Aseptic Processing – Sterile Pharmaceutical

Aseptic Processing – Overview

  • Certain pharmaceutical products must be sterile
    • injections, ophthalmic preparations, irrigations solutions, haemodialysis solutions
  • Two categories of sterile products
    • those that can be sterilized in final container (terminally sterilized)
    • those that cannot be terminally sterilized and must be aseptically prepared
  • Aseptic processing
    • Objective is to maintain the sterility of a product, assembled from sterile components
    • Operating conditions so as to prevent microbial contamination
  • Objective
    • To review specific issues relating to the manufacture of aseptically prepared products
      • Manufacturing environment
        • Clean areas
        • Personnel
      • Preparation and filtration of solutions
      • Pre-filtration bioburden
      • Filter integrity/validation
      • Equipment/container preparation and sterilization
      • Filling Process
      • Validation of aseptic processes
      • Specific issues relating to Isolators, BFS and Bulk

Manufacturing Environment

  • Classification of Clean Areas
    • Comparison of classifications (Table 1)
WHO GMPUS 209EUS CUSTOMARYISO/TC (209)
ISO 14644
EEC GMP
Grade AM 3.5Class 100ISO 5Grade A
Grade BM 3.5Class 100ISO 5Grade B
Grade CM 5.5Class 10000ISO 7Grade C
Grade DM 6.5Class 100000ISO 8Grade D
(Table 1)

  • Classification of Clean Areas
    • Classified in terms of airborne particles (Table 2)

  • “At rest” – production equipment installed and operating
  • “In operation” – Installed equipment functioning in defined operating mode and specified number of personnel present

Four grades of clean areas

  • Grade D (equivalent to Class 100,000, ISO 8):
    • Clean area for carrying out less critical stages in manufacture of aseptically prepared products eg. handling of components after washing.
  • Grade C (equivalent to Class 10,000, ISO 7):
    • Clean area for carrying out less critical stages in manufacture of aseptically prepared products eg. preparation of solutions to be filtered.
  • Grade B (equivalent to Class 100, ISO 5):
    • Background environment for Grade A zone, eg. cleanroom in which laminar flow workstation is housed.
  • Grade A (equivalent to Class 100 (US Federal Standard 209E), ISO 5 (ISO 14644-1):
    • Local zone for high risk operations eg. product filling, stopper bowls, open vials, handling sterile materials, aseptic connections, transfer of partially stoppered containers to be lyophilized.
    • Conditions usually provided by laminar air flow workstation.
  • Each grade of cleanroom has specifications for viable and non-viable particles
    • Non-viable particles are defined by the air classification (See Table 2)

Limits for viable particles (microbiological contamination)(Table 3)

  • These are average values
  • Individual settle plates may be exposed for less than 4 hours
  • Values are for guidance only – not intended to represent specifications
  • Levels (limits) of detection of microbiological contamination should be established for alert and action purposes and for monitoring trends of air quality in the facility

