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DECEMBER 2008

VALIDATION OF ASEPTIC PROCESSES

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VALIDATION OF ASEPTIC PROCESSES DECEMBER 2008

HEALTH SCIENCES AUTHORITY HEALTH PRODUCTS REGULATION GROUP Page 2 of 23

GUIDE-MQA-009-007

1. INTRODUCTION

1.1 PURPOSE

The aim of this document is to provide guidance to the currentpractice in this field by giving recommendations for the validation

of aseptic processes.

1.2 SCOPE

1.2.1 This document applies to all manufacturers involved inaseptic processing of finished dosage forms (human andveterinary) as well as manufacturers of sterile labeledbulk drug substances (active pharmaceutical ingredients).

1.2.2 At the time of issue this document reflected the currentstate of the art. It is not intended to be a barrier totechnical innovation or the pursuit of excellence.However, the Manufacturing & Quality Audit Division ofthe Health Products Regulation Group, Health SciencesAuthority, should always be referred to when determiningthe extent to which the provisions laid down in thisdocument are binding.

1.3 GENERAL INFORMATION

1.3.1 The basic principles and application of process

validation are described in the Manufacturing & QualityAudit Divisions Document GUIDE-MQA-005, 006, 007and 008 (Guidance Notes on Validation Master Plan,Installation and Operational Qualification, Non-SterileProcess Validation, Cleaning Validation Principles ofQualification and Validation in PharmaceuticalManufacture) and apply also to aseptic processing.

1.3.2 Validation of aseptic processes relies upon prospective,concurrent and retrospective as well as re-validation.

1.3.3 Prospective studies would include the installation andoperational qualifications for a new or renovated facilityas well as product simulation studies and a prospectiveprocess validation with the original product according tothe Manufacturing & Quality Audit Divisions DocumentGUIDE-MQA-006 & 007.

1.3.4 Concurrent validationincludes a process validation withthe same requirements as for prospective studies, butperformed during routine production on qualifiedequipment.

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1.3.5 Retrospective validation uses the data of earlier

manufactures, but is not a recommended technique foraseptic processes.

1.3.6 Re-validationincludes:

Regular performance of process simulation studies Monitoring of environment, disinfection procedures,

equipment cleaning and sterilization (includingcontainers and closures)

Routine maintenance and re-qualification ofequipment, e.g. autoclaves, ovens, HVAC (heating,ventilation and air conditioning) systems, watersystems, etc.

Regular integrity testing of product filters, containers,closures and vent filters

Re-validation after changes

1.3.7 It is the sum total of all validation data that provides thenecessary level of assurance for aseptically producedproducts.

1.3.8 Process simulation studies (media fills) simulate thewhole process in order to evaluate the sterilityconfidence of the process. Process simulation studiesinclude formulation (compounding), filtration and filling

with suitable media in order to evaluate the sterilityconfidence of the process. Simulations are made toensure that the regular process for commercial batchesrepeatedly and reliably produces the finished product ofthe required quality. However, each process simulationtrial is unique and so it is not possible to extrapolatethese results directly to actual production contaminationrates.

1.3.9 The methods for simulating an aseptic process varyaccording to the process used for the various types of

products, i.e. liquid, semi-liquid and solid dosage forms.

1.3.10 In these Recommendations the term should indicatesrequirements that are expected to apply unless shownto be inapplicable or replaced by an alternativedemonstrated to provide at least an equivalent level ofquality assurance.

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2. DEFINITIONS

Action level: Established criteria, e.g. microbial or particulate levels,requiring immediate follow-up and corrective action if exceeded.

Alert limits (environmental monitoring): Established microbial or

particulate levels giving early warning of potential drift from normaloperating conditions which are not necessarily grounds for definitivecorrective action but which require follow-up investigation.

Alert limits (media fill): Established levels or numbers of positivemedia filled units, the cause of which should be investigated, but whichare not necessarily grounds for definitive corrective action.

Aseptic filling: Operation whereby the product is sterilized separately,then filled and packaged using sterilized containers and closures incritical processing zones.

Bioburden: Total number of viable microorganisms on or inpharmaceutical product prior to sterilization.

Compounding: A process wherein bulk drug substance is combinedwith another bulk drug substance and/or one or more excipients toproduce a drug product.

