2IntroductionTo give an overview of the principles involved in the manufacture of sterile productsThe overall objective is to produce product that has a high assurance of sterility (and which meets all other quality parameters)This presentation:Summarises the general approachGives a framework for other detailed guides on specific aspects of sterilisation & sterile manufacturingIllustrates the underlying principlesProvides advice and gives recommendations.
3General Principles of Sterile Manufacturing Moist Heat SterilizationDry Heat SterilizationAseptic ProcessingEnvironmental MonitoringEthylene Oxide SterilizationSterile FiltrationWater systems validationSterility testingRadiation SterilizationVisual Inspection
4Fundamentals Sterility is the absence of living organisms This is an absolute definitionThe probability of achieving sterility depends on the overall processIt is generally accepted that a terminally sterilized product should have a probability of non-sterility of less than 10-6 (i.e., a lower probability than one in a million of having a non-sterile unit)This is often expressed as an SAL Sterility Assurance Level of 106This is a worst-case figure (with a challenge more resistant than product bioburden). Real confidence levels are generally very much higherA figure that has sometimes been quoted for aseptically filled product is probability of non-sterility of less than However, this is harder to analyse as contamination does not follow a clear statistical distribution. Potential contamination sources are not randomly distributed.
5Why Validate and Control? The test for sterility cannot confirm that the whole batch is sterileIt is performed on a sample from a batch and has statistical limitationsIt can miss contamination if only a proportion of units are non-sterileIt is thus necessary to recognize and understand every aspect that could lead to loss of sterility assuranceSuch conditions should be prevented by the application of carefully designed barriers and/or control measures.
6Development – Validation and Control It is important that the product and process are designed to maximise sterility assuranceWherever possible, the product should be developed to withstand sterilization in the final containerOnce the product design is defined, a suitable production process must be developedThis is installed and validatedThe process must then be tightly controlled to assure reliability and consistency.
7Product Design Considerations For New Products:Define product and processing requirementsConsider stability of product to the sterilization conditionsBase the process on achieving the required sterility assurance levelWhere possible choose terminal sterilization in final containerDefine process flow and the important microbiological aspectsEnsure changes are subject to strict change controlFor reviewing existing (marketed) products:Establish the process description and assess in detailPreferably, sterilization should be by compendial proceduresWhere other procedures are registered, assess SALWhere necessary (if existing SAL is too low) may need to improve process and maybe re-registerRequire justification & validation.
8Facility DesignMust be in compliance with company policies and procedures, for example:Must minimise the risk of contamination at all critical stagesRequired Grades of Clean Rooms : need to be appropriate for the process - e.g. for Terminal Sterilization or Aseptic FillPersonnel Access and Material FlowRestricted access, correct gowningMaterials flow, air locks, decontamination, segregationHVAC-SystemSegregation/Dedicated HVAC of correct standardRequires control of Filtration/ΔP/Air Flow/Temp./Pressure/HumidityAir flow patterns demonstratedNo sinks and drains in Zone A/B areas, air breaks to drains in othersSurfaces and ease of cleaning: smooth unbroken impervious surfaces
9Cleaning and disinfection of the Facility Cleaning and disinfection is important in environmental controlEfficacy needs to be validatedValidated procedures, conducted consistentlyIn class A & B areas, the cleaning and disinfectant materials must be sterilizedAnd need to minimise contamination risk in other areasOperating procedures must include, at minimum:Preparation of cleaning materials (and sterilization if applicable)Exact procedure of cleaning & disinfection.Responsibility & scheduling.Type and concentration of detergents and disinfectants.Type of cleaning tools.Training is required for cleaning and disinfection of clean roomsRoutine decontamination using formaldehyde gas should be avoided.
10Water All water systems require good design and validation Typically, for pharmacopoeial grades, validation includesTwo studies over a total of 4 weeks to assess against the acceptance criteria,Additional 11 months to verify that the system remains under controlMust demonstrate consistent production of water of the required qualityPhysico-chemical,Microbiological,Biological (endotoxin, where applicable)Water systems must be regularly monitored following a defined written monitoring plan based on results of the validation studies.
11Categories of Water Water for Injections (WFI) For injectables formulationFinal rinse water for product-contact items (for injectables)Freshly prepared or from a validated hot (e.g., >75°C) storage /distribution system or otherwise protected from microbial contaminationHighly Purified Water (HPW)To European PharmacopoeiaPurified Water (PW)For initial washing of product-contact itemsPrepared, suitably stored and distributed to maintain quality and prevent microbiological proliferation, following the relevant company procedures.
