ASEPTIC PROCESS SIMULATION STUDY

OR
MEDIA FILL STUDY
REQUIREMENTS AS PER

USFDA ASEPTIC PROCESSING

GUIDANCE AND ISO 13408-1 (ASEPTIC
PROCESSING OF HEALTHCARE
PRODUCTS)
Sterile is a powerful word, with harsh
legal implications surrounding non-
compliance
Global regulatory authorities would define sterile as "free
of viable organisms", and sterility assurance has become
one of the most scrutinised areas of pharmaceutical and
medical device manufacture.
Overview of Injectable

Media Fill
Key elements of a media fill

aseptic process:
 USFDA:
• Defines Aseptic Process Simulations: The
process simulation also known as media
fills, normally includes exposing the
microbiological growth medium to product
contact surfaces of equipment, container
closure systems, critical environments,
and process manipulations to closely
simulate the same exposure that the
product itself will undergo.
 ISO GUIDELINES (13408-1)
✓Defines Media Fill as : Method of
evaluating an Aseptic Process using a
microbial growth medium.
Why To Perform Media fill?
Regulatory Guidelines
Important Elements
 Study Design

A media fill program should incorporate the contamination

risk factors that occur on a production line, and accurately
assesses the state of process control.

Media fill studies should closely simulate aseptic

manufacturing operations incorporating, as appropriate,
worst-case activities and conditions that provide a challenge
to aseptic operations.
 Study Design
It is recommended that the media fill program shall address applicable
issues such as:
❑ Longest permitted run .
❑Normal interventions that occur with each run
❑Non interventions and events (e.g. maintenance,
stoppages, equipment adjustments)
❑Lyophilization, when applicable
❑Aseptic assembly of equipment (e.g. at start-up, during processing)
❑Number of personnel and their activities
❑Representative number of aseptic additions (e.g. charging containers
and closures as well as sterile ingredients) or transfers
 Study Design
It is recommended that the media fill program shall address applicable
issues such as:

❑Shift changes, breaks, and gown changes (when applicable)

❑Type of aseptic equipment disconnections/connections
❑Aseptic sample collections
❑Line speed and configuration
❑Weight checks
❑Container closure systems (e.g. sizes, type, compatibility with
equipment)
❑Specific provisions in written procedures relating to aseptic
processing (e.g. conditions permitted bef ore line clearance)
 Frequency and Number of Runs:
➢Initially three consecutive successful runs
➢If Qualified one run
➢Interventions in Runs
➢Authorized personnel including technicians and maintenance personnel, should
participate in a media fill at least once a year.
➢Each change to a product or line change should be evaluate
➢ Any changes or events affect aseptic process for example, facility and equipment
modifications, line configuration changes, significant changes in personnel, anomalies
in environmental testing results, container closure system changes, extended shutdowns,
or end product sterility testing showing contaminated products may be cause for
revalidation of the system.

When data from a media fill indicate the process may not be in control, an investigation
should be conducted to determine the origin of the contamination and the scope of the
problem.

Once corrections are instituted, process simulation run(s) should be performed to

confirm that deficiencies have been corrected and the process has returned to a state of
control.
 Revalidation other than scheduled
validation
Special media fills are required incase of following
conditions, but not limited to,
• Changes in the process, facility and major equipment
modification
• Abnormalities in environmental monitoring results
• Sterility test failures
• After major construction activities such as demolition of
adjacent to or surrounding controlled areas
• Line Configuration changes
• Extended shut down
• Containers/ Closure changes
• Major changes in production personnel
• Initiation of additional production shifts
Duration of Runs:
➢Full batch size and duration
➢For lyophilization operations, it is recommended that
unsealed containers be exposed to partial
evacuation of the chamber in a manner that simulates the
process.
Vials should not be frozen,
and precautions should be taken that the medium remains
in an aerobic state to avoid potentially inhibiting the
growth of microorganisms.
Size of Runs:

➢ The simulation run sizes should be adequate to mimic commercial production

conditions and accurately assess the potential for commercial batch
contamination.

➢Run size is in the range of 5,000 to 10,000 units. For operations with
production sizes under 5,000, the number of media filled units should at least
equal the maximum batch size

➢When the possibility of contamination is higher based on the process design

(e.g., manually intensive filling lines), a larger number of units, generally at or
approaching the full production batch size, should be used.
Line Speed:
➢As speeds employed during production. Each media fill run
should evaluate a single line speed, and the speed chosen should
be justified
➢High line speed is often most appropriate in the evaluation of
manufacturing processes characterized by frequent interventions or
a significant degree of
manual manipulation.
➢Slow line speed is generally appropriate for evaluating
manufacturing processes with prolonged exposure of the sterile
drug product and containers/closures in the aseptic area.
 Environmental Monitoring During Media Fill

• Environmental monitoring for Viable and Non-Viable Particle

counts shall be monitored before, during and at the end of Media
Fill Run.

• Temperature, Differential Pressures & Relative Humidity shall be

monitored during the process.
Environmental Conditions:
Media fills should be adequately representative of the conditions
under which actual manufacturing operations are conducted.
➢ An inaccurate assessment (making the process appear cleaner
than it actually is) can result from conducting a media fill under
extraordinary air particulate and microbial quality, or under
production controls and precautions taken in preparation for the
media fill.

