The Value of Plant Isolates in Pharma Quality (Myatt & Morgan, PDA Letter, JulAug 2011)

7/25/2019 The Value of Plant Isolates in Pharma Quality (Myatt & Morgan, PDA Letter, JulAug 2011)

1/530 Letter July/August 2011

The Value of Plant Isolates in Pharma QualityDavid Myatt, PhD and Charlotte Morgan, PhD, BTF, a bioMrieux company

What value are plant isolates in microbiological quality in the pharma industry?

Increasingly, pharmaceutical companies are including their own isolates in the battery of microorganisms that theyuse for media growth promotion testing and validation studies. These plant isolates are wild-type strains isolated

during environmental monitoring, sterility and bioburden testing, and routine testing for contamination or spoilage. In so

doing, these companies seek best microbiology practice, but it remains somewhat controversial. Some commentators

argue that compendial methods do not mandate such an approach, others challenge its scientic merit, and some

query the practicality. Notwithstanding a level of public debate, many companies are implementing standard operating

procedures and grappling with the practicalities of strain selection, culture maintenance that sustains the cultura

characteristics of wild plant isolates, a degree of regulatory uncertainty and, certainly, a paucity of guidance on

how to achieve the desired outcome, whether that is simply compliance or genuine commitment to more challenging

tests in pharmaceutical quality management.


2/5Letter July/August 2011

Trends in Use of Wild Isolates

Lets begin by agreeing that this reallyisnt anything new! Authorities on qual-ity in pharmaceutical microbiology havebeen suggesting the merit of includingwild-type isolates in media QC testing

for many years (1, 2, 3) and auditorsnow issue FDA 483 observations in re-lation to this expectation.(4)Certainly,its become a topical matter in recentyears, with periodic debate in industrydiscussion forums and blogging sites.(5,6, 7)While perspectives on the scientificmerits vary, and whether its a functionof regulatory attention or best micro-biological practice, use of plant isolates(or whatever you choose to call them)is now commonplace in pharmaceutical

microbiology.

One authors own insight, gleaned frommany conversations with practicingpharmaceutical microbiologists, clearlyindicates that many big pharma com-panies and smaller ones alike are imple-menting (or already have) the use of a fewof their own isolates to complement thecompendial reference strains in growthpromotion testing of environmentalmonitoring and sterility testing media,

and sometimes in validation studies fornew methods such as rapid microbio-logical methods (RMM) for sterility as-surance testing. In most cases, these labsintend to make an annual assessment ofthe frequency of species amongst theirenvironmental isolates and select eitherthe two or three with highest frequencyor the highest frequency isolate fromeach of the Gram positive, Gram nega-tive and fungal isolate groups. Their in-tention is usually articulated in terms of

compliance (i.e., what auditors want) orbest laboratory practice, even if they donot subscribe to the view that the use ofthese strains is a valuable exercise in veri-fying the performance of their culturemedia or test methods. It certainly seemsthat there is now a widely-perceivedneed for compliance here (in the absenceof an FDA audit citation) given that theuse of environmental isolates is stronglyrecommended in a number of compen-dial references and other authoritativedocuments.

It is also commonplace to see manufac-turers of personal care products and nu-traceuticals include extensive batteries ofcontaminant organisms (isolated fromtheir raw materials or spoilage of their

products) in studies to verify the efficacyof their preservative systems. Of course,conceptually, this is akin to the testingof non-sterile pharmaceuticals for ob-jectionable organisms that often origi-nate as contaminants in raw materials orfrom the manufacturing environment.Whatever the case, these practices arefounded on the idea that these micro-organisms are a better challenge to themicrobiological method than the stan-dard compendial strains.

Applications, Regulations and

Recommendations

The compendial references for sterilitytests, enumeration tests, specified micro-organisms, and antimicrobial effective-ness tests (USP chapters , , and respectively) and thecorresponding sections of the EuropeanPharmacopoeia do not prescribe the useof environmental or other wild isolates.However, a number of compelling rec-

ommendations in this regard are made inguidelines issued by several authorities:

Concerning the microbiological evalu-ation of controlled environments, USP says for the Growth Promotiontest, representative microflora isolatedfrom the controlled environmentmay also be used to test media.

