microbiological control and monitoring of aspectic environment and processes

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Relatedness: The extent of relationship or similarity oftwo (or more) organisms on a Phylogenetic Tree or a Den-drogram.

rRNA Sequence: The DNA sequences that encoderRNA used in protein synthesis are highly conserved amongmicroorganisms of a common ancestry. They are used todetermine the phylogenetic distance between organismsand are useful in microbial taxonomy and identification.

Strain: A specific isolate of a species that is maintainedin pure culture and is characterized. The type strain is repre-sentative of the species that provides a reference for the spe-cies based on its historic isolation, characterization, anddeposition in recognized culture collections.

Strain typing: Strain typing is an integral part of epide-miological investigations in clinical and public health micro-biology. Methods including pulsed-field gel electrophoresis,riboprinting, arbitrarily primed polymerized chain reaction,and whole genome ordered restriction or optical mappingcan be used to demonstrate that microbial species are thesame strain and most likely are from a common source.

1116MICROBIOLOGICALCONTROL AND MONITORING

OF ASEPTICPROCESSING ENVIRONMENTS

Microbiologically controlled environments are used for avariety of purposes within the healthcare industry. This gen-eral information chapter provides information and recom-

mendations for environments where the risk of microbialcontamination is controlled through aseptic processing.Products manufactured in such environments include phar-maceutical sterile products, bulk sterile drug substances,sterile intermediates, excipients, and, in certain cases, medi-cal devices. Aseptic processing environments are far morecritical in terms of patient risk than controlled environmentsused for other manufacturing operationsfor example,equipment and component preparation, limited bioburdencontrol of nonsterile products, and processing of terminallysterilized products. In this chapter, the type of aseptic pro-cessing is differentiated by the presence or absence of hu-man operators. An advanced aseptic process is one in whichdirect intervention with open product containers or exposed

product contact surfaces by operators wearing conventionalcleanroom garments is not required and never permitted.[NOTEA description of terms used in this chapter can befound in the Appendix at the end of the chapter.]

The guidance provided in this chapter and the monitoringparameters given for microbiological evaluation should beapplied only to clean rooms, restricted-access barrier sys-tems (RABS), and isolators used for aseptic processing. ISO-classified environments used for other purposes are not re-quired to meet the levels of contamination control requiredfor aseptically produced sterile products. The environmentsused for nonsterile applications require different microbialcontrol strategies.

A large proportion of products labeled as sterile are man-ufactured by aseptic processing rather than terminal sterili-zation. Because aseptic processing relies on the exclusion ofmicroorganisms from the process stream and the preventionof microorganisms from entering open containers duringprocessing, product bioburden as well as the bioburden ofthe manufacturing environment are important factors gov-erning the risk of unacceptable microbial contamination.The terms asepticand sterileare not synonymous. Sterilemeans having a complete absence of viable microorganismsor organisms that have the potential to reproduce. In thepurest microbiological sense, an asepticprocess is one thatprevents contamination by the exclusion of microorganisms.In contemporary aseptic healthcare-product manufacturing,asepticdescribes the process for handling sterilized materialsin a controlled environment designed to maintain microbialcontamination at levels known to present minimal risk.

In any environment where human operators are present,microbial contamination at some level is inevitable. Even themost cautious clean-room environment design and opera-

tion will not eliminate the shedding of microorganisms ifhuman operators are present. Thus, an expectation of zerocontamination at all locations during every aseptic process-ing operation is technically not possible and thus is unrealis-tic. There are no means to demonstrate that an aseptic pro-cessing environment and the product-contact surfaces with-in that environment are sterile. Monitoring locations shouldbe determined based upon a assessment of risk. Althoughmanufacturers should review environmental monitoring re-sults frequently to ensure that the facility operates in a vali-dated state of control, monitoring results can neither provenor disprove sterility. Because of the limitations of monitor-ing, manufacturers cannot rely directly on monitoring, sta-tistics, or periodic aseptic-processing simulations to ensure a

sterility assurance level.Environmental monitoring is usually performed by person-nel and thus requires operator intervention. As a result, envi-ronmental monitoring can both increase the risk of contami-nation and also give false-positive results. Thus, intensivemonitoring is unwarranted, particularly in the ISO 5 envi-ronments that are used in the most critical zones of asepticprocessing.

A number of sampling methods can be used to assess andcontrol the microbiological status of controlled environ-ments for aseptic processing. At present, nearly all of thesemethods rely on the growth and recovery of microorgan-isms, many of which can be in a damaged state caused byenvironmental stress and therefore may be difficult to recov-

er. The numerical values for air, surface, and personnel mon-itoring included in this chapter are not intended to repre-sent limits or specifications but are strictly informational. Be-cause of the variety of microbiological sampling equipmentand methods, it is not scientifically reasonable to suggestthat the attainment of these values guarantees microbialcontrol or that excursions beyond values in this chapter indi-cate a loss of control. The assessment of risks associatedwith manufacturing environments must be made over a sig-nificant period; and in each case, the contamination recov-ery rate metric should be established on the basis of a re-view of actual findings within the facility. The objective ofeach user should be to use contamination recovery rates to

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track ongoing performance and to refine the microbiologi-cal control program to foster improvements. When opti-mum operational conditions are achieved within a facility,contamination recovery rate levels typically become relative-ly stable within a normal range of variability.

There are no standard methods for air sampling, andavailable literature indicates that air-sampling methods arehighly variable. It should not be assumed that similar samplevolumes taken by different methods will produce similarrates of recovery. Many factors can affect microbial recoveryand survival, and different air sampler suppliers may havedesigned their systems to meet different requirements. Also,sample-to-sample variation in microbial sampling can be ex-tensive. Limited data are available regarding the accuracy,precision, sensitivity, and limits of detection of monitoringmethods used in the aseptic processing of healthcare prod-ucts.