Environmental monitoring programme: Defined documentedprogramme, which describes the routine particulate and microbiological

monitoring of processing and manufacturing areas, and includes acorrective action plan when action levels are exceeded.

Growth promotion test: Test performed to demonstrate that mediawill support microbial growth.

High efficiency particulate air (HEPA) filter: Retentive matrixdesigned to remove a defined percentage of particulate matter of adefined size.

HVAC:Heating, ventilation and air conditioning.

Integrity test:Test to determine the functional performance of a filtersystem.

Media fills:Method of evaluating an aseptic process using a microbialgrowth medium. (Media fills are understood to be synonymous tosimulated product fills, broth trials, broth fills etc.).

Sampling frequency:Established period for collecting samples.

Shift: Scheduled periods of work or production, usually less than 12

hours in length, staffed by alternating groups of workers.

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Sterile: Free of any viable organisms. (In practice, no such absolutestatement regarding the absence of microorganisms can be proven,see sterilization.)

Sterilization:Validated process used to render a product free of viableorganisms. Note: In a sterilization process, the nature of

microbiological death of reduction is described by an exponentialfunction. Therefore, the number of microorganisms, which survive asterilization process, can be expressed in terms of probability. Whilethe probability may be reduced to a very low number, it can never bereduced to zero.

Sterility assurance level (SAL):Probability that a batch of product issterile. (SAL is expressed as 10 -n).

Sterility test: Test performed to determine if viable microorganismsare present.

Vent filter:Non-shedding porous material capable of removing viableand non-viable particles from gases passing in and out of a closedvessel.

3. PROCESS SIMULATION TEST PROCEDURES

3.1 GENERAL COMMENTS

3.1.1 The media fill should emulate the regular product fill

situation in terms of equipment, processes, personnelinvolved and time taken for filling as well as for holding.

3.1.2 Where filling takes place over extended periods, i.e.longer than 24 hours, the process simulation test shouldextend over the whole of the standard filling period. Inorder to prevent excessively high numbers of unitsbeing filled it is usually acceptable to just run themachine for a reasonable time, if the validity of thesimulation is not diminished by this procedure.

3.1.3 It should be considered that inert gases would preventthe growth of aerobic microorganisms. Therefore forprocess simulations, sterile filtered air should be usedinstead of inert gases, and also for breaking a vacuum.Where anaerobes are detected in the environmentalmonitoring or sterility testing, the use of an inert gasshould be considered for a process simulation, as inertgas supports the growth of anaerobes.

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3.1.4 Before enumerating the different process simulation test

procedures some preliminary explanations arenecessary for the preparation of liquid media as it isused for the majority of the process simulation tests.Where a liquid nutrient medium is used it should beprepared in a similar manner to the product. The

medium should be dissolved in Water for Injection in astandard manufacturing vessel. If heat is required todissolve it then only minimal heat should be used. ThepH of the medium should be measured and, ifnecessary, adjusted to bring it into the required range.

3.1.5 The medium should be aseptically filtered into anaseptic receiving holding vessel using the normalproduction filter and processing procedure. In justifiedcases it may be also acceptable to sterilize the media.All aseptic holding vessels should be covered by aprocess simulation test on a regular basis unless avalidated, pressure hold or vacuum hold test is routinelyperformed.

3.1.6 The following chapter illustrates the test procedures forthe various simulation tests for aseptically producedsolutions, lyophiles, suspensions, ointments andpowders and summarizes the considerations to bemade.

3.2 LIQUID PRODUCTS

3.2.1 Vial Products

The liquid growth medium for the simulation test isprepared as above and kept in a sterile holding vesselfor the maximum permitted holding time before startingthe simulation test. If the bulk solution is stored underrefrigerated conditions during the holding time then thisshould also be performed for the medium. Vials andclosures should be prepared as in regular production.

3.2.2 Sterile Products in Plastic Containers

Ear and eye drops are typically marketed in plasticcontainers. Containers, inserts, closures and whereapplicable, overseals are washed and sterilized as inregular production. Instead of sterilization with heat,irradiation or ethylene oxide are used.

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Whilst clear plastic containers are frequently used forprocess simulation trials, the plastic is usually slightlyopaque and thus hinders identification of contaminatedunits that show only a slight haze. In such caseexamination under natural or room lighting would notsuffice. Where opaque containers are used for process

simulation trials the whole contents should be removedfor examination.

