Asepting procesing


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  • Aseptic processing and sterilization by filtration Aseptic processing We have already discussed the fact that terminal sterilization of a product is preferable as it reduces the risk of and provides more assurance of sterility. However, for some types of products, this is not possible. Another method to prepare a sterile products then is to maintain the sterility of a product, assembled from sterile components. All operating conditions should be such to prevent microbial contamination. What do you think are the aspects that require careful attention?
  • Personnel play an important part in ensuring the quality of manufacture. It is also relevant (perhaps in particular) in the manufacture of sterile products. Only a minimum number of personnel should work in clean areas, especially during aseptic processing. As far as possible, all inspections and controls should be done from outside the production rooms. Training should be given to all including cleaning and maintenance staff, and should include initial and regular training on manufacturing, hygiene, and microbiology. Look at the procedure for training, training program, training material and assessment of the personnel. In special cases, when outside staff have to enter the clean areas, they should be supervised. Remember also the previous discussion on decontamination procedures (e.g. staff who worked with animal tissue materials).
  • Personnel working in clean areas should maintain high standards of hygiene and cleanliness. They should undergo periodic health checks, wear clothing that do not shed particles, and should take care not to introduce microbiological contaminants in the areas. No outdoor clothing should be brought into clean change rooms. Personnel should follow changing and washing procedures, wear no watches, jewellery and cosmetics
  • The WHO GMP text specifies the type of clothing that is appropriate for the different grades of rooms.
  • Garments should be changed at every working session, or once a day (if supportive data exist through validation studies). Gloves and masks should be changes at every working session Personnel should disinfect their gloves frequently during operations to prevent possible introduction of contaminants (micro) into the areas where they work or touch. Arrangements must be in place for the laundering and sterilization of clean-room clothing. This should be carried out in a controlled environment. If fibres are damaged due to inappropriate cleaning or sterilization, an increased risk for contamination may develop as clothing could shed particles. The use of contract laundries for this purpose, requires an audit by the company to ensure that appropriate procedures are in place.
  • Asepting procesing

    1. 1. By SUNILBOREDDY M.Pharmacy
    2. 2.  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
    3. 3. Aseptic processing  Objective is to maintain the sterility of a product, assembled from sterile components  Operating conditions so as to prevent microbial contamination
    4. 4. 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
    5. 5. Classification of Clean Areas ◦ Comparison of classifications 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 10 000 ISO 7 Grade C Grade D M 6.5 Class 100 000 ISO 8 Grade D Table 1
    6. 6. Classification of Clean Areas ◦ Classified in terms of airborne particles (Table 2) Grade At rest In operation maximum permitted number of particles/m3 0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm > 5 µ A 3 500 0 3 500 0 B 3 500 0 350 000 2 000 C 350 000 2 000 3 500 000 20 000 D 3 500 000 20 000 not defined not defined “At rest” - production equipment installed and operating “In operation” - Installed equipment functioning in defined operating mode and specified number of personnel present
    7. 7. 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.
    8. 8.  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)
    9. 9.  Limits for viable particles (microbiological contamination) Grade Air sample (CFU/m3) Settle plates (90mm diameter) (CFU/4hours) Contact plates (55mm diameter) (CFU/plate) Glove print (5 fingers) (CFU/glove) A < 3 < 3 < 3 < 3 B 10 5 5 5 C 100 50 25 - D 200 100 50 - 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
    10. 10. Environmental Monitoring  Physical ◦ Particulate matter ◦ Differential pressures ◦ Air changes, airflow patterns ◦ Clean up time/recovery ◦ Temperature and relative humidity ◦ Airflow velocity
    11. 11. 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
    12. 12. Environmental Monitoring - Physical  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
    13. 13. Environmental Monitoring - Physical  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 ususally 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
    14. 14. Environmental Monitoring - Physical  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)
    15. 15. 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)
    16. 16. Personnel (2)  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
    17. 17. Personnel (3)  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
    18. 18. Personnel (4)  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
    19. 19.  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 containers ◦ May 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
    20. 20. 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
    21. 21. Preparation and Filtration of Solutions (2)  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
    22. 22. Preparation and Filtration of Solutions (3)  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)
    23. 23. Preparation and Filtration of Solutions (4)  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
    24. 24. 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
    25. 25. Prefiltation Bioburden (2)  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
    26. 26. 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
    27. 27. 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
    28. 28. Equipment/container preparation and sterilization (2)  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
    29. 29. Equipment/container preparation and sterilization (2)  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)
    30. 30. 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
    31. 31.  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
    32. 32.  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.