Surface sampling methods are also not standardized. Dif-ferent media are employed, and in the case of swabs, differ-ent results have been reported for wet and dry swab meth-

ods and contact plates. Replicate sample contact platesshould be expected to give similar results under identicalconditions, but rates of recovery have been reported to beboth lower than expected and highly variable. In general,surface monitoring has been found to recover


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may differ. By diluting and removing contaminants, largevolumes of air are likely to reduce airborne contamination inaseptic production. Optimum conditions vary considerably,depending on process characteristics, particularly theamount of contamination derived from personnel. Thesespecifications should be used only as a guide in the designand operation of clean rooms, because the precise correla-tions among air changes per hour, air velocity, and microbi-al control have not been satisfactorily established experi-mentally.

Manufacturers should maintain a predominantly unidirec-tional flow of air (either vertical or horizontal) in a staffedClass 5 clean room environment, particularly when prod-ucts, product containers, and closures are exposed. In theevaluation of air movement within a clean room, studyingairflow visually by smoke studies or other suitable means isprobably more useful than using absolute measures of air-flow velocity and change rates. Risk assessment models areanother useful way of reducing contamination risk andshould be considered.

Air velocity and change rates are far less important in iso-lators or closed RABS than in clean rooms because personnelare more carefully separated from the product, product con-tainers, and closures. Air velocities substantially lower thanthose used in human-scale clean rooms have proved ade-quate in isolator systems and may be appropriate in RABS aswell. In zones within isolators where particulate matterposes a hazard to product quality, predominantly vertical orhorizontal unidirectional airflow can be maintained. Experi-ence has shown that well-controlled mixing or turbulent air-flow is satisfactory for many aseptic processes and for sterili-ty testing within isolators (see Sterility TestingValidation ofIsolator Systems 1208).

IMPORTANCE OF A MICROBIOLOGICALEVALUATION PROGRAM FOR CONTROLLEDENVIRONMENTS

Monitoring of total particulate count in controlled envi-ronments, even with the use of electronic instrumentationon a continuous basis, does not provide information on themicrobiological content of the environment. The basic limi-tation of particulate counters is that they measure particlesof 0.5 mm or larger. While airborne microorganisms are notfree-floating or single cells, they frequently associate withparticles of 1020 mm. Particulate counts as well as microbi-al counts within controlled environments vary with the sam-pling location and the activities being conducted during

sampling. Monitoring the environment for nonviable partic-ulates and microorganisms is an important control functionbecause they both are important in achieving product com-pendial requirements for Foreign and Particulate MatterandSterilityin Injections 1.

Total particulate monitoring may provide a better meansof evaluating the overall quality of the environment in isola-tors and closed RABS than in most conventional cleanrooms. The superior exclusion of human-borne contamina-tion provided by an isolator results in an increased propor-tion of nonviable particulates. Total particulate counting inan isolator is likely to provide an immediate indicator ofchanges in contamination level. Microbial monitoring pro-

grams should assess the effectiveness of cleaning and sani-tization practices by and of personnel who could have animpact on the bioburden. Because isolators are typically de-contaminated using an automatic vapor or gas generationsystem, microbial monitoring is much less important in es-tablishing their efficiency in eliminating bioburden. Theseautomatic decontamination systems are validated directly,using an appropriate biological indicator challenge, and arecontrolled to defined exposure parameters during routineuse to ensure consistent decontamination.

Microbial monitoring cannot and need not identify andquantify all microbial contaminants in these controlled envi-ronments. Microbiological monitoring of a clean room istechnically a semiquantitative exercise, because a trulyquantitative evaluation of the environment is not possible,given the limitations in sampling equipment. Both the lackof precision of enumeration methods and the restrictedsample volumes that can be effectively analyzed suggestthat environmental monitoring is incapable of providing di-rect quantitative information about sterility assurance. Ana-

lysts should remember that no microbiological samplingplan can prove the absence of microbial contamination,even when no viable contamination is recovered. The ab-sence of growth on a microbiological sample means onlythat growth was not discovered; it does not mean that theenvironment is free of contamination.

Routine microbial monitoring should provide sufficient in-formation to demonstrate that the aseptic processing envi-ronment is operating in an adequate state of control. Thereal value of a microbiological monitoring program lies in itsability to confirm consistent, high-quality environmentalconditions at all times. Monitoring programs can detectchanges in the contamination recovery rate that may be in-dicative of changes in the state of control within the envi-

ronment.Environmental microbial monitoring and analysis of databy qualified personnel can assist in ensuring that a suitablestate of control is maintained. The environment should besampled during normal operations to allow the collection ofmeaningful, process-related data. Microbial sampling shouldoccur when materials are in the area, processing activitiesare ongoing, and a full complement of personnel is workingwithin the aseptic processing environment.

Microbial monitoring of manufacturing clean rooms,RABS, and isolators should include compressed gases, surfa-ces, room or enclosure air, and any other materials andequipment that might produce a risk of contamination. Theanalysis of contamination trends in an aseptic environment

has long been a component of the environmental controlprogram. In aseptic processing environments and particular-ly in ISO Class 5 environments, contamination is infrequent-ly observed. In isolator enclosures, contamination is rarerstill because of superior exclusion of human-borne contami-nation. Because of the criticality of these environments,even minor changes in the contamination incident ratesmay be significant, and manufacturers should frequentlyand carefully review monitoring data. In less critical environ-ments, microbial contamination may be higher, butchanges in recovery rates should be noted, investigated,and corrected. Isolated recoveries of microorganisms shouldbe considered a normal phenomenon in conventional clean






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rooms, and these incidents generally do not require specificcorrective action, because it is almost certain that investiga-tions will fail to yield a scientifically verifiable cause. Becausesampling itself requires an aseptic intervention in conven-tional clean rooms, any single uncorrelated contaminationevent could be a false positive.

