New Frontiers in the Quality of Medicines · E.Charton, Strasbourg, June 2007 How should users/agencies cope with the chapters during this transition period? •Proposal from « Group

New Frontiers in theQuality of Medicines

WorkshopInternational Harmonisation - Microbiological

Techniques

Moderators:Prof. Dr Henning G. KristensenDr Sylvie Guyomard Devanlay

EDQM International Conference13-15 June 2007

Strasbourg, France

1

The background andimplementation programme forthe new chapters

Emmanuelle Charton, Ph. D.Deputy Head, European Pharmacopoeia Department

E.Charton, Strasbourg, June 2007

International Harmonisation ofthe microbiological chapters

• Signed-off by PDG: November 2005• Adopted by Ph. Eur. Commission: November 2005• Ph. Eur. Commission submitted a proposal to QWP

for a special implementation: December 2005• QWP approved proposal: spring 2006• -> Publication on EDQM website (as FAQ on EDQM

Helpdesk)

2


Symposium organised by EDQM(October 2006)• Identification of a need for a more detailed

recommendation on howmanufacturers/regulators should apply thenew chapters


Milestones

• 1 January 2007: official Ph. Eur. implementation ofChapters 2.6.12, 2.6.13 and 5.1.4 with 2 sets oftests:

– 1st set (“old”)– 2nd set (“harmonised”)

• 1 January 2009: official implementation ofmonographs prescribing chapters 2.6.12 and2.6.13 (Presently published in Pharmeuropa 19.2)

• 1 January 2007-> 1 January 2009: transition period

3


How should users/agenciescope with the chapters duringthis transition period?

• Proposal from « Group 1 », Group of Expertsof the European Pharmacopoeia to QWP

• Proposal refined by QWP


2.6.12 Harmonised <=> Present• New application:

– transition period: either old or harmonised(harmonised recommended)

– from 1 January 2009: reference= harmonised• Substance covered by monograph

– change to be made within 6 months of official date• Already authorised products not covered by

monograph– transition period: either old or harmonised– from 1 January 2009: reference= harmonised

4


2.6.13 Harmonised ≠ Present !• New application:

– transition period: either old or harmonised (harmonisedhighly recommended since it might give differentpass/fail results in exceptional, borderline cases)

– From 1 January 2009: reference = harmonised• Substance covered by monograph

– change to be made within 6 months of official date• Already authorised products not covered by

monograph– transition period: either old or harmonised– from 1 January 2009: reference= harmonised


5.1.4

The set of criteria should alwayscorrespond to the set of test methods used,and hence the time schedule of applicationof criteria is dependent on the timeschedule applied to change to the new setof tests.

5


Should industry demonstrate thesuitability of the harmonised methodsfor every registered product?

Industry can apply a scientifically justifiedstrategy to reduce work:

• Risk-based approach in which not allproducts are tested

• Bracketing for a product line


Validation of alternativemethods to 2.6.12/2.6.13 duringthe transition period

• Validation against either set• If validation already made against 1st set,

no revalidation is necessary

6


Should industry apply for approval of2nd set of test methods?

• No notification and variation application forthe products concerned will be required

• However, during an inspection by EUcompetent authority compliance withPh. Eur. as well as suitability of theharmonised methods should bedemonstrated upon request


Should industry apply for approval of2nd set of criteria?

• No notification and variation application forthe products concerned will be required

• However, when submitting a variationapplication for another reason it isrecommended to include the updatedspecification

7


Publication of thisimplementation timeline• Submission of this proposal to EMEA:

March 07 (QWP)

• QWP to discuss proposal during June 07meeting

• Publication on EMEA Website


Implementation in other regions

• USA: May 2009• Japan: October 2007

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Pharmeuropa 19.2 enquiry

• Over 80 monographs being revised forintroduction of TAMC and TYMC

• Comments welcome until end of June!

Thank you for your attention

1

1

Questions from the usersResponses to frequently asked questions

Hans van DoorneUniversity Centre for Pharmacy,

University of Groningen,The Netherlands

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Major changes in chapter 2.6.12

Text easier to read Headings and subheadings Words like “suitable” and “such as” no longer used No cross references

Section on growth promotion and suitability of the test Negative control Growth promotion in absence of product

Same test organisms as in sterility test Growh promotion in presence of product

Interpretation Total aerobic microbial count (bacterial count) Total yeast and mould count (total viable aerobic count) Maximum acceptable count is 2x criterion (5x)

2

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4. Growth promotion and suitability of the counting method(Effectiveness of culture media and validity of the counting method) 4.2 Preparation of test strains

Harmonized Grow each of the bacterial and

fungal test strains separatelyas described in Table 1.

