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Microbial Testing

Microbial Testing

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5
Pharmaceutical Technology 
JUNE 2004 
www.pharmtech.com
Microbial Testing in Support ofAseptic Processing
Anthony M.Cundell
Anthony M. Cundell
is the director ofmicrobiological development and statistics,Global Quality Technology, at WyethPharmaceuticals, 401 N. Middletown Road,Pearl River, NY 10965-1299,tel. 845.602.2497, cundela@wyeth.com.
icrobial testing is conducted in the sterile pharma-ceutical industry in support ofsterile product de-velopment;for in-process monitoring during asep-tic processing and filling operations;and for testingfinished products.The role that microbial testing plays in pro-moting sterility assurance ofaseptically filled sterile productswill be discussed.
Product development
The objective ofthe product development process is to takesuccessfully progressing discovery leads from preclinical trialsthrough development with the purpose ofdefining the formu-lation,delivery system,manufacturing process,and productspecifications.The following microbial tests can be used dur-ing sterile product development and scale-up:
microbial limits and bioburden testing
bacterial endotoxin testing
antimicrobial effectiveness testing
container and closure integrity testing
bacterial challenge testing for sterilizing filters
aseptic processing validation using media fills.
Pharmaceutical ingredient and packaging component evaluation.
Microbial considerations play a key role in the successful de-velopment ofnew sterile drug products.During formulationdevelopment,the potential microbial and endotoxin content of the active pharmaceutical ingredients and excipients should beconsidered.The testing used to evaluate the ingredients shouldcomply with
USP
General Tests
61
“Microbial Limit Tests”and
82
“Bacterial Endotoxins Tests”(1–2).Typically,USP- orNF-grade raw materials are selected for use in the formulationand the possible contibution each ingredient would make theproduct bioburden are evaluated.Recently,United States Phar-macopeia (USP) has begun adding bacterial endotoxin re-quirements—on the basis ofmaximum human dosage—formonograph ingredients that may be used in sterile products.In some cases,the blanket compendial Microbial Limits for theTotal Aerobic Microbial Count not
1000 cfu/g or mL,andTotal Combined Yeast and Mold Count not
100 cfu/g or mLfound in the draft
USP
General Chapter
1111
may be too loosefor some sterile products (3).The contribution that each indi-vidual ingredient may make to the presterile filtration biobur-
M
The role of microbialtesting to ensure thesterility of asepticallyfilled sterile productsis explained,from theproduct developmentphase to in-processmonitoring tofinished producttesting.
DSM PHARMACEUTICALS
 