When contamination recovery rates increase from an es-tablished norm, process and operational investigationshould take place. Investigations will differ depending onthe type and processing of the product manufactured in theclean room, RABS, or isolator. Investigation should include areview of area maintenance documentation; sanitization/decontamination documentation; the occurrence of non-routine events; the inherent physical or operational parame-ters, such as changes in environmental temperature and rel-ative humidity; and the training status of personnel.

In closed RABS and isolator systems, the loss of glove in-tegrity or the accidental introduction of material that hasnot been decontaminated are among the most probablecauses of detectable microbial contamination. Following the

investigation, actions should be taken to correct or eliminatethe most probable causes of contamination. Because of therelative rarity of contamination events in modern facilities,the investigation often proves inconclusive. When correctiveactions are undertaken, they may include reinforcement ofpersonnel training to emphasize acceptable gowning andaseptic techniques and microbial control of the environ-ment. Some additional microbiological sampling at an in-creased frequency may be implemented, but this may notbe appropriate during aseptic processing because intrusiveor overly intensive sampling may entail an increased con-tamination risk. When additional monitoring is desirable, itmay be more appropriate during process simulation studies.Other measures that can be considered to better control mi-

crobial contamination include additional sanitization, use ofdifferent sanitizing agents, and identification of the microbi-al contaminant and its possible source.

In any aseptic environment, conventional or advanced,the investigation and the rationale for the course of actionchosen as a result of the investigation must be carefully andcomprehensively documented.

PHYSICAL EVALUATION OF CONTAMINATIONCONTROL EFFECTIVENESS

Clean environments should be certified as described inISO 14644 series in order to meet their design classificationrequirements. The design, construction, and operation of

clean rooms vary greatly, so it is difficult to generalize re-quirements for parameters such as filter integrity, air veloci-ty, air patterns, air changes, and pressure differential. In par-ticularly critical applications such as aseptic processing, astructured approach to physical risk assessment may be ap-propriate.

One such method has been developed by Ljundqvist andReinmller. This method, known as the L-R method, chal-lenges the air ventilation system by evaluating both airflowand the ability of an environment to dilute and remove air-borne particles. In the L-R method, a smoke generator al-lows analysts to visualize the air movements throughout aclean room or a controlled environment, including vortices

or turbulent zones, and the airflow pattern can be fine-tuned to minimize these undesirable effects. Following visu-al optimization of airflow, particulate matter is generatedclose to the critical zone and sterile field. This evaluation isdone under simulated production conditions but withequipment and personnel in place. This type of test can alsobe used to evaluate the ability of RABS and isolator systems,particularly around product exit ports in these systems, toresist the effects of contamination.

Visual evaluation of air movement within clean rooms is asubjective process. Complete elimination of turbulence orvortices is not possible in operationing clean rooms thatcontain personnel and equipment. Air visualization is simplyone step in the effort to optimize clean room operations andis not a definitive pass/fail test, because acceptable or unac-ceptable conditions are not readily definable.

Proper testing and optimization of the physical character-istics of the clean room, RABS, or isolator are essential beforeimplementation of the microbiological monitoring program.Assurance that the clean room, RABS, or isolator is in com-

pliance with its predetermined engineering specificationsprovides confidence that the ability of the facility systemsand operating practices to control the bioburden and nonvi-able particulate matter are appropriate for the intended use.These tests should be repeated during routine certificationof the clean room or advanced aseptic processing systems,and whenever significant changes are made to the opera-tion, such as personnel flow, equipment operation, materialflow, air-handling systems, or equipment layout.

TRAINING OF PERSONNEL

Good personnel performance plays an essential role in thecontrol of contamination, proper training and supervision

are central to contamination control. Aseptic processing isthe most critical activity conducted in microbiological con-trolled environments, and manufacturers must pay close at-tention to details in all aspects of this endeavor. Rigorousdiscipline and strict supervision of personnel are essential inorder to ensure a level of environmental quality appropriatefor aseptic processing.

Training of all personnel working in controlled environ-ments is critical. This training is equally important for per-sonnel responsible for the microbial monitoring program,because contamination of the clean working area could in-advertently occur during microbial sampling. In highly auto-mated operations, monitoring personnel may be the em-ployees who have the most direct contact with the critical

surfaces and zones within the processing area. Microbiologi-cal sampling has the potential to contribute to microbialcontamination caused by inappropriate sampling techni-ques or by placing personnel in or near the critical zone. Aformal training program is required to minimize this risk.This training should be documented for all personnel whoenter controlled environments. Interventions should alwaysbe minimized, including those required for monitoring ac-tivities; but when interventions cannot be avoided, theymust be conducted with aseptic technique that approachesperfection as closely as possible.