3.2.3 Ampoule Products

Open or closed ampoule types may be used. Theyshould be sterilized by dry heat and afterwards used inthe simulation test as per the regular production run.

Ampoules should be prepared as in regular production.

3.3 INJECTABLE POWDER PRODUCTS

There are two possibilities for simulation of this process. It maybe done either by filling a sterilized liquid growth medium intothe sterile container or adding a powder (inert or growthmedium) before or after a sterile diluent (WFI or growthmedium). Inert materials commonly used include: polyethyleneglycol 8000 and carboxymethyl cellulose. These materials areusually sterilized by irradiation.

3.4 SUSPENSION PRODUCTS

This procedure is comparable to the filling of liquid products,except for the process step of maintaining suspension of theingredients. The stirring or recirculation should be part of thesimulation. If aseptic additions are made to the bulk solutionthese should be simulated by the use of inert sterileliquids/powders.

3.5 FREEZE DRIED (LYOPHILIZED) PRODUCTS

3.5.1 Crystallization of the medium should be preventedbecause it may reduce the likelihood of recovery oforganisms.

3.5.2 Two simulation methods are commonly used. In the firstone a dilute medium is subject to a cycle that removeswater until a determined medium strength is obtained,but is not subject to freezing. The second method usesfull strength medium and requires only a partial vacuumbe drawn whilst the chamber be kept at ambienttemperature. There is a risk that the medium may boil

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over and contaminate the chamber unless conditions aretightly controlled. The absence of boiling under thedefined cycle conditions should be confirmed.

3.6 SEMI-SOLID PRODUCTS (e.g. sterile ointments)

For this simulation test the liquid growth medium is thickened tothe appropriate viscosity, used as in the routine productionprocedure. Suitable thickening agents are agar andcarboxymethyl cellulose. Other agents would need to bevalidated with regard to lack of their bacteriostatic andfungistatic properties. Metal and plastic ointment tubes preventthe examination of the medium in-situ. Usually the whole contentof the tube should be examined and this is usually achieved bysqueezing the contents into a plate (petri dish), and afterwhirling it is examined for turbidity and fungal colonies under

defined light conditions or by performing a sterility test. Ifproperly validated, an alternative method for detection ofcontamination of semi-solid products could be the use of media,which changes color in the presence of contamination.

3.7 CLINICAL TRIAL MATERIALS AND SMALL BATCH SIZEPRODUCTS

As processes for smaller quantities (less than 3000 units) do notallow an interpretation according to chapter 4 of this guidancenote, any presence of microbial contamination should beregarded as an alert limit. The simulation of such processes forsmaller quantities (less than 3000 units) necessitates a differentinterpretation of the test results (Chapter 4), which are notsufficient as compared to a normal batch size. Monitoring andtest conditions, like incubation or media selection remain thesame as for commercial production runs.

The size of media fills for small batch size products should atleast equal the number of containers filled for the commercialproduct.

3.8 BIOLOGICAL AND BIOTECHNOLOGY PRODUCTS

The manufacture of these products vary, such that there is notone single process. It may be more practical to validate thevarious segments of the process individually. The frequency ofthe revalidation should relate to the one of regular, commercialproduction.

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3.9 STERILE BULK PHARMACEUTICALS

3.9.1 Whenever possible a growth medium should be usedand the process should be simulated as closely aspossible to the normal route of manufacturing the sterilebulk drug substance.

3.9.2 The aseptic manufacture of a sterile bulkpharmaceutical chemical drug substance is a difficultprocess, which may have numerous individualsegments that need to be validated. The possibility ofmicrobial ingress into the system has to be consideredafter each step of the routine production.

3.9.3 The validation may include segments, where the use ofgrowth media is not feasible.

4. PROCESS SIMULATION TEST CONDITIONS

4.1 TEST PERFORMANCE

4.1.1 The process simulation test should follow as closely aspossible the routine aseptic manufacturing process andinclude all critical subsequent manufacturing steps. Allequipment should remain the same whereverpracticable as for the routine process. Appropriatecombinations of container size and opening as well as

speed of the processing line should be used (preferablyat the extremes).

4.1.2 The process simulation test should represent a worstcase situation and include all manipulations andinterventions likely to be represented during a shift.

