國(guó)外藥典關(guān)于無(wú)菌檢驗(yàn)的規(guī)范

1、1sterility testingprinciple & regulations : ep 5.5 & usp29boualem rafa -technology manager europewhat is sterility?definitions?classical:|the completeabsence of life|absoluteterm?practical:|absence of living material|demonstratedby growthand reproduction?f&b industry define ,commercial s

2、terility“:|products not containing organisms of a kind or a number likely to cause harm under conditions of its anticipated use.sterility testingwhat is the test for sterility ?presence / absence test?pass/fail test result?represents one set of data which contributes to the decision of whether or no

3、t the product lot meets the stated claims.?not intended as a sole product release test2pharmaceutical microbiologymicrobiologylaboratoryin-processprograms in pharmaceutical microbiologywaterenvironmentalmonitoringfinal productrawmaterialssupport dataproduct releasesterilitytestingsterility testingli

4、mitations?not for viruses or mycoplasma?destructivetest?long incubation period: 14 days?only 2 media to enable growth of bacteria, fungi and yeasts?probability-based|not statisticallyvalid, notrepresentative|lot homogenity: a lot is defined as homogeneous closed set, prepared in such a way that the

5、risk of contamination is the same for each of the component units of this lot.|the probability to discover microorganisms increases with their number in the sample and varies according to the capacity of growth of the different typessterility testinglimitations?ep 5.5|“the probability of detecting v

6、ery low levels of contamination even when it is homogeneous throughout the batch is very low.”|? a satisfactoryresult only indicatesthatno contaminatingmicro-organismhas been foundin the sampleexaminedin the condition of the test?validate and perform the test for sterility in the best conditions in

7、order to get the most reliable data3sterility testingwhy to conduct a sterility test ?regulations and global qualityapproach?cgmp requirements|“for each batch of drug product purporting to be sterile and/or pyrogen free , there shall be appropriate labtesting to determine conformance to such require

8、ments ”21cfr211.167 (a)?ep 5.5|,the test is applied to substances, preparationsorarticles which, accordingto the pharmacopoeia, arerequired to be sterile.“sterility testingwhy to conduct a sterility test ?european pharmacopoeia(ep 5.5)?us pharmacopoeia(usp29-nf24)?japanesepharmacopoeia(jp)?internati

9、onal commityharmonisation (since1990)?regulatory guidelinesor guidance|fda: food and drug administration|emea: european agency for the evaluation of medicinal products |cfr: code of federalregister(21cfr 211.167 -a - ) |pda: parenteraldrug administration|aami: association for the advancement of medi

10、cal instrumentation |iso: international organization for standardization |who: world healthorganizationsterility testinghow to perform a sterility test?ep, usp and otherpharmacopeia?international harmonisation : pharmacopeialdiscussion group (pdg) |set up in 1990|with the pharmacopoeias of europe, j

11、apan and the united states.?this group meets regularly (twice a year), with the location of the meetings rotating among europe, japan and the united states.?monographs and general methods of analysis proposed by national associations of manufacturers of pharmaceutical products are selected for conve

12、rgence and harmonisation among the three pharmacopoeias. ?each pharmacopoeia is therefore responsible for a programme of international harmonisation. 4sterility testingpharmacopoeia structure?general chapters |are the source of many of the official test methods and procedures that are specified in t

13、he monographs.?monographs|describe active substances and preparations that are within the pharmacopoeia. |monographs show the chemical structure, and packaging and storage, identification, standards and tests for purity and content, and other pertinent information.sterility testingusp 29-nf24 2st su

14、p. valid from 1.8.2006 until 31.12.2006?general chapters| antimicrobial effectiveness testing| biological indicators| microbial limit tests| sterility testsharmonized !| microbiological evaluation of clean rooms and other controlled environments| sterility testing-validation of isolator systems| val

15、idation of microbial recovery from pharmacopeial articles| water for pharmaceutical purposes?monographs|water for injections, bacteriostaticwfi, sterile wfi|purified water, sterile purified watermandatory sterility testingep 5.5 valid from 1.7.2006 until 31.12.2006?general chapters|2.6.1 sterilityha

16、rmonized !|2.6.8 pyrogens|2.6.12 total viable aerobic count |2.6.13 microbiological examination of non sterile product|2.6.14 bacterial endotoxins?monographs|water for injections |water, purified |water, highly purified5sterility testinghistory?1957: membrane filtration (mf) for products with antimi

