aseptic lecture

8/13/2019 Aseptic Lecture

http://slidepdf.com/reader/full/aseptic-lecture 1/133

Aseptic Processing

The Community College of Baltimore County – Continuing Education & Economic Development

&The Northeast Biomanufacturing Center & Collaborative (NBC 2 )

Tom Burkett, [email protected]



Schedule – Friday, October 26 th 7:30 – 9:30

Introduction to aseptic processing, contamination:type and sources, environmental monitoring

9:45-11:45Aseptic Processing vs. Terminal Sterilization

11:45-12:15 Lunch

12:15-2:15Maintaining an Aseptic Environment, Filter sterilization,Media fills, sterility testing, principals of sanitary design



What is AsepticProcessing?



Asepsis- ― A state of control attained by using anaseptic work area and performing activities in amanner that precludes microbiological contaminationof the exposed sterile product‖

Guidance for industry: Sterile Drug Products Produced byAseptic Processing-Current Good Manufacturing Practice.FDA, September 2004



Asepsis is the practice to reduce or eliminatecontaminants (such as bacteria , viruses , fungi ,and parasites ) from entering the operative field

in surgery or medicine to prevent infection .Ideally, a field is "sterile" — free ofcontaminants — a situation that is difficult toattain. However, the goal is elimination of

infection, not sterility.http://en.wikipedia.org/wiki/Asepsis

http://en.wikipedia.org/wiki/Bacteria

http://en.wikipedia.org/wiki/Virus

http://en.wikipedia.org/wiki/Fungi

http://en.wikipedia.org/wiki/Parasites

http://en.wikipedia.org/wiki/Surgery

http://en.wikipedia.org/wiki/Medicine

http://en.wikipedia.org/wiki/Infection


http://en.wikipedia.org/wiki/Medicine

http://en.wikipedia.org/wiki/Surgery

http://en.wikipedia.org/wiki/Parasites

http://en.wikipedia.org/wiki/Fungi

http://en.wikipedia.org/wiki/Virus

http://en.wikipedia.org/wiki/Bacteria



Aseptic Processing is the processing ofdrug components ( drug product,containers, excipients, etc.) in a mannerthat precludes microbiologicalcontamination of the final sealed product.



“Ster i le d rug m anu fac tu re r s s ho u ldhave a keen aw areness of th e pub l icheal th imp l icat ions o f d is t r ibut ing ano ns te r il e p rod uc t . Poo r CGMP

cond i t i ons a t a m anu fac tu r ing fac i li tycan u l t imately p os e a l ife-threateningheal th r i sk to a pa tien t .”

FDA Guidance “Sterile Drug Products Produced by Aseptic Processing-CurrentGood Manufacturing Practice” 2004.



Progression of Symptoms

FeverDecreased Blood PressureRapid Breathing and Heart RateSkin LesionsSpontaneous Blood ClottingOrgan FailureDeath

https://reader010.{domain}/reader010/html5/0623/5b2e44b604572/5b2e44ba1d6cf.jpg

“Sepsis is a serious medical condition characterized bya whole-body inflammatory state caused by infection .”

http://en.wikipedia.org/wiki/Sepsis

http://en.wikipedia.org/wiki/Inflammation






Recalls Lack of Sterility Assurance

Lack of Sterility Assurance is the #1 reason for drug recalls in last 5 years Nearly all drugs recalled due to Lack of Sterility Assurancein last 20 years were produced via aseptic processing

Numberof

Recalls

Fiscal Year



Producing sterile drug products

Terminal sterilizationProduct containers are filled andsealed under high-qualityenvironmental conditionsdesigned to minimizecontamination, but not toguarantee sterility.Product in its final container issubject to a sterilization processsuch as heat or irradiation.

Aseptic processingDrug product, container, andclosure are subject to sterilizationseparately, and then broughttogether.Because there is no process tosterilize the product in its finalcontainer, it is critical thatcontainers be filled and sealed inan extremely high – qualityenvironment .



Terminal Sterilization

DrugProduct

Container /Closure

Excipiants

SterilizationProcess

SterileDrug

Product !

Sterilization Process must be compatible with all components !



