In September 2004, after 17 years in existence, the FDAreplaced older guidelines with an updated document,‘Guidance for Industry – Sterile Drug Products Producedby Aseptic Processing – Current Good ManufacturingPractices’. The world was a very different place when theoriginal guidance was issued. AIDS was a relatively newillness, few had heard about ‘Mad Cow Disease’, theInternet was unheard of, and genetic fingerprinting wasnot yet in use in criminal investigations. Throughout the1980s and 1990s, technological advances continued to bemade, but the pharmaceutical industry kept a conservativeapproach to their processes. Also in September 2004,Europe made some changes to the ‘EC Guide to GoodManufacturing Practice, Annex I, Manufacture of SterileMedicinal Products’. Together, these documents initiatedmeetings, conferences, discussions and debate. Many areashave seen little amendments or increased regulatoryclarity; however, aseptic process simulations haveundergone significant changes.

Within a short time of the new guidance being issued,a UK vaccine manufacturer found itself in trouble with theregulators. The production facility was shut down, causinga huge shortfall in flu vaccines available to patients. Thiswas a timely reminder of the importance of process control in critical manufacturing environments.

STERILITY‘Sterile’ is a powerful word, with harsh legal implicationssurrounding non-compliance. Global regulatory authoritieswould define sterile as ‘free of viable organisms’, and sterilityassurance has become one of the most scrutinised areas ofpharmaceutical and medical device manufacture. Thefavoured method of production of sterile pharmaceuticalproducts includes a terminal sterilisation process, such asautoclaving or irradiation. Since it is not practical toexamine every unit for confirmation of sterility, terminalsterilisation processes use biological indicators (BIs) toprovide levels of sterility assurance. BIs are substrates

Biotechnology

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The Media Fill Approach: An UpdateThe design and execution of rugged process simulations – together with the use of high quality growth media – will help ensure that the risk ofcontamination of aseptic processes is kept within acceptable limits.

By Phil Smith at Oxoid Ltd

carrying high loads of resistant micro-organisms, at levels fargreater than the bioburden of the load being sterilised. Ifeverything on the BI is killed, it is reasonable to assume thatthe load is also free of viable organisms and can be deemedsterile. However, many therapeutic agents would notwithstand terminal sterilisation, so aseptic manufacture andaseptic filling processes are required.

Aseptic processing used to produce sterile parenteraldrug products and Active Pharmaceutical Ingredients(APIs) involves the handling of pre-sterilised products in a highly controlled environment. Using the BIcorrelation approach is not applicable here, as asepticprocessing involves ensuring a great deal of processcontrol, with sensitive handling of products until theyare sealed within their final containers.

All efforts are made to minimise the risk ofcontamination:

� Filling and support areas are engineered to minimise contamination

� Air in critical areas is supplied at point-of-use as high-efficiency particulate air (HEPA) filtered,laminar flow air at a velocity sufficient to sweepparticles away from the filling and closing areas

� Positive air pressure is used to prevent ingress of airborne contamination: anything that can be sterilised must be rendered sterile before it canbe taken into the clean area where the process is performed

� Human intervention is kept to a minimum� Cleaning is thorough and validated� Disinfection practices are tight and validated� Monitoring is done to prove the process

and environment are under control

Despite such measures, contamination is an ever-presentthreat, since there will always be a risk that materials andsurfaces may carry organisms, and inefficiencies in air

Phil Smith is Pharmaceutical Marketing Manager at Oxoid Ltd (Basingstoke, UK). One of his responsibilities at Oxoid is to ensurethat the company’s products meet the changing needs of the pharmaceutical industry and the constraints imposed by regulatoryrestrictions. Mr Smith has 15 years’ experience in the pharmaceutical and regulated industries, working largely on sterility issues andaseptic applications. Prior to joining Oxoid in 2004, he worked for STERIS Corporation, where he was involved in developing theirdisinfection and critical cleaning portfolio for the pharmaceutical industry in Europe, the Middle East and Africa.

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� Type of products being filled� Lot/Batch sizes� Container and closure configuration� Fill volume� Line speed� Operator shifts and fatigue� Filling line configuration� Sterile hold times� Number of units filled (production

vs simulation)� Number and frequency of runs� Acceptance criteria� Run duration� Interventions – atypical and typical� Other elements that could impact upon

sterility assurance

Also, worst-case conditions are used in many forms ofvalidation, including process simulations. This does notmean waiting for a tornado to rip off the cleanroom roofbefore the media fill, but undertaking the simulation atthe limits of a normal process.

GROWTH MEDIAThe selection of the correct growth medium to be usedin the process simulation is a very important step. Themedium needs to support the growth of a wide variety ofmicro-organisms, including aerobic bacteria, yeasts andmoulds. The broad range of organisms being looked for is consistent with organisms tracked through thefirm’s environmental monitoring programme. The FDA

filtration may pose a risk. The largest source of potentiallyviable contamination comes from people – the operatorsrunning the filling process. Aseptic processing is a processbeing operated in a controlled – but not sterile –environment; the probability of non-sterility cannot becalculated. The industry works to recognised, acceptedcontamination levels, so the probability of viablecontamination is recognised and calculated. Routinesampling for sterility testing is not sensitive enough todetect such low level contamination. Sample numbers aretoo small, and only gross contamination is likely to bedetected. Pharmaceutical manufacturers, therefore, needother means of guaranteeing the quality of their product.This is why process simulations (media fills) – supportedby environmental monitoring and other related processes– are required. These are used to demonstrate control ofthe process to the industry standard for allowablecontamination levels.