17、crobial activity (fda)?1966: membrane filtration method improvement with filtration manifold (fda). usage of rinsing fluids?1974: closed concept system steritest(millipore) ?since the early 80s: first isolators (la calhne) for sterility testing?1983: specificmembrane(durapore, millipore) forantibiot

18、ics?1998: incubationtime for the test increasedfrom7 to 14 days (4thedition ep)sterility testingregulatory requirements : ep 5.5 / usp29?sterility testing requirements|environment|media|test method|minimum quantityto use for eachmedium / minimum numberof items to betested|sterilebuffers|validation t

19、est : bacteriostasis& fungistasis|observation and interpretationsterility testingregulatory requirements : general considerations?operator training|“because sterility testing is a very exacting procedure, where asepsis of the procedure must be ensured for a correct interpretation of results, it

20、is important that personnel be properly trained and qualified”(usp 29)?usp microbial best laboratory practices“ training curriculashould be established for each laboratory staff member specific for their job function. they should not independently conduct a microbial test until they are qualified to

21、 run a test.”6sterility test environmentsterility test environmentregulatory requirements : ep 5.5 / usp29?aseptic conditions?class a lfh in class b?isolator (iso5)avoid false positivesterility test environmentregulatory requirements : usp29? sterilitytestingvalidation of isolatorsystems|“isolators

22、for sterility testing need not be installed in a classified clean room , but it is important to place the isolator in an area that provides limited access to nonessential staff. the appropriate location provides adequate space around the isolator for moving transfer isolators, staging of materials,

23、and general maintenance. no environmental monitoring of the surrounding room is required.“?note:|operators are not required to wear special clean-room clothing for conducting sterility tests within isolators; standard laboratoryclothing is adequate 7sterility test environmentprecautionsagainstmicrob

24、ialcontamination?the working conditions in which the tests are performed are monitored regularlyby appropriate sampling and by carrying out appropriate controls(ep 5.5/ usp 29) such as those indicated in the appropriate european community directives and associated notes for guidance on gmp (ep 5.5)?

25、the precautionstaken to avoid contaminationare such that they do not affect any micro-organismswhich couldbe revealedin the test.sterility test environmentlaminar flow hood (lfh)?corrugated box : generates a high amount of particles?particles = vehicles and vector for development of bacteria and fun

26、gi (storage in humid and dirty areas)high level of particles on blister exteriorshigh concentration of gram + bacteria / fungi on the blister exteriorpotential risk of poor (alcohol) decontamination risk of false positiveclass b roomlfh (class a)unclassified roomsteritest boxin storage areabox elimi

27、nation blister exterior decontamination (alcohol solution)blister transfersecond blister exterior decontamination (alcohol solution)“aseptic”transfer in lfhsterility test environmentlaminarflow hood (lfh)8sterility test environmentisolatorswithout transferchambervhp/paa decontamination 1-2 time/day(

28、for each loading)unclassified room(with access control)steritest boxes in storage areatransfer in theunclassified roomboxblisterstransferboxsterility testingisolator (class a)blisterstransferchamber(class a)unclassified room(with access control)vhp/paa decontamination (1-2 time/day)storage of bliste

29、rs with exterior sterilevhp/paa decontamination (1 time/month)steritest boxes in storage areatransfer in theunclassified roomboxblisterstransferboxsterility testingisolator (class a)blisterssterility test environmentisolatorswith transfer chambersterility test environmentisolator -bio-decontaminants

30、?isolatorsare not sterilized. theyare bio-decontaminatedwith followingagents:|h2o2as vapourof hydrogeneperoxide(vphp)from a solution of 31% ou 35%|peraceticacid (paa) from a stabilizedmixture of peraceticacid (2 to 5%) and of hydrogeneperoxideh2o2(10 to 20%)?sterilization:|autoclave|steam sterilizat

31、ion|sterilisation withhot air|eo sterilisation|gamma irradiation9sterility test environmentaccessto theworkspacesterility test environmentsterility testing in isolators- access to the isolator workspacegloveshalf-suitfull-suitsterility test environmentsterility testing in isolators- gloves: material

32、and how to usethe handinnerisolatorprotectionglovesglovesglovesno watchlatexlatexlatexno ringscottonbutylchirurgicalshort nailspvcneoprenesandwich gloves from 3 different materials optimum?stretching units for the decontamination cycle10sterility test environmentsterility testing : materiallogistic?