    33. 33.  Bulk Processing (2) • 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
    34. 34. Environmental MonitoringEnvironmental Monitoring ConsiderationsConsiderations
    35. 35.  Airborne nonviable particulate monitoring  Airborne viable contaminant monitoring  Viable contaminant monitoring of surfaces  Viable contaminant monitoring of personnel  Temperature and humidity monitoring  Pressure differential monitoring Environmental MonitoringEnvironmental Monitoring ComponentsComponents
    36. 36.  Water monitoring: ◦ Total organic carbon ◦ Conductivity ◦ Microbial Contaminants ◦ Endotoxin Environmental MonitoringEnvironmental Monitoring ComponentsComponents
    37. 37.  Monitoring frequencies and strategies ◦ Establishment of a meaningful and manageable program  Sampling and testing procedures  Establishment of effective alert and action limits  Trending of results General EnvironmentalGeneral Environmental Monitoring ConsiderationsMonitoring Considerations
    38. 38.  Investigation and evaluation of trends as well as excursions from alert and action limits  Corrective actions to be implemented in response to environmental monitoring excursions  Personnel training - sampling, testing, investigating excursions, aseptic technique General EnvironmentalGeneral Environmental Monitoring ConsiderationsMonitoring Considerations
    39. 39.  Should include monitoring of all environments where products and their components are manufactured ◦ All areas where there is a risk of product contamination  Should include monitoring of all water used for product manufacturing as well as feed water to the final water purification system (WFI System) Scope of EnvironmentalScope of Environmental Monitoring ProgramMonitoring Program
    40. 40.  CFR GMP regulations  FDA Guidance Documents  USP Informational Chapter Regulatory Basis forRegulatory Basis for Environmental MonitoringEnvironmental Monitoring ProgramProgram
    41. 41.  Aseptic processing areas: ◦ Easy to clean and maintain ◦ Temperature and humidity controlled ◦ HEPA filtered air ◦ Environmental monitoring system ◦ Cleaning and disinfecting procedures ◦ Scheduled equipment maintenance and calibration 21 CFR 211.4221 CFR 211.42
    42. 42.  Ventilation, air filtration, air heating and cooling: ◦ Adequate control over microorganisms, dust, humidity and temperature. ◦ Air filtration systems including prefilters and particulate matter air filters for air supplies to production areas. 21 CFR 211.4621 CFR 211.46
    43. 43.  Defines critical and controlled manufacturing areas  Recommends airborne nonviable and viable contaminant limits  Provides some guidance on monitoring frequencies for critical areas Guideline on Sterile DrugGuideline on Sterile Drug Products Produced by AsepticProducts Produced by Aseptic ProcessingProcessing
    44. 44.  Recommendations for air pressure differentials  Includes guidance on aseptic media fills  Note: This guidance document was written in 1987 and is in need of revision Guideline on Sterile DrugGuideline on Sterile Drug Products Produced by AsepticProducts Produced by Aseptic ProcessingProcessing
    45. 45.  USP General Information Chapter <1116>  Establishment of clean room classifications ◦ Federal Standard 209E  Importance of EM program  Personnel training in aseptic processing  Establishment of sampling plans and sites ◦ suggested sampling frequencies Microbial Evaluation andMicrobial Evaluation and Classification of Clean Rooms andClassification of Clean Rooms and Clean ZonesClean Zones
    46. 46.  Establishment of alert and action limits  Suggests limits for airborne, surface and personnel contaminant levels.  Methods and equipment for sampling  Identification of isolates  Aseptic media fills  Emerging technologies - barrier; isolator Microbial Evaluation andMicrobial Evaluation and Classification of Clean Rooms andClassification of Clean Rooms and Clean ZonesClean Zones
    47. 47.  “Airborne Particulate Cleanliness Classes in Clean Rooms and Clean Zones  Approved by the GSA for use by all Federal Agencies  Frequently referenced for controlled environment particulate requirements: Classes 100, 10,000 and 100,000 (based on particles > 0.5µ) Federal Standard 209EFederal Standard 209E
    48. 48.  Scope limited to final drug product manufacturing and data required for application submission (NDA, BLA)  Requests information on: ◦ Buildings and facilities ◦ Manufacturing operations for drug product  Filter validation  Validation of hold times Guidance for Industry for Sterile ValidationGuidance for Industry for Sterile Validation Process Validation in Applications for HumanProcess Validation in Applications for Human and Veterinary Drug Productsand Veterinary Drug Products
