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[ Microbiology Topics.Coordinated by Scott Sutton

Does nternationalarmonization of the USPMicrobial Limits Tests RequireRe Validation of FinishedProduct TestsScott SuttonWelcome to Microbiology Topics.

This column discusses various topics in microbiologyof practical use in val idation and compliance. Weintend this column to be a useful resource for dailywork applications.

Considerations associated with microbiology arepart of every pharmaceutical and medical deviceproduct. Microbiology and applications thereof arefundamental to daily operations, quality assuranceand control, compliance, validation, and so on l i t -erally all aspects of manufacturing and compliance.These topics require knowledge and understandingof basic technical principles for sound decisions ineveryday work situations.

Microbiology principles are complex. New technology is evolving, and the field is becoming increasinglyspecialized. These considerations are relevant to awide range of circumstances including synthesis ofbiotech products, sterile product and device processing, microbial and environmental monitoring offacilities and utilities, sterilization processes, microbialanalytical methods, oral and topical product manufacturing, and so on diverse range of applications.Further, the language of microbiology associated withthe aforementioned may be esoteric and int imidating. These considerations make discussion of relevant

microbiology topics a challenging task. This columnaddresses microbiology topics with these difficultiesin mind. It is our challenge to present microbiologytopics clearly, using understandable language to provide useful information for daily work applications.

Reader comments, questions, and suggestions areneeded to help us fulfill our objective for this column. ase studies from readers are also most welcome. Please send your comments and suggestionsto column coordinator Scott Sutton at [email protected] or coordinating editor Susan Haigney [email protected] POINTSThis article discusses the finalized harmonization ofth e Microbial Limits Tests. Changes to th e respec-tive tests and the ramifications of these changes areaddressed. Specific key points discussed include thefollowing:

The final versions change the requirement forvalidation to verification of suitability of themethod

The Microbial Limits Tests are a collection oftests and specifications. United States harmaco-peia USP -National ormulary NF chapter looks to bioburden testing total aerobic microbial

For more AUlhorinformalion

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ABOUT THEAUTHORSco tt Su t ton Ph.D., is a consultant Vectech Pharmaceutical Consultants and operates The Mi-crobiology Network (www.mlcrobiol.orgJ, which provides services to microbiology-related user s groups.Dr Sutton can be reached at [email protected].

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count [TAMCJ and total yeast and mold count[TYMCD while USP describes tests for theabsence of seven different specified organ-isms. USP is an informational chapterfor setting microbial quality standards for nonNF materials.

USP describes bioburden testing TAMC andTYMC). Specific details of these tests have beenchanged including inoculum broth, preparation,dilution requirements, and growth promotion

USP describes seven separate tests for thefollowing: Bile tolerant gram-negative bacteria,Clostridia, Candida albicans, Staphylococcus aureus,Pseudomonas aeruginosa, Salmonellaspp, and Esch-erichia coli. In addition to new organisms, thereare changes in media requirements. Product risk analysis including product use androute of administration, growth potential, preservation, and other considerations is recommendedin USP . There have been product recallsdue to the presence of objectionable organisms.

Re-validation of existing tests to current harmo-nized standards and level of detail using a matrixapproach is discussed

The fundamental shortcomings of these testsin regards to the current good manufacturingpractice CGMP) requirements for absence ofobjectionable organisms is discussed.

INTRODUCTIONThe international harmonization of the MicrobialLimitsTests in the United States Pharmacopeia USP ,Japanese Pharmacopeia IP , and the European Pharma-copoeia EP, Pharm Eur) was finalized in 2009. Thenear-final drafts of these chapters were published in2003 as part of the harmonization process 1,2,3) tominimal comment. These drafts, with minor revision, were approved in 2005 by the three participating pharmacopeia. The final drafts were publishedin 2006 4,5,6), originally with an implementationdate of May 2007 USA), which was later changedto May 1 of 2009 7). Since then, minor changes weremade in 2007 and 2008 before implementation of thefinal draft versions 8).

