1117

794 1116 Aseptic Processing Environments / General Information USP 36REFERENCES

cepted methods and adherence to good laboratoryAgalloco J, Akers J. Risk analysis for aseptic processing: The practices.

Akers-Agalloco method. Pharm Technol. 2005; 29(11):7488.Agalloco J, Akers J. The simplified Akers-Agalloco method

MEDIA PREPARATION AND QUALITYfor aseptic processing risk analysis. Pharm Technol. 2006;31(7)6072. CONTROL

Akers, J. The proper role of environmental monitoring inaseptic processing. Am Pharm Rev. 2006; 9(4):2428.

CDC, Healthcare Infection Control Advisory Committee. Media PreparationGuidelines for environmental control in healthcare facilities.MMWR 2003; 52(No. RR-10):142. Culture media are the basis for most microbiological tests.Favero MS, Puleo JR, Marshall JH, Oxborrow GS. Microbi- Safeguarding the quality of the media is therefore critical toological sampling of surfaces. J Appl Bacteriol. 1968; the success of the microbiology laboratory. Media prepara-31:336346. tion, proper storage, and quality control testing can ensureHussong D, Madsen R. Analysis of environmental microbi- a consistent supply of high-quality media.ology data from clean room samples. Pharm Technol. 2004; It is important to choose the correct media or compo-Aseptic Processing Suppl:1015. nents in making media based on the use of acceptedInternational Organization for Standardization (ISO). sources or references for formulas. The manufacturer's14644-1, Clean rooms and associated environments, part 1: formula and instructions for preparation routinely accom-classification of air cleanliness. Geneva: ISO; 1999. pany dehydrated media and ready-made media. BecauseInternational Organization for Standardization (ISO). different media types may have different preparation re-14644-2, Clean rooms and associated environments, part 2: quirements (e.g., heating, additives, and pH adjustment), itspecifications for testing and monitoring to prove continued is important to follow these instructions to ensure prepara-compliance with 14644 part 1. Geneva: ISO; 2000. tion of acceptable media quality. A certificate of analysisJensen PA, Todd WF, Davis GN, Scarpino PV. Evaluation of describing expiration dating and recommended storageeight bioaerosol samplers challenged with aerosol of free conditions accompanies ready-made media, as well as thebacteria. Am Ind Hyg Assoc J. 1992; 53:660667. quality control organisms used in growth-promotion and se-Ljungqvist B. Active sampling of airborne viable particu- lectivity testing of that media.late in controlled environments: a comparative study of Water is the universal diluent for microbiological media.common instruments. Eur J Parenter Sci. 1998; 3:5962. Purified Water is most often used for media preparation, butLjungqvist B, Reinmuller B. Interaction between air move- in certain cases the use of deionized or distilled water mayments and the dispersion of contaminants: clean zones with be appropriate. Water of lesser quality should not be usedunidirectional air flow. J Parenter Sci Technol. 1993; for microbiological media preparation. The volume of the47(2):6069. water used should be recorded.Ljungqvist B, Reinmuller B. Airborne viable particles and Consistent preparation of media requires accurate weigh-total number of airborne particles: comparative studies of ing of dehydrated media or media constituents. A calibratedactive air sampling. PDA J Sci Technol. 2000; 54:112116. balance with the appropriate weight range for the ingredi-Maruyama M, Matsuoka T, Deguchi M, Akers J. The appli- ents should be used (See Weighing on an Analytical Balancecation of robotics to aseptic surface monitoring. Pharm 1251). Clean weighing containers and tools (such asTechnol. 2007; 32(7):4044. spatulas) should be used to prevent foreign substancesProcess simulation testing for sterile bulk pharmaceutical from entering the formulation. The weight of the compo-chemicals. PDA Technical Report No. 28. J Parenter Sci nents should be recorded.Technol. 1998; 52 S3. Dehydrated media should be thoroughly dissolved inReinmuller B. Dispersion and risk assessment of airborne water before dispensing and sterilization. If heating is neces-contaminants in pharmaceutical cleanrooms. Building Serv sary to help dissolve the media, care should be taken not toEng Bull (Sweden). 2001; Bulletin No. 56. overheat media, because all culture media, to a greater or Stewart SL, Grinshpun SA, Willeke K, Terzieva S, Ulevicius lesser extent, are heat-sensitive. Equipment used in theV, Donnelly J. Effect of impact stress on microbial recovery preparation of media should be appropriate to allow foron an agar surface. Appl Environ Micro. 1995; controlled heating, constant agitation, and mixing of the61:12321239. media. Darkening of media (Maillard-type reaction orWhyte W. Reduction of microbial dispersion by clothing. nonenzymatic browning) is a general indication of overheat-J Parenter Sci Technol. 1985; 39(1):5160. ing. When adding required supplements to media, adequate

mixing of the medium after adding the supplement shouldbe performed.