Use the suspensions within 2 hor within 24 h if stored at 2-8°C.

The stable spore suspensionmay be maintained at 2-8 °C fora validated period of time.

Test organisms: Staph. aureus,Ps. aeruginosa, B. subtilis, C.albicans, A. niger

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Question:

What sort of strain is it preferableto use? Could I use otherstrains than that strain that arecited in the Ph. Eur.?

Answer:

You must use the micro-organisms that are cited in this chapter, but you can use strains from other culture collections. The culture collections are just given as examples.

4

4. Growth promotion and suitability of the counting method(Effectiveness of culture media and validity of the counting method) 4.3 Negative control

Harmonized

To verify testing conditions anegative control is performedusing the chosen diluent inplace of the test preparation.There must be no growth ofmicro-organisms.

Question

What is the purpose of thenegative control

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Answer

The purpose of this negative control is to show that there is no contamination during the test. It is tho show that the conditions of sterility (aseptic precautions andhandling) are respected

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4. Growth promotion and suitability of the counting method(Effectiveness of culture media and validity of the counting method) 4.3 Negative control

Harmonized

To verify testing conditions anegative control is performedusing the chosen diluent inplace of the test preparation.There must be no growth ofmicro-organisms.

Question

When are you actualy supposedto do the negative control:when testing the suitability ofthe method, or when testingthe product, or in bothsituations?

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Answer

You are supposed to do the negative control (preferably) at the same time as when you are testing the product

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4. Growth promotion and suitability of the counting method(Effectiveness of culture media and validity of the counting method) 4.4 Growth promotion of the media

Harmonized Test each batch of ready-prepared

medium and each batch of medium,prepared either from dehydratedmedium or from the ingredientsdescribed.

QuestionIs it necessary to test the

growth promotion onall received batches ordoes it serve just formicrobiologicalvalidation?

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AnswerYes it is necessary.

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Harmonized Test each batch of ready-

prepared medium and eachbatch of medium, preparedeither from dehydrated mediumor from the ingredientsdescribed.

QuestionDo we have to test

systematically in parallel aprevious and approved batchin order to compare with thenew batch?

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Answer

You do not have to testa previous batch inparallel. You do thecomparison on paper.

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Harmonized Inoculate portions/plates of

medium with a smallnumber (not more than 100CFU) of the micro-organisms indicated inTable 1. Incubate in theconditions described inTable 1.

QuestionWhat is the sufficient volume of

the microbial suspension ofnot more than 100 cfu. Whatdoes cfu refer to?

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AnswerThe micro-organisms are to beadded to the diluted/suspended product. The 100 cfu refers tothe inoculum (i.e. what will beon the filter or plate).

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QuestionIs there a method that allows

counting faster in order toverify that there is 100 cfuin the inoculum?

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AnswerYes, you must(spectrophotometrically)establish a calibrationcurve. You can also usecommercially availablestandardized inocula.

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Harmonized For solid media, growth

obtained must not differby a factor greater than 2from the calculated valuefor a standardizedinoculum.

QuestionWhat does the factor 2

mean? How is thisfactor calculated?

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AnswerIt means that the result can be 2times higher or lower than theinoculum. For example with aninoculum of 100 cfu acceptablecounts are 100/2 = 50 cfu,to 100 x 2 = 200 cfu

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QuestionWhy have growth promotion

tests to be performed onCSDA for C. albicans and A.niger? “Why are bacteria nottested on SDA?”

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AnswerThis is a matter of definition.TAMC includes yeasts andmoulds. TYMC is meant foryeasts and moulds only. SDA with antibiotics may be usedas alternative.

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4. Growth promotion and suitability of the counting method(Effectiveness of culture media and validity of the counting method) 4.5 Suitability of the counting method in the presence of the product

Harmonized If (inactivators are) used, their

efficacy and their absence oftoxicity for micro-organismsmust be demonstrated bycarrying out a blank withneutraliser and withoutproduct.

