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[ Microbiology Topics.Scott Sutton

Qualification ofan EnvironmentalMonitoring ProgramScott Sutton

"fvlicrobiology Topics" discusses various topics inmicrobiology of practical use in val idation and compliance. We intend this column to be a usefulresource for dai Iy work applications.Reader comments, questions, and suggestions a.re

needed to help us fulfi!1 our objective far this column. Please send your comments and suggestionsto column coordinator Scott Sutton at [email protected] or journal coordinating editor SusanHaigney at [email protected].

KEY POINTSThe following key points are discussed in this article: The number of sites to be used in qualifying cleanrooms for non-viable particulate measurementscan be found in International Organization forStandardization (ISO) 14644-1. However, thereare no recognized standards for determination ofthis number for viable air (passive and active) norfor surface monitoring. Amethod is suggested inthis article

Recommendations for the selectionof sample sitesto beused in the qualificationprogram are provided.These recommendations are directed at providingdata to allowcreation of a program useful in determination of the state of control of the facility

The frequency of samplingduring a qualificationstudy of this type should minimallybe at least therate of the eventual routine monitoring programfor each area

The qualification study should providedata to allowdetermination ofmeaningful alert and action lev-els for that facility. It must be noted that there aresignificant technical and scientific issues with theregulatory guidelines for the areas ofan aseptic coreregion. A suggestion consistent with the proposedrevisions to United States Pharmacopeia chapter "Microbiological Control and MonitoringEnvironments Used for theManufacture ofHealthcare Products" is provided

The qualification program is an excellent opportunity to begin the study of the microbial flora inyour facility.

INTRODUC'I'lONThe qualification, or requalification, of an asepticmanufacturing facility depends in large part on thedemonstration of controlled microbial conditions.The following are several areas where this is especially true:

Cleaning studies Contamination control planning (1) Equipment hold-time studies (Le., establishmentofclean and dirty hold times-process hold timesare process-specific)

Selection of sample sites for environmentalmonitoring

Establishment of facility-relevant alert and actionlevels for controlled environments.

For more Authorinformation,

go to

ABOUT THE AUTHORScott Sutton, Ph.D., is owner and operator of The Microbiology Network (www.microblol.org),which provides services to microbiology-related user's groups. Dr. Sutton may be reached bye-mail [email protected].

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This article focuses on amethod to qualify and justify the selection of the sample sites within a facilityused for routine environmental monitoring. The discussion presentedby the author is notmeantto describethe onlypossible approach to this selection but ratherone that the author has used in the past with success.Due to the limitations of space, this discussion doesnot include sampling of the water system, gasses, orpersonnel that have distinct considerations.NUMBER OF SITES FOR QUALIFICATIONSTUDIESlSO 14644-1 describes amethod to determine the number of sampling sites for site qualification. Annex Bstates thatwe should determine theminimum numberof sample sites by the following equation (2):

Where, NL is the minimum number of samplinglocations (rounded up to a whole number).A is the area of the clean room orzone in meters2 This might work well enough for non-viable par

ticulate measures (which is the intent and scope oflSO 14644-1), but we also wish to consider viable airsampling (both passive and active) and viable surface monitoring. Frequently, the sample site studyis worked into the facility HVAC performance qualification study for ease of documentation and logisticconsiderations. Let's make this a bi t easier and arguethat for the initial facility HVAC qualification protocol,both viable and non-viable active air sampling sitesshould be done a t the same locations (or as close aspractical to avoid compromising the othermeasureor the product integrity). This leaves determinationof the number of sites for passive air sampling andsurface sampling.Passive air sampling (i.e., settle plates) is a frequent

ly-used measure of clean room (or controlled zone)monitoring. Settle plates have several advantages inthis regard, chiefamong them the ability to remainin continuous exposure for up to four hours. Fourhours is cited in the European Union (EU) guidance(3)-extended exposure times must be demonstratedvia demonstration of the growth promotingcapabilitiesof the aged and exposed media. In addition, passiveviable monitoring (settle plates) is not disruptive tothe immediate environment and may possibly samplesites very near product exposure points (4). In addition, settle plates are not as prone to variation amonggxponcljvt.com

Scott Sutton.