USP concerning Microbiolog-ical Best Laboratory Practice suggestsmicroorganisms used in growth-pro-motion testingmay include repre-sentative environmental isolates (butthese latter are not to be construed ascompendial requirements).

FDA Guidance for Industry for SterileDrug Products Produced by Aseptic Pro-cessing Current Good Manufacturing

Practice (September 2004) says TheQC laboratory should determine ifUSP indicator organisms sufficientlyrepresent production-related isolatesEnvironmental monitoring and steril-ity test isolates can be substituted (asappropriate) or added to the growthpromotion challenge.

FDA Guidance for Industry concerningValidation of Growth-Based Rapid Microbiological Methods for Sterility Testingof Cellular and Gene Therapy Products(draft guidance, February 2008) sug-gests, in relation to selecting a panel ofappropriate challenge microorganismfor validating an RMM, the inclusionof isolates detected in starting materi-als, isolates detected by in-process test-

Wild-type Strains

By denition, strains found in nature. But in our context, we mean to discuss

strains that are recently-isolated in a manufacturing context, either from a con-

trolled manufacturing environment or, perhaps, a contaminant of raw materials

or nished pharmaceutical product. These are strains that are not conditioned

through serial subculture to growth on rich laboratory culture media and may ex-

hibit unstable phenotypic characteristics associated with oligotrophy, desiccation

or biolm formation, namely traits that have enabled survival in harsh conditionsand may not persist in strains that are serially passaged in rich culture media un-

characteristic of the environment from where they were isolated.

Isolated Look at this Article

Testing of plant isolates, or wild-type

strains, is a regulatory expectation

Arguments against such test-ing include practicalities related

to repeatability, reproducibility in

validations and cost

The real value derives from sig-

nicantly greater condence in

media, methods and systems

that are validated and tested us-

ing strains that are more typical of

target organisms than those refer-

enced in compendial methods



ing or during preliminary product test-ing, isolates detected by environmentalmonitoring of your manufacturing fa-cility, [and] isolates from your produc-

tion areas which represent low nutrientor high stress environments.

USP concerning Disinfectantsand Antiseptics suggests surface chal-lenge tests using standard test micro-organisms and microorganisms thatare typical environmental isolates.

The Japanese Pharmacopoeia (XV,General Information section 11.4.1concerned with Media Fill Tests) saysin relation to selection of growth pro-

motion testing organisms one or tworepresentative microorganisms whichare frequently isolated in environmen-tal monitoring should be used.

While none of these can be construed asa mandatory requirement, here are manycalls to consider the relevance of plantisolates in growth promotion testing,validation studies and challenge testing.Presumably, this selection of referencesrepresents a much greater number of

experienced individuals on expert panelswhove co-authored these documents inconjunction with the regulatory agenciesthat have published them. So, it seemsfair to say that there are widely-heldviews that plant isolates are relevant.

Costs and Value

Arguments against the inclusion of plantisolates in pharmaceutical microbiologyare varied and include the practicalitiesof standardizing such isolates for repeat-

ability and reproducibility in valida-tions, and the challenge of preservingthese strains in a culture collection.(7)We would add to that list the consider-able challenges related to expertise andspecialized resources needed to managea culture collection of plant isolates sothat theyre minimally compromisedby subculture and preservation. This isan increasingly acute issue in pharmawhere everyone, including microbiologi-cal quality laboratories, is asked to do

much more with much less in toughereconomic times.