➢ To the extent standard operating procedures permit stressful

conditions (e.g., maximum number of personnel present and
elevated activity level), it is important that media fills include
analogous challenges to support the validity of these studies.
Media:
-soybean casein digest medium, should be used.
-Anaerobic growth media -fluid thioglycollate medium
❑Demonstrated to promote growth of gram-positive and gram-negative
bacteria, and yeast and mold (e.g., USP indicator organisms).
❑Environmental monitoring and sterility test isolates can be substituted (as
appropriate) or added to the growth promotion challenge.
Growth promotion units should be inoculated with a <100 CFU challenge.

❑ If the growth promotion testing fails, the origin of any contamination ,

investigated and media fill must be repeated.
❑Each unit should be filled with an appropriate quantity and type of microbial
growth medium to contact the inner container closure surfaces
(when the unit is inverted or thoroughly swirled) and permit visual detection of
microbial growth.
Incubation and Examination of Media-Filled Units
Media units should be incubated under conditions adequate to detect microorganisms

• Incubation temperature should be suitable for recovery of bioburden and

environmental isolates and should at no time be outside the range of 20-35 ºC.
Incubation temperature should be maintained within ±2.5 ºC of the target
temperature.

• Incubation time should not be less than 14 days.

❑ If two temperatures are used for the incubation of the media filled units, the units should
be incubated for at least 7 days at each temperature (starting with the lower temperature).

Each media-filled unit should be examined for contamination by personnel with

appropriate education, training, and experience in inspecting media fill units for
microbiological contamination.
To allow for visual detection of microbial growth, it is recommend to substitute clear
containers (with otherwise identical physical properties) for amber or other opaque
containers.
 Media selection, GPT for Media, Identification,
Incubation and Inspection of Media Filled Units –
requirements contd…

• Incubation of media filled containers

All media filled containers shall be incubated excluding the
containers with physical defects and containers that would be
discarded during normal production. Examples of Containers that
should be discarded during Media Fill Study are:
Volume/Wt. checked Containers
Broken Containers
Cracked Containers
Unsealed Containers those had fallen on the floor
Leak test failed Containers
❑ Identification of media filled Containers
Media filled containers should be chronologically identified within
the batch to help identify the time at which a contaminated unit
was filled.
Incubation and Examination of Media-Filled Units

❖Units found to have defects not related to integrity (e.g., cosmetic

defect) should be incubated; units that lack integrity should be
rejected.
❖After incubation is underway, any unit found to be damaged
should be included in the data for the media fill run, because the
units can be representative of drug product released to the market.
❖Any decision to exclude such incubated units (i.e., non-integral)
from the final run tally should be fully justified and the deviation
explained in the media fill report.
❖If a correlation emerges between difficult to detect damage and
microbial contamination, a thorough investigation should be
conducted to determine its cause .
Incubation and Examination of Media-Filled Units

❑Written procedures regarding aseptic interventions should be clear and specific

(e.g., intervention type; quantity of units removed.
❑Recording
❑To assess contamination risks during initial aseptic setup (before fill),
incubating these units
❑These units are typically incubated separately, and would not necessarily be
included in the acceptance criteria for the media fill.
❑Appropriate criteria should be established for yield and accountability
(reconciliation of filled units).
❑Media fill record reconciliation documentation should include a full accounting
and description of units rejected from a batch.
Interpretation of Test Results:

❑Reconciliation
❑Video recording of a media fill may serve as a useful aide in identifying personnel
practices that
could negatively affect the aseptic process
❑ Any contaminated unit should be considered objectionable and investigated. The
microorganisms should be identified to species level.
❑In addition, any failure investigation should assess the impact on commercial drugs
produced on the line since the last media fill.
❑Whenever contamination exists in a media fill run, it should be considered indicative
of a
potential sterility assurance problem, regardless of run size.
❑The number of contaminated units should not be expected to increase in a directly
proportional manner with the number of vials in the media fill run.
❑ Test results should reliably and reproducibly show that the units produced by
an aseptic processing operation are sterile.