The final draft versions have a change that is ofinterest to the general topic of validation. Referencesin the text of both USP and to methodvalidationwere replaced by verification of suitabilityofthe method 1,2). This was done at the request ofUSP for the harmonized chapters, because the mandatory chapters of USP are provided to test productsgxpandjvl com

cott utton

to monograph requirements see USP General Notices:2.10 Official Text and are validated for this purposeby definition. Validating a validated method seemsredundant. Ifwe wish to use an alternate method totest an official article Section 2.20) to monographspecification, the non-USP method must be validated.The actual question we are interested in answeringis whether or not the method is suitable for testing aparticular product. The harmonized methods includesomewhat detailed instructions on this procedure foreach test . These method suitability studies are thefocus ofthis article.DIFFERENCES BETWEEN THENEW AND OLD TESTSThe Microbial Limits Tests, as the name implies, areactually a collection of tests and specifications. Themajor divisions are along chapter organization. USPchapter looks to bioburden testing both TAMCand TYMC) while describes tests for the absenceof seven different specified organisms. The specifications these tests support are described in at leastone monograph of at least one of the three regionalpharmacopeia. Finally, USP is an informational chapter to assist in setting microbial qualitystandards for those products and raw materials notcovered by monograph in the National Formulary.Tests or Specified OrganismsThe first tests we can look at are the tests for specifiedorganisms USP


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you determine that you must comply fully, additionalwork will be in order at least for these organisms.Thequestion of re-validation or demonstration ofmethod suitability) might not be as obvious for theother tests i n the absence of specified organismsbattery of tests. The absence of specified organismstests continue to provide procedures for demonstration of the absence of the following: Staphylococcus aureus Pseudomonas aeruginosa almonella spp Escherichia coliDo the method suitability tests for absence of

these organisms have to be repeated? The first stepof both old and new methods is an enrichment toencourage growth of low numbers of the specifiedorganism, followed by subculture of the microbialgrowth suspensions on selective and/or differentialmedia. The intentof the incubation on selective mediais to depress the growth ofmicroorganisms, allowinga selective advantage to the more resistant specifiedmicroorganisms. The differential mediawas formulated to distinguish th e colony morphology of thespecified organisms from others, allowing visualidentification. As stated previously, this fundamentalapproach is unchanged in the harmonized versions.However, the enrichment broth has changed in someof the new tests and the selective/differential schemehas changed in most. Whi le it might be possible toargue that i fthe old test worked the new should also,this is a high-risk approach. This assertion is likely tobe challenged at some point and data will be requiredas part of the justification.

Should you decide to justifyexisting method suitability studies as meeting current requirements, thereis at least one more consideration. The new tests havefar more detail as to challenge organism i.e., identity,growth conditions, and inoculum size) than were inth e PreparatoryTesting sections ofthe previousUSP tests. This section in th e previous U described the method suitability study designs forboth the enumeration and the absence of tests. Theinoculum concentration especially might be incompatible between th e two versions, as th e previouspreparation instructions were to add 1 mL of notless than 10-3 dilution of a 24-hour broth culture ofthe microorganism to the first dilution ... As this 24hour broth culturewill consist of approximately 107- 108 CFU/mL, the inoculum is therefore about 10'- 105 CFU. The current version specifies not more

JOURN L O V LID TION TE HNOLOGY [SUMM R 20091

than 100 CFU total . In addition, the inocula are nowrequired to be used fresh where before there wereno specifications of this type in the USP prior to theharmonized method. In otherwords, if you were incompliance with the old tests, those same studiescannot be in total compliance with the harmonizedtests. Whether this difference is significant to theoutcome of the test is for the company to determineand justify their decision (although a 1000-fold to1O,000-fold difference in the inoculum seems ratherlarge to ignore).ioburden stThe second Microbial Limits test group is the bioburden test USP


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Scott Sutton.

Start

Figure 1: Overview of previous preparatory testingprocedure for USP microbial limits tests.

1 of 1 3 dilution of a 24 hourculture in Phosphate Buffer,SeDM or Fluid LactoseSo aureus

.. - ol- P. aeruginosa SalmonellaInto test media by relevantprocedure

YesQbNo

Incorporateneutralizersor modifyprocedure

What are th e microbiology quality requirementsfor non-sterile products? Going back to the GeneralNotices and Requirements section 3.10.10, USP 32(2009) states, The applicable US or NF standardapplies to any article marketed in the United Statesthat 1 is recognized in the compendium and (2) isintended or labeled foruse as a drug or as an ingredien t of a drug. Section 3.10.20 extends this to medical devices if they are labeled as complyingwith USP.This seems to be stating plainly that the US chapterslabeled under 1000 are required only for those products for which there are monographs in the National ormulary or those medical devices labeled USP.For most products then, and are not a USPrequirement. Even for those monograph productsth e standard seems to be i f tested shaH comply aspresented in section 3.10, Applicability of Standardsthat states the following;