33、 set up a material flowcharttestbuffersteritestunitsempty containerssteridilutorsamplemediasolid waste(e.g. packaging)liquid waste(e.g. sample, buffer)sterility test environmentsterility testing : material logistic9sterilizedbags withtubing system (sterile connectors)sterility test environmentsteril

34、ity testing in isolator : waste removal11?rtp includinga system for liquid removal? vacuumsteritest pump?sterile filtersterile connectorsterilizedbottleexample for a waste removal system for liquids:isolatorsterility test environmentsterility testing in isolator : waste removalsterility test environ

35、mentenvironmental monitoring?viable particules|surface|personalattire, protective clothing|air?non-viable particulescontact platesrisk of remainingmedia on the surfacessterility test environmentenvironmental monitoring?microbial air monitoring|active samplingmethod|passive samplingmethod50100200ndnd

36、dndndnd50100 100255010020 0003 500 000c8100 000100 000510 2055102 000350 000b710 00010 0003729335 20061 00011 (c) 3 1 1 113 500a5100100ufc/4h90 mmcfu/m3cfu/m3cfu/4h55mmcfu/4h90mm (b)cfu/m35m0.5 meu gmpisouspfdacgmpfdacgmpuspeu gmp (a)eu gmpin operation(a): average value(b): individual settle plates

37、may be exposed for less than 4 hours(c): samplesfrom class 100 (iso 5) environmentsshould normallyyield no microbiologicalcontaminants12m air t isolatorm air t isolatorparticle monitoringnote:residual levels of vapor hydrogen peroxide can remain in the barrier and inhibit the growth of micro-organis

38、ms after concentration on agar media, rendering microbiological air monitoring difficult.use m air t pyruvatecassetteto avoidany false negativeresultssterility test environmentenvironmental monitoring?microbial air monitoring in isolator?the isolatortechnologyis a solution to improvethe aseptic mani

39、pulationsand the protection of the operators.?isolators reduce the risk of havingfalse positiveresultsduring sterility testing . sterility test environmentculture media13culture media regulatory requirements : ep 5.5 / usp29?soybean casein digest broth (sbcd, tsb)|aerobic bacteria and fungi|incubate

40、d at 20-25c (ep) and 22.5c +/-2.5 c (usp)?fluid thioglycollate medium (ftm)|primarily intended for the culture of anaerobic bacteria. it will also detect aerobic bacteria|resazurin sodium solution as an indicative of oxygen|ration surface to depth : not more than 1/3 pink colour during the storage a

41、nd 1/2 at the end of the incubation period|incubated at 30-35c (ep) and 32.5c +/-2.5 c (usp)avoidfalse negativeculture media regulatory requirements : ep 5.5 / usp29?other media|ep :other media can be used provided that they pass the growth promotion and the validation tests|usp : alternative thiogl

42、ycolate medium (without agar and rezasurin sodium solution) and media for penicillins or cephalosporins. scdb and ftm supplemented with lactamase.?sterilization|using a validated processep /uspculture media regulatory requirements : ep 5.5 / usp29?storage|store at a temperature between 2and 25 c in

43、a sterile, airtight container|ep : do not usedthe medium for a longer storageperiod thanhas been validated|usp :unsealedcontainers : 1 monthgrowth promotion within2 weekscolor indicator(ftm)tight containers :1 yeargrowth promotion within3 monthscolor indicator(ftm)14?suitability tests|the suitabilit

44、y test have to be carried out before, or in parallel, with the test on the product to be examined|sterility|pharmacopoeias require that sterility testing should be performed to confirm the sterility of the microbiological medium.|growth promotion test|to confirm the ability of the test medium to sup

45、port the growth and reproduction of selected microorganisms.|test each lot of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients culture media regulatory requirements : ep 5.5 / usp29culture media regulatory requirements : ep 5.5 / usp29?sterili

46、ty|the medium is considered sterile if no microbial growth(visually determined by lack of turbidity) is observed within the incubation period(14 days)|no more than half of the ftm is red/pink (oxygenated)culture media regulatory requirements : ep 5.5 / usp29?growth promotion test : strainsftm incuba