QuestionWhy is it necessary to

demonstrate the efficacy…….If recovery of micro-organisms in the presence ofproduct is given. (It is verytime consuming)

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Answer.. It seems like good practice to test the efficacy and toxicity of the neutralizing agent…

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4. Growth promotion and suitability of the counting method(Effectiveness of culture media and validity of the counting method) 4.5 Suitability of the counting method in the presence of the product

Harmonized Membrane filtration. Use

membrane filters having anominal pore size not greaterthan 0.45 µm.

QuestionWhy is the use of 0.45µ deemed

appropriate, in view of thefact that a 0.22µ is regardedrequired for sterilization?

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AnswerThis is the same pore size as used in the sterility test. The retention of such filters is sufficient. •Filtration is easier (viscous products)•Diffusion of nutrients is faster.

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5 . Testing of productsInterpretation of the results

Harmonized The total aerobic microbial count

(TAMC) is considered to be equal tothe number of CFU found usingcasein soya bean digest agar;colonies of fungi …. are counted aspart of TAMC.

The total combined yeasts/mouldcount (TYMC) is considered to beequal to the number of CFU foundusing Sabouraud-dextrose agar;colonies of bacteria….are countedas part of TYMC.

QuestionIf colonies of bacteria are detected

on SDA they are counted as partof the TYMC. The bacteria arecounted twice.

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AnswerFew bacteria will grow. In anycase you have the possibility touse SDA with antibiotic if a highcount of bacteria would lead toout of specification.

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15

5 . Testing of productsInterpretation of the results

Harmonized When an acceptance criterion for

microbiological quality isprescribed it is interpreted asfollows:

101 micro-organisms: maximumacceptable count = 20,

102 micro-organisms: maximumacceptable count = 200,

103 micro-organisms : maximumacceptable count = 2000, and soforth.

QuestionWhat is the signification of < 102

cfu: maximum acceptablecount: 200.

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AnswerIt means that if the specification in a product monograph is 102 micro-Organisms, the product could be released if up to 200 micro-organisms are counted.

16

Any questions?

1

1

Questions from the usersResponses to frequently asked questions

(FAQ) on chapter 2.6.13

Sylvie Guyomard DevanlayExpert for France in group 1

Sanofi Aventis R&DQuality control/Microbiology,

France

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Outline of presentation General considerations on the 2 sets of tests:

methods A & B Review of different sections of this chapter

and FAQs related to each section 1-Introduction 2- General procedure 3- Growth promoting and inhibitory properties of

the media and suitability of the test 4-testing of products 5- recommended solutions and culture media

(section for information) Conclusion

2

3

Microbiological examination of non-sterile products:2.6.13-Test for specified micro-organisms

Harmonised method Method B

Reference methodsof the Eur PhApplication for

Marketingauthorisation

intended to replacemethod A as soon asmonographs replaced

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Non harmonisedmethod: Method A

Reference methodsof the Eur Phcompliance with

monographs waitingrevision of thesemonographs (ongoing)

4

1. Introduction

Harmonised: Set B Alternative

microbiologicalprocedures, includingautomated methods,may be used, providedthat their equivalence tothe Pharmacopoeialmethod has beendemonstrated.

Ph. Eur : Set ANot mentioned

here but ingeneral notices

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2. General procedures

Harmonised: Set B If surface-active

substances are usedfor samplepreparation, theirabsence of toxicity formicro-organisms andtheir compatibilitywith inactivators usedmust bedemonstrated.

Ph. Eur : Set A

Not mentioned

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3. Growth promoting and inhibitoryproperties of the media and suitability of thetest: general considerations

Harmonised: Set B The ability of the test to

detect micro-organismsin the presence of theproduct to be testedmust be established

Suitability must beconfirmed if a changein testing performance,or the product, whichmay affect the outcomeof the test isintroduced.

Ph. Eur : Set A Not given

Not given

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3. Growth promoting and inhibitory propertiesof the media and suitability of the test:

Preparation of test strains

Harmonised: Set B

Use standardized stablesuspensions of test strainsor prepare as stated below .

Seed lot culturemaintenance techniques(seed-lot systems) are usedso that the viable micro-organisms used forinoculation are not morethan 5 passages removedfrom the original masterseed-lot.