Preparation of media in poorly cleaned glassware can al-low inhibitory substances to enter the media. Inhibitory sub-stances can come from detergent residue after cleaningglassware or from prior materials used in the glassware. Be1117 MICROBIOLOGICAL BEST sure that the cleaning process removes debris and foreignmatter, and that the detergent is thoroughly rinsed out withLABORATORY PRACTICES Purified Water. See Cleaning Glass Apparatus 1051 for addi-tional guidance.

Sterilization of media should be performed within the pa-rameters provided by the manufacturer or validated by theuser. Commercially prepared media should provide docu-

INTRODUCTION mentation of the sterilization method used. Autoclaving bymoist heat is the preferred sterilization technique, except in

Good laboratory practices in a microbiology laboratory instances when boiling is required in order to avoid deterio-consist of activities that depend on several principles: aseptic ration of heat-labile components of the media. Sterilizationtechnique, control of media, control of test strains, opera- by filtration may also be appropriate for some formulations.tion and control of equipment, diligent recording and evalu- The effects of the sterilization method and conditions onation of data, and training of the laboratory staff. Because of the media should be validated by sterility and growth-pro-the inherent risk of variability in microbiology data, reliabil- motion testing of the media. In addition, if sterilized byity and reproducibility are dependent on the use of ac- moist heat, the autoclave cycle should be validated to en-

Official from December 1, 2013Copyright (c) 2014 The United States Pharmacopeial Convention. All rights reserved.

Accessed from 220.225.196.130 by anware31 on Wed Mar 05 06:53:16 EST 2014

USP 36 General Information / 1117 Microbiological Best Laboratory Practices 795

sure proper heat distribution for selected loads and volumes. should be held in a monitored water bath at a temperatureTypically, manufacturers recommend using an autoclave cy- of 45 to 50 for not more than 8 hours. Caution should becle of 121 for 15 minutes using a validated autoclave. taken when pouring the media from a container immersedThese conditions apply to time at temperature of the media. in a water bath to prevent water from the bath commin-As container size and the load configuration of the auto- gling with the poured sterile media. Wiping the exterior ofclave will influence the rate of heating, longer cycles may be the container dry before pouring may be advisable.required for larger loads. However, the sterilization time will Disposal of used cultured media (as well as expired me-be dependent on the media volume and autoclave load. dia) should follow local biological hazard safety procedures.Sterilization cycles in which the autoclave is slow to comeup to temperature may result in overheating of the media. Quality Control TestingTherefore, care must be taken to validate a sterilization cy-cle, balancing the need for sterile media against the ten- Although growth media can be prepared in a laboratorydency of the media to degrade under excessive heating. from individual components, many laboratories, for ease ofStorage of the media in the autoclave after the liquid cycle use, use dehydrated media or purchase commercially pre-is completed is not recommended after cooling, as it may pared media in plastic plates or glass containers. Manufac-damage the media. Improper heating or sterilizing condi- turers of media attempt to standardize raw materials fromtionsfor commercially prepared or internally prepared me- biological sources, but must constantly deal with unavoida-diamay result in a difference in color change, loss of clar- ble differences in raw materials obtained from naturality, altered gel strength, or pH drift from the manufacturer's sources, and therefore, lot-to-lot variability of media must berecommended range, as well as reduced growth-promotion considered. In addition, the performance of media preparedactivity and/or selectivity. in a laboratory or by a manufacturer is highly dependent onThe pH of each batch of medium should be confirmed preparation and storage conditions. Improper media prepa-after it has cooled to room temperature (2025) by asepti- ration can cause unsatisfactory conditions for microbialcally withdrawing a sample for testing. Refrigerated pur- growth or recovery and unreliable results.chased media should be allowed to warm up to ambient Therefore, quality control tests should be performed on allroom temperature if it is to be checked for pH confirmation. prepared media, including media associated with swabs orA flat pH probe is recommended for agar surfaces, and an media in strips and other nontraditional formats. Tests rou-immersion probe is recommended for liquids. See pH 791 tinely performed on in-house prepared media should in-for guidance with pH measurement and instrument calibra- clude pH, growth promotion, inhibition, and indicativetion. The pH of media should be in a range of 0.2 of the properties (as appropriate), and periodic stability checks tovalue indicated by the manufacturer, unless a wider range is confirm the expiration dating.acceptable by the validated method. When in-house prepared microbiological media are prop-Prepared media should be checked by appropriate inspec- erly prepared and sterilized using a validated method, thetion of plates and tubes for the following: growth-promotion testing may be limited to each incoming Cracked containers or lids lot of dehydrated media, unless otherwise instructed by the Unequal filling of containers relevant compendial method. If the media preparation pro- Dehydration resulting in cracks or dimpled surfaces on cedure was not validated, then every batch of media shouldsolid medium be subjected to growth-promotion testing. Test organisms Hemolysis may be selected from the appropriate compendial test Excessive darkening or color change chapter. In addition, microorganisms used in growth-pro- Crystal formation from possible freezing motion testing may be based on the manufacturer's recom- Excessive number of bubbles mendation for a particular medium, or may include repre- Microbial contamination sentative environmental isolates (but these latter are not to Status of redox indicators (if appropriate) be construed as compendial requirements). Lot number and expiration date checked and recorded Expiration dates on media should have supporting Sterility of the media growth-promotion testing to indicate that the performance Cleanliness of plates (lid should not stick to dish) of the media still meets acceptance criteria up to and in-

