EVALUASI SEDIAAN INJEKSI

Quality Control

• Samples for sterility testing should be representative

• From parts of the batch, most at risk
– Aseptic filling – at beginning and end of batch
filling, and after interruptions
– Heat sterilized – coolest part of the load
• Sterility of the batch ensured through validation
– Validated sterilization cycle
– Media fill
• Sterility test procedure as per pharmacopoeia, and
validated for each product
• Batch processing records, sterility testing records,
environmental records should be reviewed

2.1 -2.2
 Quality Control

• Endotoxin testing for injectable products

– Water for injection, intermediate and finished product
• Always for large volume infusion solutions
• Pharmacopoeia method, validated for each
product
• Failure of the test – investigation
• Corrective action

2.3
 Finishing of products

• Containers closed by means of validated methods

• Samples checked for integrity
• Maintenance of vacuum (where applicable) checked
• Parenteral products inspected individually
• Visual inspection under suitable and controlled
conditions:
– illumination and background
– eyesight checks of operators
– allowed frequent breaks
• Other methods:
– validated, and equipment performance checked
at intervals
– results recorded
11.1 – 11.3
 Microbiological Control Tests

Mrs Robyn Isaacson

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 Microbiological Testing
Objectives
• To review microbiological environmental and
quality contol testing
– Microbiological Environmental Monitoring
– Container integrity testing
– Pre-sterilization bioburden testing
– Media fill medium growth promotion
testing
– Sterility Testing
– Other microbiological laboratory issues

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 Environmental Monitoring
Limits for Viable Particles
Grade Air sample Settle plates (90mm Contact plates Glove print
(CFU/m3) diameter) (55mm (5 fingers)
(CFU/4hours) diameter) (CFU/glove)
(CFU/plate)
A <3 <3 <3 <3
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -

Table 3
– These are average values
– Individual settle plates may be exposed for less than 4 hours

• Values are for guidance only - not intended to represent specifications

• Levels (limits) of detection of microbiological contamination should be
established for alert and action purposes and for monitoring trends of
air quality in the facility

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 Environmental Monitoring
Methods
• Surface monitoring
– Product contact surfaces, floors, walls, and equipment should be
tested on a regular basis

– Touch plates - used for flat surfaces

• sample area of 25cm2
• medium protrudes above sides
• medium contains neutralisers

– Surface Swabs - used for irregular surfaces

• area approx 25cm2 is swabbed
• qualitative or quantitative

Surface monitoring should be performed at conclusion of aseptic

processing (to minimise risk of contaminating critical surfaces during
production)

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 Environmental Monitoring
Methods
– Active Air Monitoring
• impaction, centrifugal and membrane (or gelatin) samplers
• a certain volume of air is sampled (volume and location
should be meaningful)
• instruments should be calibrated
– Passive Air Monitoring
• Settle plates exposed for 30-60 minutes (longer may result in
agar drying out) and replaced for duration of filling
• Media should be capable of growing a range of bacteria and
moulds (e.g. Soybean Casein Digest Agar (SCDA)/Trypticase
Soy Agar (TSA)
• Should consider use of medium specific for moulds if
shown to be a problem in the environment
• Only give qualitative or semi-quantitative results
• Data generated considered in combination with active air
sampling results

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 Environmental Monitoring
Sampling Locations
– Should be based on risk of microbiolgical
contamination
– Should be clustered around areas where product or
components are exposed e.g.
• at filling heads on filling lines
• loading of product into lyophilizers
• stopper bowls
• where aseptic connections are made
• where there are high levels of operator activity (but
without impacting on production)
– Lower grade areas are monitored less frequently and
trends monitored

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 Environmental Monitoring
Personnel
• For each session - gloves
should be monitored (but not
immediately after sanitising!)
• Periodic sampling for other
locations on gown
• Clean room operators should
be regularly validated to
demonstrate that they do not
contaminate gowns during
gowning up (gowning
qualification)

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 Environmental Monitoring
Levels and Trends
• Limits in Code of GMP are for guidance only
• Manufacturers should set alert and action limits
appropriate to the location
• Individual results should be considered - averaging can
mask unnacceptable localised conditions
• There should be written procedures (SOPs) for data review
and action to be taken if limits are exceeded
• Trend Reports
– Short and long term reports on environmental and
personnel monitoring
– Results of EM should be included in Batch Records
– Significant changes in microbial flora should be
considered

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 Environmental Monitoring
Disinfectants
• Suitablility, efficacy, limitations of disinfectants and procedures
should be assessed
– minimum contact time established
• Disinfectants in Grade A/B areas should be sterile, supplied in
sterile containers and used for a defined period