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IS THIS TEST REALLY NECESSARY?As we look at the opportunities provided to us by theharmonized Microbial LimitsTests, a legitimatequestionhas to be: Is this test doing anygood? Is there a benefitto the patientor to the company in performing this test?I realize this sounds like heresy, but it is a legitimatequestion. As we discuss this topic, understand that theauthor is not a lawyer, nor does he speakforUSP, FDA, orany otherregulatory organizationwith an acronym. Theadvantage to having written regulations is that anyonecan read them. The following discussion is presentedonly for consideration and is no t meant as a specificrecommendation for any courseof action.gxpflndjvt com

detailed in the harmonizedprocedures. Thepreviousver-sion of the Microbial LimitsTests for specified organismsbarelydiscusses media quality control at all, and so it isextremely unlikely that media used in existing methodsuitability studies meetcurrent requirements. Again, anyclaimthat an existingmethod suitabilitystudyis adequatemust be able to withstand challenge in an audit.

Overviews of the method suitability studydesigns arepresented in Figure 1 (olderrequirementsof the regional[US] pharmacopeia) and Figure 2 (the current, harmonized test requirements).

In the end, it is possible to useexisting method suitability studies for the new tests iftheyare doneaccordingto thedetailed instructions present in the harmonizedUSP and harmEurchapters 2.6.12 and 2.6.13)and the tests have sufficientlydetailed documentation toshow this. If eitherof theseconditions is not true (which isthe overwhelminglylikelysituation), the author s recommendation is to repeat the method suitability studies tocurrent standards if you wish to claim compliance withthe USP method.CAN THESE PRODUCTS BE MATRIXED?Ifit is determined that the method suitabilitytests mustbe repeated, then the next question is one oflogistics.Some companies have reported hundreds of productsaffected by this harmonization and the resultantchangesinthe test methods. Many products can be thought ofas existing in families, with minorvariations in formulation or unit fill volume. One approach to the issueof having to perform method suitability testing on somanyproducts is to tryto put togethera testing plan thatconsidersthese productfamily groups. All productsmayeventually have to be re-validated; this depends on theproduct line and the company (and the US Food andDrugAdministration) philosophy. However, this is oneway to at least begin to organize the task.

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Figure 2: Method suitability flowcharts for harmonized method suitability testing

Enumerat ion Specified Grow Cultures ofrow Cultures of S. aureus P aeroginosa B subtilis C. albicans A. ni r now A. brasiliensisFrom national stock cultures passages,

seed-lot culture at 30-35C in SCD mediafo r 18-24 hours bacteria) or 20-25C in SDAmedia for 2-3 days fungi)

Dilute in Saline-peptone orPhosphateBuffer, use within 24

hours refrigerated) orvalidated period

E coli B subtilis S aureus C. alb/cans Salmoneffa enterica C. sporogenesFrom national stock cultures NMT 5 passages,seed-lot culture) at 30-35C in SCD mediafor 18-24 hours bacteria) or 2o-25C in SDAmedia for 2-3 days Yeast) or anaerobically inReinforced Medium for Clostridium at 30-35Cin SCD mediafor 18-24 hours

Dilute in Saline-peptone or Phosphate Buffer;use within 24 hour refrigerated) or validated periodInoculate testmedium a negativeproduct testmedium controlwith s100CFUIncubate at indicatedtemperature for 5 days

Incorporateneutralizers ormodify pressureNo

Yes

Sample preparation as described in test Inoculate s1 CFU of test organism Perform relevant test using shortest timeframe allowable

Incorporatemodifyneutralizers ormodify pressureNo

YesStop The manufacturer s specifications, and goodmanufacturing practices generally, are developedand followed

to ensure that the article will comply with compendial standards until its expiration date, when stored asdirected. Thus, any official article tested as directed inthe relevantmonograph shall comply (General Notices3.10 second paragraph, USP 2009 .

What about the GMP? One aspect is the recognition ofUSP by CGMP, which states in 21 CFR 211.194Laboratory Records (a)(2):A statementofeachmethodusedin the testing of thesample. The statement shall indicate the location ofdata

that establish that the methods used in the testing ofthe sample meet proper standards of accuracy and reli-

4 JOURNAL OF VALIDATION TECHNOLOGY [SUMMER 2009]

ability as applied to the product tested. Ifthemethodemployed is in the current revision of the United StatesPharmacopeia-National Formulary Association ofOfficial Analytical Chemists, Book Methods or in otherrecognized standard references, or is detailed i n anapproved new drug application and the referencedmethod is not modified, a statement indicating themethod and reference will suffice). The suitability ofall testing methods used shall beverified under actualconditions of use 9).

