microbiological quality control

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

*Microbiological Control TestsMrs Robyn Isaacson



*Microbiological TestingObjectivesTo review microbiological environmental and quality contol testingMicrobiological Environmental MonitoringContainer integrity testingPre-sterilization bioburden testingMedia fill medium growth promotion testingSterility TestingOther microbiological laboratory issues



*Environmental MonitoringTable 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 facilityLimits for Viable Particles


Grade

Air sample (CFU/m3)

Settle plates (90mm diameter) (CFU/4hours)

Contact plates (55mm diameter) (CFU/plate)

Glove print (5 fingers) (CFU/glove)

A

< 3

< 3

< 3

< 3

B

10

5

5

5

C

100

50

25

-

D

200

100

50

-


*Environmental MonitoringMethodsSurface monitoringProduct contact surfaces, floors, walls, and equipment should be tested on a regular basisTouch plates - used for flat surfacessample area of 25cm2medium protrudes above sidesmedium contains neutralisers

Surface Swabs - used for irregular surfacesarea approx 25cm2 is swabbedqualitative 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



*Environmental MonitoringMethodsActive Air Monitoringimpaction, centrifugal and membrane (or gelatin) samplersa certain volume of air is sampled (volume and location should be meaningful)instruments should be calibratedPassive Air MonitoringSettle plates exposed for 30-60 minutes (longer may result in agar drying out) and replaced for duration of fillingMedia 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 resultsData generated considered in combination with active air sampling results



*Environmental MonitoringSampling LocationsShould be based on risk of microbiolgical contaminationShould be clustered around areas where product or components are exposed e.g.at filling heads on filling linesloading of product into lyophilizersstopper bowlswhere aseptic connections are madewhere there are high levels of operator activity (but without impacting on production)Lower grade areas are monitored less frequently and trends monitored



*Environmental MonitoringPersonnelFor each session - gloves should be monitored (but not immediately after sanitising!)Periodic sampling for other locations on gownClean room operators should be regularly validated to demonstrate that they do not contaminate gowns during gowning up (gowning qualification)



*Environmental MonitoringLevels and TrendsLimits in Code of GMP are for guidance onlyManufacturers should set alert and action limits appropriate to the locationIndividual results should be considered - averaging can mask unnacceptable localised conditionsThere should be written procedures (SOPs) for data review and action to be taken if limits are exceededTrend ReportsShort and long term reports on environmental and personnel monitoringResults of EM should be included in Batch RecordsSignificant changes in microbial flora should be considered



*Environmental MonitoringDisinfectantsSuitablility, efficacy, limitations of disinfectants and procedures should be assessedminimum contact time establishedDisinfectants in Grade A/B areas should be sterile, supplied in sterile containers and used for a defined periodShould be shown to be effective against facility microbial floraShould be sporicidal (if spores found in the environment) and for spraying in of components and equipmentDisinfection SOPs should include sufficient detail to enable reproducibilitypreparation, work sequence, contact timeOrganisms identified from adverse trends should be tested for their sensitivity to the disinfectants used



*Environmental MonitoringWatermicrobiological quality of water very importantShould be an extensive, comprehensive water testing programmeFeed water, pre-treatment, reverse osmosis (RO), deionized (DI), purified/highly purified and water for injection (WFI) should be testedAlert 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 pharmacopoeiapurified


*Environmental MonitoringSuggested 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


*Environmental MonitoringWaterWater should also be tested for presence of coliforms and/or pseudomonads if appropriate (may cause biofilm)Water used for parenterals should be tested for pyrogenslimit is not more than 0.25 EU/mLWater should be tested using R2A agar (low nutrient for the recovery of water borne organisms) incubated for at least 5 days at 30-35CSampling procedures should follow those used in production

Compressed Air/Nitrogen/CO2Should be tested for non-viables and viablesPressure reduction orifices should be used to provide a steady stream of air, validation of media should be ensured with consideration of validation



*Container Integrity TestingIntegrity 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 microorganismcontainers sealed under a vacuum should be periodically tested to demonstrate that vacuum is maintained over shelf lifeprocedures in place to detect faulty containers during manufactureoperators involved in visual inspection should have frequent breaks and regular eye-sight tests



*Bioburden/IPC TestingShould be written procedures for pre-sterilization bioburden, in-process control and raw material testingmethod should be validated for the recovery of low numbers of organismsuse of anaerobic medium should be considered if shown to be present in environmenttarget, alert and action limits should be documented and include action taken if limits exceeded



*Growth Promotion TestingMedia used for microbiolgical testing should be tested for its ability to support microbial growthmedia 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 presenceAfter 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 TestingMediaThe inoculated media should be capable of showing growth within 3 days of incubationMedia 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 testedMedia used for media fills and environmental monitoring should be pre-incubated to demonstrate sterility prior to useMedia should have a validated shelf life