Processing

Drug

ProductSterilization

Process

Container

Closure

Excipient




SterileClosure

SterileExcipient

AsepticProcessing

Sterile

DrugProduct

SterileContainer

SterileFinal

Product

Can use multiple sterilization processes each optimized for the individual component



Global Scene European Agency for the Evaluationof Medicinal Products (EMEA)

From: Decision Trees for the Selection of Sterilization Methods (10/1999)

Aseptic Processing

“Adjunct” Processing Fo > 8 minutes, and PNSU > 1 in 10 6

Terminal SterilizationFo > 15 minutes



Terms

PNSU - Probability of a Non-Sterile UnitThe probability of a unit (product container) being non-sterile after the application of a lethal agent.

PNSU of 1 in 106

-- the probability that a unit is non-sterile is one in a million

FO - Sterilization Process Equivalent TimeThe equivalent number of minutes at 121.1

°

C delivered

to a unit by a sterilization process.F O = 8 minutes -- the cycle delivered a microbiallethality equivalent to 8 minutes at 121.1

°

C



Probability of aNon-Sterile Unit (PNSU)

Aseptic ProcessingImpossible to scientifically determine a PNSU*Many critical systems involved

Processing roomEquipmentPersonnel

―Contamination Rate‖ assessed with media fill. Simulated production run with media that promotes growth of microbialorganisms.

* PNSU - Probability of a Non-Sterile Unit: The probability of a unit (product container) being non-sterile after the application of a lethal agent. PNSU of 1 in 10 6 -- the probability that a unit is non-sterile is one in a million



The four pillars of arobust * aseptic process

Personnel training & monitoringEnvironmental monitoringFacilities design & HVAC validation

Process simulation (media fills)



Personnel Training & Monitoring

Avoiding contamination means knowing the potential sources of contamination

PersonnelEquipment

Air/liquidsDrug productContainers/closuresOutside environment

Anything Brought in contact with, or in the vicinity of, the product is a potential source of contamination!



Types of Contamination



Particles of dust, fibers, or other material aresuspended in the air and may contaminate

product. These particles may, or may not, containliving organisms (bacteria and their spores).

The more particles in the air surrounding the productthe more likely the product will be contaminatedwith those particles.

Standards for particulate contamination were initially developed by NASAfor moon exploration, those same standards were later adopted by the

pharmaceutical and semiconductor industry.

Viable & Nonviable particles



Sneezing produces 100’s ofthousands of aerosol dropletsthat can then attach to dust

particles. In the absence of anyfiltration system these particleswhich may contain bacterialspores, or viruses may bepresent in the air for weeks.



Humans and bacteriaOver 200 different species of bacteria are found associatedwith humans.Bacteria are found in the intestines, eyes, nares, mouth, hairand skin.Dry skin can have 1000’s of microbes / mm 2 !

Staphylococcus epidermidis Scanning EM. CDC.



Progression of Symptoms

FeverDecreased Blood PressureRapid Breathing and Heart RateSkin LesionsSpontaneous Blood ClottingOrgan FailureDeath

https://reader010.{domain}/reader010/html5/0623/5b2e44b604572/5b2e44c051e9d.jpg

“Sepsis is a serious medical condition characterized bya whole-body inflammatory state caused by infection .”

http://en.wikipedia.org/wiki/Sepsis







Endotoxins



https://reader010.{domain}/reader010/html5/0623/5b2e44b6

Endotoxin: a pyrogenic (fever inducing) substance (e.g. lipopolysaccharide)present in the bacterial cell wall. Endotoxin reactions range from fever to death.

Extremely heat stable – recommended conditions for inactivation are 180 0 C for 3 hours.



Endotoxin MACROPHAGE

Tumor Necrosis Factor

Interleukins (1,6,8)

Endotoxin effects



What are SPORES

Why are they a MAJOR CHALLENGE!!!!

http://www.samedanltd.com/members/archives/PMPS/Spring2003/graphics/f1_p12.gif http://micro.med.harvard.edu/faculty/rudner.html

Heat alone will not inactivate spores!



Viral Contamination

Viruses are small(nm) non-livingentities that ―hijack‖the machinery of ahost cell

https://reader010.{domain}/reader010/html5/0623/5b2e44b604572/5b2e44c39d10f.jpg

http://www.scq.ubc.ca/.../2006/08/viralreplication.jpg

http://www.scq.ubc.ca/.../2006/08/viralreplication.jpg



Sources of Contamination



http://www.rit.edu/

http://www.imi.org.uk/

Would you let these people into your processing area ?



Sick people aren’t the onlysource of contamination!