    49. 49.  Requests information on: ◦ Sterilization and depyrogenation ◦ Media fills and actions taken when they fail ◦ Microbiological monitoring of the environment  Airborne microorganisms, personnel, surfaces, water system, product component bioburden ◦ Yeasts, molds, anaerobes ◦ Exceeded EM limits Guidance for Industry for Sterile ValidationGuidance for Industry for Sterile Validation Process Validation in Applications for HumanProcess Validation in Applications for Human and Veterinary Drug Productsand Veterinary Drug Products
    50. 50. Viable and NonviableViable and Nonviable Contaminant LimitsContaminant Limits Classifi- cation Nonviable (>0.5µ) Viable ft3 m3 ft3 m3 Class 100 100 3,530 0.1 3.5 Class 10,000 10,000 353,000 0.5 18 Class 100,000 100,000 3,530,000 2.5 88
    51. 51.  Preparation or manufacturing area where nonsterile product, in-process materials and product-contact equipment surfaces, containers and closures are exposed to the environment  Control nonviable and viable contaminants to reduce product /process bioburden  Class 100,000 or Class 10,000 Controlled AreaControlled Area
    52. 52.  Capping areas are now considered controlled manufacturing areas ◦ Should be supplied with HEPA filtered air ◦ Should meet class 100,000 conditions during static conditions Controlled AreaControlled Area
    53. 53.  Aseptic processing area where sterile products, components or in-process products are exposed to the environment and no further processing will occur.  Air quality must be Class 100 during processing  Local Class 100 areas are often utilized during open processing steps during drug substance manufacture. Critical AreaCritical Area
    54. 54.  The area just preceding the sterile core should be one classification higher than the core. Critical AreaCritical Area
    55. 55.  Airborne cleanliness classifications should be met during operations  Nonviable monitoring should occur routinely during operations  Monitoring during static conditions is done as part of HVAC qualification and may be done periodically after that to insure area meets acceptable conditions before use or following cleaning Nonviable Particulate MonitoringNonviable Particulate Monitoring
    56. 56.  Locations for monitoring should be established during performance qualification; probes placed close to work surface  Monitoring frequencies vary: ◦ For aseptic processing areas, during each use ◦ For other, controlled areas, varies from each use to weekly or less depending on use of area Nonviable Particulate MonitoringNonviable Particulate Monitoring
    57. 57.  HVAC Validation and Maintenance Considerations: ◦ Air velocity, airflow patterns and turbulence should be validated; smoke studies to determine flow patterns during static and dynamic conditions ◦ HEPA filter integrity testing ◦ HEPA filter efficiency testing ◦ Air pressure differentials Nonviable Particulate MonitoringNonviable Particulate Monitoring
    58. 58.  Airborne viable contaminants  Surface contaminants ◦ walls ◦ equipment surfaces ◦ countertops ◦ floors  Personnel contaminants Microbial MonitoringMicrobial Monitoring
    59. 59.  Monitoring methods should be capable of detecting molds and yeasts  Should also be able to detect anaerobes ◦ Most often, this is an issue associated with products filled anaerobically (with nitrogen overlay)  All lots of media for EM sampling should be growth promotion tested Microbial MonitoringMicrobial Monitoring
    60. 60.  Routine microbial monitoring should take place during operations (for airborne contaminants) and immediately following operations (for surfaces and personnel).  Airborne monitoring frequencies: ◦ Each use for aseptic processing areas ◦ Varies from daily to weekly to less frequently for controlled areas depending on use Microbial MonitoringMicrobial Monitoring
    61. 61.  Personnel and surface monitoring frequencies vary: ◦ Aseptic processing - after every fill ◦ Other controlled areas - varies from daily to weekly or less for surfaces ◦ Personnel monitoring often restricted to aseptic area personnel and personnel working in Class 100 hoods performing tasks such as inoculation Microbial MonitoringMicrobial Monitoring