Management of the facility must ensure that personnel in-volved in operations in clean rooms and advanced aseptic

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processing environments are well versed in relevant micro-biological principles. The training should include instructionabout the basic principles of aseptic technique and shouldemphasize the relationship of manufacturing and handlingprocedures to potential sources of product contamination.Those supervising, auditing, or inspecting microbiologicalcontrol and monitoring activities should be knowledgeableabout the basic principles of microbiology, microbial physi-ology, disinfection and sanitation, media selection and prep-aration, taxonomy, and sterilization. The staff responsible forsupervision and testing should have academic training inmedical or environmental microbiology. Sampling person-nel as well as individuals working in clean rooms should beknowledgeable about their responsibilities in minimizing therelease of microbial contamination. Personnel involved inmicrobial identification require specialized training about re-quired laboratory methods. Additional training about themanagement of collected data must be provided. Knowl-edge and understanding of applicable standard operatingprocedures are critical, especially those procedures relating

to corrective measures taken when environmental condi-tions require. Understanding of contamination control prin-ciples and each individual's responsibilities with respect togood manufacturing practices (GMPs) should be an integralpart of the training program, along with training in con-ducting investigations and in analyzing data.

The only significant sources of microbial contamination inaseptic environments are the personnel. Because operatorsdisperse contamination and because the ultimate objectivein aseptic processing is to reduce end-user risk, only healthyindividuals should be permitted access to controlled envi-ronments. Individuals who are ill must not be allowed to en-ter an aseptic processing environment, even one that em-ploys advanced aseptic technologies such as isolators,

blow/fill/seal, or closed RABS.The importance of good personal hygiene and a carefulattention to detail in aseptic gowning cannot be overem-phasized. Gowning requirements differ depending on theuse of the controlled environment and the specifics of thegowning system itself. Aseptic processing environments re-quire the use of sterilized gowns with the best available fil-tration properties. The fullest possible skin coverage is desir-able, and sleeve covers or tape should be considered tominimize leaks at the critical glovesleeve junction. Exposedskin should never be visible in conventional clean rooms un-der any conditions. The personnel and gowning considera-tions for RABS are essentially identical to those for conven-tional clean rooms.

Once employees are properly gowned, they must be care-ful to maintain the integrity of their gloves, masks, and oth-er gown materials at all times. Operators who work with iso-lator systems are not required to wear sterilized clean-roomgowns, but inadequate aseptic technique and employee-borne contamination are the principal hazards to safe asep-tic operations in isolators, as well as RABS, and in conven-tional clean rooms. Glove-and-sleeve assemblies can devel-op leaks that can allow the mechanical transfer of microor-ganisms to the product. A second glove, worn either underor over the primary isolator/RABS glove, can provide an ad-ditional level of safety against glove leaks or can act as a hy-gienic measure. Also, operators must understand that asep-

tic technique is an absolute requirement for all manipula-tions performed with gloves within RABS and isolator sys-tems.

The environmental monitoring program, by itself, cannotdetect all events in aseptic processing that might compro-mise the microbiological quality of the environment. There-fore, periodic media-fill or process simulation studies arenecessary, as is thorough ongoing supervision, to ensurethat appropriate operating controls and training are effec-tively maintained.

CRITICAL FACTORS IN THE DESIGN ANDIMPLEMENTATION OF A MICROBIOLOGICALENVIRONMENTAL MONITORING PROGRAM

Since the advent of comprehensive environmental moni-toring programs, their applications in capturing adversetrends or drifts has been emphasized. In a modern asepticprocessing environmentwhether an isolator, RABS, or con-ventional clean roomcontamination has become increas-

ingly rare. Nevertheless, a monitoring program should beable to detect a change from the validated state of controlin a facility and to provide information for implementing ap-propriate countermeasures. An environmental monitoringprogram should be tailored to specific facilities and condi-tions. It is also helpful to take a broad perspective in the in-terpretation of data. A single uncorrelated result on a givenday may not be significant in the context of the technicallimitations associated with aseptic sampling methods.

Selection of Growth Media

A general microbiological growth medium such as soy-beancasein digest medium (SCDM) is suitable for environ-

mental monitoring in most cases because it supports thegrowth of a wide range of bacteria, yeast, and molds. Thismedium can be supplemented with additives to overcomeor to minimize the effects of sanitizing agents or of antibiot-ics. Manufacturers should consider the specific detection ofyeasts and molds. If necessary, general mycological mediasuch as Sabourauds, modified Sabourauds, or inhibitorymold agar can be used. In general, monitoring for strictanaerobes is not performed, because these organisms areunlikely to survive in ambient air. However, micro-aerophilicorganisms may be observed in aseptic processing. Shouldanoxic conditions exist or if investigations warrant (e.g.,identification of these organisms in sterility testing facilitiesor Sterility Tests 71results), monitoring for micro-aero-

philes and organisms that grow under low-oxygen condi-tions may be warranted. The ability of any media used inenvironmental monitoring, including those selected to re-cover specific types of organisms, must be evaluated fortheir ability to support growth, as indicated in 71.

Selection of Culture Conditions

Time and incubation temperatures are set once the ap-propriate media have been selected. Typically, for generalmicrobiological growth media such as SCDM, incubationtemperatures in the ranges of approximately 2035 have






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been used with an incubation time of not less than 72hours. Longer incubation times may be considered whencontaminants are known to be slow growing. The tempera-ture ranges given above are by no means absolute. Meso-philic bacteria and mold common to the typical facility envi-ronment are generally capable of growing over a widerange of temperatures. For many mesophilic organisms, re-covery is possible over a range of approximately 20. In theabsence of confirmatory evidence, microbiologists may in-cubate a single plate at both a low and a higher tempera-ture. Incubating at the lower temperature first may compro-mise the recovery of Gram-positive cocci that are importantbecause they are often associated with humans.