So the perception is that if you follow the USP youare following GMP. While this is true in many cases,we also have to consider the issue of objectionableorganisms.

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Thephrase (or concept) of objectionable organismsappears threetimes in 21 CFR 211:

.21 CFR 211.84(d)(6). Each lot of a componentdrug product container, or closure with potential for microbiological contamination that isobjectionable in view of its intended use shallbe subjected to microbiological tests before use.[The changes in GMP effective December 8, 2008included changing the phrase that is liable to forwith potential for in this section.]

.21 CFR211.113(a). uAppropriatewrit tenprocedures,designed to prevent objectionablemicroorganismsin drug products not required to be sterile, shall beestablished and followed..21 CFR 2I1.165(b). There shall be appropriatelaboratory testing, as necessary, of each batch ofdrug product required to be free of objectionablemicroorganisms.

It has been suggested that the Microbial limits Testswill meet these requirements and demonstrate theabsence ofobjectionableorganisms 10). However, thisposition is not supported by FDA 11) nor by USP 198212) and obviouslyhasn t beenfor decades. In fact, addition characterization of isolates from non-sterile productsand theiranalysis is now an explicit expectation of theharmonized chapters. This direct instruction can befound in the harmonizedinformational chapter WHERE DOES T S LEAVE US?I would urge the re-validation of existing tests to currentstandards and level ofdetail. It is extremely unlikelytha tmethod suitability studies performed years ago will bedocumented in sufficient detail to showcompliancewiththe expectationswritten intothe harmonizedprocedureseven if the tests were performed identically. The firststep in this process would be to perform an analysis ofcurrently marketed products for relationships amongthem. Aplancan then bedeveloped from this productanalysis to perform method suitability studies as efficiently as possible.Whatever the situation with the re-evaluation of thesuitabilityof the testmethod, it must also be dearlyheldinmind that this is not the microbialqualityexpectationfor marketed, non-sterile product in the United Statesas described by 21 CFR 211, FDA regulatory precedent.and the internationally harmonized USP . Themanufactureris expected to knowwhatmicroorganismsare present in their non-sterile medications and haveconfidence that those microorganisms will not imperileither the patient using the medication nor the qualityof the medication on long-term storage.

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REFERENCES1 USP, 2003a, Microbiological Examination OfNonsterile Products: Microbial Enumeration Tests:

Pharm Forum, 29 5 :1714-1722.2. USP, 2003b, Microbiological Examination of

Nonsterile Products: Tests for Specified Microorganisms, Pharm Forum, 29 5 :1722-1733.

3. USP, 2003c, Microbiological Quality ofNonsterile Pharmaceutical Products: Pharm Forum,29 5):1733-1735.

4. USP 2006a, Microbial Examination of Nonsteri le Products: Microbial Enumeration Tests, USP SuppI2:3759-3765.

5 USP, 2006b, Microbiological Examination OfNonsterile Products: Tests For Specified Microorganisms, USP 29 Suppl 2: 3759-3765.

6 USP, 2006c, Microbial Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for PharmaceuticalUse, USP 29 SuppI2:3801.

7 USP 2007, Harmonized Microbiology General Chapters: NoticeOfPostponement, Pharm Forum 33 2 :168169.

8 USP, 2009, USP32 - NF 27, The United States Pharmacopeia Convention. Rockville, MD USA, 2008.

9 FDA, 21 CFR 211, Volume 4, Revised as of April 12008.

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10. Sutton, S The Harmonization of the Microbial LimitsTests, Pharm Technol. 30 12):66-73,2006.11 FDA, Guide to Inspections ofMicrobiological Pharmaceutical Quality Control Laboratories, 1993. http://www.fda.gov/ICECI/lnspections/lnspectionGuides/ucm074914.htm.

12. USP, Microbial Contamination of Sterile and NonSterlile Articles, with Special Reference Pseudomonas cepacia, Pharm Forum 8 4):2239, 1982.

13. Additional discussion of this point can be found in aseries of articles written by the author in 2006 for thePMF Newsletter. http://www.microbiologyforum.org/news.htm.

14. Jimenez, 1. , Microbial Diversity in PharmaceuticalProduct Recalls and Environments, PDA J Pharm SciTech., 61 5):383-398, 2007. V

ARTICLE ACRONYM LISTINGCGMP urrent Good Manufacturing PracticeP European PharmacopeiaFD US Food and Drug dministrationJP Japanese PharmacopeiaN National FormularyT MC Total erobic Microbial ountTYMC Total Yeast and Mold ountUSP United States Pharmacopeia

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