    62. 62.  Monitoring of surfaces and airborne contaminants during rest periods (following cleaning) ◦ Important for confirming adequacy of cleaning procedures ◦ Indicates whether HVAC system is operating properly ◦ NOTE: Disinfectant effectiveness studies also required for cleaning agents used in the facility Microbial MonitoringMicrobial Monitoring
    63. 63.  Monitoring frequencies and procedures are influenced by a number of factors: ◦ Stage of manufacturing ◦ “Open” or “closed” manufacturing step ◦ Single or multiple product manufacturing Microbial MonitoringMicrobial Monitoring
    64. 64.  Establishment of monitoring locations should be based on performance qualification studies during dynamic conditions ◦ gridding study to determine worst case locations/most meaningful locations  Should also establish common flora - will aid in investigations Microbial MonitoringMicrobial Monitoring
    65. 65.  Action limits (for the most part) have been established in a variety of guidance documents  Alert limits ◦ Lower than action limits ◦ Reflect actual historical results under normal processing conditions Setting Alert and ActionSetting Alert and Action LimitsLimits
    66. 66.  Alert limits are designed to provide some warning that environmental quality is approaching action limit and allow you time to correct.  Exceeding alert limit triggers a warning response - i.e., alert affected area personnel  Exceeding multiple alerts - triggers action level response Exceeding LimitsExceeding Limits
    67. 67.  Action limit excursions require investigations ◦ Speciation of organism(s) ◦ Review batch records from date of excursion ◦ Review other recent EM data (trends) ◦ Review cleaning records ◦ Interview personnel ◦ Product impact - must quarantine until determined Exceeding LimitsExceeding Limits
    68. 68.  Excursions from action limits require corrective actions that may include: ◦ More rigorous or additional monitoring ◦ More rigorous cleaning ◦ Retraining of personnel ◦ Procedural changes - change to or addition of disinfection procedures, for example ◦ HVAC maintenance Exceeding LimitsExceeding Limits
    69. 69.  The investigation procedures to be followed should be pre-established and included in SOPs  Depending on the outcome of the investigation, corrective actions should be pre-established to the extent possible Investigations and CorrectiveInvestigations and Corrective ActionsActions
    70. 70.  Imperative that EM results be linked to product release so that affected products are not released until investigation completed  Material Review Board or equivalent should be consulted prior to releasing product that was potentially affected by adverse environmental conditions Investigations and CorrectiveInvestigations and Corrective ActionsActions
    71. 71.  Should trend monitoring results (environmental and water) ◦ Periodic (quarterly or monthly) review by QA and others ◦ Re-evaluation of action and alert limits on an annual basis ◦ This trending information is generally included in the Annual Product Review TrendingTrending
    72. 72.  Control of temperature and humidity required for aseptic processing areas ◦ 21 CFR 211.42(c)(10)(ii)  Generally 65°F and 35-50% humidity are average ◦ Too high - Increases personnel shedding ◦ Too low - Increase static electricity Temperature and HumidityTemperature and Humidity
    73. 73.  Temperature should be controlled throughout all manufacturing areas  Temperature and humidity should be monitored and controlled in warehouse areas where temperature/humidity sensitive raw materials are stored ◦ If not able to control humidity, need procedure to follow if humidity exceeds limit Temperature and HumidityTemperature and Humidity
    74. 74. Water RequirementsWater Requirements Test Potable Water Purified Water WFI TOC none 500 ppb 500 ppb Conduc- tivity none See USP Table Micro. Purity 500 CFU/ml 100 CFU/ml 10 CFU/ 100 ml Endo- Toxin none none 0.25 EU/ml
    75. 75.  Water purified by distillation or reverse osmosis  Prepared from water complying with the U.S. EPA National Primary Drinking Water Regulations  Contains no added substance Water For InjectionWater For Injection
    76. 76.  Obtained by a suitable process, usually one of the following: ◦ deionization ◦ reverse osmosis ◦ combination Purified WaterPurified Water
    77. 77.  Meets National Drinking Water Regulations  40 CFR Part 141  Periodic monitoring in-house as well as periodic certificates from municipality (if applicable) Potable WaterPotable Water
    78. 78.  WFI Systems ◦ Microbial quality and endotoxin  Daily system monitoring  Each use point at least weekly ◦ TOC and Conductivity  Weekly system monitoring  can be taken from worst case point (end of loop, return to tank) Water System MonitoringWater System Monitoring