Sterilization processes for preparing growth media shouldbe validated. When selective media are used for monitoring,incubation conditions should reflect published technical re-quirements. Contamination should not be introduced into amanufacturing clean room as a result of using contaminatedsampling media or equipment. Of particular concern is theuse of aseptically prepared sampling media. Wherever possi-

ble, sampling media and their wrappings should be termi-nally sterilized by moist heat, radiation, or other suitablemeans. If aseptically prepared media must be used, analystsmust carry out preincubation and visual inspection of allsampling media before introduction into the clean room.The reader is referred to Microbiological Best Laboratory Prac-tices 1117for further information regarding microbiologylaboratory operations and control.

ESTABLISHMENT OF SAMPLING PLAN ANDSITES

During initial startup or commissioning of a clean room orother controlled environment, specific locations for air and

surface sampling should be determined. Locations consid-ered should include those in proximity of the exposed prod-uct, containers, closures, and product contact surfaces. Inaseptic processing, the area in which containers, closures,and product are exposed to the environment is often calledthe critical zonethe critical zone is always ISO 5. For asep-tic operations the entire critical zone should be treated as asterile field. A nonsterile object, including the gloved handsof clean room personnel or an RABS/isolator glove, shouldnever be brought into contact with a sterile product, con-tainer closure, filling station, or conveying equipment beforeor during aseptic processing operations. Operators and envi-ronmental monitoring personnel should never touch sterileparts of conveyors, filling needles, parts hoppers, or any oth-

er equipment that is in the product-delivery pathway. Thismeans that surface monitoring on these surfaces is bestdone at the end of production operations.

The frequency of sampling depends on the manufactur-ing process conducted within an environment. Classified en-vironments that are used only to provide a lower overall lev-el of bioburden in nonsterile product manufacturing areasrequire relatively infrequent environmental monitoring.Classified environments in which closed manufacturing op-erations are conducted, including fermentation, sterile APIprocessing, and chemical processes, require fewer monitor-ing sites and less frequent monitoring because the risk ofmicrobial contamination from the surrounding environment

is comparatively low. Microbiological monitoring of environ-ments in which products are filled before terminal steriliza-tion is generally less critical than the monitoring of asepticprocessing areas. The amount of monitoring required in fill-ing operations for terminal sterilization depends on the sus-ceptibility of the product survival and the potential for pro-liferation of microbial contamination. The identification andestimated number of microorganisms that are resistant tothe subsequent sterilization may be more critical than themicrobiological monitoring of the surrounding manufactur-ing environments.

It is not possible to recommend microbial control levelsfor each type of manufacturing environment. The levels es-tablished for one ISO Class 7 environment, for example,may be inappropriate for another ISO Class 7 environment,depending on the production activities undertaken in each.The user should conduct a prospective risk analysis and de-velop a rationale for the sampling locations and frequenciesfor each controlled environment. The classification of aclean room helps establish control levels, but that does not

imply that all rooms of the same classification should havethe same control levels and the same frequency of monitor-ing. Monitoring should reflect the microbiological controlrequirements of manufacturing or processing activities. For-mal risk assessment techniques can result in a scientificallyvalid contamination control program.

Table 2suggests frequencies of sampling in decreasing or-der of frequency and in relation to the criticality or productrisk of the area being sampled. This table distinguishes be-tween aseptic processing where personnel are asepticallygowned and those where a lesser gowning is appropriate.Environmental monitoring sampling plans should be flexiblewith respect to monitoring frequencies, and sample plan lo-cations should be adjusted on the basis of the observed rate

of contamination and ongoing risk analysis. On the basis oflong-term observations, manufacturers may increase or de-crease sampling at a given location or eliminate a samplinglocation altogether. Oversampling can be as deleterious tocontamination control as undersampling, and careful con-sideration of risk and reduction of contamination sourcescan guide the sampling intensity.

Table 2. Suggested Frequency of Sampling for AsepticProcessing Areasa

Sampling Area/LocationFrequency of

Sampling

Clean Room/RABS

Critical zone (ISO 5 or better)

Active air sampling Each operational shift

Surface monitoring At the end of the operation

Aseptic area adjacent critical zone

All sampling Each operating shift

Other nonadjacent aseptic areas

All sampling Once per day

Isolators

Critical zone (ISO 5 or better)

Active air sampling Once per day

a All operators are aseptically gowned in these environments (with the ex-ception of background environments for isolators). These recommenda-tions do not apply to production areas for nonsterile products or other clas-sified environments in which fully aseptic gowns are not donned.






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of colonies recovered from a given sample. Table 3providesrecommended contamination recovery rates for aseptic pro-cessing environments. The incident rate is the rate at whichenvironmental samples are found to contain microbial con-tamination. For example, an incident rate of 1% wouldmean that only 1% of the samples taken have any contami-nation regardless of colony number. In other words, 99% ofthe samples taken are completely free of contamination.Contamination recovery rates that are higher than thoserecommended in Table 3may be acceptable in rooms ofsimilar classification that are used for lower-risk activities. Ac-tion should be required when the contamination recoveryrate trends above these recommendations for a significanttime.

Table 3. Suggested Initial Contamination Recovery Rates inAseptic Environmentsa

RoomClassification

ActiveAir

Sample(%)

SettlePlate(9 cm) 4h Expo-sure (%)

ContactPlate orSwab(%)

Glove orGarment(%)

Isolator/ClosedRABS (ISO 5 orbetter)


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ty of a lot of product. Environmental monitoring can onlyassure those responsible for a process that a production sys-tem is in a consistent, validated state of control. Care shouldbe taken to avoid drawing inappropriate conclusions frommonitoring results.

SAMPLING AIRBORNE MICROORGANISMSAmong the most commonly used tools for monitoring

aseptic environments are impaction and centrifugal sam-plers. A number of commercially available samplers are lis-ted for informational purposes. The selection, appropriate-ness, and adequacy of using any particular sampler are theresponsibility of the user.