Modern aseptic processing operations in suitably designed facilities have

demonstrated a capability of meeting contamination levels approaching zero and
should normally yield no media fill contamination.
Recommended criteria for assessing state of aseptic line control are as follows:
•When filling fewer than 5000 units, no contaminated units should be detected.
-- One (1) contaminated unit is considered cause for revalidation, following an
investigation.
• When filling from 5,000 to 10,000 units:
-- 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.
•When filling more than 10,000 units:
-- One (1) contaminated unit should result in an investigation.
-- Two (2) contaminated units are considered cause for revalidation, following
investigation.
For any run size, intermittent incidents of microbial contamination in media filled runs can be
indicative of a persistent low-level contamination problem that should be investigated.
Accordingly, recurring incidents of contaminated units in media fills for an individual line,
regardless of acceptance criteria, would be a signal of an adverse trend on the aseptic processing
line that should lead to problem identification, correction, and revalidation.
A firm's use of media fill acceptance criteria allowing infrequent contamination does not mean
that a distributed lot of drug product purporting to be sterile may contain a nonsterile unit.
The purpose of an aseptic process is to prevent any contamination.
As with any process validation run, it is important to note that invalidation of a media fill run
should be a rare occurrence. A media fill run should be aborted only under circumstances in
which written procedures require commercial lots to be equally handled. Supporting
documentation and justification should be provided in such cases.
 Acceptance Criteria and Interpretation of
Results contd…
The investigation should include but not limited to, consideration of the following:
• Microbial environmental monitoring data
• Particulate monitoring data
• Personnel monitoring data (glove prints, gowns);
• Sterilisation cycles of media, accessories, equipment, etc.;
• HEPA filter evaluation (airborne particulate levels, filter integrity testing, velocity
measurements, etc.);
• Room air flow visualization studies, pressure differentials;
• Operator techniques and training;
• Unusual events that occurred during media fill runs;
• Storage conditions of sterile accessories;
• identification of the contaminant organisms as a clue to the source of
contamination;
• house keeping procedures and training;
• calibration of sterilisation equipment;
• pre- and post-filter integrity test data and/or filter housing assembly;
• product and/or process defects, and/or limitation of inspectional processes; and
documented disqualification of samples for obvious reasons prior to final reading
 Post Media Fill Study

❑ Product manufacturing may resume while the media

filled inuts are being incubated; however, product
release shall not occur until acceptable media-fill data
are obtained.(ISO)
Thanks
Comparison of Aseptic Process
Simulation
Media fill Guidance’s

Personnel
 Comparison of Aseptic Process Simulation

S.No. Category PDA FDA PIC/S WHO/EU SOP

1 Frequency and ➢ At least three consecutive ➢ At least three ➢ A “start-up” Process
number of runs successful process consecutive simulation test simulation tests
simulations are performed separate consists of three should be
when qualifying a new successful runs consecutive performed as part
facility, filling line or be performed satisfactory of validation by
process during initial line simulation tests running three
➢ At least semi-annual qualification per shift and consecutive
simulations for a qualified ➢ Subsequently, should be carried satisfactory
facility, line or process routine semi- out before routine simulation tests
➢ Interventions should annual manufacturing can
include the activities which qualification start. These tests
occur during an aseptic conducted for ➢ On-going” should be
filling process that would each processing simulation tests repeated at
affect the sterility of the line will evaluate should be defined intervals
product as well as any the state of performed with and after any
permitted corrective control of the each shift of each significant
interventions. aseptic process. process line at modification to
➢ Personnel should Activities and least twice per the heating,
demonstrate their interventions year under the ventilation and
proficiency in aseptic representative of condition that air-conditioning
technique by successfully each shift, and there were no (HVAC) system,
performing a qualification shift changeover, changes in the equipment or
test entailing manual media should be normal production process.
manipulation not associated incorporated into procedures and no
with an APS or participate the design of the action limits were Process
in a successful aseptic semi-annual exceeded simulation tests
process simulation run in qualification ➢ Exceeding an should
which they perform the program action level incorporate
same functions to the extent ➢ All personnel demands a re- activities and
that they will perform it who are validation. interventions
during actual production at authorized to Depending on the known to occur
least once a year. enter the aseptic result of the during normal
➢ Additional process processing room follow-up production as
simulations required should during investigation this well as the worst-
be based on a risk manufacturing, re-validation may case situation.
assessment to assist in the including require the The process
evaluation of any major technicians and inclusion of one to simulation tests
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 Comparison of Aseptic Process Simulation
changes to procedures, maintenance three satisfactory should be
practices or equipment personnel, process simulation representative of
configuration. should tests. each shift and
➢ If there is a container participate in a ➢ The media fill shift changeover
closure system or a unique media fill at least should emulate to address any
system, different from once a year. the regular time-related and
others in the room or on the Participation product fill operational
fill machine, then a process should be situation in terms features
simulation at six-month consistent with of equipment,
intervals should be the nature of processes,
performed for each unique each operator’s personnel
process. duties during involved and time
routine taken for filling as
production. well as for
➢ Each change to a holding.
product or line ➢ Where filling
change should be takes place over
evaluated using a extended periods,
written change i.e. longer than 24
control system. hours, the process
Any changes or simulation test
events that have should extend
the potential to over the whole of
affect the ability the standard
of the aseptic filling period. In
process to order to prevent
exclude excessively high
contamination numbers of units
from the being filled it is
sterilized product usually acceptable
should be to just run the
assessed through machine for a
additional media reasonable time, if
fills. the validity of the
➢ When data from simulation is not
a media fill diminished by this
indicate the process
process may not
be in control, an
investigation
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 Comparison of Aseptic Process Simulation
should be
conducted to
determine the
origin of the
contamination
and the scope of
the problem.
Once corrections
are instituted,
process
simulation run(s)
should be
performed to
confirm that
deficiencies have
been corrected
and the process
has returned to a
state of control.
➢ When an
investigation
fails to reach
well-supported,
substantive
conclusions as to
the cause of the
media fill failure,
three consecutive
successful runs
in tandem with
increased
scrutiny of the
production
process may be
warranted.