4.1.3 Worst-case conditions are often thought to be thelargest container with the widest mouth as it is exposedlonger to the environment. However, there areexceptions to this and one of them is small ampoules

run at the highest speed as the ampoules may beunstable and cause frequent jams thus necessitatingfrequent operator intervention.

4.1.4 The fill volume of the containers need not be filled to thenormal volume but should be sufficient to enablecontact of all the container-closure seal surfaces whenthe container is inverted and also sufficient to allow thedetection of microbial growth.

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4.1.5 If batches smaller than 3000 units are produced, the

minimum number of containers used for the processsimulation should be equal to that of the commercialbatch size.

4.1.6 Simulation tests should be performed on different days

and hours during the week and not only at thebeginning of a workday.

4.1.7 If the same process is conducted in a separate cleanroom, this should also be validated.

4.1.8 In order to find the possible source of contamination itmay be a good advise to videotape the aseptic fill andalso number the individual vials or segregate vials inchronological order during incubation.

4.2 SELECTION OF GROWTH MEDIUM

4.2.1 The criteria for the selection of growth medium include:low selectivity, clarity, medium concentration andfilterability.

4.2.2 Ability to support growth of a wide range ofmicroorganisms: The medium should have a lowselectivity, i.e. be capable of supporting growth of awide range of microorganisms such as Bacillus subtilis,

Staphylococcus aureus, Candida albicans, Aspergillusnigerand Clostridium sporogenes(e.g. Soybean CaseinDigest).

4.2.3 The selection of the medium has to be based also onthe in house flora (e.g. isolates from monitoring etc.).

4.2.4 Growth promotion tests should demonstrate that themedium supports recovery and growth of low numbersof microorganisms, i.e. 10-100 CFU/unit or less.

4.2.5 Growth promotion testing of the media used insimulation studies should be carried out on completionof the incubation period to demonstrate the ability of themedia to sustain growth if contamination is present.Growth should be demonstrated within 5 days at thesame incubation temperature as used during thesimulation test performance.

4.2.6 Clarity:The medium should be clear to allow for ease inobserving turbidity.

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4.2.7 Medium Concentration: Recommendations of the

supplier should be followed unless alternativeconcentrations are validated to deliver equal results.

4.2.8 Filterability: If a filter is used in the asepticmanufacturing process, the medium should be capable

of being filtered through the same grade as used inproduction.

4.3 INCUBATION CONDITIONS

4.3.1 It is generally accepted to incubate at 20-25C for aminimum of 14 days without having collected data tosupport this incubation schedule. It is similarlyacceptable for firms who prefer a two-temperatureincubation schedule to incubate at 20-25C for aminimum of 7 days followed immediately by incubationat a higher temperature range not to exceed 35C for atotal minimum incubation time of 14 days. Otherincubation schedules should be based on supportingdata.

4.3.2 Prior to incubation the containers with themicrobiological growth medium should be inverted orotherwise manipulated to ensure that all surfaces,included the internal surface of the closure, arethoroughly wetted by the medium. The containers

should not be completely filled with medium in order toprovide sufficient oxygen for the growth of obligateaerobes.

4.3.3 The microorganisms present in the containers of thesimulation test should be identified to genus butpreferably species level to aid determination of thepossible sources of the contamination.

4.4 READING OF THE TEST

When inspecting the containers they should be compared to aknown sterile container for comparison as some microbialgrowth shows up as a faint haze, which is difficult to detectunless there is a control container to compare against.Personnel should be trained for this task.

4.5 TEST FREQUENCY

4.5.1 The manufacturer, based on his individualcircumstance, should ultimately decide if more of morefrequent tests are required than requested by regulatory

authorities in this chapter.

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4.5.2 It should be distinguished between start-up and on-

going simulation tests.

4.5.3 A start-up simulation testconsists of three consecutivesatisfactory simulation tests per shift and should becarried out before routine manufacturing can start.

4.5.4 Start-upsimulation tests are performed, for example,for new processes, new equipment or after criticalchanges of processes, equipment or environment.

4.5.5 An on-going simulation test consists of onesatisfactory simulation test per shift and is mainlyperformed for the periodic monitoring of asepticconditions during routine manufacturing but also, forexample, after less critical changes of processes,equipment or environment or if processing lines standidle for more than 6 months.

