Media Fill Protocol
Luisa Stoppa
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
Nanjing, the People’s Republic of China – 16-20 November 2009
Objectives of the hands-on
 To evaluate the aseptic process simulation (media fill or
broth fill)
 To know the key points in designing a media fill protocol
to meet regulatory expectations
 To learn how failures have to be evaluated and which
consequences they have
 To discuss in a team
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
2 | Nanjing, the People’s Republic of China, 16-20 November 2009
Objectives of the hands-on
aseptic process:
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
3 | Nanjing, the People’s Republic of China, 16-20 November 2009
Principles of media fill
 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 such measures, 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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
4 | Nanjing, the People’s Republic of China, 16-20 November 2009
Principles of media fill
 Pharmaceutical regulations:
- Regulations: FDA “guidance for industry, sterile drug products
produced by aseptic processing – cGMP” - EU GMP Part I annex 1 –
- PIC/S PI 007-2 “recommendations on the validation of aseptic
process”
 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
What medium? How many units? How long?
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
5 | Nanjing, the People’s Republic of China, 16-20 November 2009
Principles of media fill
 In aseptic processing, the greatest risk comes from the
personnel working in the clean room: the operators have
to participate in media fills
Which qualifications to the operators need and when can
operators be considered qualified?
 Environmental monitoring activities are required during
aseptic filling operations
Are there additional monitoring activities necessary or not?
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
6 | Nanjing, the People’s Republic of China, 16-20 November 2009
Principles of media fill
 It is usual to include the “worst case” conditions that can
occur in production runs
Which kind of interventions have to be considered ?
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Hands-on: aseptic liquid filling line
capping machine
washing machine
depirogenating tunnel
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stoppering machine
filling machine
Hands-on: Media Fill Protocol
 Key elements to be taken into account include:
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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-routineIncubation method
Acceptance criteria
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
9 | Nanjing, the People’s Republic of China, 16-20 November 2009
Hands-on: Media Fill Protocol
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
10 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: 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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
11 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: medium culture (1/3)
The medium needs to support the growth of a wide variety of
microorganisms, including aerobic bacteria, yeasts and moulds (nonselective medium).
Guidance notes the use of Soybean Casein Digest Medium (SCD)
also known as tryptone soya broth (TSB).
If the product is being filled in anaerobic conditions, usually in a
nitrogen environment, an anaerobic medium is used: fluid
thioglycollate medium (FTM).
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
12 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: medium culture (2/3)
The media have to support the growth of microorganisms (growth
promotion test). The organisms to be tested are stated by
pharmacopoeia (USA, EU, Japan). Moreover the use of one or two of
the most common environmental isolates is recommended. 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).
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
13 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: medium culture (3/3)
About prion contamination from component of animal origin found
within media, it is important that medium supplier can provide the
certification for confirming materials are sourced from “BSE-free”
countries.
An alternative approach would be to use medium TSB derived from
vegetable materials.
SDC powder is generally subjected to mycoplasmi contamination that
are not removed by sterile filtration; in this case previous sterilised
powder (with gamma rays) can be used.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
14 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: 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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Media fill Protocol
 Key element: container size (1/2)
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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
16 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: container size (2/2)
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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
17 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: 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).
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
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Media fill Protocol
 Key element: 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.
In the initial validation of a filling line, one run might be performed at the
lowest speed and two at the highest speed.
In routine evaluation of the line, the speeds would be alternated
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
19 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: duration of fill
Even if the most accurate model would simulate the actual production run,
other models cab be justified.
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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
20 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: 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)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
21 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: 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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
22 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: interventions –both routine and non-routineThe interventions should simulate what occurs in a production run; 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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
23 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: 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 2035°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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
24 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: 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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
25 | Nanjing, the People’s Republic of China, 16-20 November 2009
Media fill Protocol
 Key element: acceptance criteria
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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Health Canada
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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
27 | Nanjing, the People’s Republic of China, 16-20 November 2009
Contamination
Contamination rate
 Contamination rate increasing during filling
Filling chronology (time or units)
This could indicate a
contamination originated in the
liquid media path (i.e. wrong
aseptic connection to the media
tank, contamination in the
recirculation loop). It is
important to keep under aseptic
conditions the media bulk tank
at the end of filling, while waiting
the media fill results, for
verifying this hypothesis
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
28 | Nanjing, the People’s Republic of China, 16-20 November 2009
Contamination
 Contamination rate decreasing during filling
Contamination rate
This could be indicative of a
contamination occurred during
the line set-up (stopper bowl /
guides, dosing pumps / needles),
partially or totally “washed out”
during filling.
Filling chronology (time or units)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
29 | Nanjing, the People’s Republic of China, 16-20 November 2009
Contamination
 Contamination spike during filling
Contamination rate
A spike (some contaminated
units in a short time interval
could be linked to a not correctly
performed critical intervention
during filling. For this reason it
is crucial to have an accurate
time traceability of the filled
units and the interventions
performed (videotaping the
filling operations can be helpful)
Filling chronology (time or units)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
30 | Nanjing, the People’s Republic of China, 16-20 November 2009
Contamination
 Contamination spike followed by an increase
Contamination rate
Similar to the previous example,
but in this case the wrong
intervention could have affected
the liquid path/loop (i.e. change
of media tank, replacement of a
pump / needle)
Filling chronology (time or units)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
31 | Nanjing, the People’s Republic of China, 16-20 November 2009
Contamination
Contamination rate
 Isolated events (few contaminated units per run)
Filling chronology (time or units)
Unfortunately, this occurrence is
the most common and also the
most difficult to be investigated.
If repeated on multiple runs, it
could indicate that this profile is
representative of the
“background noise” of the
process, so requiring radical
actions (i.e. change of
garments/dressing code,
disinfectants, air flow patterns)
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32 | Nanjing, the People’s Republic of China, 16-20 November 2009
Contamination
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33 | Nanjing, the People’s Republic of China, 16-20 November 2009
Contamination
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Conclusion
 Even if all media fills are negative in a Company, it does not
necessarily indicate that no products were ever possibly
contaminated because the frequency requirement of media fill testing
is so minimal that it is not statistically significant
 A robust media fill programme is a necessary step to validate process
 Media fill testing is just one part of a necessary overall quality
assurance program (HVAC qualification, cleaning procedure,
sanitization, personnel training, …)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
35 | Nanjing, the People’s Republic of China, 16-20 November 2009
l.stoppa@aifa.gov.it
36 |
Workshop on WHO prequalification requirements for reproductive health medicines,
Jakarta, October 2009
Reference documents
 WHO GMP guidelines – Technical Report series n. 937
 EU GMP guidelines, Part I annex 1 & 15
 ICH Q7A or EU GMP Part II chapter 13
 FDA Guidance for Industry: sterile drug products produced by
aseptic processing cGMP
 PIC/S Recommendations PI 007-5
 USP <797> ‘media fill testing’ / <71> ‘growth promotion test’
 EP 2.6.1 ‘sterility’
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors
37 | Nanjing, the People’s Republic of China, 16-20 November 2009