• Should be shown to be effective against facility microbial flora

• Should be sporicidal (if spores found in the environment) and for
“spraying in” of components and equipment

• Disinfection SOPs should include sufficient detail to enable

reproducibility
– preparation, work sequence, contact time
• Organisms identified from adverse trends should be tested for
their sensitivity to the disinfectants used

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 Environmental Monitoring
Water
• microbiological quality of water very important
• Should be an extensive, comprehensive water testing programme
• Feed water, pre-treatment, reverse osmosis (RO), deionized (DI),
purified/highly purified and water for injection (WFI) should be
tested

• Alert and Action limits set by manufacturer (with action to be

taken if limits are exceeded)
• WHO recommendations (next slide)
• For purified/highly purified water and WFI, limits defined in
pharmacopoeia
– Purified <100CFU/mL
– Highly purified and WFI 10CFU/100mL (but is usually kept at
high temperatures)

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 Environmental Monitoring
Suggested Microbial Limits (CFU/mL) for
facility water

Sampling Location Target Alert Action

Raw water 200 300 500
Post multimedia filter 100 300 500
Post softener 100 300 500
Post activated carbon filter 50 300 500
Feed to RO 20 200 500
RO permeate 10 50 100
Point of use 1 10 100

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 Environmental Monitoring
Water
• Water should also be tested for presence of coliforms and/or
pseudomonads if appropriate (may cause biofilm)
• Water used for parenterals should be tested for pyrogens
– limit is not more than 0.25 EU/mL
• Water should be tested using R2A agar (low nutrient for the
recovery of water borne organisms) incubated for at least 5 days
at 30-35°C
• Sampling procedures should follow those used in production

Compressed Air/Nitrogen/CO2
• Should be tested for non-viables and viables
• Pressure reduction orifices should be used to provide a steady
stream of air, validation of media should be ensured with
consideration of validation

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 Container Integrity Testing
• Integrity of container/closure system

– is intitally validated by filling container with

sterile growth medium then inserting container
in broth containing 106 CFU/mL of suitable
microorganism
– containers sealed under a vacuum should be
periodically tested to demonstrate that vacuum
is maintained over shelf life
– procedures in place to detect faulty containers
during manufacture
– operators involved in visual inspection should
have frequent breaks and regular eye-sight
tests
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 Bioburden/IPC Testing
• Should be written procedures for pre-sterilization
bioburden, in-process control and raw material
testing.
• Method should be validated for the recovery of
low numbers of organisms.
• Use of anaerobic medium should be considered if
shown to be present in environment.
• Target, alert and action limits should be
documented and include action taken if limits
exceeded.

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 Growth Promotion Testing
• Media used for microbiolgical testing should be tested for
its ability to support microbial growth
– media used for media fills should be able to support the
growth of a wide range of microorganisms (bacteria and
moulds)
– Soybean Casein Digest Medium is usually used. An
anaerobic medium may also be substituted occasionally
if environmental monitoring indicates presence
– After the media fill has been completed, it is important
to demonstrate that the media would have been able to
support the growth of organisms if they had been
present
– containers with media should be inoculated with 10-100
CFU of organims such as Bacillus subtilis,
Staphylococcus aureus, Candida albicans, Aspergillus
niger. Environmental isolates should also be included

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 Growth Promotion Testing
Media
– The inoculated media should be capable of showing
growth within 3 days of incubation
– Media used in environmental monitoring should also
be tested for its growth promoting properties.
Validation of recovery of organisms under test
conditions should be carried out to demonstrate
neutralization of disinfectant residuals (media should
contain neutralisers).
– Media purchased externally should also be tested
– Media used for media fills and environmental
monitoring should be pre-incubated to demonstrate
“sterility” prior to use
– Media should have a validated shelf life

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 Sterility Testing
• Sterility test is a quality control test used as part of
product release for product required to be sterile
– Has significant statistical limitations - will really only
detect gross contamination
• Sampling
– No of containers and volume to be tested defined in
Pharmacopoeia
– Samples from aseptically manufactured product
should be taken from beginning, middle and end of
batch fill and also after interventions and stoppages
– Samples from terminally sterilized product should be
taken from previously identified cool spots within
load
– Sampling should be sufficient to allow for retests if
needed