    79. 79.  Purified Water Systems ◦ Weekly monitoring of system for:  microbial quality  TOC  conductivity Water System MonitoringWater System Monitoring
    80. 80.  WFI ◦ Solvent for preparation of parenteral solutions ◦ Formulation of mammalian cell culture media ◦ Formulation of purification buffers ◦ Final product formulation ◦ Vial and stopper washing ◦ Final rinse for product equipment Water UseWater Use
    81. 81.  Purified Water ◦ Preparation of terminally sterilized microbiological media ◦ Initial rinsing/cleaning ◦ Laboratory use ◦ Feed for WFI system Water UseWater Use
    82. 82.  Potable Water ◦ Non-product contact uses ◦ Feed for purified water system Water UseWater Use
    83. 83.  Slit-to-Agar (STA) - Powered by vacuum, air taken in through a slit below which is a slowly revolving plate.  Sieve impactor - Vacuum draws in air through perforated cover which is impacted onto petri dish containing nutrient agar Microbial Monitoring DevicesMicrobial Monitoring Devices
    84. 84.  Centrifugal Sampler - consists of a propeller that pulls a known volume of air into the unit and then propels the air outward to impact on a nutrient agar strip  Sterilizable Microbiological Atrium (SMA)- similar to sieve impactor; cover contains uniformly spaced orifices; vacuum draws in air which is impacted on agar plate Microbial Monitoring DevicesMicrobial Monitoring Devices
    85. 85.  Surface Air System Sampler - An integrated unit containing an entry section with an agar contact plate; behind is a motor and turbine that pulls air in through the perforated cover and exhausts it beyond the motor.  Settle plates - qualitative; may be useful in worst case locations Microbial Monitoring DevicesMicrobial Monitoring Devices
    86. 86.  Surface contaminant monitoring devices: ◦ Contact Plates - plates filled with nutrient agar; for regular surfaces ◦ Swabs - useful for hard to reach or irregular surfaces; swab placed in suitable diluent and inoculated onto microbiological plate Microbial Monitoring DevicesMicrobial Monitoring Devices
    87. 87.  Remote sampling probes - validate use of tubing  Must sample adequate quantity of air to be statistically meaningful. ◦ 80-100 ft3/min  Must validate growth promotion after exposure of settle plates (or other plates) for prolonged time periods. Monitoring ConsiderationsMonitoring Considerations
    88. 88. Contamination Control
    89. 89. Methods to AchieveMethods to Achieve CleanlinessCleanliness Positive Pressure / Airflow ◦ Keeps contamination out of the work area ◦ Depends on clean air input  Filtration ◦ Development of effective filtration revolutionized industry ◦ HEPA (High Efficiency Particulate Air) and ULPA (Ultra Low Particulate Air) Filters  Materials Selection  User Protocols  Cleaning
    90. 90. Facility DesignFacility Design  Complete cleanroom created with centralized air handling or fan filter units  Keeps entire room clean  Requires complete gowning, careful materials and equipment selection to maintain class  Costly, often unnecessary
    91. 91. Facility DesignFacility Design  Can use localized clean areas  Clean Benches: Horizontal and Vertical Laminar Flow (HLF on left, VLF on right)
    92. 92. Facility designFacility design  Isolators, Glove boxes provide better protection from outside contamination
    93. 93. Contamination Control byContamination Control by LayoutLayout  Isolation between processes prevents cross contamination; separate rooms, air showers, door interlocks  “Onion” concept: cleanest areas are inside, have to pass through successively cleaner areas to reach these areas
    94. 94. Air Flow & TurbulenceAir Flow & Turbulence  Most airflow is turbulent—no clear relation between velocity vectors at different points •Particles can be trapped in eddies for long time •Not optimal for contamination control!! Long path length for contamination to leave the room
    95. 95. Laminar (Unidirectional) AirLaminar (Unidirectional) Air FlowFlow  Concept of laminar airflow  In cleanrooms, often called uni-directional flow (UDF) • Ideal for contamination control—shortest path to sweep particles out of clean areas; complete room air change in shortest period of time
    96. 96. High level cleanrooms designed for laminar flow in most areas Cost means that for most, clean areas are some combination of laminar and turbulent flow Not always a simple tradeoff—with turbulent flow, require higher air velocities, which require larger air handlers.