cluding the expiration date. The length of shelf life of aMedia Storage batch of media will depend on the stability of the ingredi-

ents and formulation under specified conditions, as well asIt is prudent to consider how the manufacturer or supplier the type of container and closure.

transports and stores media before distribution to the end When a batch of media does not meet the requirementsuser. Manufacturers of media should use transport and stor- of growth-promotion testing, an investigation should be ini-age conditions that minimize the loss of moisture, control tiated to identify the cause. This investigation should includethe temperature, prevent microbial contamination, and pro- a corrective action plan to prevent the recurrence of thevide mechanical protection to the prepared media. problem. Any batch of media that fails growth-promotion

Media should be labeled properly with batch or lot num- testing is unsuitable for use. [NOTEFailed growth-promo-bers, preparation and expiration dates, and media identification test results may not be used to negate positive testtion. Media should be stored according to the manufactur- results.]er's instructions. Media prepared in house should be stored Some reagents are used for diagnostic purposes to helpunder validated conditions. Do not store agar at or below support identification of microbial organisms, e.g., Gram0, as freezing could damage the gel structure. Protect stain and oxidase test reagents. These may have attributesstored media from exposure to light and excessive tempera- that can be quality control tested similar to microbiologicalture. Before prolonged storage, agar plates should be placed media. Select the correct quality control standard microor-into a sealed package or container to retard moisture loss. ganisms, following the manufacturer's instructions, and per-

Remelting of an original container of solid media should form the testing before unknown sample diagnostic testing.be performed only once to avoid media whose quality is All relevant diagnostic reagents should be subjected to in-compromised by overheating or potential contamination. It coming quality confirmation before use.is recommended that remelting be performed in a heated Special care should be taken with media that is used inwater bath or by using free-flowing steam. The use of mi- sterility tests (see Sterility Tests 71 for requirements) and incrowave ovens and heating plates is common, but care environmental monitoring studies. Media used for environ-should be taken to avoid damaging media by overheating mental monitoring of critical areas should preferably beand to avoid the potential injury to laboratory personnel double-wrapped and terminally sterilized. If terminal sterili-from glass breakage and burns. The molten agar medium zation is not performed, media should be subjected to



796 1117 Microbiological Best Laboratory Practices / General Information USP 36

100% pre-incubation and inspection before use within a portance of that equipment to the generation of data in thecritical area. [NOTEGrowth-promotion testing for this me- laboratory.dia must be performed after the pre-incubation stage.] This Equipment that is difficult to sanitize (such as refrigeratorswill prevent extraneous contamination from being carried and incubators) should be dedicated to aseptic operationsinto controlled environments and will prevent false-positive (such as storage of media for testing and incubation of ste-results. A raised agar level for surface contact plates should rility test samples) and live culture operations to minimizebe verified. the potential for inadvertent contamination of the tests.