Ph. Eur : Set A

Not given

Not given

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3. Growth promoting and inhibitory properties ofthe media and suitability of the test:

Preparation of teststrains (2)

Harmonised: Set B Aerobic bacteria and

clostridia Details in this section on

description of the Proposed strain (ATCC..) Culture conditions :

Incubation T° and Time,aerobic/anaerobic

Media and diluents to use

Use the suspensions within 2h orwithin 24 h if stored at 2-8 °C.

The stable spore suspension maybe maintained at 2-8 °C for avalidated period of time

Ph. Eur : Set A

Described less precisely

Not given

Not given

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3. Growth promoting and inhibitory propertiesof the media and suitability of the test:

Preparation of test strains FAQs

FAQ - Preparation of test strains

What sort of strain is it preferableto use? Could I use otherstrains than that strain that arecited in the Ph. Eur.?

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Answer:

You must use the micro-organisms that are cited inthis chapter to makereference to thepharmacopoeial method,but you can use strainsfrom other culturecollections. The culture collectionsare just given as examples.

10

3. Growth promoting and inhibitory properties of themedia and suitability of the test: FAQ Negative control

Harmonized

Section 3.2: negativecontrol To verify testing

conditions a negativecontrol is performed usingthe chosen diluent in placeof the test preparation.There must be no growthof micro-organisms.

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FAQ 1- Negative control What is the purpose of the

negative control in thissection 3 ?

Answer The purpose of thisnegative control is to showthat the conditions of asepticprecautions andhandling are respected.BUT

Negative control forvalidation and notfor section 4-testingof product ???

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3. Growth promoting and inhibitory properties of themedia and suitability of the test: FAQ Negative control

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Answer

You are supposed to dothe negative control(preferably) atthe same time as whenyou are testing theproduct so in section 4. One by serie ofspecified microorganismstest (risk analysis for OOS)

FAQ 2 - Negative control

When are you actualysupposed to do thenegative control: when testing the

suitability of themethod,

when testing theproduct

in both situations Every time the product

is tested periodically?

12

Harmonized meth B Test each batch of ready-prepared

medium and each batch of medium,prepared either from dehydratedmedium or from ingredients.

Ph. Eur meth A Not covered.

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3.3 Growth promoting and inhibitoryproperties of the media

review

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Harmonized meth B Growth promotion section expanded:

which micro-organism on which medium Verification of

Nutritive properties (solid or liquid): comparable to thatpreviously obtained with a previously batch of medium

Incubation time: ≤ shortest period of time specified in thetest

Selective properties : No growth observed Incubation time: ≥ longest period of time specified in the test

indicative properties of media: Colonies are comparable(appearance and indication reactions) to those previouslyobtained.

Incubation time within the range specified in the test

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3.3 Growth promoting and inhibitoryproperties of the media

review

14

3.3 Growth promoting and inhibitory properties ofthe media

FAQs

FAQ 1 Is it necessary to test

the growth promotionon all received batchesor does it serve just formicrobiologicalvalidation?

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Answer Yes, the growth promotiontest must be performed onall batches.

- For ready to use media, ifthe supplier has beenaudited (transportationincluded), certificate ofanalysis with the growthpromotion test isacceptable. Periodic testingis recommended in the site.

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3.3 Growth promoting and inhibitory properties ofthe media

FAQs

FAQ 2

Do we have to testsystematically in parallel aprevious and approvedbatch in order to comparewith the new batch?

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Answer

You do not have to testa previous batch inparallel. You do thecomparison on paper.

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3.3 Growth promoting and inhibitory propertiesof the media

FAQs

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FAQ 3

What are thespecifications when wecompare a freshlybatch with a previousbatch for growthpromotion properties?Do we need to take afactor 2 in account?

Answer

The factor of 2 asdescribed in 2.6.12 has notto be used. No strictprescription was deliberatelygiven in this chapterbecause the test isqualitative, not quantitative.You should define thecomparability criterionyourself for example colonysize at the shortestincubation time prescribed .

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17


FAQs

FAQ 4 « You should not

incubate more thanthe shortest period oftime specified in thetest ».

How exactly are thegiven times (e.g. 18-24 h) to be followed?

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Answer For growth promotingproperties of media you mustincubate not more than 18h( worst case conditions).

For inhibitory properties ofmedia you must incubate notless than 24h ( worst caseconditions).