Environmental Monitoring

  • Physical
    • Particulate matter
    • Differential pressures
    • Air changes, airflow patterns
    • Clean up time/recovery
    • Temperature and relative humidity
    • Airflow velocity
Environmental Monitoring – Physical
  • Particulate matter
    • Particles significant because they can contaminate and also carry organisms
    • Critical environment should be measured not more than 30cm from worksite, within airflow and during filling/closing operations
    • Preferably a remote probe that monitors continuously
    • Difficulties when process itself generates particles (e.g. powder filling)
    • Appropriate alert and action limits should be set and corrective actions defined if limits exceeded
  • Differential pressures
    • Positive pressure differential of 10-15 Pascals should be maintained between adjacent rooms of different classification (with door closed)
    • Most critical area should have the highest pressure
    • Pressures should be continuously monitored and frequently recorded.
    • Alarms should sound if pressures deviate
    • Any deviations should be investigated and effect on environmental quality determined
  • Air Changes/Airflow patterns
    • Air flow over critical areas should be uni-directional (laminar flow) at a velocity sufficient to sweep particles away from filling/closing area
    • for B, C and D rooms at least 20 changes per hour are usually required
  • Clean up time/recovery
    • Particulate levels for the Grade A “at rest” state should be achieved after a short “clean-up” period of 20 minutes after completion of operations (guidance value)
    • Particle counts for Grade A “in operation” state should be maintained whenever product or open container is exposed
  • Temperature and Relative Humidity
    • Ambient temperature and humidity should not be uncomfortably high (could cause operators to generate particles) (18°C)
  • Airflow velocity
    • Laminar airflow workstation air speed of approx 0.45m/s ± 20% at working position (guidance value)
  • Personnel
    • Minimum number of personnel in clean areas
      • especially during aseptic processing
    • Inspections and controls from outside
    • Training to all including cleaning and maintenance staff
      • initial and regular
      • manufacturing, hygiene, microbiology
      • should be formally validated and authorized to enter aseptic area
    • Special cases
      • supervision in case of outside staff
      • decontamination procedures (e.g. staff who worked with animal tissue materials)
    • High standards of hygiene and cleanliness
      • should not enter clean rooms if ill or with open wounds
    • Periodic health checks
    • No shedding of particles, movement slow and controlled
    • No introduction of microbiological hazards
    • No outdoor clothing brought into clean areas, should be clad in factory clothing
    • Changing and washing procedure
    • No watches, jewellery and cosmetics
    • Eye checks if involved in visual inspection
    • Clothing of appropriate quality:
      • Grade D
        • hair, beard, moustache covered
        • protective clothing and shoes
      • Grade C
        • hair, beard, moustache covered
        • single or 2-piece suit (covering wrists, high neck), shoes/overshoes
        • no fibres/particles to be shed
      • Grade A and B
        • headgear, beard and moustache covered, masks, gloves
          • not shedding fibres, and retain particles shed by operators
    • Outdoor clothing not in change rooms leading to Grade B and C rooms
    • Change at every working session, or once a day (if supportive data)
    • Change gloves and masks at every working session
    • Frequent disinfection of gloves during operations
    • Washing of garments – separate laundry facility
      • No damage, and according to validated procedures (washing and sterilization)
    • Regular microbiological monitoring of operators

Aseptic Processing

  • In aseptic processing, each component is individually sterilised, or several components are combined with the resulting mixture sterilized.
    • Most common is preparation of a solution which is filtered through a sterilizing filter then filled into sterile containers (e.g active and excipients dissolved in Water for Injection)
    • May involve aseptic compounding of previously sterilized components which is filled into sterile containersMay involve filling of previously sterilized powder
      • sterilized by dry heat/irradiation
      • produced from a sterile filtered solution which is then aseptically crystallized and precipitated
        • requires more handling and manipulation with higher potential for contamination during processing
  • Preparation and Filtration of Solutions
    • Solutions to be sterile filtered prepared in a Grade C environment
    • If not to be filtered, preparation should be prepared in a Grade A environment with Grade B background (e.g. ointments, creams, suspensions and emulsions)
    • Prepared solutions filtered through a sterile 0.22μm (or less) membrane filter into a previously sterilized container
      • filters remove bacteria and moulds
        • do not remove all viruses or mycoplasmas
    • filtration should be carried out under positive pressure
    • consideration should be given to complementing filtration process with some form of heat treatment
    • Double filter or second filter at point of fill advisable
    • Fitlers should not shed particles, asbestos containing filters should not be used
    • Same filter should not be used for more than one day unless validated
    • If bulk product is stored in sealed vessels, pressure release outlets should have hydrophobic microbial retentive air filters
    • Time limits should be established for each phase of processing, e.g.
      • maximum period between start of bulk product compounding and sterilization (filtration)
      • maximum permitted holding time of bulk if held after filtration prior to filling
      • product exposure on processing line
      • storage of sterilized containers/components
      • total time for product filtration to prevent organisms from penetrating filter maximum time for upstream filters used for clarification or particle removal (can support microbial attachment
    • Filling of solution may be followed by lyophilization (freeze drying)
      • stoppers partially seated, product transferred to lyophilizer (Grade A/B conditions)
      • Release of air/nitrogen into lyophilizer chamber at completion of process should be through sterilizing filter
  • Prefiltration Bioburden (natural microbial load)
    • Limits should be stated and testing should be carried out on each batch
    • Frequency may be reduced after satisfactory history is established
    • and biobuden testing performed on components
    • Should include action and alert limits (usually differ by a factor of 10) and action taken if limits are exceeded
    • Limits should reasonably reflect bioburden routinely achieved
    • No defined “maximum” limit but the limit should not exceed the validated retention capability of the filter
    • Bioburden controls should also be included in “in-process” controls
    • particularly when product supports microbial growth and/or manufacturing process involves use of culture media
    • Excessive bioburden can have adverse effect on the quality of the product and cause excessive levels of endotoxins/pyrogens
  • Filter integrity
    • Filters of 0.22μm or less should be used for filtration of liquids and gasses (if applicable)
      • filters for gasses that may be used for purging or overlaying of filled containers or to release vacuum in lyphilization chamber
    • filter intergrity shoud be verified before filtration and confirmed after filtration
      • bubble point
      • pressure hold
      • forward flow
    • methods are defined by filter manufacturers and limits determined during filter validation
  • Filter Validaton
    • Filter must be validated to demonstrate ability to remove bacteria
      • most common method is to show that filter can retain a microbiological challenge of 107 CFU of Brevundimonas diminuta per cm2 of the filter surface
      • a bioburden isolate may be more appropriate for filter retention studies than Brevundimonas diminuta
      • Challenge concentration is intended to provide a margin of safety well beyond what would be expected in production
      • preferably the microbial challenge is added to the fully formulated product which is then passed through the filter
      • if the product is bactericidal, product should be passed through the filter first followed by modified product containing the microbial challenge (after removing any bactericidal activity remaining on the filter)
      • filter validation should be carried out under worst case conditions e.g. maximum allowed filtration time and maximum pressure
      • integrity testing specification for routine filtration should correlate with that identified during filter validation