Autoclaves are central to the operation of the laboratoryand must have proper validation in place to demonstrate

MAINTENANCE OF MICROBIOLOGICAL adequate sterilization for a variety of operations. AutoclaveCULTURES resources must be available (and validated) to sterilize waste

media (if performed in that laboratory) as well as the mediaBiological specimens can be the most delicate standards prepared in that laboratory. The choice of one or several

to handle because their viability and characteristics are de- autoclaves is not driven by a need to separate aseptic andpendent on adequate handling and storage. Standardizing live operations (everything in the properly maintained auto-the handling and storage of cultures by the user laboratory clave is sterile after the cycle) but rather driven by resourceshould be done in a way that will minimize the opportunity considerations (see below).for contamination or alteration of growth characteristics.The careful and consistent treatment of stock cultures is crit-

LABORATORY LAYOUT AND OPERATIONSically important to the consistency of microbiological testresults. Cultures for use in compendial tests should be ac-

Laboratory layout and design should carefully consider thequired from a national culture collection or a qualified sec-requirements of good microbiological practices and labora-ondary supplier. They can be acquired frozen, freeze-dried,tory safety. It is essential that cross-contamination of micro-on slants, or in ready-to-use forms. Confirmation of the pu-bial cultures be minimized to the greatest extent possible,rity of the culture and the identity of the culture should beand it is also important that microbiological samples beperformed before its use in quality control testing. Ready-to-handled in an environment that makes contaminationuse cultures should be subjected to incoming testing for pu-highly unlikely.rity and identity before use. The confirmation of identity for

In general, a laboratory should be divided into clean orcommonly used laboratory strains should ideally be done ataseptic areas and live culture areas. Areas in which environ-the level of genus and species.mental or sterile product samples are handled and incu-Preparation and resuscitation of cultures should follow thebated should be maintained completely free of live cultures,instructions of the supplier or a validated, establishedif possible. If complete separation of live and clean culturemethod. The Seed-Lot technique is recommended forzones cannot be accomplished, then other barriers andstorage of stock cultures.aseptic practices should be employed to reduce the likeli-The original sample from the national culture collection orhood of accidental contamination. These barriers includea qualified secondary supplier is resuscitated and grown inprotective clothing, sanitization and disinfection procedures,an appropriate medium. Aliquots of this stock culture (theand biological safety cabinets designated for clean or asepticfirst transfer or passage) are suspended in a cryoprotectiveoperations only. Procedures for handling spills or mishapsmedium, transferred to vials, and frozen at 30 or below,with live cultures should be in place, and all relevant techni-until use. If stored at 70, or in lyophilized form, strainscal personnel should be trained regarding these methods.may be kept indefinitely. These frozen stocks can then be

Some samples will demonstrate microbial growth and re-used to inoculate monthly or weekly working cultures. Oncequire further laboratory analysis to identify the contami-opened, do not refreeze unused cell suspensions after cul-nants. When growth is detected, the sample should beturing a working suspension. The unused portion should betaken from the clean section of the laboratory to the livediscarded to minimize the risk of loss of viability and con-culture section without undue delay. Subculturing, staining,tamination of the stock.microbial identification, or other investigational operationsThe number of transfers of working control culturesshould be undertaken in the live culture section of the labo-should be tracked to prevent excessive subculturing that in-ratory. If possible, any sample found to contain growingcreases the risk of phenotypic alteration or mutation. Thecolonies should not be opened in the clean zone of thenumber of transfers allowable for specific compendial testslaboratory. Careful segregation of contaminated samplesmay be specified in that test. One passage is defined as theand materials will reduce false-positive results.transfer of organisms from a viable culture to a fresh me-

Staff engaged in sampling activities should not enter ordium with growth of the microorganisms. Any form of sub-work in the live culture handling section of a laboratory un-culturing is considered to be a transfer/passage.less special precautions are taken, including wearing protec-tive clothing and gloves and careful sanitizing of hands

LABORATORY EQUIPMENT upon exiting. Ideally, staff assigned to sampling activities,particularly those in support of aseptic processing, should

