Validation of Sterile Filtration article

Pharmaceutical Technology FILTRATION 2004 s13

he Parenteral Drug Association(PDA) published the authorita-tive summary of best practices

in sterile filtration and validation ofsterile filtration in its 1998 technicalreport, “Sterilizing Filtration of Liq-uids” (1). It highlights the history ofsterile filtration, explains how filterswork, details selection criteria, andexplains validation considerationsand integrity testing methods.Much of this article is based on thatmonograph.

The purpose of sterile filtrationvalidation is to prove that a particu-lar filtration process generates a ster-ile filtrate. This is achieved by choos-ing a sterilizing grade filter that iscompatible with the process, non-toxic, integrity testable, sterilizable,that does not adsorb formula com-ponents or add extractables to theprocess and can remove the biobur-den associated with the product.The filter then is challenged with.107 colony forming units (cfu) ofBrevundimonas diminuta (ATCC19146) per cm2 under process con-ditions and demonstrated by testingto produce a sterile filtrate.

Validation of SterileFiltrationLeesa McBurnie and Barry Bardo*

TThis article discusses thebasics of sterile filterqualification and validation,with emphasis on bacterialchallenge protocoldevelopment and testing.Reference is made toTechnical Report no. 26 ofthe Parenteral DrugAssociation.

Leesa McBurnie is seniormicrobiologist and BarryBardo is director of businessdevelopment at MeissnerFiltration Products, Inc.,Camarillo, CA [email protected].

*To whom all correspondenceshould be addressed.

s14 Pharmaceutical Technology FILTRATION 2004 www.pharmtech.com

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Sterilizing grade filtersThe removal rating of a given mem-brane filter type refers to the size, ornarrower dimension, of microor-ganisms and particles removed bythe filter, rather than the actual sizeor shape (i.e., morphology) of thefilter pore structure. The industry-accepted rating for a sterilizinggrade filter is 0.2 or 0.22 mm, de-pending on the manufacturer, whichis validated as capable of removing.107 cfu/cm2 B. diminuta undercertain extreme processing condi-tions. Tighter filters such as 0.1 mmfilters that demonstrate the samebacterial retention also may be ratedas sterilizing grade.

In validating and performingsterile filtration, it is essential toidentify the bioburden or endemicmicroorganism(s) in a givenprocess, to use the grade of filterthat quantitatively removes the mi-croorganism(s), and to demonstratequantitative removal by test beforeusing the filter in production. Thisis the essence of filter validation.

For filter manufacturers, a criticalrequirement is to provide users witha reasonably convenient, safe, easy-to-perform integrity test which con-firms the integrity of the filter, theseals, and contiguous process equip-ment. Liquid-sterilizing filters canbe integrity tested by the bubblepoint, forward flow or diffusion/diffusive flow test, or the pressurehold test. Hydrophobic filters can betested with any of those methods orby the water intrusion test. Filter in-tegrity tests are explained in anotherarticle in this issue.

Qualifying pharmaceutical filtersSafety and purity. Before they can beused in a sterilizing filtrationprocess, filters must meet or exceedminimum safety standards.

The first step is to qualify the filterin several important areas of safety.To qualify as a pharmaceutical filter,the filter must be nontoxic, accordingto USP-specified tests (e.g., USP ^87&“Biological Reactivity Tests,” in vitro,USP ^88& “Biological Reactivity Tests,”in vitro, USP ^88& “Biological Reactiv-ity Tests,” in vivo, including the ClassVI Plastics Tests) and be tested free ofpyrogen or endotoxin to acceptablylow levels.

Pharmaceutical-grade filters alsomust have very low extractables levels,although neither USP nor the Foodand Drug Administration have speci-fied minimum or maximum levels. Itis the responsibility of pharmaceuticalmanufacturers to set allowable specifi-cations for extractables.

As a further indicator of safetyand purity, filters also should rinsequickly when exposed to high-purity water, must be compatiblewith pharmaceutical process fluidsand pharmaceutical products, andbe both sterilizable and integritytestable.

All this pharmaceutical filter safetyqualification information typically isprovided in the filter manufacturers’validation guide and product litera-ture for a specific filter.

Performance qualification. Filtersmust be qualified by the user todemonstrate that their performancein processing will meet or exceedminimum process requirements.