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 Sterility Testing
• Facilities
– Sterility testing should be carried out under the same
conditions as aseptic manufacture
• In a Grade A laminar air flow cabinet in a Grade B
background (may also be carried out in an
isolator)
• Air supply through HEPA filters, pressures should
be monitored and alarmed
• Access to area should be through airlocks
• Operators should be appropriately gowned is
sterile garments
• Operators should be appropriately trained and
validated
• Appropriate cleaning, sanitisation and disinfection
procedures should be in place
• Environmental monitoring should be conducted

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 Sterility Testing
• Methods are defined in Pharmacopoeia
– membrane filtration is the preferred method if product is
filterable
– direction innoculation is alternative
• Media types
– Soybean Casein Digest medium (SCD), (also knows as
Trypticase Soy Broth(TSB)) and Fluid Thioglycollate medium
(FTM) is usually used (to detect aerobic and anaerobic
organisms)
– validation studies should demonstrate that the media are
capable of supporting growth of a range of low numbers of
organisms in the presence of product. May need to
incorporate inactivators
• growth should be evident after 3 days (bacteria), 5 days
(moulds)
– media may be purchased or made in-house using validated
sterilization procedures

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 Sterility Testing
• Media
– should be tested for growth promoting qualities prior to
use (low number of organisms)
– should have batch number and shelf life assigned
• Incubation Period
– At least 14 days incubation
– 20-25°C for SCD/TSB, 30-35°C for FTM
– Test containers should be inspected at intervals
– temperatures should be monitored and temperature
monitoring devices should be calibrated
– if product produces suspension, flocculation or deposit
in media, suitable portions (2-5%) should be transferred
to fresh media, after 14 days, and incubated for a futher
7 days

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 Sterility Testing
• Negative Contols
– media should be incubated for 14 days prior to use, either a
portion or 100% of batch (may be done concurrently with
test)
– negative product controls - items similar in type and
packaging to actual product under test should be included
in each test session
• facilitate interpretation of test results
• negative control contamination rate should be
calculated and recorded
• Positive Test Controls
– bactiostasis/fungistasis test
• should demonstrate that media are capable of
supporting growth of a range of low numbers of
organisms in the presence of product. May need to
incorporate inactivators
– growth should be evident after 3 days (bacteria), 5
days (moulds)
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 Sterility Testing
• Positive Controls
• should be performed on all new products and when
any changes are made.
• Should be repeated annually
– Stasis test recommended particularly for product with
antibiotics or preservatives or slow release tested by
direct innoculation
• demonstrates that media can support growth at the
end of the incubation period and has not been
affected by product
• Results
– Any growth should be identified (genotypic)
– Automated/Semi-automated systems used for
identification should be periodically verified using
reference strains

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 Sterility Testing
• Interpretation and Repeat Tests
– No contaminated units should be found
– A test may only be repeated when it can be
demonstrated that the test was invalid for causes
unrelated to the product being examined
– European Pharmacopoeia criteria
(a) the data of the micro monitoring of the sterility test
facility show a fault
(b) a review of the testing procedure used during the
test in question reveals a fault
(c) microbial growth is found in negative controls
(d) after determination of the identity of the
microorganisms isolated from the test, the growth of
this species or these species may be ascribed
unequivocally to faults with respect to the material
and/or technique used in conducting the sterility test
procedure
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 Sterility Testing
• Interpretation and Repeat Testing
• When conditions (a), (b) or (c) apply the test should
be aborted
• If a stasis test performed at the end of the test shows
no growth of challenge organisms, this also
invalidates the test
• For conditions (d) to apply must demonstrate that
the orgamisms isolated from the sterility test is
identical to an isolate from materials (e.g. media)
and/or the environment
– must use genotypic identification methods
– Repeat test is carried out with same number of samples
as first test
– Any contamination detected in repeat test, product does
not comply

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 Other Microbiological Laboratory Issues
Reference Culture Collections
• Reference cultures may be used for
• Quality contol of media
• Test method validation
• Control of test reagents
• Must remain genetically stable to retain characteristics
for which they have been selected.
• Cultures of microorganisms tend to undergo variation/
genetic change that can affect characteristics of a
culture - unsuitable for further use.
• Probability of variation/genetic change increases with
frequency of repeated subculture of reference culture –
working culture must be no more than 5 generations
removed from original source culture.

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 Other Microbiological Laboratory Issues
Reference Cultures (2)
• Laboratory must have a system for preserving
and maintaining reference cultures with their
original characteristics.
• Laboratory should:
– maintain suitable reference cultures for QC of
culture media and test reagents and for test
method validation;
– ensure reference cultures are traceable to a
recognised culture collection eg. ATCC, NCTC;
– ensure reference cultures are uniquely
identified within laboratory, with traceability to
recognised culture collection.

