18 www.cleanroom-technology.co.uk October 2006 CLEANROOM TECHNOLOGY

Where it is not possible to terminallysterilise, to be confident ofproducing a sterile product

manufacturers of parenteral drugs mustensure that a robust environmentalmonitoring programme is in place withinaseptic processing areas.

According to the US Food & DrugAdministration, environmental monitoringshould promptly identify potential routes ofcontamination, allowing for implement-ation of corrections before productcontamination occurs.1 It is one of the mostimportant laboratory controls in asepticprocessing, providing crucial information onthe quality of the aseptic manufacturingenvironment; preventing the release of apotentially contaminated product; andpreventing future contamination bydetecting adverse trends.

Ideally, a monitoring programme providesimmediate feedback, allowing any problemto be rectified promptly. Environmentalmonitoring by microbiological means doesnot provide this because of the incubationtime required to detect micro-organisms.Nevertheless, the microbiological status ofthe cleanroom is a valuable tool that canindicate a loss of environmental control notpicked up by other means. It also serves as a

reminder to personnel of the importance ofaseptic techniques.

Environmental monitoring is only a partof ensuring a sterile manufacturingenvironment. Other elements include: ■ good cleanroom design; ■ effective air handling; ■ cleaning and disinfection; ■ staff training for adherence to aseptictechniques; ■ properly fitted cleanroom gowns;sterilisation of materials brought into theaseptic environment; ■ adequate monitoring of controlledconditions (such as air flow and air pressure)along with alert settings that draw attentionto a developing problem.

Contaminant sourcesThere are many routes bywhich a cleanroom canbecome contaminated. In awell-designed and controlledcleanroom, personnel are themost likely source ofcontamination. This mayoriginate in the changingroom where they shed theirown clothes and put on sterilesuits. If this process is not

performed correctly, contaminants canenter the cleanroom on gowns, gloves,overshoes etc. The garments worn in thecleanroom must fit well, so that skinparticles are retained within the suit, and bemade of a material that does not shed fibresor particles. Aseptic techniques should betaught so that staff do not contaminategloves after, for example, touching theirfaces.

Water is a potential vector of contamin-ation, enabling organisms to spread in waysthey cannot on solid surfaces. Regulationsstate that there should be no water outlets incleanroom areas; however, water is used incleaning and is probably the mostcommonly used raw material inmanufacture. Water-based fluids must besterilised before they are taken into the fillingrooms. As water is also used in cleanroomsupport areas, pharmaceutical grade watershould be used wherever possible, and itshould be microbiologically controlled andmonitored.

Materials of animal, plant or naturalmineral origin are considered to be higherrisk than materials produced by chemicalsynthesis. However, any materials

introduced to an asepticprocessing area should bemonitored for signs ofdamage, and attention paid tothe microbiological quality ofthe material content.

Disruption of airflow, airmovement and inadequatemaintenance of HEPA filterscould all cause contamin-ation. HEPA filters should be

Microbe monitoring

Figure 1: Typical settle and contact plates

Key to any cleanroom or aseptic drugs operation isa planned programme of monitoring. Colin Booth,vice-president of science and technology at Oxoid,outlines a microbial monitoring strategy forpharmaceutical cleanrooms

Contact

Oxoid LimitedWade Road, BasingstokeHampshire RG24 8PW UKT +44 1256 841144F +44 1256 463388oxoid@oxoid.com

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M O N I T O R I N G

free from leaks around the sealing gaskets,frames and fabric. A high degree of airmovement can pick up skin or dust particles,so cleanroom design should ensure thatairflow sweeps particles away from thepoint-of-fill, product or other critical areas.Proper design will ensure unidirectionalairflow with no turbulence or stagnant air.

Although it is well known that the designand construction of the cleanroom shouldinvolve smooth surfaces made of easy-to-clean materials, special attention must begiven to areas that are difficult to clean anddisinfect. Different control measures shouldbe in place to minimise contamination fromthese sources.

The aim of an environmental monitoringprogramme is to detect a change in count ofcolony forming units (cfu), or a change ofspecies, outside the “norm”. Minimumacceptable standards for clean areas are laiddown in guidance, such as the USPharmacopoeia (USP), the EU (2003) guideto Good Manufacturing Practice, Annex 1,Manufacture of Sterile Medicinal Products,(see table 1) and the Manufacture of SterileProducts 2002 (Therapeutic Goods Agency,Australia).