"The skin is home to a virtual zoo of bacteria "Martin J. Blaser New York University Medical Center



If people are a major source ofcontamination how do we

avoid contaminating the product while we process it?

www.cellgenix.com/rundgang/pix/rg_7b.jpg



1 st step – eliminate the

source of contamination !―A Well designed, maintained, and operatedaseptic process minimizes personnel

intervention . As operator activities increase inan aseptic processing operation, the risk tofinished product sterility also increases.‖

FDA Guidance “Sterile Drug Products Produced by Aseptic Processing- Current Good Manufacturing Practice” 2004.



Gowning

http://www.coleparmer.com/techinfo/techinfo.asp?htmlfile=CleanroomGarments.htm&ID=63



http://www.engenderhealth.org/IP/surgical/sum4.html

Personnel : GlovesWhen are gloves worn?

What compromises gloves?How often should gloves be examined and sanitized?



Qualifying Personnel

Assess after gowning/glovingMicrobiological surface sampling of severallocations

Glove fingersFacemaskForearmChestPeriodic requalification is necessary



Gowning QualificationWritten ( photographic) procedures describingmethods used to don each gown component in anaseptic manner and the creation of barriers by

overlapping gown components.

Initial training and periodic assessment. Annualrequalification in the case of automated operationsand environmental control.



Environmental

Monitoring: Surface MonitoringTouch or Contact plates

- RODAC Plates

http://www.blood.co.uk/hospitals/services/Micro/Bact2.htm

Swabs

www.esa.int

http://images.google.com/imgres?imgurl=http://www.esa.int/images/sample_swabs400.jpg&imgrefurl=http://www.esa.int/SPECIALS/Delta_Mission/SEMFEK1PGQD_1.html&h=400&w=394&sz=21&tbnid=fiM1sjDZnlLmGM:&tbnh=120&tbnw=118&hl=en&start=1&prev=/images%3Fq%3Dmicrobiology%2Bswabs%26svnum%3D10%26hl%3Den%26lr%3D



Particles >= 0.3µm emitted per minute !

PersonnelActivity

SnapSmock

MembraneCoverall

No Movement 100,000 10Light Movement 500,000 50

Heavy Movement 1,000,000 100

Change Position 2,500,000 250

Slow Walk 5,000,000 500

Personnel: Behavior

Austin Contamination IndexSource: Encyclopedia of Clean Rooms, Bio-Cleanrooms and Aseptic Areas, Dr. Philip Austin, PE, 2000

Minimize movement: Work slowly and purposefully

Note: Light /heavy mo vement re fer to par t ia l body mo vements (mot ionin g wi th arm, tappingtoes , etc . ). Change of pos i t ion refers to wh ole body mo t ion (s tanding u p , s i t t ing do wn, e tc .).



Aseptic TechniqueContact sterile materials only with sterile instruments:

Sterile instruments should be held under Class 100conditions between uses and placed in sterile

containersOperators should not contact sterile products,containers, closures, or critical surfaces with any partof their gown or gloves



CORRECT



Aseptic Technique

Keep the entire body out of the path ofunidirectional airflow

Unidirectional airflow design is used to protect sterile equipment surfaces, container-closures, and product. Disruption of the pathof unidirectional flow in the critical area can

pose a risk to product sterility.



Horizontal airflow

Vertical airflowwww.ors.od.nih.gov/ds/pubs/bsc/graphics/fig3.gif

Unidirectional airflowThe operator should

never come between theair source and the product.



Aseptic Technique

Approach a necessary manipulation in amanner that does not compromise sterility ofthe product

Proper aseptic manipulations should beapproached from the side and not above the

product (in vertical unidirectional flowoperations).Operators should refrain from speaking when indirect proximity to the critical area.



What’s wrong with this picture?



CORRECT



What’s wrong with this picture?



CORRECT



Personnel: Hygiene

Avoid cleanrooms when illFrequent bathing and shampooing

Avoid getting sunburnedAvoid cosmetics such as face powder, hair sprays,

perfumes and aftershaveClothing should be clean, nonfrayed and nonlinting

Avoid smoking



FOREHEAD



ISOPROPYL ALCOHOL (70%)

Powerful disinfectant and antiseptic

Mode of action: denatures proteins,dissolves lipids and can lead to cellmembrane disintegration

Effectively kills bacteria and fungiBut does not inactivate spores!