For indicative properties ofthe media you could incubatewithin the specified range(here from 18 to 24h

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FAQs

FAQ5

In the growth promotiontest of rappaportvassiliadis Salmonellaenrichment broth there isno visible growth after theincubation time, but aftersubculturing on selectiveagar there is typicalgrowth. Is this the caseonly in our laboratory?

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Answer Yes it could be observed in thelab since for rappaport vassilidisSalmonella enrichment broth, weinoculate low numbers ofSalmonella sp (usually theinoculum is around 20 CFU per10 mL rappaport vassilidisSalmonella enrichment broth).

Even if the enrichment brothseems clear, you must confirmrecovery of Salmonella by sub-culturing the rappaportvassilidis Salmonella enrichmentbroth to solid agar.

10

19

3.4 Suitability of the test method

reviewHarmonized method B Sample prep as in section 4 Add each strain at the time of mixing in the

prescribed growth medium Inoculate the test strains individually (no pool) Incubation time: the shortest incubation period

prescribed Detection of the prescribed micro-organism with the

indication reactions as described in section 4 If the antimicrobial activity could not be neutralised,

then it is assumed that the inhibited micro-organismwill not be present in the product

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FAQs

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FAQ 1

Must suitabilityalso be carriedwith it aninhibitory strainor not?

Answer

No you do not haveto use an inhibitorystrain in order to testthe suitability of themethod

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FAQs

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FAQ 1bis

Which test micro-organisms should oneuse?

Just the same micro-organisms as used fortesting the growthpromoting properties ofthe respective media,

or also the micro-organisms used fortesting inhibitoryproperties of themedia?

Answer

No you do not have touse an inhibitory strain inorder to test the suitabilityof the method .

For example if youtest the suitability of themethod for E. coli, youshould use only E. colias test micro-organismfor growth promoting

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FAQs

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FAQ 2

Does it mean thatfor each test strainindividualsuitability testshave to beperformed, or is itpossible to use amixed inoculum ofall 4 strains?

Answer

The method states thatthe strains areinoculated individually.No mixed inoculum ispermitted.

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4 Testing of products

review

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Harmonised met B

For each test: Sample preparation and pre-incubation Test for absence:

Selection and subculture Quantitative test if applicable Interpretation

Characteristic colonies + confirmation byidentification tests

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Harmonised met Bexample 1

Test complies = Absence in 1g or 1 mL =No colony onViolet red bile glucose agar

Interpretation

18 h to 24h30-35°CViolet red bileglucose agar

- subcultureFrom P2i

24 h to 48h30-35°CMossel BrothTest forabsence -Enrichment:1g from P1 tomedia: P2- incubation

≥ 2h but ≤ 5h20-25°CCasein Soya BeanDigest Broth

Samplepreparation10g/100 mL ofmedia: P1 andPre-incubation

Bile-tolerantgramnegative

INCUBATION TIMETEMPE-RATURE

MEDIUM /DILUENT

STEPTEST

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4 Testing of products Harmonised met Bexample 2

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Test complies = Absence in 1g or 1 mL =No colonyon Mac Conkey Agar

Interpretation

18 h to 72h30-35°CMacConkeyagar

Subculturefrom P3i

24 h to 48h42-44°CMacConkeybroth

Selection:-Enrichment: 1mL of

P2i /100 mL ofbroth P3

18 h to 24h30-35°CCasein SoyaBeanDigestBroth

Pre-incubation: 1 g or 1 mL from P1

to medium P2

NANANaCl B1 pH7Diluantas per2.6.12

Sample preparation :P1

10g or mL /100mL(2.6.12)

at least 1g or mLof product/10mL

Test for E.coli

INCUBATION TIMETEMPE-RATURE

MEDIUM /DILUENT

STEPTEST

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FAQs

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FAQ 1- 4.1 Bile-tolerantGram-negativebacteria

What are “the Bile-tolerant Gram-negativebacteria”?

Answer

These are Gram-negativemicro-organisms whichresist to the biliary salts.They were previously calledthe Enterobacteria.

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FAQs

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FAQ 2- 4.2 Escherichiacoli

In 4-2-2 selection andsubculture, what is thepurpose of the elevatedtemperature 43-45°C?

Can one utilise analternative temperaturerange, eg. 35-37C providedthat one demonstrates therecovery of E.coli duringqualification?

