欧洲药典的无菌方法.docx
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欧洲药典的无菌方法.docx
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欧洲药典的无菌方法
04/2011:
20601
2.6.1.Sterility2.6.1.Sterility►
(1)(SEENOTE)◄
Thetestisappliedtosubstances,preparationsorarticleswhich,accordingtothePharmacopoeia,arerequiredtobesterile.However,asatisfactoryresultonlyindicatesthatnocontaminatingmicro-organismhasbeenfoundinthesampleexaminedintheconditionsofthetest.
PRECAUTIONSAGAINSTMICROBIALCONTAMINATION
Thetestforsterilityiscarriedoutunderasepticconditions.Inordertoachievesuchconditions,thetestenvironmenthastobeadaptedtothewayinwhichthesterilitytestisperformed.Theprecautionstakentoavoidcontaminationaresuchthattheydonotaffectanymicro-organismswhicharetoberevealedinthetest.Theworkingconditionsinwhichthetestsareperformedaremonitoredregularlybyappropriatesamplingoftheworkingareaandbycarryingoutappropriatecontrols.
CULTUREMEDIAANDINCUBATIONTEMPERATURES
Mediaforthetestmaybepreparedasdescribedbelow,orequivalentcommercialmediamaybeusedprovidedthattheycomplywiththegrowthpromotiontest.
Thefollowingculturemediahavebeenfoundtobesuitableforthetestforsterility.Fluidthioglycollatemediumisprimarilyintendedforthecultureofanaerobicbacteria;however,itwillalsodetectaerobicbacteria.Soya-beancaseindigestmediumissuitableforthecultureofbothfungiandaerobicbacteria.
Fluidthioglycollatemedium
l-Cystine
0.5 g
Agar
0.75 g
Sodiumchloride
2.5 g
Glucosemonohydrate/anhydrous
5.5 g/5.0 g
Yeastextract(water-soluble)
5.0 g
Pancreaticdigestofcasein
15.0 g
Sodiumthioglycollateor
0.5 g
Thioglycollicacid
0.3 mL
Resazurinsodiumsolution(1 g/Lofresazurinsodium),freshlyprepared
1.0 mL
Water R
1000 mL
pHaftersterilisation7.1 ± 0.2
Mixthel-cystine,agar,sodiumchloride,glucose,water-solubleyeastextractandpancreaticdigestofcaseinwiththewater Randheatuntilsolutioniseffected.Dissolvethesodiumthioglycollateorthioglycollicacidinthesolutionand,ifnecessary,add1 Msodiumhydroxidesothat,aftersterilisation,thesolutionwillhaveapHof7.1 ± 0.2.Iffiltrationisnecessary,heatthesolutionagainwithoutboilingandfilterwhilehotthroughmoistenedfilterpaper.Addtheresazurinsodiumsolution,mixandplacethemediuminsuitablevesselswhichprovidearatioofsurfacetodepthofmediumsuchthatnotmorethantheupperhalfofthemediumhasundergoneacolourchangeindicativeofoxygenuptakeattheendoftheincubationperiod.Steriliseusingavalidatedprocess.Ifthemediumisstored,storeatatemperaturebetween2 °Cand25 °Cinasterile,airtightcontainer.Ifmorethantheupperone-thirdofthemediumhasacquiredapinkcolour,themediummayberestoredoncebyheatingthecontainersinawater-bathorinfree-flowingsteamuntilthepinkcolourdisappearsandcoolingquickly,takingcaretopreventtheintroductionofnon-sterileairintothecontainer.Donotusethemediumforalongerstorageperiodthanhasbeenvalidated.
Fluidthioglycollatemediumistobeincubatedat30-35 °C.
Forproductscontainingamercurialpreservativethatcannotbetestedbythemembrane-filtrationmethod,fluidthioglycollatemediumincubatedat20-25 °Cmaybeusedinsteadofsoya-beancaseindigestmediumprovidedthatithasbeenvalidatedasdescribedingrowthpromotiontest.
Whereprescribedorjustifiedandauthorised,thefollowingalternativethioglycollatemediummaybeused.Prepareamixturehavingthesamecompositionasthatofthefluidthioglycollatemedium,butomittingtheagarandtheresazurinsodiumsolution,steriliseasdirectedabove.ThepHaftersterilisationis7.1 ± 0.2.Heatinawater-bathpriortouseandincubateat30-35 °Cunderanaerobicconditions.
Soya-beancaseindigestmedium
Pancreaticdigestofcasein
17.0 g
Papaicdigestofsoya-beanmeal
3.0 g
Sodiumchloride
5.0 g
Dipotassiumhydrogenphosphate
2.5 g
Glucosemonohydrate/anhydrous
2.5 g/2.3 g
Water R
1000 mL
pHaftersterilisation7.3 ± 0.2
Dissolvethesolidsinwater R,warmingslightlytoeffectsolution.Coolthesolutiontoroomtemperature.Add1 Msodiumhydroxide,ifnecessary,sothataftersterilisationthesolutionwillhaveapHof7.3 ± 0.2.Filter,ifnecessary,toclarify,distributeintosuitablevesselsandsteriliseusingavalidatedprocess.Storeatatemperaturebetween2 °Cand25 °Cinasterilewell-closedcontainer,unlessitisintendedforimmediateuse.Donotusethemediumforalongerstorageperiodthanhasbeenvalidated.
Soya-beancaseindigestmediumistobeincubatedat20-25 °C.
Themediausedcomplywiththefollowingtests,carriedoutbeforeorinparallelwiththetestontheproducttobeexamined.
