposure, Air sampling and surface monitoring procedures
and its SOP’s.
• Qualified and validated manufacturing equipments, system
facility (i.e. HVAC, water, compressed gases) CIP and SIP
procedures.
• Trained operating personnel’s.
• Approved BMR for media fill trial.
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29. Check and ensure that-
The equipment and system facility is validated.
 The HVAC system, compressed air, CIP and SIP
procedures are qualified.
All operations, cleaning/sanitization procedures are
established and operating personnel are trained.
Media used for Process Simulation is passed for GPT.
The WFI used for preparation of batch is complied to
USP/IP
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IDENTIFICATION OF CRITICAL CONTROL
MONITORING PARAMETER

30. VALIDATION PROCEDURE
 Main steps for the Validation of the integrated line
by media fill test
1. Cleaning of the line
2. Dispensing of Soybean Casein Digest Medium for 150 L
batch size
3. Batch Preparation 150 L
4. Filling And Sealing
5. Incubation and Examination of Media Filled Units
6. Interpretation of Results
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31. VALIDATION PROCEDURE
1. Cleaning of the SVP line
 Carry out cleaning of SVP mixing tank and holding tank along
with product line and bottle pack machine as per respective SOP
for CIP.
 At the end of cleaning, collect last rinses sample from sampling
point and send to QC department with written information for
testing of previous product traces.
 After getting approval report from QC, affix status label on the
tank “READY FOR STERILIZATION”.
 Immediately carry out the sterilization of SVP holding tank
along with final filter and product line of bottle pack machine as
per respective SOP.
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32. 2. Dispensing of Soybean Casein Digest Medium for 150 L batch
size
 Enter to dispensing room as per SOP for entry exit procedure to
dispensing area.
 Check for the clearance of the area from any unwanted
materials. Check for the cleanliness of the area, LAF, weighing
pan as per checklist. Put “ON” the reverse LAF unit 15 minutes
before dispensing of material.
 Check the availability of clean containers, pressure differentials,
and temperature & humidity should be not more than 250
C and
45 to 60% RH respectively.
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33.  Calibrate the balance as per SOP of Balance Calibration.
 Take the Approved Soybean Casein Digest Medium in pre-
dispensing room, place on SS pallet and check the label of
container for correctness and Approval of material.
 Transfer the material to Dispensing room, place the empty clean
container on the balance and record the tare weight. Press
“ZERO” of the balance and weigh the required quantity of
material, note the weighed material and then remove the
container from balance and press Zero.
 Close the dispensed material, affix the weighing tag and transfer
the material in dispensed material storage room.
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34.  After dispensing, put “OFF” the balance and LAF. Clean the
surrounding area, balance and spray with 70% IPA solution.
 Reseal the original container and shift to their original
place.
• Batch Preparation 150 L:
 Ensure that the area and product line is clean and free from
the traces of previous product.
 Recheck gross weight of Soybean Casein Digest Medium
(SCDM) to be used for manufacturing and ensure that they
match as per entries made in the BMR weighing sheet.
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35.  Check the status board affixed on the tank “READY FOR
USE”, also verify the records and ensure that the bottom
outlet valve of the mixing tank is closed.
 Send the entry point sample of WFI from the user point to QC
department for testing along with BMR.
 On approval of WFI sample from QC department, affix a
status board on the Mixing tank “UNDER
MANUFCTURING” with Product name and B. No.
 Collect approx 50 L water for injection at 80 to 850
C in a
manufacturing tank fitted with stirrer.
 Start the stirrer and add SCDM through the mainhole of the
tank.
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36.  Continue stirrer for complete dissolution of
ingredients.
 Stop the stirrer.
 Make up the volume to the 150 L with water for
injection.
 Start the stirring for complete dissolution of SCDM
and homogeneous bulk solution (generally required 10
minutes).
 Collect sample of bulk solution in a sterile sampling
bottle and send it to QC for testing of color clarity, pH
and bioburden along with bulk intimation slip.
 After getting clearance of bulk analysis from Quality
Control, start the filtration from mixing tank to
Holding tank of line with the help of pump.
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37.  After getting clearance of bulk analysis from Quality Control,
start the filtration from mixing tank to Holding tank of line
with the help of pump.
 Perform the bubble point test of the final filter after holding
tank as per SOP of Bubble point test.
4.Filling And Sealing:
 Start the filtration from holding tank to FFS machine using
pump.
 Drain one buffer tank approx 1.3 liters of bulk solution from
filling nozzle to eliminate any possibility of dilution of bulk
by condensates in product line of the machine post SIP.
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38.  Check online cartridge filter integrity test as per its respective
SOP.
 Start Machine line and discard initial 15 shots.
 Collect first cassette of vials from next shot and send the
sample with written information to QC for testing.
 Arrange the out coming cassettes of vials sequentially in
vacuum chamber tray and verify the results of testing from QC
department.
 Now start the filling and sealing continuously as per SOP for
Filling and sealing.
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39.  Collect the filled and sealed containers coming out of
the filling area in plastic crates.
 During filling operation keep the filled ampoules
separately for each breakdown, shift change, power
breakdown, stoppage etc and assign lot number.
 Arrange the cassettes of vials lot wise in SS trays
vertically in vacuum leak testing chamber tray and
carry out the leak testing at 650 – 720 mm Hg for 30
minutes. Do not use the leak vials for further media fill
study.
 After leak test, transfer the goods vials in the clean
plastic crates horizontally in cassette from one above
the other, lot wise separately
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40. 5. Incubation and examination of filled units:
a. Incubate all media filled units in normal position after leak
test at of 20 to 250
C for 7 days. Incubation temperature
should be maintained within 22.5 ± 2.50
C .
b. After completion of 7 days Incubation at 20 to 250
C, invert
the units and incubate them at 30-350
C for next 7 days.
Incubation temperature should be maintained within
32.5±2.50
C .
c. Each media filled unit should be examined by trained
Microbiologist after 3rd
day, 7th
day, 10th
day and 14th
day.
d. All suspect units identified during the observation should be
brought to the immediate attention of the QC Microbiologist.
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41. Interpretation of Results:
When filling fewer than 5,000 units, no contaminated units
should be detected.
oWhen filling 5,000 to 10,000 units :
One contaminated unit should result in an investigation,
including consideration of a repeat media fill
Two contaminated units are considered cause for
revalidation, following investigation.
oWhen filling more than 10,000 units :
One contaminated unit should result in an investigation;
Two contaminated units are considered cause for
revalidation, following investigation.
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42. References
1. R. A. Nash and A. H. Wachter “Pharmaceutical Process
validation”; Third edition
2. Agallo James, Carleton J. Fredric “Validation of
Pharmaceutical Processes”; Third edition
3. P.P.Sharma “How to practice GMP’s” ;Third edition.
4. USP <797> ‘media fill testing’ / <71> ‘growth promotion
test’
5. FDA “guidance for industry, sterile drug products produced
by aseptic processing – cGMP”
6. PIC/S PI 007-2 “recommendations on the validation of
aseptic process
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