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The filter must be tested to verifythat it provides the flow rates re-quired by the pharmaceuticalprocess. The filter system must besized to provide flow rates and vol-umes adequate to keep pace withfilling machines or other productionequipment requirements, with somereserve capacity for use in case ofbatch contaminant variability andpremature plugging. The total liquidvolume passed through the filters,its throughput, should be adequateto process a complete batch withoutinterruption.

Small-scale sizing or filterabilitytests are used as the basis for extrap-olating or scaling-up filtration sys-tems. Sizing of final, sterilizing fil-ters, and any upstream prefiltersused to remove coarse contaminantsand thereby extend the life of thefinal filters, is based upon antici-pated flow rates and throughput in agiven pharmaceutical liquid.

Sterilizing filters and filter hous-ings, stainless steel, or disposableplastic, must be rugged enough towithstand the pressures and temper-

atures of normal process conditions,occasional runaway process condi-tions, and the temperature ranges ofprocessing (minimum to maxi-mum) and steam or autoclave steril-ization, including both temperatureranges and duration of the steriliza-tion cycle(s).

Sterilizing-grade filters for aque-ous pharmaceutical liquids are nor-mally hydrophilic, or water-wettable, membrane filters. In thecase of solvent or chemical liquidsto be filter-sterilized, hydrophobic,or nonwater-wetting, filters may beused. They can be wetted by a low-surface-tension liquid.

Compatibility. The filter systemmust be qualified to ensure that allproduct-contact surfaces of the filterand its constituent parts (mem-brane, support layers, core, cage,and end caps), o-rings, piping,hoses, seals, pumps, gaskets, and anyother components of the sterilizingfiltration system can withstand thehydraulic, thermal, and chemicalchallenges of the sterilization andproduction processes. None of theseshould extract into the filtered phar-maceutical product in any signifi-cant amount.

Chemical compatibility questionsgenerally are resolved by referenceto compatibility tables generated bymanufacturers of elastomers orpolymers used in o-rings, gaskets,and seals. Membrane compatibilitiesgenerally are well established for thecommonly used membrane materi-als. Any specific questions can read-ily be resolved by testing during thequalification stage.

• flow rates• throughput • pressure and temperature resistance• hydrophilic or hydrophobic• membrane composition• compatibility

• membrane support layers, core, or cage• o-rings• housings

Performance qualification requirements


Sterilization of sterilizing filters.Sterilizing grade filters can be steril-ized in a number of ways. Capsulefilters can be gamma irradiated orautoclaved. Disk filter holders areautoclaved with the wetted filter inplace. Cartridge filter installationsfrequently are sterilized by steam-in-place (SIP) operations.

Common steaming temperaturesused in the United States are 121–135 8C, sustained for 30–60 min inthe filter installation. Whatevertime–temperature parameters arespecified, it is critical that these pa-rameters be validated by the phar-maceutical manufacturer under op-erating conditions.

ValidationThere are four major elements ofthe filtration validation process:

• physical/chemical compatibility,usually established during thequalification phase before valida-tion, is confirmed during the val-idation process

• binding and adsorption filtercharacteristics are measured inthe qualification phase

• bacteria retention capability of thefilter, which is established by chal-lenging the filter with B. diminuta

• integrity of the process filtrationinstallation, as verified by the fil-ter integrity test.

Concerning integrity testing, theuser must demonstrate that theyknow how to install, sterilize, andintegrity test the filters. Filter in-tegrity test values provided by thefilter manufacturer are correlated tothe bacterial challenge in the manu-

facturer’s validation guide. Productintegrity test values are correlated tothe water or model solvent values.

Bacterial retention. The bacterialchallenge test validates the ability ofa filter to provide sterile effluent in aspecific pharmaceutical liquid. It isalso the ultimate compatibility test,because the bacterial challenge si-multaneously tests the physical-chemical interaction of the liquidproduct and the filter, under processconditions. Any filter inadequacycaused by this interaction will bedetected by the bacterial challenge.

Validation of bacterial retentionnormally is performed by the filtermanufacturer or an independent lab-oratory, using 47-mm diameter disksto minimize the volume of pharma-ceutical product required. Larger sur-face area filters also can be used.

Bacterial challenge tests are usu-ally performed with an industrystandard concentration of 107 cfu ofB. diminuta per cm2, using pharma-ceutical product, whenever possible,for the most realistic validation. Thehigh bacterial concentration used inthe challenge test constitutes aworst-case scenario. The manufac-turer qualifies the filter using a simi-lar challenge.