Equipment/container preparation and sterilization

  • All equipment (including lyophilizers) and product containers/closures should be sterilized using validated cycles
    • same requirements apply for equipment sterilization that apply to terminally sterilized product
    • particular attention to stoppers – should not be tightly packed as may clump together and affect air removal during vacuum stage of sterilization process
    • equipment wrapped and loaded to facilitate air removal
    • particular attention to filters, housings and tubing
  • CIP/SIP processes
    • particular attention to deadlegs – different orientation requirements for CIP and SIP
  • heat tunnels often used for sterilization/depyrogenation of glass vials/bottles
    • usually high temperature for short period of time
    • need to consider speed of conveyor
    • validation of depyrogenation (3 logs endotoxin units)
      • worst case locations
    • tunnel supplied with HEPA filtered air
  • equipment should be designed to be easily assembled and disassembled, cleaned, sanitised and/or sterilized
    • equipment should be appropriately cleaned – O-rings and gaskets should be removed to prevent build up of dirt or residues
  • rinse water should be WFI grade
  • equipment should be left dry unless sterilized immediately after cleaning (to prevent build up of pyrogens)
  • washing of glass containers and rubber stoppers should be validated for endotoxin removal
  • should be defined storage period between sterilization and use (period should be justified)

Process Validation

  • Not possible to define a sterility assurance level for aseptic processing
  • Process is validated by simulating the manufacturing process using microbiological growth medium (media fill)
    • Process simulation includes formulation (compounding), filtration and filling with suitable media using the same processes involved in manufacture of the product
    • modifications must be made for different dosage formats e.g. lyophilized products, ointments, sterile bulks, eye drops filled into semi-transparent/opaque containers, biological products
  • Media fill program should include worst case activities
    • Factors associated with longest permitted run (e.g. operator fatigue)
    • Representative number, type, and complexity of normal interventions, non-routine interventions and events (e.g. maintenance, stoppages, etc)
    • Lyophilisation
    • Aseptic equipment assembly
  • Media fill program should include worst case activities
    • Factors associated with longest permitted run (e.g. operator fatigue)
    • Representative number, type, and complexity of normal interventions, non-routine interventions and events (e.g. maintenance, stoppages, etc)
    • Lyophilisation
    • Aseptic equipment assembly
  • Worst case activities (cont)
    • No of personnel and their activities, shift changes, breaks, gown changes
    • Representative number of aseptic additions (e.g. charging containers, closures, sterile ingredients) or transfers
    • Aseptic equipment connections/disconnections
    • Aseptic sample collections
    • Line speed and configuration
  • Worst case activities (cont)
    • Weight checks
    • Container closure systems
    • Specific provisions in processing instructions
  • Written batch record documenting conditions and activities
  • Should not be used to justify risky practices
  • Duration
    • Depends on type of operation
    • BFS, Isolator processes – sufficient time to include manipulations and interventions
    • For conventional operations should include the total filling time
  • Size
    • 5000 – 10000 generally acceptable or batch size if <5000
    • For manually intensive processes larger numbers should be filled
    • Lower numbers can be filled for isolators
  • Frequency and Number
    • Three initial, consecutive per shift
    • Subsequently semi-annual per shift and process
    • All personnel should participate at least annually, consistent with routine duties
    • Changes should be assessed and revalidation carried out as required
  • Line Speed
    • Speed depends on type of process
  • Environmental conditions
    • Representative of actual production conditions (no. of personnel, activity levels etc) – no special precautions (not including adjustment of HVAC)
    • if nitrogen used for overlaying/purging need to substitute with air
  • Media
    • Anaerobic media should be considered under certain circumstances
    • Should be tested for growth promoting properties  (including factory isolates)