    97. 97. UDF More Important forUDF More Important for Cleaner AreasCleaner Areas
    98. 98. Practical ConsiderationsPractical Considerations for UDFfor UDF  Any objects in path of laminar flow will deflect airflow—this usually results in turbulence; USER BEHAVIOR HAS LARGE IMPACT •Most critical for laminar flow benches situated in non-clean areas; not as critical if located in larger clean area
    99. 99. Types of ContaminationTypes of Contamination  Particulate—encompasses most contamination  Chemical—films, vapors, etc.  Biological—bacteria, viruses, etc.; for our purposes, treat as particles  Similar concerns for rooms & equipment as for substrates
    100. 100. Airborne ContaminationAirborne Contamination From Cleanrooms Magazine, 2000 Invisible to naked eye below ~50um without special illumination
    101. 101. Particulate ContaminationParticulate Contamination  Biggest concern for LCI cleanroom users  Basis for classification of cleanrooms  Does include biological contamination as a subset of total particulates  Many sources: personnel, equipment, etc.
    102. 102. Microbial ContaminationMicrobial Contamination  Outer layer of human skin can host up to 1 million microorganisms per square cm  Human saliva up to 1 billion per mL  Bacteria is usually primary concern, but foreign organic matter, viruses, fungi, algae are all included here  Cross contamination can be a big problem.
    103. 103. ContaminationContamination MeasurementMeasurement  Particulate contamination typically measured with laser particle counter  Microbial contamination can be measured in several ways ◦ Centrifugal sampler ◦ Settle plate method ◦ Contact plate method ◦ Swabbing
    104. 104. Usage of MeasurementsUsage of Measurements  Complementary to yield tracking  Can use measurements to isolate problem areas  Regular measurements can help to track changes, which can then be tied back to protocol, personnel, or material changes ◦ Don’t depend upon room to maintain itself.
    105. 105. RealityReality  In a perfect world, could monitor many points on a very regular basis  In reality, this is usually not practical, due to personnel time and financial constraints  Important to identify a realistic test & measurement program
    106. 106. Contamination Control andContamination Control and Its RelationshipsIts Relationships  All sources of contamination and control are interrelated
    107. 107. CleaningCleaning  Critical to remove contaminants that cannot be removed by air handling  Important to follow procedures appropriate to your application  What is appropriate for one industry may not be appropriate for another  Most important thing is to develop standard procedures and FOLLOW THEM
    108. 108. Surfaces are importantSurfaces are important  The efficiency of these cleaning methods depends on the surface being cleaned  Rough or pitted surfaces are more difficult to clean  Sharp corners are difficult to clean  That’s why inside surfaces of clean rooms are smooth.
    109. 109. VacuumingVacuuming  Dry and wet ◦ Dry has low (<25% ) efficiency for particles smaller than 10 microns (about .0005 inches) ◦ wet uses liquids which result in greater force on the particles and hence better cleaning
    110. 110. Wet wipingWet wiping  Can be very efficient  Liquid breaks some bonds between surface and particles and allows particles to float off  Those adhering on surface can be rubbed off and retained in wiper.  Must be careful not to redeposit particles  Efficiency varies
    111. 111. Tacky rollersTacky rollers  Efficiency depends of tackiness of roller, cleanliness of tacky surface and softness of roller are also very important
    112. 112. Cleaning liquidsCleaning liquids  No ideal cleaning liquid  Most facilities use DI water or isopropyl alcohol with disinfectant  Water with surfactant and disinfectant may be used as well as alcohol-water solutions  The choice depends on what works, cost, history, etc.
    113. 113. Materials SelectionMaterials Selection  Choice of materials for supplies, equipment, gowning, etc. is important  “Clean” materials can become dirty!!  Look for easy-to-clean materials  Triboelectricity can cause static problems, as can low humidity—this exacerbates contamination problems  Biofilms!!
    114. 114. General RequirementsGeneral Requirements  Minimize sources of contaminants ◦ No smoking ◦ No cosmetics ◦ Avoid high particulate clothing, such as wool sweaters ◦ Cover up! Uncovered skin can lead to more contamination