Post on 05-Jun-2020
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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Microbiological Control Tests
Mrs Robyn Isaacson
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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Environmental Monitoring
Grade Air sample (CFU/m3)
Settle plates (90mm diameter)
(CFU/4hours)
Contact plates (55mm
diameter) (CFU/plate)
Glove print (5 fingers) (CFU/glove)
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
Limits for Viable Particles
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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)
• swabs and contact plates can be used
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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Environmental Monitoring
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
Suggested Microbial Limits (CFU/mL)
for facility water
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
29
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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
30
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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
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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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
32
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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
33
Other Microbiological Laboratory Issues
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.
Endotoxin Testing (3)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
34
Other Microbiological Laboratory Issues
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 spectrophotometrically and compared to a standard curve.
Endotoxin Testing (4)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
35
Other Microbiological Laboratory Issues
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.
• Can be kinetic or endpoint
Endotoxin Testing (5)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
36
Other Microbiological Laboratory Issues
* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)
Sensitive down
to .001 EU/ml *
Sensitive down
to .005 EU/ml
Sensitive down
to 0.1 EU/ml
Sensitive down
to 0.03 EU/ml
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Manually read and recorded
Requires
incubating plate or
tube reader
Requires
incubating plate or
tube reader
Requires
spectrophotometer
or plate reader
Simple Least expensive,
Requires 37 degree bath
Quantitative Quantitative Quantitative Semi-
quantitative
Turbidimetric Chromogenic
Kinetic
Chromogenic
Endpoint
Gel Clot
Endotoxin Testing (6)
Manufacture of sterile medicines – Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009
37
Questions?