Disinfection efficacy

Suitability, efficacy & limitations ofdisinfectant agents and procedures should

be assessed.The disinfection program should includethe use of a sporicidal agent usedaccording to a written schedule and when

environmental data suggests presence ofspore forming agents ( Baccilus spp.).



EndosporesMycobacteriaFungal Spores

Small Non-enveloped viruses (polio, rotavirus, rabies)Vegetative Fungal Cells

Enveloped Viruses (Herpes, Hepatitis B, Hepatitis C, HIV)Vegetative Bacteria

Most Resistant

Least Resistant



Sporicidalagents

GlutaraldehydeFormaldehydeOther aldehydesChlorine-releasing agentsIodine and iodophorsPeroxygensEthylene oxideP-Propiolactone

A. D. RUSSELL, 1999. Bacterial Spores and Chemical Sporicidal Agents.CLINICAL MICROBIOLOGY REVIEWS Vol. 3, No. 2 p. 99-119 .



Equipment and bacteria

Even seemingly smoothsurfaces can harbor

bacteria !

Scanning electron micrograph of Listeria monocytogenes forming a biofilmin soy on a stainless steel chip. Courtesy of Professor Amy Wong.

http://www.rit.edu/~jadsbi/asmbiofilm/listeria.html

http://www.rit.edu/~jadsbi/asmbiofilm/listeria.html



Critical Surfaces

Critical surfaces – ―Surfaces that may come incontact with or directly affect a sterilized

product or its containers or closures. Criticalsurfaces are rendered sterile prior to the startof the manufacturing operation, and sterility ismaintained throughout processing.‖

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice, FDA, September 2004



Isolators

Advantage:No direct contact betweenoperator & product

Critical that meticulous

aseptic practices befollowed including the useof sterile tools formanipulations



Isolators

Gloves, half suits, seals,gaskets and transfersystems should be

covered by P.M. programJustified replacementfrequency



Vertical airflow

www.ors.od.nih.gov/ds/pubs/bsc/graphics/fig3.gif

Open Isolators

Require laminar airflow over criticalareas

Use pressure differential to insureseparation of critical area from

external environment (17.5-50 Pa0.07-0.20 water gauge)

Local protection of opening toguard against turbulent airflow and

pressure waves that couldcompromise critical area



Isolator Decontamination

Vaporized agents often usedGlutaraldehyde, Formaldehyde, etcUse indicator organisms to demonstrate effectiveness ofdecontamination

Should be able to achieve a 4-6 log reduction in titerBiological indicator should be placed in multiple, justifiedlocations throughout the isolatorHard to reach areas (between fingers on gloves) should beaddressed

Must show that defined concentration of decontamination agent isuniformly reached in validation studies



ENVIRONMENTALMONITORING

―In aseptic processing, one of the most

important laboratory controls is theenvironmental monitoring program‖

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice, FDA, September 2004

A i



AsepticProcessing

The key is a ―high quality environment‖



1.

2.

3.4.

5.

6.7.

8.

9.10.

11.

13.

12.

Environmental Monitoring

Critical (processing) areas

Where do we sample?



1.

2.

3.4.

5.

6.7.

8.

9.10.

11.

13.

12.

Environmental Monitoring

Critical (processing) areas

Where do we sample?

Sampling of adjacent classifiedareas (aseptic corridors, gowning

rooms, etc) will provide trenddata and may help identifysources of contamination.



Items to include in environmental

monitoring SOP’s Frequency of samplingWhen the samples are taken (during or at conclusion ofaseptic operations)Duration of samplingSample size (surface area, air volume)Specific sampling equipment and techniquesAlert and action levelsAppropriate response to deviations from alert or action levels



EnvironmentalMonitoring: Particulate Air Monitoring

Use of remote systems recommended in laminar flow areas

http://www.apcportable.com/



EnvironmentalMonitoring: Surface Monitoring

Touch or Contact plates- RODAC Plates

http://www.blood.co.uk/hospitals/services/Micro/Bact2.htm

Swabs

www.esa.int

http://images.google.com/imgres?imgurl=http://www.esa.int/images/sample_swabs400.jpg&imgrefurl=http://www.esa.int/SPECIALS/Delta_Mission/SEMFEK1PGQD_1.html&h=400&w=394&sz=21&tbnid=fiM1sjDZnlLmGM:&tbnh=120&tbnw=118&hl=en&start=1&prev=/images%3Fq%3Dmicrobiology%2Bswabs%26svnum%3D10%26hl%3Den%26lr%3D