12Gases and Vacuum Gases Vacuum Systems Specification equivalent to the room air quality where it is to be usedIn aseptic applications, gases are to be filter sterilizedConsider sterile filtering non-product contact gases for aseptic applications. (But, note safety considerations, e.g. avoidance of leakage)All gas filters to be integrity tested on installation and at defined intervalsVacuum SystemsSometimes used for cleaning and dust controlMay be mobile units, fitted with exhaust HEPA filtersOr may have central dust collectionOn these, use dedicated vacuum pumps’ protected against back-flowDesign to prevent unprotected route into the aseptic suite.
13Equipment (1) Equipment Qualification To include the critical aspects for sterile product processingQualification of critical aspects of moist heat sterilization, aseptic processing, dry heat sterilization etc.Cleaning and Sanitization of EquipmentEquipment designed for easy cleaning and sanitizationFor Terminal Sterilization applications, low microbial challenge. Where possible, critical surfaces should be sterilizedFor aseptic work, the critical (product contact) surfaces must be sterilized before use. In exceptional cases where this is not possible (e.g., some stopper bowls), they should be sanitized by a validated methodCleaning validation must show effectiveness and absence of residues.
14Equipment (2) Equipment Sterilization and handling Sterilization must follow a validated procedureAseptic processes designed to minimise aseptic assembly and interventionUnavoidable aseptic assembly needs clear & precise proceduresAseptic assembly must be simulated (worst-case) in media fill simulation trialsSterilization In Place is a good method where possible – must be validated.
15PersonnelTraining - personnel appropriately trained for sterile processing, including assessment and documentation:Basic GMPFundamentals of microbiologyPersonal hygiene, health and cleanlinessBehaviour and aseptic working techniquesGowning and entry proceduresCleaning and disinfectionSterilization procedures, validation and routine operationEmergency procedures to protect product quality (e.g. loss of HVAC System, loss of power, equipment interventions etc.)Personnel participating in aseptic processing must have practical training in aseptic techniques before doing aseptic manipulationsThey must have participated in a successful media fill run.
16Gowning and Aseptic Technique Personnel must correctly wear appropriate clean room garmentsDetailed, easily understood, gowning procedure (preferably illustrated)Aseptic TechniquesPersonnel in the aseptic manufacturing area, must understand the principles of aseptic proceduresThey must only be considered qualified after appropriate training, working under supervision and demonstration of competenceThe supervisor should observe technique & correct as necessaryAll personnel directly involved in aseptic processing must participate in a media fill at least once per yearGlove disinfectionSterile disinfectants must be available (e.g., alcohol based)Glove disinfection must be reasonably frequent, defined in SOP.
17Environmental Monitoring (1) The scope of environmental monitoring includes:Non-viable particulates,Viable (microbial) countsDifferential pressuresTemperaturesHumiditiesAir flows
18Environmental Monitoring (2) Monitoring During Room QualificationOperational Qualification (OQ) at rest conditions to verify operationPerformance Qualification (PQ) in worst case operational conditionsAction levels should meet USP or Euro GMP as applicableAlert levels tight enough to detect deterioration, but not so tight that they become meaningless due to frequent transgressionPQ must cover a sufficient period to establish consistencyRoutine MonitoringEnsures area remains satisfactory. Results should be within alert levelResults above alert levels need review and perhaps corrective actionsAbove action levels, must trigger appropriate actions (described in guide),Results must be assessed for trends so that progressive or sudden changes in the results may be observed. This should be reviewed regularly.
19Environmental Monitoring (3) Deviation Reports and Failure InvestigationsThe data must be analysedWhere necessary further investigations initiatedPossible contamination sources to be assessed and, eliminatedOutcome and detail must be reportedRecommended Methods for Routine MonitoringPhysical measurements of the air supplyPhysical and microbiological monitoring of the environmentParticles (viable and non-viable) in the airMicro-organisms settling out of the airMicro-organisms contaminating surfacesPresence of micro-organisms on the hands and garmentsMonitoring PlanDefined monitoring plans: tests, locations, alert/action levels & frequenciesMay contain details of water, compressed gas clean steam testingA review of environmental data is a requirement for batch release.