B. diminuta is grown to producemonodispersed cells capable of pen-etrating a 0.45-mm filter, typically inaccordance with ASTM StandardF838. Following that standard, theorganism is cultured in saline lac-tose broth (SLB) and either usedfreshly cultured or concentratedinto a frozen cell paste that isthawed immediately prior to use. A


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0.4-µm rated membrane filter isused as a positive control; if the or-ganisms pass through this controlfilter, it proves that they are alive,small, and nonaggregated, thus veri-fying that the challenge is a strin-gent test.

Validation parameters for bacterialchallenge using pharmaceutical prod-uct. Key considerations for usingpharmaceutical product liquid tovalidate sterile filters are listed in thefollowing chart.

Product contact time.The bacterialchallenge using pharmaceuticalproduct must be run for at least thesame duration as a product batchwill be run in processing. If thebatch requires eight hours to filter,the challenge must be run for atleast eight hours. It is good practiceto run the challenge for a littlelonger to anticipate unusual pro-cessing circumstances.

Differential pressure and flow rates per unitarea. Maximum process differentialpressures and flow rates should beincorporated into the protocol.Often, it is virtually impossible tomatch both simultaneously. At thestart of the challenge, when the filteris clean, extremely high flow ratesper unit area are necessary to gener-ate process-level differential pres-sures (pressure drop across the fil-ter). But, as the test filter disksaccumulate bacteria and pressurebuilds, the flow drops. One solutionis to match flow rates at the start ofthe challenge and pressures near theend. Another solution is to decidewhether pressure or flow is more rel-evant, and then develop a technically

sound rationale to support the deci-sion and use it in challenge testing.

Temperature. If the liquid tempera-ture is outside the viable range ofthe challenge bacteria, it may benecessary to recirculate the productat process temperature, condition-ing the filter first, and then chal-lenge the filter at a temperature atwhich the bacteria survive.

Bioburden. Bioburden levels can in-fluence process filtration efficacy.The probability of passage increaseswhen the bioburden is high. Thearea-specific bioload (Ba) is thebioburden per unit area of filter(cfu/cm2) or Ba = BV/A, where B isthe bioburden in cfu/mL, V is thetotal volume to be filtered (mL) andA is the surface area of the filter incm2. It is therefore best to controlthe bioburden of the raw materialsto avoid approaching or exceedingthe validated limit.

Integrity test correlation.Filters used inthe bacterial challenge must be in-tegrity tested to form the correlationto retention. Because the user can-not use a destructive challenge testin processing, the filter manufac-turer must supply a correlated, non-destructive integrity test that reli-ably assesses the integrity of a givenfilter installation. In performing the

• product contact time• differential pressure• flow rates per unit area• temperature• bioburden• integrity test correlation

Validation parameters


challenge test, three lots of filterstypically are chosen, including alow-bubble-point lot, one with abubble point specification that ap-proximates the manufacturer’s pro-duction limit. This is important be-cause the end user is limited tousing filters in their process thatmatch or exceed the lowest bubblepoint validated in product.

Establishing the challenge protocol.The above chart highlights the stepsneeded to develop a suitable chal-lenge protocol.

After the filter is qualified and se-lected, filter extractables and micro-bial retentivity are validated. Physi-cal parameters such as sterilizationand integrity test methods also arevalidated. Viability of the test mi-croorganism, B. diminuta, in the

pharmaceutical product is comparedto a control over time. If there is lessthan one log difference in the counts,the organism is considered viableunder those conditions. Results aredocumented. If a pharmaceuticalproduct is toxic, has high potentialfor abuse, or is in limited supply, anonbactericidal surrogate fluid withvery similar properties may be devel-oped. It is recommended that theuser consult with local FDA inspec-tors before developing this approach.

If the microbe is viable. If B. diminutais found to be viable in the pharma-ceutical product, it is directly inocu-lated into the product as the chal-lenge microorganism.

If the microbe is nonviable. If B. dimin-uta is found to be nonviable underprocess conditions, the test filter is

Select filter

Filter extractable validation

Microbial retentivity validation

Physical parameters validation

Determine and document viability of B. diminuta

in product underprocess conditions

Develop nonbactericidal surrogate with same product

characteristics for toxic, high abuse potential

or limited supply drugs

B. diminuta viable in product under process

conditions

B. diminuta not viable in product under process

conditions

Direct inoculation of B. diminuta into product under process conditions

Precondition filter with product–followed by microbial challenge.