Media must be qualified to supportgrowth of USP indicator organisms

To support growth of bacteria and fungiInoculated with < 100 cfu challengeUpper and lower limits for incubation 20-35 0 C, and

not less than 14 days (if two temps 7 days at eachtemp)



Environmental Monitoring:



Environmental Monitoring:Trending Data

Why is it important?Evaluate the disinfection efficiencyAre certain microbes migrating into the aseptic

processing area from a lesser controller area?Long and short termData by:

RoomShiftOperatorProductContainer type

Filling lineSamplingTesting personnelIsolate



Environmental Monitoring:Trending Data

Averages of data can be misleading and maskunacceptable localized conditions.Alert and action levels should be set for eachsample siteIndividual sample results should be evaluatedagainst the action and alert levels

Environmental Monitoring:



Microbial identification shouldextend to the species level.Routine identification can bedone using traditional phenotypicand biochemical techniques.Genotypic techniques aresuggested for failureinvestigations

Environmental Monitoring:Identification



Gram Stain

QC Micro: Identifying Microbes



http://www.arches.uga.edu/~kristenc/cellwall.html

Environmental Monitoring:Identification



Metabolic Based Assays

Vitek





QC Micro: Identifying MicrobesMetabolic Based Assays



Staphylococcus xylosus

Reduction ofTetrazolium Violet


Metabolic Based Assays

QC Mi Id if i Mi b



QC Micro: Identifying MicrobesMetabolic Based Assays



Genotypic Methods

Use DNA sequence (often ribosomal RNAgenes rDNA) to identify organismFaster, and more accurate then traditional

biochemical and phenotypic techniques



QC Micro: Identifying MicrobesGenotype Based Assay: MicroSeq



QC Micro: Identifying MicrobesGenotype Based Assay: MicroSeq

PCR: Polymerase Chain Reaction




DNA SEQUENCE DATA

Q y gGenotype Based Assay: MicroSeq



Endotoxin Testing



https://reader010.{domain}/reader010/html5/0623/5b2e44b



Endotoxin Testing



ENDOTOXIN LIMIT FOR WFI IS

0.25EU/ml

QC Micro: LAL Assay(Limulus amebocyte lysate)



https://reader010.{domain}/reader010/html5/0623/5b2e





The Lysate

QC Micro: LAL Assay



QC Micro: LAL Assay

Types of LAL assays: Gel Clot: a clot forms and stays intact at the bottom of theassay tube.

Turbidity: An increase in turbidity (cloudiness) is seen.Chromogenic: a color indicator is used to signal a positiveresult.Endpoint - Measure turbidity/absorbance after a definitive

time period.Kinetic - Measure rate of increased turbidity/absorbance.

l



Facilities

Establishing and Maintaining an aseptic environmentUse clean-rooms of various classes to establish an aseptic areaClean rooms use combinations of filtration, air exchange, and positive

pressure to maintain ―clean‖ environment

Lower quality clean areas should not be placed next to high quality areas



Facility Design: Clean Area Classification

FS209 Cleanroomclassification

ISO 14644-1 Cleanroomclassification ≥ 0.5um particles/m3

Microbiological Active Air Action Levels

(cfu/m3)

Microbiological SettlingPlates Action Levels (diam.

90mm; cfu/4hours)100,000 8 3,520,000 100 5010,000 7 352,000 10 51000 6 35,200 7 3100 5 3,520 1 1



Facilities: General Cleanroom Design

HEPA/ULPA filters on ceilingExhaust vents on floorDrains in aseptic processing areas are inappropriateAirlocks and interlocking doors to control air balance

Seamless and rounded floor to wall junctionsReadily accessible cornersFloors, walls, and ceilings constructed of smooth hard surfaces that can beeasily cleanedLimited equipment, fixtures and personnel

Layout of equipment to optimize comfort and movement of operators



Facilities: HEPA Filters

http://people.deas.harvard.edu/~jones/lab_arch/nano_facilities/hepa.gif

High Efficiency Particulate AirMinimum particle collection efficiency:99.97% for 0.3µm diameter particles.

