or Gram-negative species are surviving.Finally, there were also considerations forgowning, e.g. should more be spent onundergarments?Chris Wells, Director, Global Key

Accounts at bioMérieux, spoke on the useof plant (facility) isolates in QC testingand pharmaceutical microbiologyvalidation. Increasingly, pharmaceuticalcompanies are including their own isolatesin the battery of micro-organisms that areused for media growth promotion testingand validation studies. These are wild-type strains isolated during EM, sterilityand bioburden testing, and routine testingfor contamination or spoilage. Some industry commentators, however,

point out that compendial methods do notcurrently mandate such an approach,while others have challenged the meritand practicality of their use. Despite their use being optional, Wells

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MEETING REGULATORYEXPECTATIONSBest practise and meeting the expectations of regulators on theuse of plant isolates, bioburden strategies, rapid ID methods,auditing environmental controls and deviation management aretopics taxing microbiologists. Susan Birks reports on these andother issues discussed at the recent Pharmig conference

Research is revealing moreinformation about microbes found onhumans and in the manufacturingenvironment, changing the waymicrobiological strategies and tests aredevised. Pharmig’s annual conference,held in Nottingham, UK, highlighted someareas where pharmaceutical regulatorshave embraced these new concepts buthave not, as yet, mandated or specifiedprocedures in the standards. Tim Sandle, Site Microbiologist, BPL

began the proceedings by looking at recentresearch from the Human MicrobiomeProject (HMP) and its implications forcleanroom microbiology. Research on theHMP has revealed that there are some1,000 different species of microbes foundon men and women, and it is nowrecognised that a human adult housesabout 1012 bacteria on the skin, 1010 in themouth and 1014 in the gastro-intestinaltract. There is also a considerablediversity of species and a variationbetween individuals.The skin is now regarded as an

ecosystem with different areas populatedby different species. Knowing what isthere is important because people canshed as many a billion skin cells a day, andup to 10% of those may carry on averagefour microbes per skin cell. Today, we can identify those microbes

through the use of genotypic techniques(such as 16s rRNA genes in lysedmicrobial DNA) and whereas previousculture methods tended to detectMicroccocus spp., Staphylococcus spp., anda low incidence of Gram positive rods, newresearch indicates the prevalence of otherspecies such as: more Gram-negativebacteria (Acinetobacter is the dominantgenus) under the arms and between toes,more Gram-positive and anerobic bacterialinked to arms and torsos, and more fungigenerally distributed all over the body.For microbiologists concerned with

bioburden, this difference between what isfound in the cleanroom using culturemethods compared with what we nowknow resides on most people poses thequestion, is the media and its incubationregime (time and temperature) suitablefor the recovery of the types of microbelikely to be shed from the skin? In the light of this, Sandle considered

various implications: Is there a need tochange environmental monitoring (EM)methods? Is anaerobic or fungalmonitoring needed? Does the disinfectantefficacy panel need expanding? Doesmedia growth promotion need to change?What can be learned for micro datadeviations? And do the findings inform

about gowning practices? One of Sandle’s conclusions was that

culture-based methods remain useful as aspot check for indicators of cleanroomcontamination as they pick up enoughspecies to show a trend. However, EM mayneed to be adjusted for anerobic micro-organisms, and given the relatively highlevels of Propionibacterium spp. associatedwith hair follicles, he suggested, this isparticularly relevant where nitrogen gasor compressed air lines are used as part ofthe filling process and have contact withthe product. The findings also have a bearing on

disinfection studies. Perhaps considerationshould be given to broadening the ‘testpanel’ and checks made as to whichspecies are most resistant, he said. Interms of trending, it may be advisable toreview efficacy of cleaning and disinfectionregimes to check whether spore-formers

said more ‘challenge testing’ was beingcarried out to check if disinfectants areworking. He said that there was ‘aperception’ among large pharmacompanies that regulators in the mostdeveloped markets expect the use of plantisolates despite the lack of definitive andclear guidance on what is required. In thepast 10–15 years some 10 or morecompanies were cited in 483 warningletters for not using wild isolates, saidWells. Their use was also driven by anaspiration towards best practice orcorporate edict, he said.While many companies currently select

two (or more) of their high frequencyisolates based on annual review of EMdata (reassessed annually) there is nocorrespondence between common plantisolate species and those prescribed byPharmacopoeial methods, raisingquestions about the relevance ofprescribed strains, said Wells. Patrick Nieuwenhuizen, Manager, QC,