20Bioburden and Components Active Ingredients, Excipients, AdditivesAll ingredients should have appropriate biological specificationsAny limitations to sterilization must be definedDescription of origin (e.g. virological / prion risk)Materials Used in the ProcessWhere appropriate, determine bioburden (e.g., ion exchange materials)Primary Packaging ComponentsContainer and the closure and cleaning / sterilization to be clearly specifiedSteps such as siliconization may need monitoringIf cleaning/sterilization is by supplier, same exigencies applyContainer-closure integrityThe integrity must be validatedSimulate, where appropriate: stress from processingMethod appropriate to container/closure system
21Weighing, Compounding and Sterilization Weighing and compounding must be carried out in suitably classified roomsVessels must be cleaned, and sterilized or sanitised as appropriate and stored dry in a way to prevent microbial contaminationStorage of pre-sterilization intermediates to be controlled & time limitedFollowing aspects to be considered:Pre-filtration bioburden (filter sterilized material)Pre-sterilization bioburdenAppropriate in-process controlsSterilization of product and product contact materialsSelection of a suitable sterilization protocol must be based on SALMethod must also consider the stability of the productValidation always requiredChange control is vital; even apparently minor change must be assessed
22Terminal Sterilization Steam SterilizationBy far the most common method for aqueous-based pharmaceuticalsPreferred cycle is the Pharm Eur reference cycle is 15 minutes at 121°CThe sterilization cycle chosen must be compatible with product stabilitySterilization parameters clearly definedIn conjunction with other controls, the required SAL must be demonstratedValidation to confirm sterilization conditions consistently throughout the loadSterilization by Ionizing RadiationCommon for medical devices, but not for pharmaceuticals.Pharm. Eur. reference condition, 25 KiloGray (kGy), has been widely accepted. Other conditions may be used if validated and accepted by the regulatorImportant to consider susceptibility of the product to radiation damageDry Heat SterilizationLower antimicrobial efficacy than moist heat, thus higher temperatures and/or longer exposures. Pharm Eur reference cycle is 2 160°CRarely used for terminal sterilization of pharmaceuticals; in rare cases heat resistant non-aqueous products may be terminally sterilized.
23Sterilization of Items for Aseptic Fill (1) Steam SterilizationWidely used, but careful validation needed – particularly complex itemsBroadly similar to terminal steam sterilization, but two aspects are criticalQuality of saturated steamRemoval of air and subsequent steam penetrationSterilization by Ionizing RadiationMay be used for temperature sensitive primary packaging or componentsUsed for disposables for sterile areas and sterility testing areasValidation includes dosimetry, - correct, even, irradiation of the itemsDry Heat Sterilization/DepyrogenationSterilization/ depyrogenation of heat resistant primary packaging materialsPharm Eur notes that temperatures in excess of 220 oC have been frequently used, the USP suggests 250 ± 15 oCValidation must include endotoxin challenge studiesDry heat may be used to sterilize non-aqueous preparations (e.g. Ointment bases) at lower temperature/time relationships, without depyrogenation.
24Sterilization of Items for Aseptic Fill (2) Ethylene Oxide SterilizationQuite widely used to sterilize heat labile componentsEuropean Pharmacopoeia and the European GMP guide indicate that this method should only be used where there is no suitable alternativeHazardous - toxic, potentially carcinogenic, flammable, potentially explosiveGenerally conducted by specialized contractorsThere are strict regulatory limits on maximum permissible product residuesBulk packs for sterilization must be gas permeable, but sealed against microbial ingressSterilization must consider packaging, load pattern, gas penetration (ethylene oxide & water vapour), bulk pack integrityValidation and routine monitoring must include Biological indicators.
25Sterilization by Filtration (Liquids) Principle:Contaminating organisms are not killed, but are retained on the filters. Any faults in the filter structure, may compromise thisValidation includes:Retention of bacterial challenge: B. diminuta at 107 per cm2This is correlated with an integrity test valueValidation should address:Filter suitability - toxicity, extractables, shedding of particlesAdsorption of productCompatibility with product solventsThe required filter size and suitability of the filtration equipmentRetention of B.diminuta in the actual product under process conditionsParameters for the physical integrity testRoutine FiltrationConducted in line with the validated parametersCheck integrity testing, process time, differential pressure, flow rates, sterilization and reuse of filters.
26Performance Qualification of Aseptic Manufacturing Based on simulating the risk of contamination in all aseptic operationsFor a new process, a minimum of three consecutive satisfactory media filling trialsFor aqueous liquid products, simulation trials use a liquid microbiological mediumFor solid dosage forms, a powder ‘placebo’ is used, followed by aseptic reconstitution into a liquid microbiological mediumThe following slide gives a general overview....
27Aseptic Process Simulation (Media Fill Trial) Media Fill Trials (MFTs)All process stages simulated as closely as possibleParticularly interventions and manual manipulationsMust follow routine procedures and include all interventionsRegular interventions simulated with the same frequency as actual processIn each case, the worst-case eventuality must be coveredProcess must be successfully validated before product filling is permittedRevalidation by media fill must be conducted every half year (each line)Manufacturing EnvironmentMicrobiological monitoring must be performed during the trialFilling Conditions and EquipmentAll according to routine operating conditions and at normal times of dayContainers must be passed through all stages.