Disposable

Filter made of pleated borosilicate glass



Class 10,000 cleanroom

http://www.americancleanrooms.com/am/photogallery_08.html

Class 100 cleanroom

Facilities: Cleanroo m Classification

http://www.americancleanrooms.com/am/photogallery.html





Facilities: Cleanroom Classification

FS209Cleanroom

classification

ISO 14644-1Cleanroom

classification≥0.5um

particles/m3

ViableMicrobes(cfu/m3)

Ave AirflowVelocity

(fpm)Air

changes/hr

100,000 8 3,520,000 100 5-10 5-48

10,000 7 352,000 10 10-15 60-90

1000 6 35,200 7 25-40 150-240

100 5 3,520 1 40-80 240-480

Facilities:



Facilities:Pressure Differentials

Used to maintain airflow in the direction of highercleanliness to adjacent less clean areasA minimum of 10-15 Pascals should be maintained

between the aseptic area and an adjacent rooms withdiffering cleanroom classifications (doors open)



http://news.thomasnet.com/images/large/451/451402.jpg

Facilities: Air Lock

Permits the passage of objects andpeople into a cleanroom.

Consists of two airtight doors in series

which do not open simultaneously.

Spray down materials with 70% IPAbefore placing in the airlock

http://en.wikipedia.org/wiki/Hermetic_seal

http://en.wikipedia.org/wiki/Door

http://en.wikipedia.org/wiki/Door

http://en.wikipedia.org/wiki/Hermetic_seal



Fiber-shedding materials such as cardboard and paperCardboard packaging must be removed and items placed into non-cardboard containers.

Wood (i.e. wooden pallets)Undesignated charts

Facilities:

Material NOT permitted in a Cleanroom



Facilities: Cleaning

Water should be changed FREQUENTLY

1. Vacuum all accessible surfaces

3. Mop floors using a lint freepolyester mops attached tostainless steel handles

2. Wipe surfaces with a cleaning solution

Isolators



Isolators

The use of isolators prevents direct contactwith product

However, the use ofisolators can lead torelaxation of aseptic

practices!



Sterile Filtration

Sterilizing grade filters (0.22 μm pore size orsmaller)Use of redundant sterilizing filters should beconsideredFilters should be validated including the use ofmicrobial challenge



Dead End (Perpendicular Flow) Filters

Plate & Frame Depth



Materials

polyvinylidenedifluoride (PVDF)Polytetrafluoroethylene(PTFE)Polyethersulfone (PES)

Nylon 6,6 (N66)Choice depends on properties (i.e. hydrophobic orhydrophilic, and compatibility with processmaterials)

Filter Validation



Filter ValidationFactors that affect filter performance:

Viscosity & surface tension of material to be filtered pHFilter membrane compatability

PressuresFlow ratesTemperatureOsmolality

Hydraulic shock

Fil i i bi l h ll



Filtration microbial challengeShould reflect worst case scenario based on product bioburden

profileUse of Brevundimonas diminuta ( ATCC 19146) commonly used

( Acholeplasma laidlawii (0.10 µm ) and Serratia marcescens (0.45 µm ) also

used ) Small size (0.3 μm) allows testing of sterile filtration units(0.2 μm)Challenge should be at least 10 7 organisms / cm 2 of filtration

area

Fil i i bi l h ll



Filtration microbial challenge

• Direct inoculation of challenge organism intodrug product should be consideredMust consider nature of drug product(bactericidal and oil based formulations maycompromise results)Can simulate by first processing drug productunder worst case conditions – followed by

microbial challenge using drug productlacking bactericidal agent as vehicle



Number of uses

―Sterile filters should be routinely discardedafter the processing of a single lot‖

If repeated use can be justified the validation plan should include the maximum number

of lots to be processed before replacement



Filtration Time Limit

An established maximum time limit to achievefiltration should be established and adhered to.

The established maximum time limit should be based on upstream bioburden and endotoxin load



Integrity TestingFilter integrity is essential to filter performance. Integrity testing can be

performed prior to processing but must be performed after filtration.