Slit-to-Agar Air Sampler (STA): The unit is powered byan attached source of controllable vacuum. The air intake isobtained through a standardized slit below which is placeda slowly revolving Petri dish that contains a nutrient agar.Airborne particles that have sufficient mass impact the agarsurface, and viable organisms are allowed to grow. A remote

air intake is often used to minimize disturbance of unidirec-tional airflow.

Sieve Impactor: This apparatus consists of a containerdesigned to accommodate a Petri dish that contains a nu-trient agar. The cover of the unit is perforated with open-ings of a predetermined size. A vacuum pump draws aknown volume of air through the cover, and airborne parti-cles that contain microorganisms impact the agar mediumin the Petri dish. Some samplers feature a cascaded series ofsieves that contain perforations of decreasing size. Theseunits allow determination of the size range distribution ofparticulates that contain viable microorganisms based onthe size of the perforations through which the particles lan-ded on the agar plates.

Centrifugal Sampler: The unit consists of a propeller orturbine that pulls a known volume of air into the unit andthen propels the air outward to impact on a tangentiallyplaced nutrient agar strip set on a flexible plastic base.

Sterilizable Microbiological Atrium: The unit is a var-iant of the single-stage sieve impactor. The unit's cover con-tains uniformly spaced orifices approximately 0.25 inch insize. The base of the unit accommodates one Petri dish con-taining a nutrient agar. A vacuum pump controls the move-ment of air through the unit, and a multiple-unit controlcenter as well as a remote sampling probe are available.

Surface Air System Sampler: This integrated unit con-sists of an entry section that accommodates an agar contactplate. Immediately behind the contact plate is a motor and

turbine that pulls air through the unit's perforated coverover the agar contact plate and beyond the motor, where itis exhausted. Multiple mounted assemblies are also availa-ble.

Gelatin Filter Sampler: The unit consists of a vacuumpump with an extension hose terminating in a filter holderthat can be located remotely in the critical space. The filterconsists of random fibers of gelatin capable of retaining air-borne microorganisms. After a specified exposure time, thefilter is aseptically removed and dissolved in an appropriatediluent and then plated on an appropriate agar medium toestimate its microbial content.

Settling Plates: This method is still widely used as asimple and inexpensive way to qualitatively assess the envi-ronments over prolonged exposure times. Published data in-dicate that settling plates, when exposed for 4- to 5-hourperiods, can provide a limit of detection for a suitable evalu-ation of the aseptic environment. Settling plates may beparticularly useful in critical areas where active samplingcould be intrusive and a hazard to the aseptic operation.

One of the major drawbacks of mechanical air samplers isthe limited sample size of air being tested. When the micro-bial level in the air of a controlled environment is expectedto contain extremely low levels of contamination per unitvolume, at least 1 cubic meter of air should be tested in or-der to maximize sensitivity. Typically, slit-to-agar deviceshave an 80-L/min sampling capacity (the capacity of thesurface air system is somewhat higher). If 1 cubic meter ofair were tested, then it would require an exposure time of15 min. It may be necessary to use sampling times in excessof 15 min to obtain a representative environmental sample.Although some samplers are reported to have high sam-

pling volumes, consideration should be given to the poten-tial for disruption of the airflow patterns in any critical areaand to the creation of turbulence.

Technicians may wish to use remote sampling systems inorder to minimize potential risks resulting from interventionby environmental samplers in critical zones. Regardless ofthe type of sampler used, analysts must determine that theextra tubing needed for a remote probe does not reducethe method's sensitivity to such an extent that detection oflow levels of contamination becomes unlikely or even im-possible.

SURFACE SAMPLING

Another component of the microbial-control program incontrolled environments is surface sampling of equipment,facilities, and personnel. The standardization of surface sam-pling methods and procedures has not been as widely ad-dressed in the pharmaceutical industry as has the standardi-zation of air-sampling procedures. Surface sampling can beaccomplished by the use of contact plates or by the swab-bing method.

Contact plates filled with nutrient agar are used for sam-pling regular or flat surfaces and are directly incubated forthe appropriate time and temperature for recovery of viableorganisms. Specialized agar can be used for the recovery oforganisms that have specific growth requirements. Microbialestimates are reported per contact plate.

The swabbing method can be used to supplement con-tact plates for sampling of irregular surfaces, especially irreg-ular surfaces of equipment. The area that will be swabbed isdefined with a sterile template of appropriate size. In gener-al, it is in the range of 2430 cm2. After sample collectionthe swab is placed in an appropriate diluent or transportmedium and is plated onto the desired nutrient agar. Themicrobial estimates are reported per swab of defined sam-pling area.

Surface monitoring is used as an environmental assess-ment tool in all types of classified environments. In ISO 5environments for aseptic processing, surface monitoring isgenerally performed beside critical areas and surfaces. Com-






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ponent hoppers and feed chutes that contact sterile surfaceson closures and filling needles can be tested for microbialcontamination. Often in conventional staffed clean rooms,these product contact surfaces are steam sterilized and asep-tically assembled. The ability of operators to perform theseaseptic manipulations are evaluated during process stimula-tions or media fills, although true validation of operatortechnique in this manner is not possible. Surface monitoringon surfaces that directly contact sterile parts or productshould be done only after production operations are com-pleted. Surface sampling is not a sterility test and should notbe a criterion for the release or rejection of product. Becausethese samples must be taken aseptically by personnel, it isdifficult to establish with certainty that any contaminationrecovered is product related.