One or a combination of the following methods may possibly be used

Modify process–adjust temperature, etc.

Modify formulation– adjust pH, remove

bactericidal component product surrogate

Use product fortime period challenge

organism is viable

Change from B. diminuta–use bacteria isolated from formulation or environment

Figure 1: Establishing a microbial challenge protocol (1).


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preconditioned with pharmaceuticalproduct liquid, and one of severaloptions can be followed, prior toconducting the bacterial challenge.

Options are to:• modify the process by adjustingtemperature or otherwise• modify the formulation, by ad-justing pH, removing bactericidalcomponents, or using a productsurrogate liquid• use the bacteria for a brief enoughtime such that the challenge mi-croorganism retains its viability dur-ing the test period• switch the test organism from B.diminuta to a process isolate, or na-tive bioburden endemic to the ac-tual pharmaceutical process. It isrecommended to consult first withFDA inspectors on this option.If the product is bactericidal, the

volume and time required to thor-oughly rinse the filter free of theproduct must be established, afterthe product has been recirculated,prior to adding the bacteria andchallenge solution. This is docu-mented in an inhibition test.

Bacterial challenge. The challengeprocedure typically consists of in-tegrity testing the filters, autoclaving

the apparatus, integrity testing post-sterilization and recirculating prod-uct through the filters at processflow rates or pressures. Bacteria areadded according to the results of theviability study. In nonbactericidalproducts, the microorganisms areadded at intervals throughout thechallenge. Before introducing thebacteria, analysis membranes are in-stalled downstream of all test andcontrol filters. Analysis filters musthave a pore size of at least 0.45 mmor tighter. Membranes that are 0.45mm will retain 104–106 logs of Bre-vundimonas, which generally is ac-cepted as adequate for a valid test.

Factors affecting bacterial retention.The bacterial challenge procedureaddresses the possible influencesupon retention of the membranepolymer, liquid, process conditions,and bioburden level. Adverse effectsfrom any of these parameters wouldresult in bacterial passage.

The only factor not routinely ad-dressed in the filter validation ormicrobial challenge process is com-position of the bioburden. Compo-sition of bioburden should be ad-dressed prior to choosing theremoval rating of the filter, duringfilter qualification.

Bracketing.It is not always necessary

• filter type: polymer, structure• fluid properties: pH, viscosity, osmolarity,

and ionic strength• process conditions: temperature, differential

pressure, and flow rate• bioburden: quantity, composition

Factors affecting microbialretention

• challenge extreme products• carefully choose family groupings

• API concentration levels• pH, other

• validates intermediate products

Bracketing


to perform a microbial chal-lenge on every pharmaceuti-cal product. The practice ofbracketing makes this ac-ceptable. With a family ofpharmaceutical productshaving high degrees of simi-larity but only slightly differ-ing characteristics such asdiffering concentrations ofactive ingredient, we canperform the microbial chal-lenge on the extreme prod-ucts (i.e., those having thehighest and lowest concen-trations) while productswith intermediate-level con-centrations need not be in-dividually challenge-tested.However, we must generate data toshow that these product extremeshave been tested.

Extractables. Another important as-pect of sterile filter validation is ex-tractables testing. It is importantthat the filters are not themselves asource of physical (e.g., particles) orchemical contaminants.

The nonvolatile residue (NVR)test from USP ^661& Containers isused to quantify the amount of ex-tractables released by a filter in aparticular process stream. NVR typ-ically is tested by using a model sol-

vent, rather than pharmaceuticalproduct. It involves soaking the fil-ter, boiling off the solvent, dryingthe residue, and weighing it. Theweight of residue per filter is calcu-lated. Pharmaceutical products sel-domly are used for NVR assay, be-cause their constituents mask filterextractables. Various analytical tech-niques may be applied to identifyfilter extractables.

Adsorption.The flip side of the phe-nomenon of filter extractables is thephenomenon of filter adsorption.The filter should not remove activeingredients, excipients, carriers, dilu-ents, proteins, preservatives, or anyother formulation component. It isimportant to test the product to en-sure that the filter does not cause anyingredient to fall below specification.