  • Incubation, Examination
    • In the range 20-35ºC.
    • If two temperatures are used, lower temperature first
    • Inspection by qualified personnel.
    • All integral units should be incubated. Should be justification for any units not incubated.
    • Units removed (and not incubated) should be consistent with routine practices (although incubation would give information regarding risk of intervention)
    • Batch reconciliation
  • Interpretation of Results
    • When filling fewer than 5000 units:
      • no contaminated units should be detected
      • One (1) contaminated unit is considered cause for revalidation, following an investigation
    • When filling from 5000-10000 units
      • One (1) contaminated unit should result in an investigation, including consideration of a repeat media fill
      • Two (2) contaminated units are considered cause for revalidation, following investigation
    • When filling more than 10000 units
      • One (1) contaminated unit should result in an investigation
      • Two (2) contaminated units are considered cause for revalidation, following investigation
  • Interpretation of Results
    • Media fills should be observed by QC and contaminated units reconcilable with time and activity being simulated (Video may help)
    • Ideally – no contamination. Any contamination should be investigated.
    • Any organisms isolated should be identified to species level (genotypic identification)
    • Invalidation of a media fill run should be rare
  • Batch Record Review
    • Process and environmental control activities should be included in batch records and reviewed as part of batch release
      • In-process and laboratory control results
      • Environmental and personnel monitoring data
      • Output from support systems(HEPA/HVAC, WFI, steam generator)
      • Equipment function (batch alarm reports, filter integrity)
      • Interventions, Deviations, Stoppages – duration and associated time
      • Written instructions regarding need for line clearances
      • Disruptions to power supply

Additional issues specific to Isolator and BFS Technologies

  • Isolators
    • Decontamination process requires a 4-6 log reduction of appropriate Biological Indicator (BI)
    • Minimum 6 log reduction of BI if surface is to be free of viable organisms
    • Significant focus on glove integrity – daily checks, second pair of gloves inside isolator glove
    • Traditional aseptic vigilance should be maintained
  • Blow-Fill-Seal (BFS)
    • Located in a Grade D environment
    • Critial zone should meet Grade A (microbiological) requirements (particle count requirements may be difficult to meet in operation)
    • Operators meet Grade C garment requirements
    • Validation of extrusion process should demonstrate destruction of endotoxin and spore challenges in the polymeric material
    • Final inspection should be capable of detecting leakers
  • Issues relating to Aseptic Bulk Processing
    • Applies to products which can not be filtered at point of fill and require aseptic processing throughout entire manufacturing process.
    • Entire aseptic process should be subject to process simulation studies under worst case conditions (maximum duration of “open” operations, maximum no of operators)
    • Process simulations should incorporate storage and transport of bulk.
    • Multiple uses of the same bulk with storage in between should also be included in process simulations
    • Assurance of bulk vessel integrity for specified holding times.
  • Bulk Processing
    • Process simulation for formulation stage should be performed at least twice per year.
    • Cellular therapies, cell derived products etc
      • products released before results of sterility tests known (also TPNs, radioactive preps, cytotoxics)
      • should be manufactured in a closed system
      • Additional testing
        • sterility testing of intermediates
        • microscopic examination (e.g. gram stain)
        • endotoxin testing

Useful Publications

  • PIC/S Recommendation on the Validation of Aseptic Processes
  • FDA Guidance for Industry- Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Process
  • ISO 13408 Aseptic Processing of Health Care Products
    • Part 1: General Requirements
    • Part 2: Filtration
    • Part 3: Lyophilization
    • Part 4: Clean-In-Place Technologies
    • Part 5: Sterilization-In-Place
    • Part 6: Isolator Systems
  • Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors  – Nanjing, November 2009