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 Other Microbiological Laboratory Issues
Reference Cultures (3)
• Lab should have documented procedures:
– that maintain hierarchical control of reference cultures
(ie. master, stock & working cultures);
– for purchase, preservation, maintenance, identification
and frequency of subculturing of reference cultures;
– that prevent use of working cultures as replacements
for depleted stock and/or master cultures.
• Maintain records for each reference culture:
– identity, source and history and date of receipt of
master culture;
– resuscitation, preservation, maintenance and storage
conditions for master, stock and working cultures;
– results of purity and identification tests for master
and/or stock cultures; and
– dates of preparation of stock and working cultures.

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 Other Microbiological Laboratory Issues

QC of Culture Media

• Media other than sterility testing media and media fill media
must be subject to quality contol
• quantitative or semi-quantitative method/s to assess
growth promotion/fertility
• use of positive and negative controls for selective and/or
dirrerential culture media
• different levels of QC required dependent on whether
culture is

– manufactured in house (every batch should be tested)

– purchased ready to use (supplier tests media with testing
periodically verified in house)

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 Other Microbiological Laboratory Issues

QC of Culture Media (2)

• Laboratory should:

– have documented procedures for preparation, QC,

release and storage of culture media;
– have validated shelf life of culture media under
normal storage conditions;
– maintain records of preparation and QC of individual
batches of culture media;
– ensure that records of microbiological QC
performance testing are traceable to batch
preparation records; and
– that microbiological QC performance test results are
assessed against acceptance/rejection criteria.

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 Other Microbiological Laboratory Issues

Sterilization processes for Culture Media

• Sterilzation process for culture media should be
validated and monitored using same procedures
as for sterilization of product
Equipment Calibration and Checks
• Laboratory equipment (e.g. pipettes, balances,
incubators, refrigerators, thermometers,
autoclaves, laminar flow workstations etc) should
be calibrated and recalibrated and routinely
monitored (where appropriate)
Personnel
• Should be appropriately trained and authorized to
perform testing
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 Other Microbiological Laboratory Issues

Testing of Biological Indicators

• if tested in-house the method should include a heat-shock
step (this verifies that the indicators do actually contain
spores and not vegetative organisms)
• BIs should occasionally be tested in house to verify the
suppliers count
Endotoxin Testing
• Parenteral products should be free from endotoxin
• Endotoxin is a lipopolysaccharide present in the cell wall of
gram negative bacteria which can cause fever if introduced
into the body
• Raw materials, WFI used in manufacture and some finished
product must be tested for endotoxin

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 Other Microbiological Laboratory Issues

Endotoxin Testing (2)

• LAL (Limulus Amebocyte Lysate) test is used for detecting
endotoxin (previously a rabbit test)
– based on clotting reaction of horseshoe crab blood to
endotoxin

• Types of LAL test

– Gel Clot
– Turbidimetric
– Colorimetric

• Equipment used in test must be endotoxin free

• Validation of accuracy and reliability of the method for each

product is essential

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 Other Microbiological Laboratory Issues

Endotoxin Testing (3)

Gel Clot Method

• Original method
• The official “referee
test”
• The specimen is
incubated with LAL of
a known senstivity.
• Formation of a gel clot
is positive for
endotoxin.
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 Other Microbiological Laboratory Issues

Endotoxin Testing (4)

Turbidimetric Method

• A kinetic method
• The specimen is
incubated with LAL
and either the rate of
increase in turbidity
or the time taken to
reach a particular
turbidity is measured
spectrophotometricall
y and compared to a
standard curve.

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 Other Microbiological Laboratory Issues

Endotoxin Testing (5)

Colorimetric Method

• Endotoxin catalyzes
the activation of a
proenzyme in LAL
which will cleave a
colorless substrate to
produce a colored
endproduct which can
be measured
spectrophotmetrically
and compared to a
standard curve.
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 Other Microbiological Laboratory Issues

Endotoxin Testing (6)

Gel Clot Chromogenic Chromogenic Turbidimetric

Endpoint Kinetic
Semi- Quantitative Quantitative Quantitative
quantitative
Simple Least Requires Requires Requires
expensive, spectrophotometer incubating plate or tube incubating plate or tube
Requires 37 degree or plate reader reader reader
bath
Can be automated, Can be automated, Can be automated,
Manually read and
recorded allows electronic allows electronic allows electronic
data storage data storage data storage
Sensitive down Sensitive down Sensitive down Sensitive down
to 0.03 EU/ml to 0.1 EU/ml to .005 EU/ml to .001 EU/ml *
* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)

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