Genzyme, suggested ways of improvingcleanroom behaviour, aseptic practicesand operator understanding so as to avoidregulatory observations. Cleanroomtraining is key but tuition is also neededto ensure operators understand themicrobiology principles behindcleanrooms, such as: why they are cold,the difference between bacteria andviruses, why there is a pressuredifference, the difference betweencleaning and disinfection and why it isimportant. Even the rationale of using atriple bucket is important and needs to beexplained to the non-microbiologist, hesuggested.To avoid regulatory observations, he

advised cleanroom personnel trainingshould include – in addition to anintroduction to basic microbiology –courses on contamination control,cleanroom behaviour, hand washing,gowning qualification, process simulationsand media fills (if applicable). Operator performance is strongly

related to environmental excursions, hesaid. According to research, some 95% ofexcursions reported contained micro-organisms from human origin and 85% ofroot causes were attributed to inadequateaseptic handling/behaviour (Figure 1). When training in aseptic techniques it

is useful to have somewhere thatoperators can practise. Similarly, specifictraining should be carried out todemonstrate proficiency in handlingmedia fills, and both should be under thesupervision of a trained microbiologist. Because training is quickly forgotten

and old habits creep in, Nieuwenhuizen

suggested the need to develop a learningculture around cleanroom behaviour byhaving weekly communications and teammeetings: ‘Get the right people involvedand explain what the microbiologistsfound, why it is an issue, and what can bedone to correct it.’ Thereafter,communicate what the outcome was and,importantly, appreciate the inputprovided, he added.

Audit planningElaine Doyle, Senior Compliance Auditor,Abbott, looked at environmental controls,audit planning and writing auditobservations. Doyle produced a longchecklist of questions for use whenauditing suppliers, such as: whatregulations they use, what they produce,ask to see quality documents, identifytheir sites and product portfolio;determine the scope of outsourcing,identify significant changes to sites orquality systems (such as redundancies orchange in site management), review theaudit /inspection history of the site,request and review applicable procedures,determine the scope of utilities, forexample, do they use compressed gas? In addition, she suggested requesting

and reviewing the list of non-conformances and out of spec (OOS)investigations, any applicable qualityagreements, where contract services andtesting are utilised, and how they notify ofany changes (e.g. by letter, e-mail, etc).

Doyle said that many citations arisebecause sites are not carrying outprocesses as documented in their ownSOPs, so ask to see trend data, look attemperature and humidity controls – dothey have alarms? are they validated? Hasthere been a pattern to excursions andhow was it dealt with? To simply write onthe investigation ‘additional training’ isnot sufficient, she said. Ask whether thereis any evidence to show it has improved.Finally, data integrity is another big

cause of citations, she said, so verifycomputer systems that support the controlof facilities. Alan Whipple, Microbiology Director,

Product Quality at GSK UK, looked atdeviation management and identification(ID) of isolates. As technology hasimproved, rapid methods mean that aninitial ID can be carried out quickly – butbe wary of rushing to management toannounce the initial findings, Whipplewarned, as certain statements couldpersuade non-microbiologist managers tostop production when it may not benecessary. He suggested that before rushing to

fully identify an organism themicrobiologist should ask: Is the isolatelikely to present a risk to product qualityor product safety? Is there a regulatoryexpectation to identify? And is it useful toknow the potential source? For some situations the need to identify

is clear such as OOS results in producttests (sterility/microbial limit testing),Grade A (and B) recoveries in asepticprocessing environments or action levelexcursions in water and EM. But thinkcarefully before identifying other isolateshe said. ‘To comply with expectations youdon’t need to identify every organism.’ He added that traditional methods have

