Microbiological Control Tests: Mrs Robyn Isaacson

Microbiological Control Tests
Mrs Robyn Isaacson
1 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

inspectors - Nanjing, November 2009
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

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

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

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

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

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)

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

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

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)

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

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

Container Integrity Testing
• Integrity of container/closure system
– is intitally validated by filling container with sterile growth
medium then inserting container in broth containing 10 6
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

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

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

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

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

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

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

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

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)

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

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
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

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.

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.

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.

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)

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.

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

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

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


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.

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.


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

Gel Clot Chromogenic Chromogenic Turbidimetric

Endpoint Kinetic
Semi- Quantitative Quantitative Quantitative
quantitative
Simple Least Requires Requires Requires
expensive, spectrophotometer incubating plate or incubating plate or
Requires 37 degree or plate reader tube reader tube reader
bath
Manually read and Can be automated, Can be automated, Can be automated,
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)

Questions?


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Microbiological Control Tests

Mrs Robyn Isaacson

1 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

2 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

3 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

– Surface Swabs - used for irregular surfaces

• swabs and contact plates can be used

4 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

5 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

6 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

7 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

8 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

9 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

10 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Suggested Microbial Limits (CFU/mL)

11 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

12 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

13 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

14 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

15 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

16 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

17 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

18 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

19 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

20 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

21 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

22 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

24 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

25 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Reference Cultures (2)

26 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Reference Cultures (3)

27 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

28 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

QC of Culture Media (2)

29 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Sterilization processes for Culture Media

Equipment Calibration and Checks

30 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Testing of Biological Indicators

31 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Endotoxin Testing (2)

• Equipment used in test must be endotoxin free

• Validation of accuracy and reliability of the method for each

32 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Endotoxin Testing (3)

• The official “referee test”

• The specimen is incubated

33 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Endotoxin Testing (4)

• The specimen is incubated

34 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Endotoxin Testing (5)

• Endotoxin catalyzes the

• Can be kinetic or endpoint

35 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

Endotoxin Testing (6)

Gel Clot Chromogenic Chromogenic Turbidimetric

Sensitive down Sensitive down Sensitive down Sensitive down

36 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

37 Manufacture of sterile medicines – Advanced workshop for SFDA GMP

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