CULTURE MEDIA AND DILUENTS

The type of medium, liquid or solid, used for sampling orplating microorganisms depends on the procedure and

equipment used. Any medium used should be evaluated forsuitability for the intended purpose. The most commonlyused all-purpose solid microbiological growth medium issoybeancasein digest agar. As previously noted, this medi-um can be supplemented with chemicals that counteractthe effect of various antimicrobials.

IDENTIFICATION OF MICROBIAL ISOLATES

A successful environmental control program includes anappropriate level of identification of the flora obtained bysampling. A knowledge of the flora in controlled environ-ments aids in determining the usual microbial flora anticipa-ted for the facility and in evaluating the effectiveness of the

cleaning and sanitization procedures, methods, agents, andrecovery methods. The information gathered by an identifi-cation program can be useful in the investigation of thesource of contamination, especially when recommended de-tection frequencies are exceeded.

Identification of isolates from critical and immediately ad-jacent areas should take precedence over identification ofmicroorganisms from noncritical areas. Identification meth-ods should be verified, and ready-to-use kits should bequalified for their intended purpose.

CONCLUSION

Environmental monitoring is one of several key elementsrequired in order to ensure that an aseptic processing area ismaintained in an adequate level of control. Monitoring is aqualitative exercise, and even in the most critical applica-tions such as aseptic processing, conclusions regarding lotacceptability should not be made on the basis of environ-mental sampling results alone. Environments that are essen-tially free of human operators generally have low initial con-tamination rates and maintain low levels of microbial con-tamination. Human-scale clean rooms present a very differ-ent picture. Studies conclusively show that operators, evenwhen carefully and correctly gowned, continuously sloughmicroorganisms into the environment. Therefore, it is unrea-

sonable to assume that samples producing no colonies,even in the critical zone or on critical surfaces, will always beobserved. Periodic excursions are a fact of life in human-scale clean rooms, but the contamination recovery rate, par-ticularly in ISO 5 environments used for aseptic processing,should be consistently low.

Clean-room operators, particularly those engaged in asep-tic processing, must strive to maintain suitable environmen-tal quality and must work toward continuous improvementof personnel operations and environmental control. In gen-eral, fewer personnel involved in aseptic processing andmonitoring, along with reduction in interventions, reducesrisk from microbial contamination.

APPENDIX

Airborne Particulate Count(also referred to as TotalParticulate Count): The total number of particles of agiven size per unit volume of air.Airborne Viable Particulate Count(also referred to as

Total Airborne Aerobic Microbial Count): The recoverednumber of colony-forming units (cfu) per unit volumeof air.Air Changes:The frequency per unit of time (minutes,hours, etc.) that the air within a controlled environ-ment is replaced. The air can be recirculated partially ortotally replaced.Air Sampler:Devices or equipment used to sample ameasured amount of air in a specified time to quanti-tate the particulate or microbiological status of air inthe controlled environment.Aseptic:Technically, the absence of microorganisms,but in aseptic processing this refers to methods and op-erations that minimize microbial contamination in envi-

ronments where sterilized product and components arefilled and/or assembled.Aseptic Processing:An operation in which the productis assembled or filled into its primary package in an ISO5 or better environment and under conditions thatminimize the risk of microbial contamination. The ulti-mate goal is to produce products that are as free aspossible of microbial contamination.Barrier System:Physical barriers installed within anaseptic processing room to provide partial separationbetween aseptically gowned personnel and criticalareas subject to considerable contamination risk. Per-sonnel access to the critical zone is largely unrestricted.It is subject to a high level disinfection.

Bioburden:Total number and identity of the predomi-nant microorganisms detected in or on an article.Clean Room:A room in which the concentration of air-borne particles is controlled to meet a specified air-borne particulate cleanliness Class. In addition, the con-centration of microorganisms in the environment ismonitored; each cleanliness Class defined is also as-signed a microbial level for air, surface, and personnelgear.Commissioning of a Controlled Environment:Certifi-cation by engineering and quality control that the envi-ronment has been built according to the specificationsof the desired cleanliness Class and that, under condi-






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tions likely to be encountered under normal operatingconditions (or worst-case conditions), it is capable ofdelivering an aseptic process. Commissioning includesmedia-fill runs and results of the environmental moni-toring program.Contamination Recovery Rate:The contamination re-covery rate is the rate at which environmental samplesare found to contain any level of contamination. Forexample, an incident rate of 1% would mean that only1% of the samples taken have any contamination re-gardless of colony number.Controlled Environment:Any area in an aseptic proc-ess system for which airborne particulate and microor-ganism levels are controlled to specific levels, appropri-ate to the activities conducted within that environ-ment.Corrective Action:Actions to be performed that areaccording to standard operating procedures and thatare triggered when certain conditions are exceeded.Critical Zone:Typically the entire area where product

and the containers and closures are exposed in asepticprocessing.Detection Frequency:The frequency with which con-tamination is observed in an environment. Typically ex-pressed as a percentage of samples in which contami-nation is observed per unit of time.Environmental Isolates:Microorganisms that havebeen isolated from the environmental monitoring pro-gram.Environmental Monitoring Program:Documentedprogram implemented via standard operating proce-dures that describes in detail the methods and accept-ance criteria for monitoring particulates and microor-ganisms in controlled environments (air, surface, per-

sonnel gear). The program includes sampling sites, fre-quency of sampling, and investigative and correctiveactions.Equipment Layout:Graphical representation of anaseptic processing system that denotes the relationshipbetween and among equipment and personnel. Thislayout is used in the Risk Assessment Analysisto deter-mine sampling site and frequency of sampling basedon potential for microbiological contamination of theproduct/container/closure system. Changes must be as-sessed by responsible managers, since unauthorizedchanges in the layout for equipment or personnel sta-tions could result in increase in the potential for con-tamination of the product/container/closure system.

