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Microbiological Control Tests: Mrs Robyn Isaacson
Microbiological Control Tests: Mrs Robyn Isaacson
Microbiological Control Tests: Mrs Robyn Isaacson
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
Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
Environmental Monitoring
Limits for Viable Particles
Grade Air sample (CFU/m3) Settle plates (90mm Contact plates diameter) (55mm (CFU/4hours) diameter) (CFU/plate) Glove print (5 fingers) (CFU/glove)
A B C D
<3 5 50 100
<3 5 25 50
<3 5 -
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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
Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors swabs - Nanjing, and November contact 2009 plates can be used
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
Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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
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
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
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
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
Environmental Monitoring
Suggested Microbial Limits (CFU/mL) for facility water
Sampling Location
Raw water Post multimedia filter Post softener Post activated carbon filter Feed to RO RO permeate Point of use
Target
200 100 100 50 20 10 1
Alert
300 300 300 300 200 50 10
Action
500 500 500 500 500 100 100
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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 30-35C 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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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
At least 14 days incubation 20-25C for SCD/TSB, 30-35C 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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
Personnel
Should be appropriately trained and authorized to perform testing
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
Chromogenic Endpoint
Quantitative Requires spectrophotometer or plate reader Can be automated, allows electronic data storage Sensitive down to 0.1 EU/ml
Chromogenic Kinetic
Quantitative Requires incubating plate or tube reader Can be automated, allows electronic data storage Sensitive down to .005 EU/ml
Turbidimetric
Quantitative Requires incubating plate or tube reader Can be automated, allows electronic data storage Sensitive down to .001 EU/ml *
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009
Questions?
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Manufacture of sterile medicines Advanced workshop for SFDA GMP inspectors - Nanjing, November 2009