differentvendors as are active samplers (5). However,it is no t clearwhether all the advantages cited for passive sampling apply in areas of laminar air flow at therates used formodern clean rooms. In addition, settleplates may be particularly susceptible to handling,transport, and lab contamination. Howeveryouviewtheir usefulness, current regulatory expectation for airmonitoring includes the use of settle plates and thejustification of sampling sites. Aprudent measure is touse the same number of samplingsites for settle platesas used for the active viable and non-viable samplingprograms. These will not be the same sites, but similarin number.This leaves us with determination of the number of

surfacesamplingsites for the qualification study. Thereis no regulatory guidance directed to this point for theinternational pharmaceutical industry. Even the PharmaceuticallnspectionConvention and PharmaceuticalInspection Co-operation Scheme (PlC/S), which generally can be counted on to provide details on almosteverything microbiological, is silent on this point (6).Oddly enough, even the Parenteral DrugAssociation(PDA) Technical Report #13 (7) offers no help here. Weare left to our own devices. One approach to determination of the number of sites would be to address it ina manner similar to that of ISO 14644-1 for the wallsand floors (as relevant). Each surface would then betreated as a separate item and the minimum number ofsites determined for each. While this mightwork forwalls and floors, the number of surface sampling sitesfor equipment remains unanswered and is not noticeablyamenable to this approach. This, quite frankly, maywell be something thatmustbe leftto determination ateach individual site-the numbers could be driven bythe nature of the equipment and the associated manufacturing process.Havingdeterminedthe numberof sites for each room,

we now need to determine their location. It should beobvious that using the "square rootof the area" methodwill frequently yield a larger number ofsamplesites than isneeded for routine monitoring, but that is appropriate fora validation study. Use the data collected from this "oversampling" to determine the appropriate sample sites.SELECTION OF SAMPLE SITES FOR THEQUALIFICATION STUDYThe selection of sample sites should be designed toprovide useful information for eventual selection ofroutine sample sites.

PDA. PDA Technical Report #13 (7) provides the following useful guidance in this regard:

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"Factors to consider in selecting sites for routinesurveillance are:1. Atwhich sites would microbial contamination

most likely have an adverse effect on productquality?

2. What sites would most likely demonstrateheaviest microbial proliferation during actualproduction?

3. Should site selection involve a statistical design(e.g., following the calculations in Federal Standard 209E) or should site selection be made onthe basis of grid profiling? Should some sites forroutinemonitoring be rotated? [Note from author:As 20ge has been withdrawn in favor of ISO 14644,the answer is "No"}

4. What sites would represent the most inaccessible or difficult areas to clean, sanitize, ordisinfect?

5. What activities in the area contribute to thespread of contamination?

6. Would the act of sampling at a given site disturbthe environment sufficientlyto cause erroneousdata to be collected or contaminate product?"

FDA. The US Food and Drug Administration's aseptic processing guidance document (8) also providessome guidance in section IVA:"Air in the immediate proximity of exposed sterilized containers/closures and filling/closing operationswould be of appropriate particle quality when it has aper-cubic-meter particle count of no more than 3520in a size range of 0.5 pm and larger when countedat representative locations normally no t more thanone foot awayfrom the work site, within the airflow,and during filling/closing operations. This level of aircleanliness is also known as Class 100 (ISO 5)."We recommend that measurements to confirm

air cleanliness in critical areas be taken at sites wherethere is most potential risk to th e exposed sterilizedproduct, containers, and closures. The particle counting probe should be placed in an orientation demonstrated to obtain a meaningful sample. Regularmonitoring should be performed during each production shift. We recommend conducting nonviableparticle monitoring with a remote counting system.These systems are capable of collecting more comprehensive data and are generally less invasive thanportable particle counters. See Section X.E. for additional guidance on particle monitoring."Some operations can generate high levels of product (e.g., powder) particles that, by their nature, do

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no t pose a r isk of product contamination. It maynot, in these cases, be feasible to measure air qualitywithin the one-foot distance and still differentiatebackground levels of particles from air contaminants.In these instances, air can be sampled in a mannertha t, to the extent possible, characterizes the truelevel of extrinsic particle contamination to which theproduct is exposed. Initial qualification of the areaunder dynamic conditions without the actual fillingfunction provides somebaseline information on thenon-product particle generation of the operation."Further, Section XA states:"Sample timing, frequency, and location should

be carefully selected based upon their relationshipto the operation performed...