47、ted at 30-35c (ep), 32,5 + 2.5c (usp) with :|clostridium sporogenesanaerobicatcc 19404|harmonization with usporatcc 11437|pseudomonas aeruginosaaerobicatcc 9027|staphylococcus aureus aerobicatcc 6538sbcd incubated at 20-25c (ep), 22,5 + 2,5c (usp) with :|aspergillus nigerfungiatcc 16404|bacillus sub

48、tilisaerobicatcc 6633|candida albicansfungiatcc 10231?use separateportions of medium for eachmicro-organism?viable micro-organismsused for inoculation are not more thanfive passages fromthe original seed-lot.15?growth promotion test|list of microorganisms is the same in ep4.5 (valid from 1.7.2003 un

49、til 31.12.2003)as in 4.6 (valid from 1.1.2004 to 31.3.2004)and above, but |anaerobic microorganism clostridium sporogenes not yet harmonized with usp |only clostridium sporogenes atcc 19404 is allowed?ftm|inoculate. as a minimum one aerobicbacteria and one anaerobicbacteria?sbcd|. use as a minimum o

50、ne fungiand one aerobicbacteriaculture media regulatory requirements?growth promotion test : alternative strains (usp only)|alternative to: |staphylococcus aureus is bacillus subtilis atcc 6633|pseudomonas aeruginosa is micrococcus luteus (kocuria rhizophila) atcc 9341|clostridium sporogenes, when a

51、 nonspore-forming microorganism is desired, is bacteroides vulgatus atcc 8482culture media regulatory requirements : ep 5.5 / usp29culture media regulatory requirements : ep 5.5 / usp29?growth promotion test : inoculation |“inoculate portions of . medium with a small number(not more than 100 cfu)of

52、the following micro-organisms ”?growth promotion test : incubation time|,for not more than 3 days in the case of bacteriaand not more than five daysin the case of fungi . “ (since usp27)|incubate 5 days for bacteria and fungi (usp 26)?growth promotion test : result|? the media are suitableif a clear

53、lyvisible growthof the micro-organismsoccurs ?16culture media?sterility & growth promotion testsuse a vented needle for all bottles: |removes initial vacuum or pressure|gives more o2 for aerobic microorganisms (bacillus.) |prevents pressure building if/when growth inside the bottle|does not affe

54、ct efficacy of resazurinin ftmlot alot a20-25 c30-35 cventedneedlescat no tefg02525tsb / sbcdftm?media|suitability test|sterility : no growth after 14 days|growth promotion test of aerobes, anaerobes and fungi|scdb/tsb : aspergillus niger, bacillus subtilis, candida albicans|ftm : clostridium sporog

55、enes, pseudomonas aeruginosa, staphylococcus aureusor bacetroides vulgaris, micrococcus luteus (kocuria rhizophila), bacillus subtilis (usp)|alternative ftm : clostridium sporogenes (usp)|not more than 100 cfu in separate portion of medium|not more than 3 days in case of bacteria|not more than 5 day

56、s in case of fungi|the media is suitable if visible growth of the micro-organisms occurs|each batch of media is tested (ready-prepared or dehydrated media)|strains used for inoculation are not more than 5 passages removed from the original master seed-lotavoidfalse negativeavoid false positivecultur

57、e media regulatory requirements : ep 5.5 / usp29test method17?sterility test method|direct inoculation|membrane filtration?(open-) funnel method?closed system method|ep and usp require membrane filtration whenever the nature of the product permits.sterility testregulatory requirements : ep 5.5 / usp

58、29general procedure?aseptically transfer the sample directly into the culture media?sample volume less than 10% of the volume of the medium?if product with antimicrobial activity :|carry out the test afterneutralizingthe b&f effect|neutralizingsubstance / dilution?if large volume of sample, use

59、concentratedmedia preparedin suchway that it takes into account the subsequentdilution.?incubate?examine for turbiditysterility test methodsdirect inoculation : ep 5.5 / usp29advantages?direct immersion of medical devices?non-filterable products may be tested?less manipulations than open funnel meth

60、odlimitations?antimicrobial product activity may inhibit growth?intrinsic product turbidity|sub-culturing necessary?aseptic technique training and validation required?no volumes larger than 100 ml?high risk of false positives and false negativessterility test methodsdirect inoculation18sterility test methodsmembrane filt