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 GMP	US 209E	US CUSTOMARY	ISO/TC (209) ISO 14644	EEC GMP
Grade A	M 3.5	Class 100	ISO 5	Grade A
Grade B	M 3.5	Class 100	ISO 5	Grade B
Grade C	M 5.5	Class 10000	ISO 7	Grade C
Grade D	M 6.5	Class 100000	ISO 8	Grade D

• 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