"It is important that locations posing the mostmicrobiological risk to the product be a key par t ofthe program. It is especially important to monitor themicrobiological quality ofthe critical area to determine whether or no t aseptic conditions are maintained during filling and closing activities. Air andsurface samples should be taken at the locationswheresignificant activity or product exposure occurs duringproduction. Critical surfaces that come in contactwiththe sterile product should remain sterile throughoutan operation. When identifying critical sites to besampled, consideration should be given to the pointsof contamination risk in a process, including factorssuch as difficulty of setup, length of processing time,and impact of interventions."Ell. The EU guidance document "Manufacture ofSterileMedicinal Products" (3) provides the followingsite selection guidance:"18. Where aseptic operations are performed mon

itoring should be frequent using methods such assettle plates, volumetric air, and surface sampling(e.g., swabs and contact plates). Sampling methodsused in operation should no t interfere with zoneprotection."USP. Similarly, the following guidance in the pro

posed revision to United States Pharmacopeia (USP)chapter (9) is of general interest:"Microbiological sampling sites are best selected

when human activity during manufacturing operations are considered. Careful observation and mapping of a clean room during the qualification phasecan provide information concerning the movementand positioning of personnel within these rooms.Such observation can also yield important information about the most frequently conducted manipulations and interventions.

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assays (generally 25-30 CFU per plate, comparedwithregulatory guidance setting alert and action levels aslow as single digits). This concern led USP to suggesta frequency distribution approach for these areas (9).An interesting discussion of thi s approach can befound in Caputo and Huffman (10).Whichever approach is chosen to the determina

tion of the inital alert and action levels, theyshouldbe one of the deliverabIes from the EM qualificationprogram.A NOTE ON THE "MICROORGANISMCATALOG"FDA has clearly recommended establishmentof a listing of common microorganisms found in the asepticmanufacturing environment. This expectation is laidoutin section X.B. (8), as follows:"Characterization of recoveredmicroorganisms provides vital information for the environmentalmonitoring

program. Environmental isolates often correlate with thecontaminants found in a media fill or product sterilitytesting failure, and the overall environmental pictureprovides valuable information for an investigation.Monitoring critical and immediatelysurrounding cleanareasaswell as personnel should include routine identificationofmicroorganisms to the species (or, where appropriate,genus) level. In some cases, environmental trendingdatahave revealedmigration ofmicroorganisms into the aseptic processing room from either uncontrolled or lessercontrolled areas. Establishing an adequate program fordifferentiating microorganisms in the lesser-controlledenvironments, suchasClass 100,000 (ISO 8), can oftenbe instrumental in detecting such trends.At minimum,the programshould require species (or, where appropriate, genus) identification of microorganisms in theseancillary environments at frequent intervals to establish a valid, current database of contaminants presentin the facility during processing (and to demonstratethat cleaning and sanitization procedures continue tobe effective)."The EM qualification study is an excellentopportunity

to startth is catalog and to generate information on theeffectiveness of the cleaning and sanitization programfromamicrobiological perspective. Make sure thattheEM qualification program includes relevant evaluationsof all organisms isolated from air and surface samples,to the species level.

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REFERENCES1. Pharmaceutical Microbiology Forum, PMF Newsletter,Volume 13, Number 6, June 2007, http://microbiologyforum.org/PMFNews/PMFNews.13.06.0706.pdf.

2. ISO, ISO 14644-1, "Cleanrooms and associated controlled environments-Part 1: Classification of aircleanliness," Annex B, 1999.

3. EU, EudraLex The Rules Governing Medicinal Productsin the European Union Volume 4: EU Guidelines to GoodManufacturing Practice Medicinal Products for Human andVeterinary Use: Annex 1Manufacture of Sterile MedicinalProducts, 2008.

4. Whyte, W., "In Support of Settle Plates," PDA JPharmSci Tech. 50(4):201-204, 1996.5. Yao, M. and Mainelis, G., "Investigation of Cut-OffSizes and Collection Efficiencies of PortableMicrobialSamplers," Aerosol Sci Techno/. 40:595 - 606, 2006.

6. PIC/S, PI-006-2 Recommendations on Validation MasterPlan, Installation andOperational Qualification, Non-sterileProcess Validation, Cleaning Validation, 2004.

7. PDA, PDATechnical Report #13 (Revised): Fundamentalsofan Environmental Monitoring Program, 2001.

8. FDA, Guidance for Industry: Sterile Drug Products Pro-duced by Aseptic Processing-Current Good ManufacturingPractice, 2004.

9. USP, "Chapter Microbiological Control andMonitoring Environments Used for th e Manufactureof Healthcare Products," Pharm Forum. 33(3),2007.

10. Caputo, RA and AHuffman, "EnvironmentalMonitoring: Data Trending Using a Frequency ModeI," PDA JPharm Sci Tech. 58(5):254-260,2004. .IV,.

AR'T'ICLE ACRONYM LISTINGEM Environmental MonitoringEU European UnionFDA US Food a nd D ru g AdministrationISO International Organization on Standari zati onPDA Parenteral Drug AssociationPICIS Pharmaceutical Inspection Convention and

Pharmaceutical Inspection Co-OperationScheme

USP United States Pharmacopeia

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