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 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

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

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

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

Environmental Monitoring Suggested Microbial Limits (CFU/mL) for facility water 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 30-35°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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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