4.5.6 On-going simulation tests should be performed witheach shift of each process line be tested at least twiceper year under the condition that there were no changesin the normal production procedures and no action limitswere exceeded.

4.5.7 Exceeding an action level demands a re-validation.Depending on the result of the follow-up investigation

this re-validation may require the inclusion of one tothree satisfactory process simulation tests dependingupon the results.

5. INTERPRETATION OF DATA

5.1 After the incubation period of the media-filled containers, theyare visually examined for microbial growth. Contaminatedcontainers should be examined for evidence of container/closuredamage, which might compromise the integrity of the packagingsystem. Damaged containers should not be included as failures

(positives) when evaluating results.

5.2 Different approaches may be used to determine limits andacceptance criteria.

5.3 One method (Method 1) is to determine a contamination rate asan absolute value (e.g. 0.1%) with guidance on the minimumnumber of units filled, and the other method (Method 2) is to usea statistical method based on the Poisson distribution ofcontaminated filled units. However the application of Method 2guarantees a higher safety level and should therefore be applied

by manufacturers.

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5.4 Ideally the contamination rate should be zero. However currently

the accepted contamination rate should be less than 0.1% with a95% confidence limit level according to the Annex I to theEU/PIC-PIC/S Guide to GMP. In order to calculate the worstcase contamination rate for an observed frequency of failures,the following table can be used. The number indicated as the

upper 95% confidence limit describes the maximum number offailures that can be expected with a 95% certainty in the truepopulation for an observed number of failures.

Table 1

Relation between observed number of failures and upper 95%confidence limit

Observednumber of

failures0 1 2 3 4 5 6 7 8 9 10

Upper 95%Confidence

Limit3 4.74 6.3 7.75 9.15 10.51 11.84 13.15 14.43 15.71 16.96

[Ref.: The Use of Simulation Tests in the Evaluation of Processes for theManufacture of Sterile Products, Parenteral Society UK, 1993]

5.5 The maximum contamination rate that can be expected with a95% certainty for an observed frequency of failures can becalculated according to the following formula:

Contamination rate = Upper 95% confidence limit / Number of filled units x 100%

5.6 Example 1: If 3,000 units were filled and two contaminated unitsobserved, the upper 95% confidence limit for the contaminationrate would be not more than 6.3/3,000 x 100% = 0.21%. Thisrate would therefore be higher than the required value (less than0.1%) and therefore be unacceptable.

5.7 Example 2: If 3,000 units were filled and no contaminated unitobserved, the upper 95% confidence limit for the contaminationrate would be not more than 3/3'000 x 100% = 0.1%. Since 3 is

a rounded value and the true value is slightly smaller than 3, thisrate would be smaller than the required value (less than 0.1%)and therefore be acceptable.

5.8 This means on one side that the minimum number of containersto be filled during a process simulation test performed afterMethod 2 is 3,000 units, and on the other side that there shouldbe no contaminated container in case of filling of the minimalnumber of 3,000 units.

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5.9 It is the responsibility of every manufacturer to ensure that a

statistically valid number of containers are filled during a processsimulation test.

5.10 The manufacturer should establish alert and action limits foreach process simulation batch size. Auditors may get help in

judging these limits from the tables in ISO 13408.2 (AsepticProcessing of Health Care Products).

5.11 To achieve an adequate confidence level of reliable processingconditions, repeated satisfactory simulation tests are required.

Corrective Actions:

5.12 The manufacturer should act according to predetermined actionand alert limits for the different batch sizes of the simulationtests.

5.13 Contamination rates for simulation tests above the 0.1% levelshould be investigated and repeated tests are required.Exceeding an alert level twice should be considered asexceeding the action limit. The manufacturer should indicate inan SOP what has to be done in such cases.

5.14 All contaminating microorganisms whether or not an alert oraction limit has been exceeded should be identified to at leastgenus, and preferably species where practicable, to determine

the possible source of contamination.

5.15 If a process simulation test fails then due account should betaken of products filled between the last successful test and thetest failure. Recording of any deviations during the simulationtest is important to trace later on the exact cause and toevaluate the consequences. The investigation should identifybatches that could be affected during this time period and thedisposition of the affected batches should be re-assessed.