2 Duration of ➢ The APS should be of ➢ The duration of ➢ The media fill

runs sufficient duration to the media fill run should emulate
include a representative should be the regular
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 Comparison of Aseptic Process Simulation
number of interventions that determined by product fill
might occur during an the time it takes situation in terms
actual production filling to incorporate of equipment,
operation manipulations processes,
➢ Justification of the selected and personnel
number of units filled, interventions, as involved and time
duration and yield should well as taken for filling as
be included in the process appropriate well as for
simulation study design. consideration of holding.
➢ A partial vaccum, the duration of ➢ Where filling
insufficient to cause boiling the actual aseptic takes place over
of the medium is drawn on processing extended periods,
the chamber at ambient operation. i.e. longer than 24
temperature, and Interventions that hours, the process
maintained for the duration commonly occur simulation test
of a normal lyophilization should be should extend
process. The stoppered routinely over the whole of
units are removed from the simulated, while the standard
lyophilizer and sealed. those occurring filling period. In
Medium is not frozen and it rarely can be order to prevent
is a common practice to use simulated excessively high
sterile inert gases to break periodically. numbers of units
the vaccum on the chamber ➢ When aseptic being filled it is
processing usually acceptable
employs manual to just run the
filling or closing, machine for a
or extensive reasonable time, if
manual the validity of the
manipulations, simulation is not
the duration of diminished by this
the process process
simulation
should generally
be no less than
the length of the
actual
manufacturing
process to best
simulate
contamination
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 Comparison of Aseptic Process Simulation
risks posed by
operators.
➢ For
lyophilization
operations, FDA
recommends that
unsealed
containers be
exposed to
partial
evacuation of the
chamber in a
manner that
simulates the
process. Vials
should not be
frozen, and
precautions
should be taken
that ensure that
the medium
remains in an
aerobic state to
avoid potentially
inhibiting the
growth of
microorganisms
3 Size of runs ➢ Small scale : APS batch ➢ A generally ➢ If batches smaller ➢ The number of
size equal to production acceptable than 3000 units containers used
batch size starting point for are produced, the for media fills
o Mid-scale : APS batch run size is in the minimum number should be
size should be of range of 5,000 to of containers used sufficient to
comparable size to the 10,000 units. for the process enable a valid
production batch size For operations simulation should evaluation. For
o Large scale : Variety with production be equal to that of small batches
of sizes under the commercial the number of
approaches(Alternate 5,000, the batch size containers for
between media fill and number of media media fills
empty units or filled units should at least
should at least equal the size
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 Comparison of Aseptic Process Simulation
alternate between WFI equal the of the product
and media filled units) maximum batch batch
➢ Manual : same as size made on the
production batch size processing line.
➢ When the
possibility of
contamination is
higher based on
the process
design (e.g.,
manually
intensive filling
lines), a larger
number of units,
generally at or
approaching the
full production
batch size,
should be used
4 Line speed ➢ Fill speed to be used for ➢ Each media fill
most containers should be run should
set at the production filling evaluate a single
speed range for that size line speed, and
container on commercial the speed chosen
production. should be
justified.
5 Environmental ➢ Process simulation should ➢ Media fills ➢ Simulation tests
conditions be carried out using routine should be should be
environmental monitoring adequately performed on
operating procedures and representative of different days and
sampling requirements. the conditions hours during the
➢ Any changes to the routine under which week and not only
environmental monitoring actual at the beginning
requirements during process manufacturing of a work day.
simulation should be operations are ➢ If the same
explained and documented. conducted. process is
➢ To the extent conducted in a
standard separate clean
operating room, this should
procedures also be validated
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 Comparison of Aseptic Process Simulation
permit stressful
conditions (e.g.,
maximum
number of
personnel present
and elevated
activity level); it
is important that
media fills
include
analogous
challenges to
support the
validity of these
studies.
6 Media ➢ The most common medium ➢ In general, a ➢ The fill volume of
for process simulation is microbiological the containers
Soybean casein digest growth medium, should be
medium such as soybean sufficient to
➢ Aseptic processing casein digest enable contact of
conducted in strict medium, should all the container-
anaerobic environment be used. Use of closure seal
should be evaluated with anaerobic growth surfaces when the
other suitable medium, in media (e.g., fluid container is
addition to aerobic thioglycollate inverted and also
evaluation. medium) should sufficient to allow
➢ Growth promotion be considered in the detection of
properties of the incubation special microbial growth
media should be evaluated circumstances. ➢ Low Selectivity:
using pharmacopeial ➢ The media The medium
methods selected should selected should be
➢ Growth promotion studies be demonstrated capable of
are performed after 14 days to promote supporting a wide
of incubation growth of gram range of
➢ The container need not be positive and microorganisms,
filled to the normal fill gram-negative which might
volume. The fill volume bacteria, and reasonably be
must be controlled and yeast and mold encountered and
monitored as performed (e.g., USP be based also on
during routine filling the in house flora
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 Comparison of Aseptic Process Simulation
➢ There must be enough indicator ➢ Media used in the
medium in the container to organisms). evaluation must
contact all container-closure ➢ Growth pass a growth
seal surfaces when the promotion units promotion test.
container is inverted and should be The control
swirled. inoculated with a organisms used
➢ There must be enough <100 CFU should include
medium in the container to challenge. those relevant
allow detection of microbial ➢ If the growth strains of test
contamination promotion micro-organisms
➢ The volume of headspace testing fails, the identified by
should be considered in the origin of any relevant
growth promoting contamination Pharmacopoeias
capability of the media to found during the as being suitable
support aerobic simulation for use in the
microorganisms. should growth promotion
nonetheless be test.
investigated and ➢ Growth promotion
the media fill tests should
promptly demonstrate that
repeated. the medium
➢ Each unit should supports recovery
be filled with an and growth of low
appropriate numbers of
quantity and type microorganisms,
of microbial i.e. 10-100
growth medium CFU/unit or less.
to contact the ➢ Growth promotion
inner container testing of the
closure surfaces media used in
➢ (When the unit is simulation studies
inverted or should be carried
thoroughly out on completion
swirled) and of the incubation
permit visual period to
detection of demonstrate the
microbial ability of the
growth. media to sustain
growth if
contamination is
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 Comparison of Aseptic Process Simulation
present. Growth
should be
demonstrated
within 5 days at
the same
incubation
temperature as
used during the
simulation test
performance.
➢ Clarity: The
medium should be
clear to allow for
ease in observing
turbidity

