Professional Documents
Culture Documents
SUNILBOREDDY
M.Pharmacy
Certain pharmaceutical products must be
sterile
◦ injections, ophthalmic preparations, irrigations
solutions, haemodialysis solutions
Table 1
Classification of Clean Areas
◦ Classified in terms of airborne particles (Table 2)
Grade At rest In operation
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
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
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
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)
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)
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
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
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
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
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
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
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)
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
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
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
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
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
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
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)
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 (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
Environmental Monitoring
Considerations
Environmental Monitoring
Components
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 Monitoring
Components
Water monitoring:
◦ Total organic carbon
◦ Conductivity
◦ Microbial Contaminants
◦ Endotoxin
General Environmental Monitoring
Considerations
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 Environmental Monitoring
Considerations
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
Scope of Environmental Monitoring
Program
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)
Regulatory Basis for Environmental
Monitoring Program
CFR GMP regulations
FDA Guidance Documents
USP Informational Chapter
21 CFR 211.42
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 Monitoring
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 Use
Purified Water
◦ Preparation of terminally sterilized microbiological
media
◦ Initial rinsing/cleaning
◦ Laboratory use
◦ Feed for WFI system
Water Use
Potable Water
◦ Non-product contact uses
◦ Feed for purified water system
Microbial Monitoring Devices
Most airflow is
turbulent—no clear
relation between
velocity vectors at
different points
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
Airborne Contamination
From Cleanrooms
Magazine, 2000
Invisible to naked
eye below ~50um
without special
illumination
Particulate Contamination