6. ENVIRONMENTAL AND PERSONNEL MONITORING

The basis for environmental and personnel monitoring requirementsand recommendations are provided in reference documents, e.g.Annex I of the EU/PIC/S Guide to GMP. Some specific additionalguidance is given below on air borne microbial and non-viable particlemonitoring, intervention monitoring and staff training.

6.1 Air Borne Microbial and Non-Viable Particle Monitoring

It is important to state that the monitoring activity itself shouldnot compromise the product quality. Worst-case scenarios of

simulations tests should also include monitoring activities.

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6.2 Non-Viable Monitoring

The location chosen for monitoring should be checked to ensurethat the positions reflect the worst case. For room monitoring,the counts should be performed in locations where there is mostoperator activity. For the filling environment the counts should

be performed adjacent to the filling zone and where componentsare exposed in such way as to detect operator activity withinthese areas. Monitoring with sampling probes located in such away that they monitor the air from the HEPA filter rather than theair immediately surrounding the critical zones should beavoided. However the location of the sample device should notcompromise the laminarity of the airflow in the critical zone.Initial validation should be checked to confirm that worst casepositions have been adequately identified. These may bereconfirmed during process simulation tests.

6.3 Microbial Monitoring

6.3.1 It is usually expected that a combination of the methodsidentified in Annex 1 of the EU/PIC/S GMP guide formonitoring microbial levels be used in environmentalmonitoring programmes where appropriate.

6.3.2 Microbial monitoring should be performed in and aroundareas of high operator activity. It is not unusual to seesettle plates and air sample locations well away from

such areas. A typical example is where settle plates arelocated well to the rear of the filling machine wherethere is little or no operator activity. The same may betrue for air sampling. It is important, therefore, toobserve operator activity over a period of time andensure that the monitoring sites are so located as tomonitor operator activity.

6.3.3 The process simulation test provides an idealopportunity to confirm that worst case locations havebeen identified by the use of additional monitoring

during the test.

6.3.4 A useful monitoring technique is to monitor the fillingneedles at the end of the filling session.

6.3.5 Additional monitoring around the affected area prior todisinfection may provide useful information as to thecause.

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6.4 Intervention Monitoring

6.4.1 It is essential to include in a simulation test the variousinterventions that are known to occur during normalproduction runs, e.g. repair or replacement ofneedles/tubes, replacement of on-line filters, microbial

sampling by monitoring personnel and sampling device,duration of stops on the line, filling and handling ofstoppers etc.

6.4.2 The process simulation test should last long enough toaccommodate all possible interventions and a worst-case scenario, which may include several unfavorableconditions, which are occurring during routineprocessing.

7. STAFF TRAINING

7.1 The routine training of personnel who work in a controlledenvironment needs special emphasis as people are potentiallyone of the main sources of micro-organisms in the environment.

7.2 Included are not only operators but also other personnel workingin a controlled environment monitoring and engineeringpersonnel as staff responsible for monitoring, equipmentmaintenance, engineering, washing and preparation.

7.3 A formal personnel training programme is needed for allactivities in each clean room. This means the programme has tobe planned, documented and repeated at adequate intervals toensure that the once trained individual meets the ongoingrequirements for aseptic manipulations the work in a controlledenvironment.

7.4 This training encompasses subjects like basic microbiology,good manufacturing practice principles, hygiene (disinfectionand sanitization), aseptic connections, alert and action limits,and gowning procedures.

7.5 Environmental monitoring personnel need a thoroughunderstanding of the sources of contamination risks (e.g.inadequately disinfected/sterilized sampling equipment) that areinvolved with the sampling methods.

7.6 Periodic process simulation tests (at least once a year) arerequired to ensure that the training of the personnel in charge offilling is effectively maintained.

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7.7 The competence of an individual should be formally assessed

after attending the training courses and active participation of aprocess simulation test.

7.8 The evaluation of filled containers of a simulation test should bedone by personnel who are especially trained. They should have

routine eyesight tests. This training should include the inspectionof a process simulation batch that has a number of filledcontainers interspersed with contaminated units.

7.9 Staff responsible for equipment maintenance, washing andpreparation requires regular retraining.

8. IMPORTANT FACTORS IN VALIDATION OF ASEPTICMANUFACTURING

Beside the elements already described in the previous chaptersvalidation of aseptic manufacturing includes, but is not limited to otherimportant factors described in this chapter.