Isolator for Aseptic Processing:An aseptic isolator isan enclosure that is over-pressurized with HEPA filteredair and is decontaminated using an automated system.When operated as a closed system, it uses only decon-taminated interfaces or rapid transfer ports (RTPs) formaterials transfer. After decontamination they can beoperated in an open manner with the ingress and/oregress of materials through defined openings that havebeen designed and validated to preclude the transfer ofcontamination. It can be used for aseptic processing ac-tivities or for asepsis and containment simultaneously.Material Flow:The flow of material and personnel en-tering controlled environments should follow a speci-

fied and documented pathway that has been chosen toreduce or minimize the potential for microbial contami-nation of the product/closure/container systems. Devia-tion from the prescribed flow could result in increase inthe potential for microbial contamination. Material/personnel flow can be changed, but the consequencesof the changes from a microbiological point of viewshould be assessed by responsible managers and mustbe authorized and documented.Media Fill:Microbiological simulation of an asepticprocess by the use of growth media processed in amanner similar to the processing of the product andwith the same container/closure system being used.Media Growth Promotion:Procedure that referencesGrowth Promotion Test of Aerobes, Anerobes, and Fungiin Sterility Tests 71to demonstrate that media used inthe microbiological environmental monitoring pro-gram, or in media-fillruns, are capable of supportinggrowth of indicator microorganisms and of environ-mental isolates from samples obtained through the

monitoring program or their corresponding ATCCstrains.Product Contact Areas:Areas and surfaces in a con-trolled environment that are in direct contact with ei-ther products, containers, or closures and the microbio-logical status of which can result in potential microbialcontamination of the product/container/closure sys-tem.Restricted Access Barrier System (RABS):An enclo-sure that relies on HEPA filtered air over-spill to main-tain separation between aseptically gowned personneland the operating environment. It is subject to a highlevel of disinfection prior to use in aseptic process. Ituses decontaminated (where necessary) interfaces or

RTPs for materials transfer. It allows for the ingressand/or egress of materials through defined openingsthat have been designed and validated to preclude thetransfer of contamination. If opened subsequent to de-contamination, its performance capability is adverselyimpacted.Risk Assessment Analysis:Analysis of the identificationof contamination potentials in controlled environmentsthat establish priorities in terms of severity and frequen-cy and that will develop methods and procedures thatwill eliminate, reduce, minimize, or mitigate their po-tential for microbial contamination of the product/container/closure system.Sampling Plan:A documented plan that describes the

procedures and methods for sampling a controlled en-vironment; identifies the sampling sites, the samplingfrequency, and number of samples; and describes themethod of analysis and how to interpret the results.Sampling Sites:Documented geographical location,within a controlled environment, where sampling formicrobiological evaluation is taken. In general, sam-pling sites are selected because of their potential forproduct/containerclosure contacts.Standard Operating Procedures:Written proceduresdescribing operations, testing, sampling, interpretationof results, and corrective actions that relate to the oper-ations that are taking place in a controlled environment






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and auxiliary environments. Deviations from standardoperating procedures should be noted and approvedby responsible managers.Sterile or Aseptic Field:In aseptic processing or in oth-er controlled environments, it is the space at the levelof or above open product containers, closures, or prod-uct itself, where the potential for microbial contamina-tion is highest.Sterility:Within the strictest definition of sterility, anarticle is deemed sterile when there is complete ab-sence of viable microorganisms. Viable, for organisms,is defined as having the capacity to reproduce. Abso-lute sterility cannot be practically demonstrated be-cause it is technically unfeasible to prove a negative ab-solute. Also, absolute sterility cannot be practicallydemonstrated without testing every article in a batch.Sterility is defined in probabilistic terms, where the like-lihood of a contaminated article is acceptably remote.Swabs for Microbiological Sampling:Devices used toremove microorganisms from irregular or regular surfa-

ces for cultivation to identify the microbial populationof the surface. A swab is generally composed of a stickwith an absorbent tip that is moistened before sam-pling and is rubbed across a specified area of the sam-ple surface. The swab is then rinsed in a sterile solutionto suspend the microorganisms, and the solution istransferred to growth medium for cultivation of the mi-crobial population.Trend Analysis:Data from a routine microbial environ-mental monitoring program that can be related totime, shift, facility, etc. This information is periodicallyevaluated to establish the status or pattern of that pro-gram to ascertain whether it is under adequate control.A trend analysis is used to facilitate decision-making for

requalification of a controlled environment or for main-tenance and sanitization schedules.

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Agalloco J, Akers J. Risk analysis for aseptic processing: TheAkers-Agalloco method. Pharm Technol.2005; 29(11):7488.

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1117MICROBIOLOGICAL BEST

LABORATORY PRACTICES

INTRODUCTION

Good laboratory practices in a microbiology laboratoryconsist of activities that depend on several principles: aseptictechnique, control of media, control of test strains, opera-tion and control of equipment, diligent recording and evalu-ation of data, and training of the laboratory staff. Because ofthe inherent risk of variability in microbiology data, reliabili-ty and reproducibility are dependent on the use of acceptedmethods and adherence to good laboratory practices.

MEDIA PREPARATION AND QUALITYCONTROL

Media Preparation

Culture media are the basis for most microbiological tests.Safeguarding the quality of the media is therefore critical tothe success of the microbiology laboratory. Media prepara-tion, proper storage, and quality control testing can ensurea consistent supply of high-quality media.

It is important to choose the correct media or compo-nents in making media based on the use of accepted sour-



microbiological control and monitoring of aspectic environment and processes

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