Answer

The purpose of the elevatedtemperature, 43-45°C is to allowfor the best selectivity of growthcondition for E.coli.

An alternative temperature rangewould depart from the Ph. Eur.method, but you can always usealternatives methods asdescribed in the general noticesof the Ph. Eur. (chapter 1.2).

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FAQs

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FAQ 3- 4.2.3 Interpretation If 10g of sample is added

to the initial broth for atest such as E. coli (4.2),but an amount isimmediately sub sampledinto a second broth that isonly representative of 1gof actual product and thisbroth is incubated - at theend of testing, can thistest be classified, for anegative result, as 'nonedetected per 10g'. Or canit only be classified as'none detected per g.

Answer The quantity that is pre-incubated is 1g therefore theoutcome of the test is "absence in1g".For Salmonella, you will note thatan "absence in 10g" test has beenimplemented since 10g isincubated

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FAQ 3- 4.2.3Interpretation

It is observed that onselective media of S.Aureus, a black colonies ofgram positive cocci in chainis seen, but the blackcolonies are without clearzones in the test sample.Whereas positive cultureshows black colonies ofgram positive cocci inclusters surrounded byclear zones.

Answer

Product to test could becontaminated, maybe not bythe germ as described in thePh.Eur. but with some othergerm. investigate (eg identify thegerm to find out where itcomes from).

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5 Recommended solutions and

culture media review

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Harmonised :met B part for information See the introductory statement of the

description of media in chapter 2.6.13 "Thefollowing solution and culture media havebeen found to be satisfactory for thepurposes for which they are prescribed in thetest for microbial contamination in thePharmacopoeia. Other media may be used ifthey have similar nutritive and selectiveproperties for the micro-organisms to betested for."

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5 Recommended solutions andculture media

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FAQ 1 Are there alternatives

media that areacceptable?

Answer part for information See the introductorystatement of the descriptionof media in chapter 2.6.13"The following solution and culturemedia have been found to besatisfactory for the purposes forwhich they are prescribed in thetest for microbial contamination inthe Pharmacopoeia. Other mediamay be used if they have similarnutritive and selective propertiesfor the micro-organisms to betested for."

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5 Recommended solutions andculture media (for information)

FAQ

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FAQ 2- How can we do the pH

measurement for culturemedia? It is written tomeasure the pH at 25°C. Atthis temperature, agar issolid. Therefore, how canwe adjust the pH?

AnswerThere are electrodes formeasurement in semisolidsamples such as meat, cheeseand fruit. These electrodes aresuitable for measurements insolid agar. E.g Metrohm(www.metrom.com ) has onecalled "Spearhead" electrode,but other companies willprobably have similarelectrodes.

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5 Recommended solutions andculture media (for information)

FAQ

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FAQ 3- If we have growth

problems of S. aureusand inhibitories problemsof E.coli with mannitolsalt agar medium that isrecommended in theharmonised method,what is the problem?Could you provide nameof suppliers?

AnswerThe composition of mannitol saltagar should be optimised to recoverS.aureus and inhibit E.coli (pH,nutritive qualities…).May be is aproblem with the temperature ofincubation or with stability of themedia along the time even withmedium stored in adequateconditions within the period ofvalidity.Bio-mérieux has proved that itsmedium (ref 43671 for 20 plates and43679 for 100 plates) had goodrecovery rates with no growth ofE.coli certified in their CoA

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Conclusion from pharmaceuticaluser point of view

1 method available worldwide. Even

if new validations will be necessary to implementthe method in the laboratory

If many new documents should be issued If specification is going to change.

transition period of 5 years

And only 1 method to compare with alternativemethods

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Conclusion from aPharmacopoeia point of view

Harmonisation necessary to obtainconsensus between Pharmacopoeias tohave international texts. Texts where harmonisation failed as

« effectiveness of microbiological protection »lead to difficulties of interpretation from onecountry to another one.

Even if many years and numerous meetingsnecessary to obtain a text acceptable by the 3Pharmacopoeias;

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Acknowledgements

Thanks to Karine Meunier for her great helpto compile FAQs and answers to thesequestions for the preparation of this lecture.

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Thank you for yourattention

Any questions?

Documents

New Frontiers in the Quality of Medicines · E.Charton, Strasbourg, June 2007 How should users/agencies cope with the chapters during this transition period? •Proposal from « Group