8.1 Container/Closure Integrity Testing

8.1.1 The integrity of the particular container/ closureconfigurations should be assured by:

Validation of the closure system by filling the containerwith sterile growth medium and inserting the containerin a broth containing approx. 106 cfu/ml of a suitablemicro-organism. The container is removed aftersubmersion for a recognized period of time, disinfectedand then incubated for 14 days. Growth would indicatea failure of the closure system.

8.1.2 The container/closure integrity test is normally checkedduring assessment of the marketing authorization. Themachine set-up is however a critical factor. For vials,the set-up of the capping machine may be critical as theoperation can cause distortion of the stopper if thecapping force is not adequately controlled.

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8.2 Container/Closure Sterilization

8.2.1 Problems are rarely encountered with sterilization ofcontainers. However sterilization of stoppers mightcause problems.

8.2.2 Lack of air removal and adequate steam penetration stoppers should not be packed too densely into trays orbags since this may prevent adequate air removalduring the vacuum phase of the autoclave cycle.

8.2.3 During the vacuum phases of the autoclave cyclestoppers may clump together to form a tightly boundmass. Pairs of stoppers may become attached to eachother with the base of one stopper becoming attachedto the top of the other stopper.

8.3 Equipment Cleaning and Sterilization

8.3.1 Manual Cleaning (see Manufacturing & QualityAudit Document GUIDE-MQA-008, CleaningValidation) and Sterilization

8.3.1.1 Manual cleaning of equipment rarely is aproblem but procedures should be checked toensure that O-rings and gaskets are removedduring cleaning. Otherwise, there can be a

build up of product residues and/or dirt.

8.3.1.2 If equipment is steam-sterilized in an autoclave,then the following points should be addressed:

Equipment should be wrapped and loadedinto the autoclave in such a way to facilitatethe removal of air from items in the load.

Sterilization of filters, housings and tubingmight cause problems.

Problems are usually identified by slow heatup times inside the equipment compared tothe chamber temperature. If there is atemperature lag of several minutes then thisis usually indicative of entrapped air. Thesteam will heat up the entrapped air butsterilizing conditions will not be obtained, assaturated steam will not be present.

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Only porous load steam autoclaves with avacuum system to withdraw entrapped airshould be used for sterilizing equipment.

Passive displacement autoclaves (novacuum to withdraw entrapped air) would

normally not be appropriate because of thedifficulties in air removal from the load.

8.3.2 Clean-In-Place/Sterilize-In-Place (CIP/SIP)

Validation of these systems may be difficult because ofthe potential incompatibilities in requirements for thedesign of CIP and SIP facilities. All systems have deadlegs to a greater or lesser extent and the requiredorientation of the dead legs differ for CIP and SIP. The

orientation for CIP dead legs is slightly sloping so thatthe cleaning solution can enter and also drain away.The dead leg for SIP is vertically up so that steam candownwardly displace the air.

8.4 Disinfection

8.4.1 There should be documented procedures describing thepreparation and storage of disinfectants and detergents.These agents should be monitored for microbialcontamination; dilutions should be kept in previously

cleaned containers and should only be stored fordefined periods unless sterilized. Disinfectants anddetergents used in Grade A and B areas should besterile at the time of use. If spray bottles are used, theyshould be sterile before being filled and have a short in-use shelf life.

8.4.2 Sporicidal agents should be used wherever possible,particularly for spraying-in components and equipmentin aseptic areas.

8.4.3 The effectiveness of disinfectants and the minimumcontact time on different surfaces should be validated.

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8.5 Filter Validation

8.5.1 Whatever type of filter or combination of filters is used,validation should include microbiological challenges tosimulate worst-case production conditions. Theselection of the microorganisms to perform the

challenge test (e.g. P. diminuta) has to be justified. Thenature of the product may affect the filter and so thevalidation should be performed in the presence of theproduct. Where the product is bacteriostatic orbacteriocidal, an alternative is to perform the test in thepresence of the vehicle (product without the active drugsubstance). It may be possible to group similar productsand just perform the test on one. The filter integrity testlimits should be derived from the filter validation data.The filter manufacturer should also evaluate themaximum permitted pressure differential across thefilter and this should be checked against the batchdocumentation to ensure that it is not exceeded duringaseptic filtration.