Most equipment (incubators, water baths, and autoclaves) not work in the vicinity of live culture laboratory operations.is subject to standard validation practices of incoming quali- It is important to consider that microbial contamination offication, operational qualification, and performance qualifica- samples, which leads to false-positive results, is always possi-tion. Additionally, periodic calibration (generally annually) is ble unless careful aseptic precautions are taken. Facilitiescommonly required. New equipment, critical to the opera- should be designed so that raw material and excipient sam-tion of the laboratory, should be qualified according to a pling can be done under controlled conditions, includingprotocol approved by the quality assurance unit (QAU). In proper gowning and sterilized sampling equipment. It mayaddition, regular cleaning and sanitization of equipment not always be possible to sample utility systems, such assuch as incubators, refrigerators, and water baths should be water systems, under full aseptic conditions; however, itperformed to minimize the potential for contamination in should be noted that when samples are not taken asepti-the laboratory. Door seals of incubators and refrigerators cally, their reliability is inevitably compromised.should be cleaned and checked for state of repair. Environmental sampling methods should require minimal

Instruments (pH meters and spectrophotometers) used in aseptic handling in loading and unloading sampling instru-a microbiology laboratory should be calibrated on a regular ments. Whenever possible, sampling equipment should beschedule and tested to verify performance on a routine ba- loaded with its microbiological recovery media in the envi-sis. The frequency of calibration and performance verifica- ronment that is to be sampled.tion will vary based on the type of instrument and the im-



USP 36 General Information / 1117 Microbiological Best Laboratory Practices 797

All testing in laboratories used for critical testing proce- A coherent system of standard operating proceduresdures, such as sterility testing of final dosage forms, bulk (SOPs) is necessary to run the microbiology laboratory.product, seed cultures for biological production, or cell cul- These procedures serve two purposes in a training program.tures used in biological production, should be performed Firstly, these SOPs describe the methodology that the mi-under controlled conditions. Isolator technology is also ap- crobiologist will follow to obtain accurate and reproduciblepropriate for critical, sterile microbiological testing. Isolators results, and so serve as the basis for training. Secondly, byhave been shown to have lower levels of environmental tracking the procedures in which a particular microbiologistcontamination than manned clean rooms, and therefore, are has demonstrated proficiency, the procedure number or titlegenerally less likely to produce false-positive results. Proper also serves to identify what training the microbiologist hasvalidation of isolators is critical both to ensure environmen- received specific to his or her job function.tal integrity and to prevent the possibility of false-negative Training curricula should be established for each labora-results as a result of chemical disinfection of materials tory staff member specific to his or her job function. He orbrought into or used within isolators (see Sterility Testing she should not independently conduct a microbial test untilValidation of Isolator Systems 1208). qualified to run the test. Training records should be current,

documenting the microbiologist's training in the current re-vision to the particular SOP.

SAMPLE HANDLING Periodic performance assessment is a wise investment indata quality. This performance testing should provide evi-

Viable microorganisms in most microbiology samples, par- dence of competency in core activities of the microbiologyticularly water, environmental monitoring and bioburden laboratory such as hygiene, plating, aseptic technique, doc-samples, are sensitive to handling and storage conditions. umentation, and others as suggested by the microbiologist'sCritical parameters in these conditions include product (or job function.sample) composition, container composition, time of stor- Microbiologists with supervisory or managerial responsibil-age, and temperature of storage. Therefore, it is important ities should have appropriate education and in-house train-to minimize the amount of time between the sampling ing in supervisory skills, laboratory safety, scheduling,event and the initiation of testing and to control, as much budgeting, investigational skills, technical report writing, rel-as possible, the conditions of storage. If the sample is to be evant SOPs, and other critical aspects of the company'stransported to a distant location for testing, then the condi- processes as suggested in their role of directing a laboratorytions of transport (time, temperature, etc.) should be quali- function.fied as suitable for that test and sample. Guidance for water Competency may be demonstrated by specific coursetesting in this regard can be found in Water for Pharmaceuti- work, relevant experience, and routinely engaging in rele-cal Purposes 1231. Product mixing before sampling may vant continuing education. Achieving certification throughneed to be evaluated and applied in order to ensure micro- an accredited body is also a desirable credential. Further, itbial dispersement and representation in the sample aliquot. is expected that laboratory supervisors and managers have a

All microbiological samples should be taken using aseptic demonstrated level of competence in microbiology at leasttechniques, including those taken in support of nonsterile as high as those they supervise. Expertise in microbiologyproducts. If possible, all microbiological samples should be can be achieved by a variety of routes in addition to aca-taken under full aseptic conditions in specialized sampling demic course work and accreditation. Each company is ex-areas. The areas should be as close to the point of use as pected to evaluate the credentials of those responsible forpossible to minimize contamination during transit. designing, implementing, and operating the microbiology

