Tes Bioburden

1. In September 2004, after 17 years in existence, the FDA
replaced older guidelines with an updated document,
‘Guidance for Industry – Sterile Drug Products Produced
by Aseptic Processing – Current Good Manufacturing
Practices’. The world was a very different place when the
original guidance was issued. AIDS was a relatively new
illness, few had heard about ‘Mad Cow Disease’, the
Internet was unheard of, and genetic fingerprinting was
not yet in use in criminal investigations. Throughout the
1980s and 1990s, technological advances continued to be
made, but the pharmaceutical industry kept a conservative
approach to their processes. Also in September 2004,
Europe made some changes to the ‘EC Guide to Good
Manufacturing Practice, Annex I, Manufacture of Sterile
Medicinal Products’. Together, these documents initiated
meetings, conferences, discussions and debate. Many areas
have seen little amendments or increased regulatory
clarity; however, aseptic process simulations have
undergone significant changes.
Within a short time of the new guidance being issued,
a UK vaccine manufacturer found itself in trouble with the
regulators. The production facility was shut down, causing
a huge shortfall in flu vaccines available to patients. This
was a timely reminder of the importance of process
control in critical manufacturing environments.
STERILITY
‘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. The
favoured method of production of sterile pharmaceutical
products includes a terminal sterilisation process, such as
autoclaving or irradiation. Since it is not practical to
examine every unit for confirmation of sterility, terminal
sterilisation processes use biological indicators (BIs) to
provide levels of sterility assurance. BIs are substrates
Biotechnology
90 Innovations in Pharmaceutical Technology
The Media Fill Approach: An Update
The design and execution of rugged process simulations – together with
the use of high quality growth media – will help ensure that the risk of
contamination of aseptic processes is kept within acceptable limits.
By Phil Smith at Oxoid Ltd
carrying high loads of resistant micro-organisms, at levels far
greater than the bioburden of the load being sterilised. If
everything on the BI is killed, it is reasonable to assume that
the load is also free of viable organisms and can be deemed
sterile. However, many therapeutic agents would not
withstand terminal sterilisation, so aseptic manufacture and
aseptic filling processes are required.
Aseptic processing used to produce sterile parenteral
drug products and Active Pharmaceutical Ingredients
(APIs) involves the handling of pre-sterilised products
in a highly controlled environment. Using the BI
correlation approach is not applicable here, as aseptic
processing involves ensuring a great deal of process
control, with sensitive handling of products until they
are sealed within their final containers.
All efforts are made to minimise the risk of
contamination:
N Filling and support areas are engineered
to minimise contamination
N Air in critical areas is supplied at point-of-use
as high-efficiency particulate air (HEPA) filtered,
laminar flow air at a velocity sufficient to sweep
particles away from the filling and closing areas
N Positive air pressure is used to prevent ingress
of airborne contamination: anything that can
be sterilised must be rendered sterile before it can
be taken into the clean area where the process
is performed
N Human intervention is kept to a minimum
N Cleaning is thorough and validated
N Disinfection practices are tight and validated
N Monitoring is done to prove the process
and environment are under control
Despite such measures, contamination is an ever-present
threat, since there will always be a risk that materials and
surfaces may carry organisms, and inefficiencies in air
Phil Smith is Pharmaceutical Marketing Manager at Oxoid Ltd (Basingstoke, UK). One of his responsibilities at Oxoid is to ensure
that the company’s products meet the changing needs of the pharmaceutical industry and the constraints imposed by regulatory
restrictions. Mr Smith has 15 years’ experience in the pharmaceutical and regulated industries, working largely on sterility issues and
aseptic applications. Prior to joining Oxoid in 2004, he worked for STERIS Corporation, where he was involved in developing their
disinfection and critical cleaning portfolio for the pharmaceutical industry in Europe, the Middle East and Africa.

2. 91Innovations in Pharmaceutical Technology
N Type of products being filled
N Lot/Batch sizes
N Container and closure configuration
N Fill volume
N Line speed
N Operator shifts and fatigue
N Filling line configuration
N Sterile hold times
N Number of units filled (production
vs simulation)
N Number and frequency of runs
N Acceptance criteria
N Run duration
N Interventions – atypical and typical
N Other elements that could impact upon
sterility assurance
Also, worst-case conditions are used in many forms of
validation, including process simulations. This does not
mean waiting for a tornado to rip off the cleanroom roof
before the media fill, but undertaking the simulation at
the limits of a normal process.
GROWTH MEDIA
The selection of the correct growth medium to be used
in the process simulation is a very important step. The
medium needs to support the growth of a wide variety of
micro-organisms, including aerobic bacteria, yeasts and
moulds. The broad range of organisms being looked
for is consistent with organisms tracked through the
firm’s environmental monitoring programme. The FDA
filtration may pose a risk. The largest source of potentially
viable contamination comes from people – the operators
running the filling process. Aseptic processing is a process
being operated in a controlled – but not sterile –
environment; the probability of non-sterility cannot be
calculated. The industry works to recognised, accepted
contamination levels, so the probability of viable
contamination is recognised and calculated. Routine
sampling for sterility testing is not sensitive enough to
detect such low level contamination. Sample numbers are
too small, and only gross contamination is likely to be
detected. Pharmaceutical manufacturers, therefore, need
other means of guaranteeing the quality of their product.
This is why process simulations (media fills) – supported
by environmental monitoring and other related processes
– are required. These are used to demonstrate control of
the process to the industry standard for allowable
contamination levels.
MEDIA FILLS
Media fills utilise culture media in place of product to
evaluate contamination levels. However, such media fills are
a snapshot in time, and subtle changes can incur changes in
contamination levels. It is therefore of paramount
importance that process simulations are designed to
accurately represent the aseptic process. The new FDA
guidelines pay particular attention to this aspect of aseptic
processing, and it is becoming an area requiring more work
and focus to satisfy the regulators. The media fill should be
designed to mimic, as closely as possible, the aseptic
processes used in practice. The media fill design is one
element within the overall considerations to be made in the
validation of an aseptic process. Areas of focus include:
N Facility and room design
N Design of the filling machine
N Process flow
N Heating, ventilation, and air-conditioning
design and validation
N Utility design and validation
N Response to deviations
N Trends in environmental monitoring data
N Contamination control programme
N Quality assurance and quality control systems
N Process simulations
N Personnel training and qualification
An appreciation of the many factors influencing the
validation programme allows a process simulation to be
effectively designed. Key elements in the simulation to be
taken into account include:

3. Guidance notes the use of soybean casein digest medium,
also known as ‘Tryptone Soya Broth’. With concerns
about prion contamination from components of animal
origin found within such media, it is vitally important
that the medium supplier can provide the necessary
certifications and documentation for confirming
materials are sourced from ‘BSE-free’ countries (such as
Oxoid Cold Filterable Tryptone Soya Broth – a highly
nutritious general purpose medium which is ideal for
microbiological media fills). An alternative approach
would be to use a medium derived from vegetable
materials such as Oxoid’s Cold Filterable Vegetable
Peptone Broth, which is a vegetable alternative and is
suitable for use in place of Tryptone Soya Broth.
The guidance also indicates that if the product is
being filled in anaerobic conditions, usually in a nitrogen
environment, an anaerobic medium (such as fluid
thioglycollate) is used.
As already noted, the new guidelines recommend that
media fills mimic the actual aseptic process as closely as
possible. One of the main areas where this is implicated is
where the culture medium is introduced into the process.
In the past, manufacturers have made up and sterilised the
medium outside of the controlled area, and introduced it
directly into the filling line. In order to more closely mimic
the process, the culture medium should be filtered into the
process – just as would occur to a liquid pharmaceutical
product. This creates several concerns:
N Dried culture media is usually supplied in a
non-sterile form and carries a high bioburden,
preventing it from being taken directly into a
controlled area. It would be preferential to source
media that has been irradiated.
N For liquid fills, many holding vessels upstream of
filtration do not have the capability to heat culture
media to a temperature adequate to dissolve the
powder into a solution. Even those that have this
ability take up time and energy in heating and
cooling. Sourcing a medium that dissolves at
ambient temperature would negate such problems.
N Mycoplasma can be a concern with culture media;
therefore assurance of irradication of mycoplasma
would be favoured.
N Broths traditionally used for media fills do not
have good filterability characteristics, and could
‘blind’ the sterilising filters. This would invalidate
the process simulation. It would be advantageous
to understand the filterability profile (such as Vmax
or Vcap) of the microbial growth medium, to ensure
filter sizing can tolerate the said medium.
PROCESS SIMULATIONS
Growth promotion testing must be undertaken on the
growth medium used for process simulations. There is
some confusing guidance as to when to perform this.
The FDA Guidance document does not make
specification on the timing of the test and the EU Annex
1 document does not even ask for a growth promotion
test. However, both the PICS (Pharmaceutical
Inspectorate Cooperation) and ISO documents ask that
growth promotion testing is performed upon conclusion
of the incubation period (usually 14 days). Whilst the
latter initially seems to be the more sensible option, it
also increases the holding time prior to release, as
product is waiting for the growth promotion tests to be
incubated and analysed.
Many pharmaceutical manufacturers prefer to run
growth promotion testing in parallel with the media
fill samples. Randomly removing samples from the
process simulation run has little basis for detecting
contamination. Contamination of the filling line being
challenged is a random event, and such samples are
unlikely to show all of the contamination present. In
order to meet the various regulatory guidance ‘half-way’,
a compromise would be to fill additional units at the end
of the process simulation, and use these for the growth
promotion test. These are then incubated under identical
conditions as the process simulation samples. This
approach both ensures that the process simulation units
and growth promotion test units are separated, and the
overall time of the process simulation project is reduced.
Any units that are incubated should be inspected
prior to incubation. Any defects that compromise the
container closure or non-integral units are rejected. All
rejections should be documented, with reasons for
rejection and the number of units rejected. Incubation is
then performed for 14 days at 20-350
C (+/-2.50
C). These
parameters have been accepted by the global regulatory
authorities and should allow the growth of bacteria,
yeast and moulds. 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. Also, isolates that are seen in the firm’s
environmental monitoring programme need to be
picked up by a media fill run, and data confirming this
should be made available.
Through the thorough design and execution of a
rugged process simulation, and the use of a high quality
growth promotion medium, meticulous challenge of the
aseptic process is achieved.
The author can be contacted at phil.smith@oxoid.com
92 Innovations in Pharmaceutical Technology