7 Incubation and ➢ APS units should be ➢ Incubation ➢ It is generally

examination of incubated for a minimum of temperature accepted to
media filled 14 days should be incubate at 20-
units ➢ A single incubation suitable for 25°C for a
temperature in the range of recovery of minimum of 7
20-35°C may be used bioburden and days followed
➢ Data should be available to environmental immediately, or
show the show the isolates and after a first
suitability of the selected should at no time reading, by
incubation temperature for be outside the incubation at 30-
growth range of 20- 35°C for a total
➢ Clear containers of identical 35°C. minimum
configuration may be Incubation incubation time of
substituted for opaque or temperature 14 days. Other
amber containers to aid in should be incubation
the detection of maintained schedules should
contamination within +2.5°C of be based on
➢ Normal product inspection the target supporting
process should be temperature. validation data.
maintained for APS with ➢ Incubation time ➢ Prior to incubation
non integral units removed should not be the containers
during pre-incubation less than 14 with the
inspection days. If two microbiological
temperatures are growth medium
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 Comparison of Aseptic Process Simulation
➢ For the purpose of APS used for the should be inverted
cosmetic, particulate and incubation of the or otherwise
fill volume defects should media filled manipulated to
be ignored and such units units, the units ensure that all
incubated and included in should be surfaces, included
APS evaluation and incubated for at the internal
contamination rate least 7 days at surface of the
➢ Visual inspection of all each temperature closure, are
APS units for growth is (starting with the thoroughly wetted
performed to determine the lower by the medium.
outcome of aseptic process temperature). The containers
simulation ➢ If QC personnel should not be
➢ Accurate counts should be do not perform completely filled
performed at each step in the inspection, with medium in
the simulation, filling ,pre- there should be order to provide
incubation inspection and QC unit sufficient oxygen
post-incubation inspection oversight for the growth of
➢ At the conclusion of post- throughout any obligate aerobes.
incubation inspection, filled such Similarly,
units are recounted to verify examination. All containers should
pre-incubation suspect units not be overlaid
accountability identified during with inert gases
➢ In the event of a the examination even though the
discrepancy an should be product may be
investigation should be brought to the
performed to determine the immediate
source of the variance and attention of the
potential impact on the QC
validity of APS study microbiologist.
➢ To allow for
visual detection
of microbial
growth, we
recommend
substituting clear
containers (with
otherwise
identical physical
properties) for
amber or other
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 Comparison of Aseptic Process Simulation
opaque
containers.
➢ When a firm
performs a final
product
inspection of
units
immediately
following the
media fill run, all
integral units
should proceed
to incubation.
➢ Units found to
have defects not
related to
integrity (e.g.,
cosmetic defect)
should be
incubated; units
that lack
integrity should
be rejected.
➢ Erroneously
rejected units
should be
returned
promptly for
incubation with
the media fill lot.
➢ After incubation
is underway, any
unit found to be
damaged should
be included in
the data for the
media fill run,
because the units
can be
representative of
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 Comparison of Aseptic Process Simulation
drug product
released to the
market.
➢ Any decision to
exclude such
incubated units
(i.e., non-
integral) from
the final run tally
should be fully
justified and the
deviation
explained in the
media fill report.
If a correlation
emerges between
difficult to detect
damage and
microbial
contamination, a
thorough
investigation
should be
conducted to
determine its
cause
➢ Written
procedures
regarding aseptic
interventions
should be clear
and specific
(e.g.,
intervention
type; quantity of
units removed),
providing for
consistent
production
practices and
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 Comparison of Aseptic Process Simulation
assessment of
these practices
during media
fills
➢ If written
procedures and
batch
documentation
are adequate to
describe an
associated
clearance, the
intervention units
removed during
media fills do not
need to be
incubated
➢ The ability of a
media fill run to
detect potential
contamination
from a given
simulated
activity should
not be
compromised by
a large-scale line
clearance. We
recommend
incorporating
appropriate study
provisions to
avoid and
address a large
line clearance
that results in the
removal of a unit
possibly
contaminated
during an
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 Comparison of Aseptic Process Simulation
unrelated event
or intervention.
➢ Appropriate
criteria should be
established for
yield and
accountability
(reconciliation of
filled units).
Media fill record
reconciliation
documentation
should include a
full accounting
and description
of units rejected
from a batch.