Samples submitted to the microbiology laboratory should program. Companies can thus ensure that those responsiblebe accompanied by documentation detailing source of the for the program understand the basic principles of microbi-sample, date the sample was taken, date of sample submis- ology, can interpret guidelines and regulations based onsion, person or department responsible for the submission, good science, and have access to individuals with theoreticaland any potentially hazardous materials associated with the and practical knowledge in microbiology to provide assis-sample. The testing department should acknowledge receipt tance in areas in which the persons responsible for the pro-of the sample and reconcile the identity and number of gram may not have adequate knowledge and understand-samples as part of this sample documentation. ing. It should be noted that microbiology is a scientifically

based discipline that deals with biological principles substan-tially different from those of analytical chemistry and engi-MICROBIOLOGICAL MEDIA INCUBATION neering disciplines. Many times it is difficult for individuals

TIMES without specific microbiological training to make thetransition.

Incubation times for microbiological tests of less than 3days' duration should be expressed in hours: e.g., Incubateat 30 to 35 for 18 to 72 hours. Tests longer than LABORATORY RESOURCES72 hours' duration should be expressed in days: e.g., Incu-bate at 30 to 35 for 3 to 5 days. For incubation times The laboratory management is responsible for ensuringexpressed in hours, incubate for the minimum specified that the laboratory has sufficient resources to meet the ex-time, and exercise good microbiological judgment when ex- isting testing requirements. This requires some proficiency inceeding the incubation time. For incubation times expressed budget management and in determining appropriate meas-in days, incubations started in the morning or afternoon ures of laboratory performance. A measure of laboratoryshould generally be concluded at that same time of day. performance is the number of investigations performed on

tests conducted by the laboratory, but this measure alone isnot sufficient. In addition to tracking investigations, the pe-

TRAINING OF PERSONNEL riod of time between sample submission and initiation oftesting should be tracked, as well as the period of time be-

Each person engaged in each phase of pharmaceutical tween end of test and report release (or test closure). Signif-manufacture should have the education, training, and expe- icant delays in these measures are also indications of anrience to do his or her job. The demands of microbiological under-resourced laboratory staff.testing require that the core educational background of the The laboratory management should have sufficientstaff, supervisors, and managers be in microbiology or a budget to meet testing requirements. Particular measures ofclosely related biological science. They should be assigned budgetary requirements will be specific to the given labora-responsibilities in keeping with their level of skill and tory, but budgetary considerations related directly to theexperience.



798 1117 Microbiological Best Laboratory Practices / General Information USP 36

need of the laboratory for sufficient resources must be ad- INTERPRETATION OF ASSAY RESULTSdressed to ensure reliable testing results.

Analytical microbiological assay results can be difficult tointerpret for several important reasons: (1) MicroorganismsDOCUMENTATION are ubiquitous in nature, and common environmental con-taminantsparticularly organisms associated with humansDocumentation should be sufficient to demonstrate that predominate in many types of microbiological analysis; (2)the testing was performed in a laboratory and by methods the analyst has the potential to introduce contaminating or-that were under control. This includes, but is not limited to, ganisms during sample handling or processing in the labo-documentation of the following: ratory; (3) microorganisms may not be homogeneously dis- Microbiologist training and verification of proficiency tributed within a sample or an environment; and (4) Equipment validation, calibration, and maintenance microbiological assays are subject to considerable variability Equipment performance during test (e.g., 24-hour/ of outcome. Therefore, apparent differences from an ex-7-day chart recorders) pected outcome may not be significant. Media preparation, sterility checks, and growth-promo- Because of these characteristics of microbiological analysis,tion and selectivity capabilities laboratory studies should be conducted with the utmost Media inventory and control testing care to avoid exogenous contamination as previously dis- Critical aspects of test conducted as specified by a cussed in this chapter. Equally important, results must beprocedure interpreted from a broad microbiological perspective, con- Data and calculations verification sidering not only the nature of the putative contaminant, Reports reviewed by QAU or a qualified responsible but the likelihood of that organism(s) surviving in the phar-manager maceutical ingredient, excipient, or environment under test. Investigation of data deviations (when required) In addition, the growth characteristics of the microorganismshould be considered (especially in questions of the growthof filamentous fungi in liquid media).MAINTENANCE OF LABORATORY RECORDS