The inspecting body will determine theguidance that should be followed for eachsite. Each site should establish its own alertand action levels based on historical data.These must be at least as strict as in thestandard. If the data suggests that local alertand action levels should be stricter, aregulatory body would expect these stricterlevels to be adopted.

The most common micro-organismsisolated from aseptic processingenvironments are bacteria from human skin,such as Staphylococcus and Micrococcusspecies (gram-positive cocci). In addition,airborne bacterial spores (e.g. Bacillusspecies) and fungal spores (e.g. Aspergillusor Penicillium species) are found and, moreinfrequently, gram-negative cocci (e.g.Enterobacter and Burkholderia species).2

The data produced, both counts andspecies, lead to a greater overall under-standing of the microbial environmentwithin the manufacturing area and, ifperformed regularly, can reveal trends orchanges in established patterns. Thisinformation can be used to identify high-riskareas and to eliminate potential routes ofcontamination.

There are two main methods for samplingair for microbial contamination: active andpassive. Active air sampling is generallyconsidered to be quantitative as it measuresthe number of viable organisms in a definedvolume of air (e.g. cfu/m3). Several types ofactive air samplers are available, includingsieve impactors, slit-to-agar samplers,

surface vacuum samplers, centrifugalimpactors, filtration or liquid impingers andhand-held samplers.

In a slit-to-agar sampler an agar plate(usually 140mm) is placed on a turntableand spun underneath a template with fourperpendicular slits in it. Air is drawn inthrough the slits by means of a pumpattached to the unit by a flexible hose. The airimpacts on the surface of the agar plate, asdo any airborne particles. Integral, hand-held units, contain both in the body of thedevice and pump. The agar plate sits in thehead of the machine, which has a perforatedcover through which the air is drawn. The airimpacts on the surface of the agar, alongwith the airborne particles, before beingexpelled through the rear of the machine.

Passive air sampling is performed byplacing agar plates (see Figure 1) in testareas, with the agar surface exposed for a settime. In 1981, the Parenteral DrugAssociation (PDA) recommended only 30minutes, but more recently other guidelinesrecommended that this be increased to fourhours. The logic is that longer exposuretimes increase the sensitivity of the method.However, care has to be taken against

excessive desiccation of the medium, sincethis may reduce recovery rates.

Although passive air sampling is notquantitative, settle plates are relativelyinexpensive, require no extra equipment ortechnical expertise and are less likely todisturb the airflow in the cleanroom thanactive air sampling.

With either method, the frequency oftesting should take into account the fact thatany human intervention introduces anincreased risk within critical areas. Themanufacturer must weigh the benefits ofthese interventions against the disruptionscreated by performing them.

Surface samplesRegular sampling of surfaces within theprocessing environment is anotherimportant aspect of monitoring an asepticenvironment. However, monitoring ofcritical surfaces during production is notrecommended as this can increase the risk ofcontamination. Surfaces can be sampledusing contact plates (Figure 1) or swabs(Figure 2).

Contact plates are used to sample flatsurfaces on equipment, floors, walls,

The growth promotion properties of each medium used should be validated,according to plate size and fill volume, using a predefined list of organisms. This listshould include compendial organisms and environmental isolates and should be

representative of those found in manufacturing environments, including gram-positiverods, gram-positive cocci, filamentous fungi and gram-negative rods.

The laboratory must have a robust method for the storage and recovery of organisms.For standard strains, the use of convenient, commercially available organisms, such asQuanti-Cult (Oxoid), is recommended.

Once an agar plate has been inoculated with 10-100 cfu of the test organism, it should beincubated at an appropriate temperature for up to 48 hrs for bacteria or up to 3-5 days forfungi. The recovery rate for unstressed organisms on general media should be at least 70%,and for selective media it should be at least 50%.

Validating media performance

Classification of clean area Active air Settle plate Contact plate Glove print(cfu/m3) (90mm) (55mm) (cfu/glove)

(cfu/4hr exposure) (cfu/plate)

Grade A (including local <1 <1 <1 <1zones for high risk operations, eg, point of fill)Grade B (background 10 5 5 5 environments to Grade A zones in a cleanroom)Grade C (support areas, 100 50 25 N/Aeg, rooms where aseptic solutions are prepared for filtration)

Table 1: Recommended limits for microbial contamination in cleanareas levels (EU (2003) guide to GMP, Annex 1)

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countertops, etc. These speciallydesigned agar plates have a domed surfacethat is pressed gently onto the surfacecausing organisms present to be transferredto the agar. This method is mentioned withinthe pharmacopoeias, and is consideredquantitative as it measures the number ofviable organisms on a defined surface area.