8 Interpretation ➢ The ultimate goal for the • When filling

of test results number of positives in any ➢ The process ➢ When inspecting fewer than 5000
process simulation should simulation run the containers units, no
be zero. should be they should be contaminated units
➢ All positive units should be observed by the compared to a should be detected.
identified to at least the QC Unit, and known sterile • when filling 5000–
genus, and to the species contaminated container for 10 000 units:
level when practical. units should be comparison as — one
➢ When positive units are reconcilable with some microbial contaminated unit
encountered, all possible the approximate growth shows up should result in an
sources of contamination time and the as a faint haze investigation,
should be investigated activity being which is difficult including
simulated during to detect unless consideration of a
the media fill. there is a control repeat media fills
➢ Any container to — two
contaminated compare against. contaminated units
unit should be Personnel should are considered
considered be trained for this cause for
objectionable task. revalidation
and investigated. ➢ After the following
➢ The incubation period investigation;
microorganisms of the media-filled
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 Comparison of Aseptic Process Simulation
should be containers they • when filling more
identified to are visually than 10 000 units:
species level. examined for — one
➢ The investigation microbial growth. contaminated unit
should survey Contaminated should result in an
the possible containers should investigation;
causes of be examined for — two
contamination. evidence of contaminated units
In addition, any container/closure are considered
failure damage which cause for
investigation might revalidation
should assess the compromise the following
impact on integrity of the investigation
commercial packaging system. ➢ For any run
drugs produced Damaged size,
on the line since containers should intermittent
the last media not be included as incidents of
fill. failures (positives) microbial
when evaluating contamination
➢ When filling results. may be
fewer than 5000 ➢ When filling indicative of
units, no fewer than 5000 low-level
contaminated units, no contamination
units should be contaminated that should be
detected. units should be investigated.
detected. ➢ Investigation
➢ One (1) ➢ When filling of gross
contaminated 5,000 to 10,000 failures should
unit is units: include the
considered cause ➢ a)One (1) potential
for revalidation, contaminated unit impact on the
following an should result in an sterility
investigation. investigation, assurance of
When filling including batches
from 5,000 to consideration of a manufactured
10,000 units: repeat media fill; since the last
One (1) ➢ b) Two (2) successful
contaminated contaminated media fill.
unit should result units are Care should be
in an considered cause taken to ensure that
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 Comparison of Aseptic Process Simulation
investigation, for revalidation, any validation does
including following not compromise the
consideration of investigation. processes
a repeat media ➢ When filling more
fill. than 10,000 units:
➢ One (1)
➢ Two (2) contaminated unit
contaminated should result in an
units are investigation;
considered cause ➢ Two (2)
for revalidation, contaminated
following units are
investigation. considered cause
for revalidation,
➢ When filling following
more than 10,000 investigation.
units: ➢ For any run size,
intermittent
➢ One (1) incidents of
contaminated microbial
unit should result contamination
in an may be indicative
investigation. of low-level
contamination that
➢ Two (2) should be
contaminated investigated.
units are Investigation of
considered cause gross failures
for revalidation, should include the
following potential impact
investigation. on the sterility
assurance of
➢ A media fill run batches
should be manufactured
aborted only since the last
under successful media
circumstances in fill.
which written ➢ All contaminating
procedures microorganisms
require whether or not an
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 Comparison of Aseptic Process Simulation
commercial lots alert or action
to be equally limit has been
handled. exceeded should
Supporting be identified to at
documentation least genus and
and justification preferably species
should be where practicable
provided in such to determine the
cases. possible source of
contamination.
➢ If a process
simulation tests
fail then due
account should be
taken of products
filled between the
last successful test
and the test
failure. Recording
of any deviations
during the
simulation test is
important to trace
later on the exact
cause and to
evaluate the
consequences.
The investigation
should identify
batches that could
be affected during
this time period
and the
disposition of the
affected batches
should be re-
assessed
Note:
In second updated -Health Canada, Japan (PMDA)
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A Regulatory Perspective: Rationale for Hold
duration of Partially Stoppered Media Filled Vials
Updated: Feb 10

Author: Varadharaj Vijayakumar - Aseptic Technology, Isolators & Lyophilization

https://www.lyophilizationworld.com/post/a-regulatory-perspective-rationale-
for-hold-duration-of-partially-stoppered-media-filled-vials

Introduction
Aseptic process simulation, also known as a media fill trial, estimates the contamination
risk of an aseptic production process by using sterile culture media in place of the
product constituents.

Purpose of media fill

The goal of a media fill is to demonstrate that the manufacturer can follow the routine
aseptic production process using sterile media without contamination (evaluate and
justify the aseptic capabilities of the process, the people and the system).
Hence in this article, we ensure lyo media fill should primarily validate the filling,
transportation and loading and unloading aseptic operations with certain duration of
filled vials in the lyophillizers.