Integrity testing methods:Forward FlowBubble point

Based on airflow through a given filter at a specific pressure

MediaFills



Fills

Used to validate the aseptic process

Use microbial growth media instead ofdrug product-any contamination will resultin microbial growth

Media Fill Study design



The design of a media fill study should address the following factors:Any factors associated with longest run that can pose contamination riskRepresentative interventions that occur during a ―normal‖ run as well as anynonroutine interventionsLyophilization (if applicable)Aseptic assembly of equipment

Number of personnel and their activitiesA representative number of aseptic additionsShift change, breaks, gown changesAseptic equipment connections/disconnectionAseptic sample collectionLine speed and configurationWeight checksContainer closure system



Size, duration, and speed of production runs

should be mimicked in media fill studiesWhen aseptic processing employs manual fillingor closing, or extensive manual manipulations,the duration of the process should be generally noless than the length of the actual manufacturing

process Number of units filled during the media fill

should be based on the contamination risk5,000 to 10,000 units a good starting pointIf run size is less than 5,000 units then media fillshould be at least equal the maximum batch size.

M di Fill St d



Media Fill Study

designSize, duration, and speed of production runs should bemimicked in media fill studies

Media fill studies should address the range of line speeds used andeach study should evaluate one speed





Environmental conditionsShould be representative of actual manufacturingoperations

Maximum number of individuals and elevated activitylevels allowed under SOP’s

Media Fill Studyd



design

Media fills should be observed by the QC unitVideo recording should be considered




Media Fill Study design―Modern aseptic processing operations in suitably designed facilities have

demonstrated a capability of meeting contamination levels approaching zero‖

When filling <5,000 units, no contaminated units should be detected. One (1)contaminated unit is considered cause for revalidation, following aninvestigation.

When filling 5,000 to 10,000 units, One (1) contaminated unit should resultin an investigation, including consideration of a repeat media fill. Two (2)contaminated units are considered cause for revalidation, followinginvestigationWhen filling >10,000 units , One (1) contaminated unit should result in aninvestigation. Two (2) contaminated units are considered cause forrevalidation, following investigation



Revalidation

At least semi-annuallyAfter any significant change

Facility and equipment modifications, lineconfiguration changes, significant changes in

personnel, environmental testing anomalies,container closure systems, extended shutdowns,

or end product sterility testing failure, etc.



Change Control

A written change control process should be in placeAny change to the product or production line should beevaluated using this process



Sterility Testing

21 CFR 211.167― For each batch of drug product purporting to be sterile and/or pyrogen-free, thereshall be appropriate laboratory testing to determineconformance to such requirements.‖



Which test method should I use?

―USP <71> ―Sterility Tests” is principalsource of sterility test methods including

procedures and methods‖

Methods validation must address issue offalse negatives!

St ilit T ti



Sterility Testing

Be aware that sterility testing has seriouslimitations due to small sample sizes typicallyused.

Therefore the FDA considers any sterility testfailures to be ―serious‖ CGMP issues that should

be thoroughly investigated.



Sterility Testing

Microbial growth in sterility test is grounds forconsidering entire lot non-sterile.

Only if the growth can unequivocally beassigned to laboratory contamination can testresult be considered invalid.

Sterility Testing Investigations



Sterility Testing Investigations

In the investigation stemming from a negative sterility testconsideration should be given to:Speciation of the organismRecord of laboratory results and deviations

Environmental monitoring of production environmentMonitoring personnelProduct Presterilization bioburdenProduction record reviewManufacturing history



Sterility Testing

Any isolates from sterility testing should be identified tospecies level.

The use of genotyping techniques is encouraged.

Identical methodologies should be employed in speciesidentification in sterility test and environmental

monitoring program.

Thank You



Thank You

For further information see:Guidance for Sterile Drug Products Produced byAseptic Processing

http://www.fda.gov/cber/gdlns/steraseptic.pdf

Guideline for Validation of Limulus AmebocyteLysate Test as an End Product Endotoxin Test forHuman and Animal Parenteral Drugs, BiologicalProducts, and Medical Devices

http://www.fda.gov/cber/gdlns/lal.pdf





Guidance for submission of Documentation forSterilization Process Validation in applications for Humanand Veterinary Drug Products

http://www.fda.gov/cder/guidance/cmc2.pdf Guide to Inspections of Microbiological PharmaceuticalQuality Control Laboratories

http://www.fda.gov/ora/Inspect_ref/igs/micro.html Guide to Inspections of Sterile Drug SubstanceManufacturers

http://www.fda.gov/ora/inspect_ref/igs/subst.html

http://www.fda.gov/cder/guidance/cmc2.pdf

http://www.fda.gov/ora/Inspect_ref/igs/micro.html



http://www.fda.gov/ora/Inspect_ref/igs/micro.html

http://www.fda.gov/cder/guidance/cmc2.pdf

aseptic lecture

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