1. Microbiological Control Tests
Mrs Robyn Isaacson
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009

2. 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

3. 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
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009

4. 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

5. 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 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. Environmental Monitoring
Suggested Microbial Limits (CFU/mL) for facility water
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009

12. 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

13. 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

14. 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

15. 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 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

16. 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

17. 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

18. 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

19. Sterility Testing Methods are defined in Pharmacopoeia Media types
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

20. Sterility Testing Media Incubation Period
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

21. Sterility Testing Negative Contols Positive Test Controls
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

22. Sterility Testing Positive Controls Results
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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

31. 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
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.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009

34. 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 spectrophotometrically and compared to a standard curve.
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009

35. 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.
Can be kinetic or endpoint
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009

36. Other Microbiological Laboratory Issues
Endotoxin Testing (6)
* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)
Sensitive down
to .001 EU/ml *
to .005 EU/ml
to 0.1 EU/ml
to 0.03 EU/ml
Can be automated,
allows electronic
data storage
Manually read and recorded
Requires
incubating plate or tube reader
spectrophotometer
or plate reader
Simple Least expensive,
Requires 37 degree bath
Quantitative
Semi-
quantitative
Turbidimetric
Chromogenic
Kinetic
Endpoint
Gel Clot
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009

37. Questions?
Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009