Background
During aseptic process simulation for lyophilized product, filled vials are aseptically
filled in the normal way (similar to liquid filling), but the closures (which are of a single
slotted or double slotted) are not fully inserted in the filled vials and transported
aseptically through conveyor/ALUS/robotics to the lyophilizer shelves under Grade A
atmosphere.

Loading of media filled vials in the lyophilizer

Shelf to be pre-cooled to 20-25°C. The media filled vials are loaded in the sterilized
lyophilizer (using suitable in-house designs such as automatic loading/Manual loading
using fences). At the completion of the loading process, it is important to ensure all
filled, partially stoppered vials are loaded into the lyophilization chamber.

Product lyophilization process

Lyophilization or freeze drying is a process in which water is removed from a product
after it is frozen and placed under a vacuum, allowing the ice to change directly from
solid to vapor without passing through a liquid phase.

What will happen if it is simulated for longer hours of holding similar to product
lyophillization? Exact simulation of media fill as that of product lyophillization will
have possible reduction of microbiological levels after aseptic manipulation which will
not solve the purpose of aseptic process simulation.
Carrying out a lyophilization cycle and freezing the media will be same simulation as
that of product lyophillization, however this freezing of media will reduce microbial
levels of some contaminants.
In aseptic process simulation of lyo process the scope is not to check the lethality of
freezing and its effect on microorganisms that might be present.
Hence freezing of media and the formation of ice crystals is unfavorable to
microorganisms hence this should be avoided.

Unfrozen media and complete vaccum

If media is not frozen, then the lyophilizer may get contaminated due to unfrozen media
which is left under vacuum similar to the product lyophillization vacuum level.
If it’s complete vacuum drawn and it may cause the media solution to get out from the
containers and contaminate the lyophilizer as well fluid loss from the containers and
finally this may have serious effect on the viability of the microorganism and the ability
of the media to support microbial growth will be impacted this will in turn make aseptic
process simulation invalid. Hence a complete vacuum as specified for the lyophilization
process should not be drawn during the media fill.

Hold time duration in the lyophillzer

In general the course and processing time should be long enough to challenge or stress
the process, interventions, the supporting environment and the operators. All efforts
should be made to perform all routine interventions. The filled partially stoppered vials
will remain inside the lyophilizer chamber for minimum 12 hours (Author’s experience
for hold time duration).

The duration of the media filling stated above represents lyophilization cycle for one
shift.
During this we ensure the integrity of the lyophilizer chamber by testing the lyophilizer
post sterilization cycle, therefore no additional benefit is drawn by holding the media
filled vials for longer duration (such as product lyophillization cycle) since the
lyophilizer’s integrity is maintained thought out this cycle. Hence, it is not necessary to
carry out lyophilization cycle as per the actual drug product.
As compare to lyophilization hold duration for filled vials in lyophillzer the chances of
contamination for partial stoppered vials is more during the process stages such as
empty vial transportation, media filling, samplings, half stoppering, loading of vials into
lyophillizer, etc.

So how long the APS process shall be simulated? Typically 600 mbar to 700mbar, few
manufacturers used maximum of 900 mbar of partial vacuum and held for about 12
hours and few manufacturers held it for two hours and then it was broken by sterile
filtered compressed air shall be used to back fill the chamber, instead of the nitrogen and
full stoppering of vials shall be performed using the stoppering mechanism at ambient
pressure and open the door and unload the vials aseptically and transfer to sealing
machine. All the activity shall be performed in class 100 (Grade A).

Possible questions from regulatory agencies for Lyo media fills

1. Is the aseptic handling of lyophilized products validated by media fills?
2. In the aseptic process, is lyophilization simulation performed during media
fill?
3. Is the maximum amount of time the vials are held prior to lyophilization
simulated during media fills. If vials are not sealed in lyophilization
chamber, is the maximum hold time prior to stoppering simulated in media
fills?
4. During validation, what level of vacuum is pulled on the lyophilization
chamber?
5. How long do media fill vials remain in the lyophilization chamber under
vacuum? How does this compare to commercial lots?
6. Does the process simulation result in freezing of the media? Note that this
process simulation should not include freezing of the media.
7. Is environmental monitoring performed during loading of the lyophilizer
both during production and as well as during validation?
8. Does the firm have data on growth promotion of the media? Are growth
promotion tests done on vials after incubation is completed?
9. Is environmental monitoring performed during unloading of the chamber
during production as well as during media fill validation?
10. What is used to break the vacuum during media fills (nitrogen, air, other
gas)?