8.5.2 In addition to the validation of the filter type, the integrityof each individual product filter used for routineproduction should be tested before and after use.

8.6 Vent Filters

It is important that the integrity of critical gas and air vent filtersis confirmed immediately after the filling and if it fails, thedisposition of the batch determined. In practice, vent filters failthe integrity test more frequently than product filters as they aregenerally less robust and more sensitive to pressure differentialsduring steam sterilization.

8.7 Equipment Maintenance and Testing

8.7.1 Aseptic holding and filling vessels should be subject toroutine planned preventive maintenance. Gaskets and

O-rings should be checked regularly. Sight-glassgaskets are rarely checked routinely and after a numberof autoclave cycles may become brittle and allowbypass of room air. All vessels should be subject toregular leak testing (pressure hold or vacuum hold).Where glass vessels are used an alternative leak testmethod should be devised.

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8.7.2 Standard Operating Procedures should be checked so

as to ensure that any faults or failures of equipmentidentified by examination, testing or during routinecleaning of equipment are notified immediately toQuality Assurance.

8.8 Blow Fill Seal / Form Fill Seal

8.8.1 Where these machines are used for aseptic processing,the following validation aspects should be taken intoaccount:

On most machines there are three critical zones:parison formation, parison transfer and the filling zone.An open parison is equivalent to an open container intraditional terms. On most machines only the filling zoneis protected by Grade A air showers.

Thermocouples should be placed in those parts of theSIP pipework liable to blockage (steam traps and orificeplates) and where condensate may accumulate(vertically down dead legs). This point however shouldbe dealt with during IQ (Installation Qualification).

8.8.2 Concerning leak testing, it has to be considered thatsome of the techniques are subject to limitations. Forexample, manually performed pressure tests sometimes

lack sensitivity, or marginal leakers may not be detectedwhen using the dye bath method, because the use ofvacuum post autoclaving will not always detect steamleakers especially at the base of the unit. Therefore aclose examination of leaker reject rates is called for. Ifprocess simulation leak test rates are significantlyhigher than production rates, this may indicate a higherlevel of surveillance for the simulation.

8.9 Sterility Test

8.9.1 The sterility test can provide useful information on thevalidation status of aseptic process. It is important tocompare the retest rate for aseptically processedproducts against that for terminally sterilized products. Ifaseptically processed products have a higher rate thenthis may be indicative of sterility problems not identifiedduring validation. This is not an unusual situation asvalidation cannot take into account all the possiblepermutations and combinations in equipment, personneland processes. A typical example of where the sterilitytest can identify a problem is in the case of damaged O-

rings on aseptic receiving holding vessels.

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8.9.2 However the number of retests should decrease due to

the revised Sterility Test in the EuropeanPharmacopoeia. The revision has been made in orderto have a harmonized method in the European, theUnited States and the Japanese Pharmacopoeias. Itmeans that retesting is only allowed if it can be clearly

demonstrated that the sterility test was invalid forcauses unrelated to the product to be examined. Theconditions for considering the method invalid are givenin the method. If retesting is allowed it should be madewith the same number of containers as in the first test.

8.9.3 Provision should be made to sample a sufficient amountof product from the same location of the load in case ofretesting is performed.

9. REFERENCE

9.1 PIC/S document, PI 007-3: Recommendations on the Validationof Aseptic Processes.

END OF DOCUMENT

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Contact Officers:

Jessica TeoGMP Audit BranchManufacturing & Quality Audit DivisionHealth Products Regulation GroupHealth Sciences Authority

150 Cantonment Road, Cantonment Centre,Block A #01-02 Singapore 089762www.hsa.gov.sg

T: 68663509F: 64789068

Goh Choon WeeGMP Audit Branch

Manufacturing & Quality Audit DivisionHealth Products Regulation GroupHealth Sciences Authority

150 Cantonment Road, Cantonment Centre,Block A #01-02 Singapore 089762www.hsa.gov.sg

T: 68663520F: 64789068
http://www.hsa.gov.sg/http://www.hsa.gov.sg/http://www.hsa.gov.sg/http://www.hsa.gov.sg/http://www.hsa.gov.sg/http://www.hsa.gov.sg/

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