their uses. For example, he suggested thata Gram stain can be used to identify alikely source. However, the risk ofmicrobial proliferation needs to beunderstood – e.g. some bacteria inaqueous product should ring alarm bells.‘Look for developing trends, such as anincrease in spore formers or moulds...andmake causative links,’ he suggested.The difficult question is how far to take

the identification. Whipple said: ‘We don’talways need to go to species level ID’. Hisreasoning was if you give an organism aspecies name, you need to be prepared toassess and document the risk and impactof that micro-organism. ‘Identification to species level is both

prone to inaccuracy and potentiallyexpensive,’ he added, and so should not beattempted where it is not required.

January 2015 67MICROBIOLOGY

�

Figure 1: Cleanroom Flora Distribution. Slide courtesy of Patrick Nieuwenhuizen(Genzyme, a sanofi company)

Operator performance related toenvironmental excursions?• 95% of excursions reported containedmodus operandi from human origin• 85% root cause attributed to inadequateaseptic handling / behaviour

At its November annual conference Pharmaceutical Microbiology Interest Group(Pharmig) initiated plans to create a Cleanroom Action Group (CAG) to discuss andformalise best practice in cleanroom procedures. The aim is to collate best practice across many aspects, such as cleaning, gowning and

environmental monitoring, where currently none is available. Cleanroom practice is constantly evolving and standards, regulators and facility

inspectors often specify the requirement for best practice but without specific guidance ora benchmark for companies to compare. Pharmig is a non-profit making professional organisation that represents the interests

of those who have responsibility for, or work alongside microbiology within thepharmaceutical, cosmetics and healthcare sectors. At its annual conference delegates were invited to put forward particular areas of

interest where best practice is not clear. Brian Hayes (Ipsen Biopharm) is leading the CAG as Chairman, with the group also

benefiting from the expertise of Pharmig Vice-Chairman, Dr Tim Sandle. Hayes outlinedthe main aims of the group as: 1) To establish a common understanding of world class aseptic processing; 2) To work with like-minded professionals to gain common understanding of BestCleanroom Practice and provide the opportunity to learn from others; 3) To hold regular visits to cleanroom facilities and to discuss and share good cleanroomand practices.Both Hayes and Dr Sandle would like to see the CAG becoming a key influencing group

within the pharma industry and with regulators.Hayes explained that within the pharma industry there was a need for a ‘safe’ non-

commercial forum, ‘where like-minded industry professionals can discuss best practicefor all cleanroom activities, current hot topics and industry trends and updates’.The ethos of the group was well received and 43 delegates cutting across the industry

have since joined. To get involved visit the Pharmig website.

www.pharmig.com

Pharmig Cleanroom Action Group initiated

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What is always important is that riskassessments are documented in a way thata non-microbiologist (doctor or layman)can understand the risk. Mary-Anne Weatherhead, a Qualified

Person (QP) and a microbiologist at Pfizer,discussed the QP’s role and howmicrobiologists can build partnershipswith them. She looked at the duties of aQP, key considerations for QP decisionmaking, how the microbiologists can helpthe QP and why it is important for moremicrobiologists to become one. Part of the QP’s role is to ensure that

each batch of product has beenmanufactured in compliance with nationalrequirements and MarketingAuthorisation (MA), and that each batchimported from outside the community hasundergone in the EU full qualitative andquantitative analysis of the activeconstituents, and all other tests to showcompliance with the MA.QPs and microbiologists are often key in

contamination investigations and in suchan event, the QP will want to know thescope, impact, immediate actions taken,root cause, corrective and preventiveaction plans (CAPA), and will want tohave an overall summary. ‘Remember QPs are mutually

responsible,’ she said and so whenmeeting them, as well as a clear summary,bring back-up information to answer allquestions, trend information, details ofmicrobe identifications, and your idea ofwhat should happen next – otherwise theQP will come up with ideas of their own.’ She also urged more microbiologists to

become QPs: ‘A time bomb is ticking, asmore QPs are retiring than qualifying,’she said. In the UK, there are morechemist and pharmacist QPs thanmicrobiologists. She added that havingmore microbiologists QPs would give themicrobiology community greater influenceover quality procedures and regulation.