MEDIA FILLSMedia fills utilise culture media in place of product toevaluate contamination levels. However, such media fills area snapshot in time, and subtle changes can incur changes incontamination levels. It is therefore of paramountimportance that process simulations are designed toaccurately represent the aseptic process. The new FDAguidelines pay particular attention to this aspect of asepticprocessing, and it is becoming an area requiring more workand focus to satisfy the regulators. The media fill should bedesigned to mimic, as closely as possible, the asepticprocesses used in practice. The media fill design is oneelement within the overall considerations to be made in thevalidation of an aseptic process. Areas of focus include:

� Facility and room design� Design of the filling machine� Process flow� Heating, ventilation, and air-conditioning

design and validation� Utility design and validation� Response to deviations� Trends in environmental monitoring data� Contamination control programme� Quality assurance and quality control systems� Process simulations� Personnel training and qualification

An appreciation of the many factors influencing thevalidation programme allows a process simulation to beeffectively designed. Key elements in the simulation to betaken into account include:

Guidance notes the use of soybean casein digest medium,also known as ‘Tryptone Soya Broth’. With concernsabout prion contamination from components of animalorigin found within such media, it is vitally importantthat the medium supplier can provide the necessarycertifications and documentation for confirmingmaterials are sourced from ‘BSE-free’ countries (such asOxoid Cold Filterable Tryptone Soya Broth – a highlynutritious general purpose medium which is ideal formicrobiological media fills). An alternative approachwould be to use a medium derived from vegetablematerials such as Oxoid’s Cold Filterable VegetablePeptone Broth, which is a vegetable alternative and issuitable for use in place of Tryptone Soya Broth.

The guidance also indicates that if the product isbeing filled in anaerobic conditions, usually in a nitrogenenvironment, an anaerobic medium (such as fluidthioglycollate) is used.

As already noted, the new guidelines recommend thatmedia fills mimic the actual aseptic process as closely aspossible. One of the main areas where this is implicated iswhere the culture medium is introduced into the process.In the past, manufacturers have made up and sterilised themedium outside of the controlled area, and introduced itdirectly into the filling line. In order to more closely mimicthe process, the culture medium should be filtered into theprocess – just as would occur to a liquid pharmaceuticalproduct. This creates several concerns:

� Dried culture media is usually supplied in a non-sterile form and carries a high bioburden,preventing it from being taken directly into acontrolled area. It would be preferential to sourcemedia that has been irradiated.

� For liquid fills, many holding vessels upstream offiltration do not have the capability to heat culturemedia to a temperature adequate to dissolve thepowder into a solution. Even those that have thisability take up time and energy in heating andcooling. Sourcing a medium that dissolves atambient temperature would negate such problems.

� Mycoplasma can be a concern with culture media;therefore assurance of irradication of mycoplasmawould be favoured.

� Broths traditionally used for media fills do nothave good filterability characteristics, and could‘blind’ the sterilising filters. This would invalidatethe process simulation. It would be advantageousto understand the filterability profile (such as Vmax

or Vcap) of the microbial growth medium, to ensurefilter sizing can tolerate the said medium.

PROCESS SIMULATIONSGrowth promotion testing must be undertaken on thegrowth medium used for process simulations. There issome confusing guidance as to when to perform this.The FDA Guidance document does not makespecification on the timing of the test and the EU Annex1 document does not even ask for a growth promotiontest. However, both the PICS (PharmaceuticalInspectorate Cooperation) and ISO documents ask thatgrowth promotion testing is performed upon conclusionof the incubation period (usually 14 days). Whilst thelatter initially seems to be the more sensible option, italso increases the holding time prior to release, asproduct is waiting for the growth promotion tests to beincubated and analysed.

Many pharmaceutical manufacturers prefer to rungrowth promotion testing in parallel with the media fill samples. Randomly removing samples from theprocess simulation run has little basis for detectingcontamination. Contamination of the filling line beingchallenged is a random event, and such samples areunlikely to show all of the contamination present. Inorder to meet the various regulatory guidance ‘half-way’,a compromise would be to fill additional units at the endof the process simulation, and use these for the growthpromotion test. These are then incubated under identicalconditions as the process simulation samples. Thisapproach both ensures that the process simulation unitsand growth promotion test units are separated, and theoverall time of the process simulation project is reduced.

Any units that are incubated should be inspectedprior to incubation. Any defects that compromise thecontainer closure or non-integral units are rejected. Allrejections should be documented, with reasons forrejection and the number of units rejected. Incubation isthen performed for 14 days at 20-350C (+/-2.50C). Theseparameters have been accepted by the global regulatoryauthorities and should allow the growth of bacteria, yeast and moulds. Units are incubated in an invertedposition for the first half of the incubation period, and then returned to an upright position for theremainder. Also, isolates that are seen in the firm’senvironmental monitoring programme need to bepicked up by a media fill run, and data confirming thisshould be made available.

Through the thorough design and execution of arugged process simulation, and the use of a high qualitygrowth promotion medium, meticulous challenge of theaseptic process is achieved.

The author can be contacted at phil.smith@oxoid.com

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