In my view, with practical experiences and conducted more than 30 Lyo media
fills.
Some Rationales
• Vial and Stopper Size: The neck size of the vial is considered as a deciding
parameter for the media fill and other parameters like body diameter are
considered as non deciding parameters for the media fill. The vials having
neck diameters of 13 mm, 20 mm and 32 mm are in generally used in
industry. The size of the vial shall be considered while deciding the worst
case for the media fill. The size of the rubber stopper is deciding parameter
of the media fill and hence is considered as a critical parameter. The rubber
stoppers of size 13 mm, 20 mm and 32 mm are generally used in industry
shall be considered while deciding the worst cases for the media fill.
• Type of Vial and Stoppers: The tubular or molded vials are in use. For the
media fill the tubular vials are considered as worst case since the tubular
vials are having lesser weight and have larger movement or tendency to
toppl. The stoppers of the bromobutyl and chlorobutyl are generally used
and the type of the stopper is not a deciding parameter for the media fill and
stopper of any type can be used for the media fill.
• Source of Vial:The source of vials is not a deciding parameter for the media
fill and hence any source of vials can be used.
 • Finishing and colour of Vials and Stoppers: The finish of vial viz: Blow
back or non blow back and treated or non treated does not play a deciding
role in media fill and hence the vial of any finish can be used. The clear glass
vials shall be used for the media fill to facilitate the visual inspection of the
vials. The rubber stoppers coated and noncoated are used generally but not
as deciding parameter for the media fill. But design of stoppers are
important and plays a deciding factors e.g. 2-leg, 3-leg igloo or flat-teflon,
serum etc

• Fill Volume:The volume of medium must be sufficient to provide contact

with all container closure seal surface on inversion and visibility for the
detection of microbial growth. The quantity of sterile SCDM medium shall be
considered for filling in the vial with different sizes are as follows:

• Pulling and Releasing of Partial Vacuum: Pull the condenser of lyo to -

40 °C and start the vacuum pumps. The loaded vials are exposed to the
partial vacuum cycle.

• First Pull of Vacuum: Pull the vacuum to 650 mbar, after achieving the
vacuum of 650 mbar, and maintain it for one hour after that release the
vacuum gradually using filtered (0.22 micron sterile filter) compressed air
up to atmospheric pressure. The half stoppered vials will be held at
atmospheric pressure for approx one hour.

• Second Pull of Vacuum: Pull the vacuum to 650 mbar after about one
hour of first pull of vacuum and after achieving the vacuum of 650 mbar,
and maintain it for one hour after this release the vacuum gradually using
filtered (0.22 micron sterile filter) compressed air up to atmospheric
pressure. The half stoppered vials will be held at atmospheric pressure for
approx one hour after this period perform the stoppering of vials into the
chamber at atmospheric pressure. Note: In case of anaerobic media fill,
break the vacuum of lyophilizer using filtered nitrogen gas, instead of
filtered compressed air.

How many Vials shall be loaded?

1. Minimum 10000 vials shall be filled during media fill.

2. Minimum number of vials to be filled shall be decided based on equipment
constrain, where number of vials shall be filled considering the maximum
capacity of the equipment.

Note: The media fill duration shall be sufficient to cover actual duration of the filling of
the drug product. The leftover media after completion of the filling shall be measured
and discarded.
Some FDA 483 Observations for quick look....

Observation
The protocol stated that chamber for the lyophilizer must be held under slight vacuum
conditions to simulate the process. The slight vacuum conditions were not created
during the hold time when the media filled vials were in the lyophilizer chamber.

Conclusion
The duration of the media filling stated above represents overnight lyophilization cycle.
We ensure the integrity of the lyophilizer chamber by testing the lyophilizer post SIP
cycle, therefore no additional benefit is drawn by holding the filled vials for longer
duration since the lyophilizer’s integrity is maintained throughout cycle. Hence, it is not
necessary to carry out lyophilization cycle as per the actual drug product lyophilization
cycle. As compared to lyophilization hold period, probability of contamination of vials is
more during the process stages such as filling, stoppering etc and also As per regulations
from few regulatory authorities it’s clear that hold time does not need to be the actual
duration of lyophillization cycle.However there should be written justification for the
hold duration of media fill vials in the aseptic simulation process of freeze drying
process.
Hence, A balance risk and science based approach is needed to simulate the process as
closely as possible, and rationale for holding the vials in the lyophillizer need to be
presented which is the expectation from the regulators.
Finally in case of failure in the media fill it necessary to diagnose and prove the source of
contamination accurately so that robust corrective or preventive actions gets
implemented.

References:
1. Freezing should not be simulated: 24 of 26 manufacturers using
lyophilization who responded to the PDA's 1996 survey of aseptic
manufacture claimed not to freeze their media fill vials (PDA, 1996).
2. Lyophilization Validation: A Regulatory Perspective by Ellen Huang
CBER/OCBQ/DMPQ CASSS CMC Strategy Forum July 19, 2016” slide 22
states that “Hold time does not need to be the actual duration of
lyophilization cycle”
3. FDA, Guidance for Industry: Sterile Drug Products Produced by Aseptic
Processing–Current Good Manufacturing Practice, 2004
4. Medicines and Healthcare products Regulatory Agency: Rules and
Guidance for Pharmaceutical Manufacturing and Distributors, current,
Annex-1: Manufacture of Sterile Medicinal Products.
5. Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-
operation Scheme: Recommendation on the Validation of Aseptic
Processing, 01Jan 2011.
6. Health Products and Food Branch Inspectorate, Good Manufacturing
Practices (GMP) Guidelines – 2009, Edition GUI – 0001, November 8, 2009