A bioburden control strategyWhen designing a bioburden controlstrategy, Dr Kevin Wright, PrincipalScientist, Microbiology, Procter & Gamble,UK suggested prevention is better thancure. Unfortunately, for many producers(personal care, food and pharma) thebioburden risk is increasing. Wright gavemany reasons why that is, includingincreased use of natural raw andbiodegradable materials, as well as use ofmilder formulation chemistries and fewerpreservatives. All these trends together can increase

the risk to the consumer via (undesirable)usage habits, and to product quality.

Furthermore, the production facility iswarm and wet, providing the idealbreeding ground for microbes. This meansthere is an increased need for a clean rawmaterial supply chain, clean facilitydesign, in process and raw materialsmonitoring and cGMP.To enable an estimation of the

probability and severity of bioburdenrelative to the consumer and product, hesuggested collation of data that: quantifieshow much bioburden is present (dose),identifies if it is a pathogen orenvironmental, and functionalises whatthe potential risk of the bioburden is tothe consumer or product. The best strategy is to build an

‘integrated bioburden control approach’he suggested. This is already practised bythe food sector via the ‘Hurdle’ concept,which looks to combine hostility vectorsduring production – i.e. modifiedatmospheres, antimicrobials, packagingand surface pasteurisation. In FMCG, itcould involve use of hostile intermediates,thermal cycling, HACCP, monitoring andintervention, pasteurisation and kill steps. He said changes in a facility’s bioburden

may be driven by various factors: changes

by the material supplier, changes in aformulation to less hostile ingredients,changes in equipment or processimprovement, or letting the outside indue to engineering works. To improvecontrol, Wright suggested setting upsystems that alert to changes in qualityagreements with suppliers and inform ofplant changes and increased sampling,and to communicate more withformulators. Joanne Spiers, VP, Quality Operations &

Regulatory Compliance, Catalent, lookedat how to carry out effectiveinvestigations. As stated in CFR 21 Part211.192, any unexplained discrepancy orthe failure of a batch, or any of itscomponents, to meet any of itsspecifications, must be thoroughlyinvestigated whether the batch hasalready been distributed or not, she said.Investigations must extend to otherbatches of the same drug product andother drug products that may have beenassociated with the specific failure ordiscrepancy. A written record of theinvestigation must be produced andshould include both conclusions andfollow-up actions.

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‘Inspectors will ask to look at deviationsand will check to ensure you have madecorrective actions and are monitoring thesituation going forward,’ she said. ‘Theywill also want to know the effectiveness ofthose [corrective] actions.’According to the MHRA’s annual

review, poor investigation of anomalieshas been top of the list of inspectiondeficiencies for the past five years (seetable 1). As a result, the first rule of theinvestigation process, she urged, isdocument, document, document.‘If it is not documented it didn’t happen

from the inspector’s point of view.’ Thedocument should include the ‘who, what,where and when,’ she said, i.e. collect thefacts, work out the impact and risk; do aroot cause analysis and an impactassessment, and document the CAPA andtheir effectiveness.

Biofilm preventionDr Samantha Westgate, Perfectus Biomed,looked at industrial biofilms – acommunity of micro-organisms that can beboth mono or mixed species, and thatattach to a surface (or each other), encasethemselves within a matrix ofextracellular polymeric substances and arethen internally regulated by the inherentpopulation. In addition to explaining howbiofilms form and attach to surfaces, shediscussed the consequences of biofilmcontamination and management methodsto prevent or control biofilm formation. Common misconceptions about such

microbes in water systems, she said, werethat ‘maintaining water temperatureabove 80°C would kill all microbes’, that‘ozone will eradicate them’ or that‘microbes are present evenly throughout

the system’. Within a bacterial biofilm,microbes can withstand temperatures over100°C for 20 minutes, she said.The costs associated with biofilms

include the corrosion of pipework (whichincreases bacterial adhesion), a decreasein pipe diameter, which decreases flow,and shut down and cleaning costs, as wellas costs arising from lost product. The best weapon against biofilm is

prevention, she suggested. This could bestbe achieved via a well designed system;multiple treatment methods at both endsof the system; testing water coming intothe plant; and regular monitoring forearly warning signs. Dr J. Mark Sutton, Technology