5
Pharmaceutical Technology 
JUNE 2004 
www.pharmtech.com
den,in terms ofits concentration in the formulation,must beevaluated to minimize the bacterial challenge to the sterilizingfilter and the endotoxin content ofthe product.Informationabout the properties and specifications ofpharmaceutical in-gredients is available in the
 Handbook ofPharmaceutical Excipients
(4).The bioburden ofpackaging components must be evaluatedwith respect to the sterilization process that will be used in themanufacture ofsterile products.The bacterial endotoxin levelsand the potential populations ofgram-positive,spore-formingbacteria associated with stoppers and vials are a consideration.Vials must be inspected and packaged for shipment to the cus-tomer in a controlled environment.Individual vials must beseparated with non-shredding dividers and shrink-wrapped toprevent glass-to-glass contact and particulate contamination.Vials are washed to remove particulates,depyrogenated to re-move bacterial endotoxins,and sterilized before aseptic filling.The maximum temperature and belt speed are established fora depyrogenating tunnel that adequately depyrogenates andconcurrently sterilizes the vials as they move through the tun-nel into the aseptic filling area.Stopper preparation methods should physically remove bac-terial endotoxins and nonviable particulates before siliconiza-tion.The cleaning and siliconization process should not con-tribute to the bioburden.The sterilization cycle ensures that thestoppers are sterile and dry so they can be stored before theaseptic filling operation.It should be noted that steam steril-ization is not a depyrogenation step.Typically,stoppers aresteam sterilized in heat-sealed,nonwoven,high-density poly-ethylene bags that enable steam penetration and moisture re-moval during the sterilization,exhaust,and drying processeswithin a pass-through autoclave.Cleanroom personnel usuallysize the bags to replenish the stopper hopper with a single loadto reduce the potential for microbial contamination duringmultiple handling.Alternately,semi-automated stopper prepa-ration equipment can be used to prepare sterile siliconized stop-pers.Stoppers also can be purchased clean,siliconized,andsterile.Initial bioburden and endotoxin monitoring ofincom-ing packaging components should be conducted to establishwhether the challenge levels for the cleaning,depyrogenation,and sterilization processes are adequate.
Preservation system development.
Testing for antimicrobial ro-bustness is an important part ofa drug product’s developmentalphase.In general,the use ofa preservative in single-use prod-ucts to replace good manufacturing practices (GMPs) is notsupported by regulatory agencies.Multiple-use products thatare stored in stoppered vials can be contaminated during re-peated syringe needle entries.Thus,they are formulated withpreservative systems that are tested for preservative efficacy dur-ing development using
USP
General Chapter
51
,Antimi-crobial Effectiveness Test”(5).The use ofthis test,when ap-propriate,can generate a developmental history ofa formulationin terms ofits preservative effectiveness against a range ofmi-croorganisms.The test also can indicate whether a product ismicrobiologically stable even without the presence ofa preser-vative system (i.e.,self-preserving).During the developmentphase ofthe product life cycle,the lowest concentrations atwhich the preservative system is effective can be established.The proposed formulation should be tested with the antimi-crobial effectiveness test at 50,75,and 100% ofthe target preser-vative concentration to establish the shelfspecification for theproduct on the basis ofpreservative efficacy and stability.A typ-ical preservative specification for a pharmaceutical product maybe 80–120% ofthe label claim.Thus,the preservative systemmay be monitored in the research and development stage andin product stability programs using a stability-indicating chem-ical assay in place ofmore time-consuming and more-variableantimicrobial effectiveness tests.
Container–closure integrity.
The container–closure integrity of the packaging components also is addressed during productdevelopment using a sensitive and adequately validated test.Recommendations for various container–closure combinationsfrom packaging suppliers are usually helpful.A physicalcontainer–closure integrity test may be selected and validatedusing a bacterial liquid immersion or aerosolization test.In gen-eral,physical tests are more sensitive than bacterial challengetests.Therefore,the leakage observed during a physical test maynot be indicative ofsterility assurance loss.A comprehensivediscussion about leak testing ofpharmaceutical packaging sys-tems has been published (6).When selecting a test method,the container–closure typeshould be considered.Although stoppered vials are subjectedto a bacterial immersion test,prefilled syringes are subjected toa bacterial aerosolization test because the latter has a moretorturous path for container–closure integrity.Physical testmethods described in the literature include the bubble method,helium mass spectrometry,liquid tracer (dye),headspace analy-sis,vacuum and pressure decay,weight loss or gain,and high-voltage leak detection.There are two phases to the container–closure integrity assessment:the initial evaluation and selec-tion ofthe container–closure system and integrity testing withinthe premarketed stability program.Suitable testing intervals are0,3,6,9,12,18,and 24 months during the premarketed stabil-ity program and annually during the postmarketed stabilityprogram.The number ofsamples tested at each time intervalreflects the sampling requirements found in
USP
General Chap-ter
71
,“Sterility Test”(7).Whenever possible,physicalcontainer–closure integrity tests for product monitoring shouldbe substituted for sterility testing.
Sterility test development.
The development ofa sterile prod-uct requires initial and ongoing consultation with an experi-enced pharmaceutical microbiologist.The application ofthesterility test is one indicator ofthe presence or absence ofcon-taminating microorganisms in a sterile batch,but the sterilityassurance is established by process design and validation,notsimply finished product testing.Validation ofthe sterility testincludes bacteriostasis and fungistasis testing and follows a pro-cedure that is defined in
USP
General Chapter
71
,“SterilityTests.Biological products marketed in the United States thatmust meet the 21
CFR
610.12 sterility testing requirements andproducts that differ in the subculturing requirements are an ex-ception.The development microbiologist uses results from ster-ilization process validation,aseptic process simulation usingmedia fills,and congruent environmental and personnel mon-
 