Surfaces that are not flat or are difficult toaccess may be sampled using swabs. It isimportant to sample such areas as they maybe more difficult to clean. The surface area tobe tested is sampled with a moistened swab.This can be used to inoculate a plate directlyor it can be immersed in a diluent ortransport medium, which is then used toinoculate a plate.

Swabs are not mentioned in the pharma-copoeias but they are effective for samplingareas that are not suitable for contact plates.For either method, neutralisers may beincluded in the medium to counteractresidual sterilants and cleaning agents thatmay be present on test surfaces.

The effectiveness of aseptic training andprecautions can be measured by periodicallysampling staff clothing. The hands are apotential source of contamination andgloves should be tested periodically – this isdone by touching the tips of all fingers andthumbs onto the surface of an agar plate,after which the gloves are replaced with aclean set. At the end of a shift, othergarments can be tested using contact platesor swabs.

The most frequently used culture mediain environmental monitoring are TryptoneSoya Agar (TSA) – a general purpose medium

suitable for the growth of a wide range oforganisms, and Sabouraud Dextrose Agar(SDA) – ideal for the growth of yeasts andmoulds. Other key media used include: R2AAgar (a minimal medium for testing watersamples); McConkey Agar (coliforms); XLDAgar (salmonellae); Cetrimide Agar(Pseudomonas aeruginosa); Mannitol SaltAgar (staphylococci); DCA Agar (intestinalpathogens); Baird-Parker Medium(Staphylococcus aureus).

Ready-to-use prepared media productsare now popular since they provideconsistent product performance. Pharma-ceutical companies are expected to QC themedia that they use, using quantitativemethodology and low level inocula. It is,therefore, advantageous if prepared mediahas been tested in this way and is suppliedwith supporting documentation.

Prepared plate media are normallysupplied in 90mm or 140mm Petri dishes orcontact plates. A variety of irradiatedproducts are available, suitable for use incleanroom areas. Impermeable isolatorwrap is available that allows plates forenvironmental monitoring to be left in anisolator unit while it is being flushed withsterilants, such as vapour-phase hydrogenperoxide.

Programme designIn designing an effective environmentalmonitoring programme the followingshould be considered:1. A strategy – a written action plan,

including where and when to test,focusing on known weak areas in

addition to searching for new ones.2. Validation – all methods used in the

programme should be validated,including: media stability; the maximumautoclave cycle; media performance (seeFigure 3); effectiveness of neutralisers (todemonstrate that the disinfectant, at theconcentration used in the facility, isinactivated). It may also be necessary tonote the expected morphology of certainorganisms, since the morphology ofcolonies grown on irradiated agar platesmay be different from those grown onfresh agar.

3. Standard operating procedures –including the purpose of the task;frequency of assay; composition ofmedia; selection of control strains;incubation; criteria for release;documentation; action on 'out of spec'results.

4. Growing/preparing isolates –environmental isolates will often bestressed, therefore subculture is requiredto allow the organisms to recover, toobtain a pure culture and to produceenough material to perform anidentification.

5. Identification – all isolates from Grade Aenvironments should be identified tospecies level; morphologicallyrepresentative isolates from Grade Bareas should be identified to specieslevel; morphologically representativeisolates from Grade C & D areas shouldbe characterised if above alert levels.

Periodic characterisation should beperformed if levels fall outside alert levels.Identification can be performed usingbiochemical or serological methods,although genetic techniques are being usedmore frequently as they are not affected bythe stress status of the test organism. Sub-species characterisation is possible using theRiboprinter System (Oxoid), an automatedriboprinting (DNA sequencing) method.

With all elements of a good environmentalmonitoring programme in place,manufacturers have assurance that controlof the aseptic process is being maintained.Counts that fall outside acceptable levels donot necessarily mean that product will becontaminated, but it allows the process to beexamined to identify areas where control islost. Adverse trends can be identified quickly,allowing potential future problems to beaverted. Failure to do this can have seriousconsequences – from an FDA 483 report topossible product recalls. ■

References1. FDA (2004) Guidance for Industry. Sterile Drug ProductsProduced by Aseptic Processing – current Good ManufacturingPractices2. Cundell, A. M. (2004) Pharmaceutical Technology June: 56-66

Figure 2: Swabbing is used to monitor areas that are difficult to access

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