With most sterilizing grade filters,adsorptive levels are so low as to beinsignificant and well within limits

• binding of formulation components to filter• potential for OOS ingredients• identify any problem• address problem if necessary

Adsorption analysis

Figure 2: Bacterial challenge test apparatusshowing differential pressure between two inline47-mm diameter filter disk holders in series, withthe challenge filter upstream and the analysis filterdownstream, connected by sanitary piping to aperistaltic pump which recirculates the challengemicroorganism.


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set by pharmaceutical manufactur-ers. However, if an adsorption prob-lem is experienced with a certainformulation, it should be addressed.

Redundant filtration. One final cir-cumstance to be addressed in vali-dation is the use of redundant fil-ters, two final filters of identicalrating deployed in series as “insur-ance” against nonsterile filtration.This practice is common in Europeand with some American manufac-turers of serum, tissue culturemedia, and biologicals.

If both filters are needed toachieve sterility in a given process,

both must pass the post-use in-tegrity test in process operations. Ifonly one filter in the series is neces-sary to validate a sterile filtrate, thenonly one is required to pass the in-tegrity test.

Where redundant filters are in-stalled, the validation process alsomust address the impact of the sec-ond filter on extractables levels andadsorption (i.e., twice as much).

PDA Technical Report No. 26Filter validation recommendations.PDA’s Technical Report No. 26 sum-marizes the principles and best

Table I: Tests commonly performed by filter users and the filter manufactures–general industry practices.

Filter user Filter manufacturerCriteria Filter device Membrane disk DeviceBacteria retention/integrity — Q Qtest relationship dataIntegrity test

water/solvent V Q, R, L Q, R, Lproduct V — —

Integrity test methodology V R —and selectionBacterial retention

water, SLB, etc. — Q, L Q, Lproduct V, membrane disk — —

Bacterial retention/integrity V Q Qtest methodologyEffects of chemical compat- V Q Qibility on filter integrityToxicity testing — Q QExtractables V Q, R, L Q, R, LEffects of sterilization V Q Qmethods on filter integrity

Q denotes qualification testing process specific; V denotes validation testing; Rdenotes recommendation for validation; and L denotes filter lot specific releasecriteria.


practices of sterile filtration and itsvalidation. In this article, we havedescribed details of some of themore significant aspects of filter val-idation that follow the recommen-dations of “TR26.”

TR26 recommends that the phar-maceutical manufacturer perform aprocess-specific validation which includes:

• establishing an integrity testmethodology and demonstratingintegrity of the sterilizing filter• performing bacterial retentionstudies• having a correlation between bac-terial retention and the integrity testmethod • verifying chemical compatibility • performing extractables testing• evaluating the effects of steriliza-tion on filter integrity.

Summary and conclusionThe validation report summarizesthe protocols, results, and interpre-tations of all testing performed. Italso provides parameters and proto-cols for performing steam or auto-clave sterilization and integrity test-ing. The report is an importantdocumentation of the validationprocess. It should be read and un-derstood by all end users involved invalidating the specific sterile filtra-tion process. The report also servesas an important reference documentin an FDA inspection.

While filter manufacturers per-form much of the qualification andvalidation work in sterile filtrationvalidation, it is crucial for the phar-maceutical processor to remember

that the pharmaceutical or biophar-maceutical company bears completeresponsibility for:

• filter validation• use of the validated sterile filtra-tion system in compliance with cur-rent good manufacturing processesand the recommendations of thefilter manufacturer as found in thevalidation report and supportingdocumentation.Pharmaceutical users of sterile fil-

tration are urged to read PDA’sTechnical Report No. 26, to followits guidelines, and to work closelywith their filtration manufacturer in resolving any questions or issuesrelating to sterile filtration and itsvalidation.

References1. PDA Technical Report #26, “Sterilizing

Filtration of Liquids,” J.Pharm. Sci. andTechnol. 52 (1 Supp) (1998).

2. T.H. Meltzer, ed., Filtration in the Phar-maceutical Industry (Marcel Dekker, Inc.,New York, NY, 1987).

3. American Society for Testing and Ma-terials (ASTM), Standard Test Methodfor Determining Bacterial Retention ofMembrane Filters Utilized for Liquid Fil-tration, ASTM Standard F838-83,(ASTM, Philadelphia, PA, 1983).

4. P. Stinavage,“Perspective on Filtration”presented at PDA, Washington DC, 3December 1999. PT

Validation of Sterile Filtration article

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