*Sterility TestingSterility test is a quality control test used as part of product release for product required to be sterileHas significant statistical limitations - will really only detect gross contaminationSamplingNo of containers and volume to be tested defined in PharmacopoeiaSamples from aseptically manufactured product should be taken from beginning, middle and end of batch fill and also after interventions and stoppagesSamples from terminally sterilized product should be taken from previously identified cool spots within loadSampling should be sufficient to allow for retests if needed



*Sterility TestingFacilitiesSterility testing should be carried out under the same conditions as aseptic manufactureIn 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 alarmedAccess to area should be through airlocksOperators should be appropriately gowned is sterile garmentsOperators should be appropriately trained and validatedAppropriate cleaning, sanitisation and disinfection procedures should be in placeEnvironmental monitoring should be conducted



*Sterility TestingMethods are defined in Pharmacopoeiamembrane filtration is the preferred method if product is filterabledirection innoculation is alternativeMedia typesSoybean 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 inactivatorsgrowth should be evident after 3 days (bacteria), 5 days (moulds)media may be purchased or made in-house using validated sterilization procedures



*Sterility TestingMediashould be tested for growth promoting qualities prior to use (low number of organisms)should have batch number and shelf life assignedIncubation PeriodAt least 14 days incubation20-25C for SCD/TSB, 30-35C for FTMTest containers should be inspected at intervalstemperatures should be monitored and temperature monitoring devices should be calibratedif 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 TestingNegative Contolsmedia 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 sessionfacilitate interpretation of test resultsnegative control contamination rate should be calculated and recordedPositive Test Controlsbactiostasis/fungistasis testshould 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 inactivatorsgrowth should be evident after 3 days (bacteria), 5 days (moulds)



*Sterility TestingPositive Controlsshould be performed on all new products and when any changes are made.Should be repeated annuallyStasis test recommended particularly for product with antibiotics or preservatives or slow release tested by direct innoculationdemonstrates that media can support growth at the end of the incubation period and has not been affected by productResultsAny growth should be identified (genotypic)Automated/Semi-automated systems used for identification should be periodically verified using reference strains



*Sterility TestingInterpretation and Repeat TestsNo contaminated units should be foundA test may only be repeated when it can be demonstrated that the test was invalid for causes unrelated to the product being examinedEuropean 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 TestingInterpretation and Repeat TestingWhen conditions (a), (b) or (c) apply the test should be abortedIf a stasis test performed at the end of the test shows no growth of challenge organisms, this also invalidates the testFor 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 environmentmust use genotypic identification methodsRepeat test is carried out with same number of samples as first testAny contamination detected in repeat test, product does not comply



*Other Microbiological Laboratory IssuesReference Culture CollectionsReference cultures may be used forQuality contol of mediaTest method validationControl of test reagentsMust 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.



*Other Microbiological Laboratory IssuesReference 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.



*Other Microbiological Laboratory IssuesReference 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; anddates of preparation of stock and working cultures.



*Other Microbiological Laboratory IssuesQC of Culture MediaMedia other than sterility testing media and media fill media must be subject to quality contolquantitative or semi-quantitative method/s to assess growth promotion/fertilityuse of positive and negative controls for selective and/or dirrerential culture mediadifferent levels of QC required dependent on whether culture ismanufactured in house (every batch should be tested)purchased ready to use (supplier tests media with testing periodically verified in house)



*Other Microbiological Laboratory IssuesQC 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; andthat microbiological QC performance test results are assessed against acceptance/rejection criteria.



*Other Microbiological Laboratory IssuesSterilization processes for Culture Mediasterilzation process for culture media should be validated and monitored using same procedures as for sterilization of product

Equipment Calibration and ChecksLaboratory equipment (e.g. pipettes, balances, incubators, refrigerators, thermometers, autoclaves, laminar flow workstations etc) should be calibrated and recalibrated and routinely monitored (where appropriate)

PersonnelShould be appropriately trained and authorized to perform testing



*Other Microbiological Laboratory IssuesTesting of Biological Indicatorsif 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 TestingParenteral products should be free from endotoxinEndotoxin is a lipopolysaccharide present in the cell wall of gram negative bacteria which can cause fever if introduced into the bodyRaw materials, WFI used in manufacture and some finished product must be tested for endotoxin



*Other Microbiological Laboratory IssuesEndotoxin 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 endotoxinTypes of LAL testGel ClotTurbidimetricColorimetric

Equipment used in test must be endotoxin free

Validation of accuracy and reliability of the method for each product is essential



*Other Microbiological Laboratory IssuesGel Clot MethodOriginal method

The official referee test

The specimen is incubated with LAL of a known senstivity. Formation of a gel clot is positive for endotoxin.

Endotoxin Testing (3)



*Other Microbiological Laboratory IssuesTurbidimetric 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.

Endotoxin Testing (4)



*Other Microbiological Laboratory IssuesColorimetric 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 endpointEndotoxin Testing (5)



*Other Microbiological Laboratory IssuesEndotoxin Testing (6)



*Questions?


microbiological quality control

Documents

sfda gmp inspectors

microbiological contamination

environmental monitoringtable

regular basistouch plates

sampled volume

diameter cfuplateglove

quantitativesurface

critical surfaces