Development Group, PHE Porton, lookedat the difficulties of monitoring hydrogenperoxide (H2O2) decontaminationprocesses using biological indicators (BI).BIs comprise a preparation of a specificmicro-organism that provides a definedand stable resistance to a specificsterilisation process. Micro-organismswidely recognised as suitable for BIs arespore-forming bacteria, because thesemicro-organisms are significantly moreresistant than normal microflora.Geobacillus stearothermophilus, forexample, is commonly used for H2O2 (alsosteam), Bacillus subtilis var. niger forethylene oxide, and Bacillus atrophaeusfor dry heat.Sutton discussed the shortcomings of

existing Geobacillus stearothermophilusBIs and discussed research that suggestssome microbial organisms, such as MRSA,as well as biofilms are a greater challengefor decontamination with H2O2 than areGeobacillus BIs. He went on to talk about the

development of a potential alternative:thermostable adenylate kinase (tAK)indicators, isolated from thermophilicbacteria Sulpholobus acidocaldariusas involcanic springs, which can be used as arapid read-out surrogate marker forassessing decontamination processefficacy. He provided a comparison of tAKindicator and BI performance in labstudies of two commercial (H2O2)decontamination systems. Whenmonitoring H2O2 decontamination, thetAK monitoring test could give reducedturnaround time for the decontaminationprocess development and optimisation,and could potentially improve suretyaround process performance, he said. Guest speaker Professor Val Edward

Jones, Clinical Director, MelBecMicrobiology and microbiology star of UKTV series Embarrassing Bodies, gave awide-ranging view of microbial hygieneissues outside of the cleanroom. The TVseries, aimed largely at teenagers andyoung adults, reveals the often shockingmedical conditions of people who havebeen unable to get help from regulardoctors and, thus, appear on the show onthe basis that they will get the specialisthelp they need.The show also filmed various hygiene

awareness-raising projects covering arange of aspects from body odour and badbreath to the hygiene of mobile phones,handbags, make-up, toilets, playgrounds,childrens’ toys and, the lack of hand-washing in airports.Rarely cleaned, many everyday objects

such as mobiles phones carry a multitudeof undesirable organisms: touchscreenphones can carry upwards of 100,000bacterial cells on the screen, she said.Handbags, keyboards, toys andplayground furniture were also found tohave populations of undesirable microbes. While for most people this poses a small

risk, as we lose our first-lineantimicrobials, the risk for those inhospitals is much more severe. Unless newantibiotics or new antimicrobial strategiescan be found we are looking at a futurewhere invasive surgery would no longer bepossible, she said.With the cost of bringing new

antibiotics to market estimated atUS$500m–$1bn (£300–£550m), manydrugmakers are withdrawing from thisarea of research in favour of moreprofitable lifestyle drugs. Since 2009, onlya handful of antibiotics have beendeveloped or approved. While alternative antimicrobial

strategies are under investigation‘prevention is better than cure’, she said.

TABLE 1: MOST FREQUENT DEFECT CATEGORIES OBSERVED IN THEMHRA 2013 REVIEW OF GMP INSPECTIONS Rank Defect category Percentage of critical/major

deficiencies with this defect category

1 Investigation of anomalies 6.5%

2 Quality management 5.5%

3 Investigation of anomalies – CAPA 4.7%

=4 Contamination, chemical/physical (or potential for) 3.7%

=4 Supplier and contractor audit 3.7%

6 Quality management – change control 3.6%

=7 Documentation – procedures/PSF/TAs 2.7%

=7 Personnel issues – training 2.7%

=9 Design and maintenance of equipment 2.6%

=9 Documentation – manufacturing 2.6%

=9 Finished product testing – chemical 2.6%