Sterility.Incubateportionsofthemediafor14 days.Nogrowthofmicro-organismsoccurs.
Growthpromotiontestofaerobes,anaerobesandfungi.Testeachbatchofready-preparedmediumandeachbatchofmediumpreparedeitherfromdehydratedmediumorfromingredients.Suitablestrainsofmicro-organismsareindicatedinTable 2.6.1.-1.
Table 2.6.1.-1–Strainsofthetestmicro-organismssuitableforuseinthegrowthpromotiontestandthemethodsuitabilitytest
Aerobicbacteria
Staphylococcusaureus
ATCC6538,CIP4.83,NCTC 10788,NCIMB 9518,NBRC 13276
Bacillussubtilis
ATCC6633,CIP52.62,NCIMB 8054,NBRC 3134
Pseudomonasaeruginosa
ATCC9027,NCIMB8626,CIP 82.118,NBRC 13275
Anaerobicbacterium
Clostridiumsporogenes
ATCC19404,CIP79.3,NCTC 532,ATCC 11437,NBRC 14293
Fungi
Candidaalbicans
ATCC10231,IP48.72,NCPF3179,NBRC 1594
Aspergillusbrasiliensis
ATCC16404,IP1431.83,IMI149007,NBRC 9455
Inoculateportionsoffluidthioglycollatemediumwithasmallnumber(notmorethan100CFU)ofthefollowingmicro-organisms,usingaseparateportionofmediumforeachofthefollowingspeciesofmicro-organism:
Clostridiumsporogenes,Pseudomonasaeruginosa,Staphylococcusaureus.Inoculateportionsofsoya-beancaseindigestmediumwithasmallnumber(notmorethan100CFU)ofthefollowingmicro-organisms,usingaseparateportionofmediumforeachofthefollowingspeciesofmicro-organism:
Aspergillusbrasiliensis,Bacillussubtilis,Candidaalbicans.Incubatefornotmorethan3 daysinthecaseofbacteriaandnotmorethan5 daysinthecaseoffungi.
Seedlotculturemaintenancetechniques(seed-lotsystems)areusedsothattheviablemicro-organismsusedforinoculationarenotmorethan5 passagesremovedfromtheoriginalmasterseed-lot.
Themediaaresuitableifaclearlyvisiblegrowthofthemicro-organismsoccurs.
methodsuitabilityTEST
CarryoutatestasdescribedbelowunderTestforsterilityoftheproducttobeexaminedusingexactlythesamemethodsexceptforthefollowingmodifications.
Membranefiltration.Aftertransferringthecontentsofthecontainerorcontainerstobetestedtothemembraneaddaninoculumofasmallnumberofviablemicro-organisms(notmorethan100CFU)tothefinalportionofsterilediluentusedtorinsethefilter.
Directinoculation.Aftertransferringthecontentofthecontainerorcontainerstobetested(forcatgutandothersurgicalsuturesforveterinaryuse:
strands)totheculturemediumaddaninoculumofasmallnumberofviablemicro-organisms(notmorethan100CFU)tothemedium.
Inbothcasesusethesamemicro-organismsasthosedescribedaboveunderGrowthpromotiontestofaerobes,anaerobesandfungi.Performagrowthpromotiontestasapositivecontrol.Incubateallthecontainerscontainingmediumfornotmorethan5 days.
Ifclearlyvisiblegrowthofmicro-organismsisobtainedaftertheincubation,visuallycomparabletothatinthecontrolvesselwithoutproduct,eithertheproductpossessesnoantimicrobialactivityundertheconditionsofthetestorsuchactivityhasbeensatisfactorilyeliminated.Thetestforsterilitymaythenbecarriedoutwithoutfurthermodification.
Ifclearlyvisiblegrowthisnotobtainedinthepresenceoftheproducttobetested,visuallycomparabletothatinthecontrolvesselswithoutproduct,theproductpossessesantimicrobialactivitythathasnotbeensatisfactorilyeliminatedundertheconditionsofthetest.Modifytheconditionsinordertoeliminatetheantimicrobialactivityandrepeatthemethodsuitabilitytest.
Thismethodsuitabilitytestisperformed:
a)whenthetestforsterilityhastobecarriedoutonanewproduct;
b)wheneverthereisachangeintheexperimentalconditionsofthetest.
Themethodsuitabilitytestmaybeperformedsimultaneouslywiththetestforsterilityoftheproducttobeexamined.
TESTFORSTERILITYOFTHEPRODUCTTOBEEXAMINED
Thetestmaybecarriedoutusingthetechniqueofmembranefiltrationorbydirectinoculationoftheculturemediawiththeproducttobeexamined.Appropriatenegativecontrolsareincluded.Thetechniqueofmembranefiltrationisusedwheneverthenatureoftheproductpermits,thatis,forfilterableaqueouspreparations,foralcoholicoroilypreparationsandforpreparationsmisciblewithorsolubleinaqueousoroilysolventsprovidedthesesolventsdonothaveanantimicrobialeffectintheconditionsofthetest.
Membranefiltration.Usemembranefiltershavinganominalporesizenotgreaterthan0.45 µmwhoseeffectivenesstoretainmicro-organismshasbeenestablished.Cellulosenitratefilters,forexample,areusedforaqueous,oilyandweaklyalcoholicsolutionsandcelluloseacetatefilters,forexample,forstronglyalcoholicsolutions.Speciallyadaptedfiltersmaybeneededforcertainproducts,e.g.forantibiotics.
Thetechniquedescribedbelowassumesthatmembranesabout50 mmindiameterwillbeused.Iffiltersofadifferentdiameterareusedthevolumesofthedilu
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