Experience in culture collection manage-ment and culture preservation techniquesis increasingly rare when many laborato-ries opt to purchase strains from recog-nized collections or commercial suppli-ers. Beyond that, time and competenciesneeded to prepare standardized suspen-sions by serial dilution are also increas-ingly scarce. Toted up against the costs ofthese activities are lab space, acquisitionand installation costs, qualification andvalidation projects, and maintenanceand user training demanded by various

pieces of laboratory equipment like ultralow temperature freezers, freeze-dryers,spectrophotometers and data manage-ment systems needed for a competentculture collection. There are also costs ofspecialized laboratory reagents and con-sumable items and their procurement,qualification, documentation, storageand wastage. The costs mount up dra-matically. In this context, commercially-available quantitative microbiologicalcontrols produced with compendial ref-

erence strains have grown in popularity,and leading brand products are CertifiedReference Materials according to ISOGuide 34 accreditation. Such productsoffer labs the option of outsourcing la-borious, time-consuming, expensive anderror-prone activities associated withmaintaining cultures and preparing in-ocula for routine growth promotion testsand validations studies.

But, having outsourced these activities,

those wanting to incorporate plant iso-lates in their testing are now challengedto reinstate skills, time and other re-sources needed to maintain and preparethem. Additionally, it could be arguedthat the expertise and facilities neededto preserve plant isolates markedly ex-ceeds those demanded for compendialstrains. For instance, the optimal cul-turing conditions required for the com-pendial strains are well known and doc-umented within the industry, advice is

It could be argued that the expertise and facilities needed to preserve plant

isolates markedly exceeds those demanded for compendial strains

on hand. Whereas, when preserving andculturing a plant isolate, it is unknownwhether the environmental strain wilbe as robust or have the same culturing

requirements as a known culture collec-tion strain of the particular plant isolatespecies, so it can quite often be a caseof trial and error and therefore time-consuming and expensive.

Practicalities in Implementation

The contention that exists about use ofplant isolate derives from a general lackof knowledge of how these wild strainsdiffer from culture collection strainsthat have been serially subcultured to

such an extent that they are adaptedto rich culture media. The nature of thedifferences is poorly understood, as arethe mechanisms involved. Certainly, se-rial subculture drives a process of in vitroevolution where there is natural selectionfor clones that grow most luxuriantlyon rich media, but the stability of whatcould be defined as wild attributesand consequent phenotypic and physi-ological changes, and how quickly theseemerge in serially-subcultured strains, is

generally not understood.

In this context, the safest approach is tominimize the serial subculture of plantisolates. Compendial references suggestthat culture collection strains should befive or fewer subcultures (passages) fromthe culture originally sourced from areference culture collection. This fivepassages rule has been extrapolated toplant isolates, but again in the interestsof conservatism, we suggest that mini-

mizing serial subculture is the only waylikely to minimize the risk of significantstrain evolution that would compromisea strains merit as a stringent challengeto media fertility or RMM performanceSo, it could be argued that the ideal candidates will be minimally subculturedplant isolates, with phenotypic charac-teristics stabilized through sophisticatedpreservation techniques, and standard-ized to deliver a reliable inoculum for con-sistent growth promotion testing and



for repeatable and reproducible valida-

tion studies. That is an ambitious objec-

tive! Few laboratories have the compe-

tencies and resources outlined above to

accomplish this, and it is a fact that some

very useful techniques are the subject of

patents or proprietary know-how. There

and facilities now offer to acquire from

labs their minimally subcultured strains

and return standardized inocula to cover

a year (or more) of testing with mini-

mized risk of compromising the strains

wild traits. Leaders in this field use the

most sophisticated techniques available

typical of target organisms than thosereferenced in compendial methods, thatis, strains acquired from culture collec-tions where theyve been serially passagedunder atypical conditions for many yearsCertainly, the very isolation of wild-typestrains and their minimal subculture forpreservation are selective pressures tha

threaten the traits wed hope to retainin the strains we use to challenge ourmicrobiology tests, but we dont see prac-tical alternatives beyond use of the mossophisticated techniques to preservestrains as close to their primary isola-tion as possible. Accordingly, we arguethat there is real value, albeit difficult toquantify, in maximizing confidence thamedia and methods we use in pharma-ceutical microbiology are effective and

reliable to the greatest extent that werepractically able to demonstrate, andtherefore contribute more assurance othe quality of our pharmaceuticals, med-ical devices and personal care products.

