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Microbiological Testing
Objectives
To review microbiological environmental and
quality contol testing
Environmental Monitoring
Limits for Viable Particles
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
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
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
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.
Environmental Monitoring
Personnel
Environmental Monitoring
Levels and Trends
Environmental Monitoring
Disinfectants
Environmental Monitoring
Water
Environmental Monitoring
Suggested Microbial Limits (CFU/mL)
for facility water
Environmental Monitoring
Water
Compressed Air/Nitrogen/CO2
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
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)
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
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
Sterility Testing
Positive Controls
should be performed on all new products and when any changes
are made.
Should be repeated annually
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
Personnel
Should be appropriately trained and authorized to perform
testing
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
Original method
Chromogenic
Endpoint
Chromogenic
Kinetic
Turbidimetric
Semiquantitative
Quantitative
Quantitative
Quantitative
Requires
spectrophotometer
or plate reader
Requires
incubating plate or
tube reader
Requires
incubating plate or
tube reader
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Sensitive down
to 0.03 EU/ml
Sensitive down
to 0.1 EU/ml
Sensitive down
to .005 EU/ml
Sensitive down
to .001 EU/ml *
Questions?