When results are observed that do not conform to a com-pendial monograph or other established acceptance criteria,Proper recording of data and studies is critical to the suc-an investigation into the microbial data deviation (MDD) iscess of the microbiology laboratory. The over-riding princi-required. There are generally two distinct reasons for theple is that the test should be performed as written in theobservation of microbial contamination that does not com-SOP, the SOP should be written to reflect how the test isply with a target or requirement: There may be either aactually performed, and the laboratory notebook shouldlaboratory error or laboratory environmental conditions thatprovide a record of all critical details needed to reconstructproduced an invalid result, or the product contains a level ofthe details of the testing and confirm the integrity of thecontamination or specific types of contaminants outside es-data. At a minimum, the laboratory write-up should includetablished levels or limits. In either case, laboratory manage-the following:ment and, in most cases, the Quality Unit should be notified Dateimmediately. Material tested

A full and comprehensive evaluation of the laboratory sit- Microbiologist's nameuation surrounding the result should be undertaken. All mi- Procedure numbercrobiological conditions or factors that could bring about Document test resultsthe observed condition should be fully considered, including Deviations (if any)the magnitude of the excursion compared to established Documented parameters (equipment used, microbiallimits or levels. In addition, an estimate of the variability ofstock cultures used, media lots used)the assay may be required in order to determine whether Management/Second review signaturethe finding is significant.Every critical piece of equipment should be noted in the

The laboratory environment, the protective conditions inwrite-up, and all should be on a calibration schedule docu-place for sampling, historical findings concerning the mate-mented by SOP and maintenance records. Where appropri-rial under test, and the nature of the material, particularlyate, logbooks or forms should be available and supportive ofwith regard to microbial survival or proliferation in contactthe laboratory notebook records. Equipment temperatureswith the material, should be considered in the investigation.(water baths, incubators, autoclaves) should be recordedIn addition, interviews with the laboratory analyst(s) mayand traceable.provide information regarding the actual conduct of the as-The governing SOP and revision should be clearly notedsay that can be valuable in determining the reliability of thein the write-up. Changes in the data should be crossed offresult and in determining an appropriate course of action. Ifwith a single line and initialed. Original data should not belaboratory operations are identified as the cause of the non-erased or covered over.conforming test outcome, then a corrective action planTest results should include the original plate counts, al-should be developed to address the problem(s). Followinglowing a reviewer to recreate the calculations used to derivethe approval and implementation of the corrective actionthe final test results. Methods for data analysis should beplan, the situation should be carefully monitored and thedetailed in cited SOPs. If charts or graphs are incorporatedadequacy of the corrective action determined.into laboratory notebooks, they should be secured with

If assay results are invalidated on the basis of the discov-clear tape and should not be obstructing any data on theery of an attributable error, this action must be docu-page. The chart or graph should be signed by the personmented. Laboratories also should have approved proceduresadding the document, with the signature overlapping thefor confirmatory testing (retesting), and if necessary,chart and the notebook page. Lab notebooks should include

page numbers, a table of contents for reference, and anintact timeline of use.

All laboratory records should be archived and protectedagainst catastrophic loss. A formal record retention and re-trieval program should be in place.



USP 36 General Information / 1118 Monitoring Devices 799

resampling where specific regulatory or compendial guid- Infrared DeviceThis is a device based on measuringance does not govern the conduct of an assay investigation. the IR radiation from the article whose temperature is being

determined; the IR radiation varies as a function of the ob-ject's temperature. The advantage of the device is that thearticle may be at some distance from the IR sensor. How-ever, IR devices are expensive compared to other tempera-ture sensors.

Resistance Temperature Detector (RTD)This is a de-vice based on the change in electrical resistance of a mate-1118 MONITORING DEVICESrial as a function of temperature. Precision and accuracy de-pend on the quality of the electronics used to measure theTIME, TEMPERATURE, ANDresistance. Therefore, although RTDs are among the moststable and accurate temperature sensors, their accuracy mayHUMIDITYchange with the age and temperature of the device as itselectronic components are affected. A particular type of RTDuses platinum or platinum alloy wire as the sensor. TheseThis chapter provides background on the science and are referred to as platinum resistance temperature detectorstechnology of temperature and humidity monitoring. It de- (PRT or PRTD).scribes the available technologies and their performance