It seems inevitable that there will besustained or increased attention paid tocritical environment monitoring, de-tection of objectionable organisms andother microbiological practices intendedto minimize contamination and adverseoutcomes from the use of therapeuticand nutritional products. There is littleindication from regulators or any otherauthorities in the pharma industry thatvigilance will decline or expectationswill be relaxed. Increasingly litigiousdeveloped markets, growing healthcarestandards and expectations in emergingmarkets all have the effect of encouraginggreater regulation, despite economic pres-sures. We would also expect to see greateruse of methods not based on microbia

growth, where these RMMs need to beshown, through extensive validation, tobe equivalent to traditional compendiamethods, at least according to currentrequirements.(9)For instance, is a slowgrowing plant isolate (that takes >5 daysto grow in traditional culture) detectableby a RMM? This is why, if such plant iso-lates are found, they may be relevant formethod validations. Indeed, such ques-tions only serve to fuel discussions aboutthe merits of using plant isolates to

We nd few convincing arguments against the use

of plant isolates to more effectively challenge the

media and methods used in pharmaceutical quality

are three main techniques to reliablystore microbial cultures, namely ultralow freezing at below -70C, cryopreser-vation in liquid nitrogen, or freeze dry-ing (lyophilization). Each technique has

advantages and disadvantages. However,selection of a technique is more oftenbased on the availability of equipmentand expertise than on the suitability ofthe technique to the particular strain tobe preserved. Some strains survive wellin a frozen matrix, whereas others canonly be frozen in liquid nitrogen forlong term survival. Freeze drying is thetechnique of choice for long term mi-crobial preservation (8), for cells thatcan tolerate freezing and drying, but

this technique can be too harsh for morefragile microbes (e.g., mycoplasma). Itmust be kept in mind, no matter whichtechnique is used, there is still a degreeof selection happening during storage,and viability cannot be sustained indefi-nitely, and therefore the longer the stor-age, the greater the possibility of geneticor phenotypic drift. It is for this reasonthat extensive profiling of strains prior tostorage should occur, so that any changein the strain can be detected by compari-son with the original profile. The valueof experience in the cataloguing andstorage of strains in a culture collectioncan be easily under-estimated, hencethe reason why most companies thathold commercially important microbialstrains invest in back-up storage of theirstrains at off-site facilities with relevantexpertise and capabilities.

Accordingly, specialist service providerswith the appropriate focus, experience

for strain preservation, standardizationand delivery and seek to provide pre-mium service to match the regulatoryand operational context that is peculiarto microbiological quality in the pharma

industry. When assessing the capabilitiesof such service providers, we recommenda thorough review of their track-recordwith a wide range of compendial strains,the potential for their technologies tominimize the in vitroevolution or adap-tation of plant isolates, and their abilityto provide plant isolates in formats thatare consistent with and as convenient asthe compendial strains they supply.

Conclusion and Future Trends

Weve outlined here our perception thatthere is now a very strong trend to in-creased use of plant isolates to challengepharmaceutical microbiology media,methods and systems, both in rou-tine QC testing and validation. Weveobserved the regulatory pressures forgreater compliance in this area, but ac-knowledge the practical and economicchallenges that accompany a commit-ment to the routine use of plant isolates.Nevertheless, given the ethical, legal and

economic imperatives that compel rigor-ous quality management in the pharma-ceutical industry, we find few convinc-ing arguments against the use of plantisolates to more effectively challenge themedia and methods used in pharma-ceutical quality. It is not costless to doso and must therefore deliver real value.We suggest that real value derives fromsignificantly greater confidence in media,methods and systems that are validatedand tested using strains that are more



a QA management role

implementing a qual-

ity system, cGMP and

compliance with inter-

national regulatory stan-

dards. His subsequent

commercial roles in the

pharma and bioprocess-

ing markets included focus on microbiologica

culture media, pharmaceutical quality control

critical environmental monitoring, and biop

harma production. He holds a PhD in Microbiol

ogy and an MBA in International Business and

Marketing. He leads strategic marketing at BTF

a bioMrieux company in Australia. To contac

email [email protected]