6
Pharmaceutical Technology 
JUNE 2004 
www.pharmtech.com
itoring to generate an assurance level that is satisfactory forsterile product production.The use ofbiological indicators forsterilization process cycle development is another way for mi-crobiologists to help build robustness into the productionprocess.In cases in which sterile filtration is part ofaseptic pro-cessing,the microbiologist should be aware ofthe filtrationprocess capabilities by reviewing microbial challenge data gen-erated during the validation ofthe filtration,filter integrity test-ing results,filtration operating parameters,and prefiltrationbioburden levels.Because the sterility assurance level is higherwith terminally sterilized products than with aseptically filledproducts,aseptic filling is used only when justified by the heatinstability ofthe product or when the packaging systems can-not be subject to terminal sterilization.An example ofthe lat-ter are prefilled syringes used for emergency drug administra-tion or home care.Emphasis should be given to theestablishment and monitoring ofcritical operating parametersused in the sterilization process with the aim ofusing para-metric release for terminally sterilized products.Guidance isprovided in
USP
General Information Chapter
1222
,“Ter-minally Sterilized Pharmaceutical Products:Parametric Release”(8).
Manufacturing process development.
The individual manufac-turing process steps for a new sterile drug product must be re-viewed to determine their potential for sterility assurance loss.On the basis ofrisk assessment,critical control points can beestablished and,ifnecessary,monitored to minimize the risk ofmicrobial contamination.The risk assessment includes theappropriateness ofthe aseptic manufacturing environment;aseptic techniques;quality ofthe water systems;the sanitarydesign ofprocessing equipment;equipment cleaning;steriliza-tion and storage procedures;the establishment ofimmediateholding times for sterilized aseptic processing equipment andbulk solutions;the level ofexposure ofproduct to manufac-turing personnel;aseptic sampling methods for product test-ing;and the establishment and monitoring ofcritical asepticoperating parameters.
In-process monitoring
The following microbial tests may be used during in-processmonitoring:
microbial limits and bacterial endotoxin monitoring ofin-coming pharmaceutical ingredients and packaging compo-nents
presterile filtration bioburden monitoring
bacterial endotoxin monitoring
air,surface,and personnel monitoring in aseptic processingareas
disinfectant effectiveness testing.
Monitoring incoming pharmaceutical ingredients,intermediates,and packaging components.
Incoming shipments ofpharmaceu-tical ingredients used in the sterile product are routinely testedfor bioburden and bacterial endotoxin levels.The concept oobjectionable microorganisms is not useful for pharmaceuti-cal ingredients used in sterile products;it is best reserved forthe evaluation ofthe bioburden ofnonsterile pharmaceuticalproducts as used in the control ofmicrobial contamination,per21
CFR
211.113.The author does not recommend screeningpharmaceutical ingredients used in sterile products for USP-specified microorganisms.Controlling bioburden to limit themicrobial challenge to the sterilizing filter will readily controlmicrobial toxins within sterile products because ofthe physi-cal presence ofmicroorganisms.The sterility assurance re-quirements will result in the numbers oforganisms at least threemagnitudes lower that those needed to control microbialtoxins.Sterile products are manufactured using water for injection(WFI) as ingredient water.Water must be manufactured by dis-tillation.The storage tanks,loops,and points ofuse in a WFIsystem are routinely monitored for microbial content and bac-terial endotoxins.The USP-recommended specifications are asfollows:Total Aerobic Microbial Count not
10 cfu/100 mLand Bacterial Endotoxin Levels not
0.05 endotoxin units/mL(9).When a validated,well-designed water system is used,eachloop must be monitored weekly and each point ofuse must bemonitored on a weekly or biweekly rotation.Although the useofa low nutrient microbiological medium such as R2A agar in-cubated at 20–25
C for at least 7 d may yield the highest count,the USP-recommended method ofusing plate count agar in-cubated at 30–35
C for 48–72 h may be more suitable for theroutine monitoring ofa fully validated water system when theobjective is to detect adverse trends in the water system in atimely manner and not to generate the highest possible count.Two additional advantages ofusing the plate count agar aregreater growth promotion capacity for fungi and the inabilityto consistently subculture bacterial isolates from nutrient-poorR2A agar.In July 2002,the European Pharmacopeia Commis-sion (EP) adopted the use ofan R2A medium incubated at 30–35
C for at least 5 d as its official test method for water for phar-maceutical use.This medium is usually incubated at 20–25
C(10).For US-based companies that make sterile products for aglobal market,the routine monitoring with the USP-recommended method and periodic monitoring (e.g.,each loopmonthly monitoring ofall loops using the EP-recommendedmethod) is a practical monitoring program.Opportunities existfor the application ofrapid microbial methods to water mon-itoring to generate the result earlier than 48 h.Packaging components such as molded or tubular glass vialsor vulcanized rubber stoppers are manufactured with high tem-peratures and pressures and are unlikely to have an inherentbioburden or be contaminated with bacterial endotoxins.Thus,reduced testing ofthese packaging components is justified fol-lowing a supplier’s qualification through a site audit and in-coming testing.The presterilized filtration bioburden is a critical parameterfor the maintenance ofa high level ofsterility assurance ofasep-tically filled pharmaceutical products.Presterilized filtrationbioburden monitoring ofbulk solutions is a common practicein the manufacture ofsterile pharmaceutical products.Singerand Cundell indicate that real-time measurement ofthe num-ber and size ofbacteria in the bulk solution could control thebacterial challenge to the sterilizing filter (11).However,an ap-propriate bioburden level for a specified bulk solution volumeand sterilizing filter surface area first must be determined.It is

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