Solid State DeviceThis is a device based on the effectcharacteristics, and it provides recommendations for verifica-of temperature on either an integrated circuit (see Thermis-tion and validation of performance. The shelf life of a drugtor below) or a micromechanical or microelectrical system.is a function of the temperature and humidity conditionsThese devices can attain the highest precision available andunder which it is stored and transported as well as thealso have the advantage of producing a digital output. Theirchemical and physical properties of the drug substance andaccuracy is typically limited by the accuracy of the cali-preparation. For this reason, the ability to monitor thosebrating system employed.conditions is important in the shipping and storage of tem-

ThermistorThis is a semiconductor device whose resis-perature- and humidity sensitive preparations. Historic geo-tance varies with temperature. Thermistors are able to de-graphic and seasonal trends may be used as a planning tooltect very small changes in temperature. They are accuratein selecting among the types of temperature and humidityover a broad range of temperatures.monitoring devices. Meteorological forecasts are available

for any pertinent location. ThermocoupleThis is a device based on the change inthe junction potential of two dissimilar metals as a functionof temperature. Many metal pairs may be used, with eachTEMPERATURE MEASUREMENT pair providing a unique range, accuracy, and precision. Pre-

TECHNOLOGIES cision and accuracy depend on the quality of the electronicsused to measure the voltage and the type of temperature

The devices described in this section are those most com- reference used. Accuracy may be a function of temperaturemonly used to monitor temperature in the storage and dis- reference used. Thermocouples have relatively poor stabilitytribution of drugs in North America. The measurement of and low sensitivity, but are simple and cover a wide temper-temperature at extremes, such as close to absolute zero or ature range.above those reasonably expected to be experienced by Thermomechanical DeviceThis is a device based ondrugs, is not addressed. the change in volume of a solid material as a function of

Alcohol or Mercury ThermometersThese devices are temperature. For example, a mechanical spring, which ex-based on the change in volume of a liquid as a function of pands or contracts as a function of temperature, thus open-temperature. Mercury thermometers are typically used in ing and closing an electrical circuit or moving a chart pen,the ranges from 0 to 50 with a precision of about 0.1. is such a device. Precision may be as good as 0.05, but in[NOTESome local regulations apply to mercury-based ther- practice it is rarely better than 0.5. Accuracy is often in themometers. Alcohol thermometers may have a precision as range of 1.0, but it may change with the age and tem-good as 0.01, but they must be quite large to measure perature of the device.temperatures in ranges of more than a few degrees. Bothtypes of thermometers may be designed to indicate the

TIMETEMPERATURE INTEGRATORSmaximum and minimum temperatures measured. See Ther-mometers 21.]

Timetemperature integrators, commonly referred to asChemical DeviceThis is a device based on a phaseTTIs, change color or physical appearance as a result of ex-change or chemical reaction that occurs as a function ofposure to a temperature above a specific threshold for atemperature. Examples include liquid crystals, waxes, andspecific time duration, and thus accumulate heat. TTIs arelacquers that change phase, and thereby their appearance,typically single use, disposable devices that react irreversibly.as a function of temperature. Such materials represent theOnce the color changes, it will not revert to the original oneleast expensive form of temperature measurement, but theyeven if the temperature returns to the acceptable, normalmay be difficult to interpret.range. The four basic types of chemical-based TTIs are de-Other types of chemical sensors include systems in whichscribed below.a reaction rate or diffusion process is used to deduce a tem-

Table 1 lists the four types of chemical TTIs presently inperature equivalent integrated over time rather than theuse. The closer the activation energy of the TTI's colortemperature at a specific moment in time such as a spike orchange to the activation energy of the degradation processcritical threshold, for which a separate device may be pre-of the drug being monitored, the more accurately the TTIferred. Thus, chemical sensors provide a measure of accu-will reflect the status of the drug. In actual practice, themulated heat rather than instantaneous temperature. Itactivation energy for degradation of a particular drug is notshould be noted that these devices are generally irreversible;known precisely enough to enable selection of a particularonce a color change or diffusion process has taken place,type of TTI. The range of possible activation energies of aexposure to low temperatures will not restore the device toTTI is given in the table to provide a sense of the flexibilityits original state. Accuracy and precision vary widely amongof that particular technology. A TTI with a range of possibledifferent types, to differentiate often limited by their abilityactivation energies can be configured to cover a wideror their ability to visually interpret diffusion distances.range of time and temperature thresholds.



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