Charlotte Morgan has

more than 10 years expe-

rience in research related

to precise detection and

dispensing of microbial

cells and maintaining vi-ability through techniques

such as freeze drying. Her

role as a principle researcher in the developmen

of the commercial microbial reference materia

BioBall has developed into managing a team o

dedicated scientists to expand the BioBall range

and the techniques to improve long term precise

preservation of microbial cells. She holds a Mas

ters in Water Microbiology and a PhD in Microbi

ology. She is currently a R&D Manager at BTF, a

bioMerieux company in Australia. To contact, ema

[email protected].

ber 2004.

FDA 483 Observation (www.fda.gov/4.downloads/AboutFDA/CentersOfces/ORA/ORAElectronicReadingRoom/UCM210772.pdf)

S. Sutton (2004), Quality Control of5.Microbiological Culture Media, Phar-maceutical Microbiology Forum News-

letter, Volume 12, Number 1, January,2006.

PMFList (microbiol.org/PMFList_info.6.htm), operated from The MicrobiologyNetwork (microbiol.org)

Pharmaceutical Microbiology Blog7.(2010), edited by Tim Sandle (pharmig.blogspot.com/2010/01/use-of-environ -mental-isolates.html).

Y. Miyamoto-Shinohara, J. Sukenobe,8.T. Imaizumi, T. Nakahara (2006), Sur-

vival curves for microbial species stored byfreeze-drying, Cryobiology, 52, pp 27-32.

P. Newby et al. (2004), The Introduc-9.tion of Qualitative Rapid Microbiologi-cal Methods for Drug-Product Testing,Pharmaceutical Technology, Process An-alytical Technology, 2004

About the Authors

David Myatthas more than 20 years experi-

ence in diagnostic and industrial microbiology,

having held senior roles in quality manage-

ment, marketing and commercial leader-

ship in global microbiology companies. Hispharma microbiology experience began with

complement culture collection strains inimportant pharmaceutical microbiologytesting. But, it seems the tide of opinionnow shows that many have accepted thevalue of additional strains selected fortheir relevance in individual sites andproducts. The challenge remains to ex-ecute this practice well, so its value is re-

alized, either through investment in thecompetencies and skills required to reapthe value of plant isolate strains preservedas close to their original state as possible,or by partnering with service providerswhose focus, expertise and experiencein the specialized area of microbiologi-cal strain preservation can provide stableand quantitative plant isolate strains thatare minimally passaged since isolation.

References

FDA (1993), Microbiological Pharma-1.ceutical Quality Control Labs (7/93):Guide to Inspections of Microbiologi-cal Quality Control Laboratories (www.fda.gov/ICECI/Inspections/Inspection -Guides/ucm074914.htm)

A.M. Cundell (2004), Microbial Test-2.ing in Support of Aseptic Processing,Pharmaceutical Technology, June 2004,pp 56-66.

FDA (2004), Guidance to Industry:3.Sterile Drug Products Produced by Aseptic

Processing Current Good Manufactur-ing Practice, USDHHS, FDA, Septem-

Coming Soon!The July/August PDA Journal of Pharmaceutical Science and Technologywill include an article by TimSandlecalled, A Review of Cleanroom Microora: Types, Trends and Patterns

Sign up for an E-TOC Alert, go to journal.pda.org/cgi/alerts
http://journal.pda.org/cgi/alertshttp://journal.pda.org/cgi/alerts

The Value of Plant Isolates in Pharma Quality (Myatt & Morgan, PDA Letter, JulAug 2011)

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