This document discusses key considerations for the aseptic manufacturing of sterile pharmaceutical products. It covers classification of clean areas, environmental monitoring, preparation and filtration of solutions, personnel requirements, equipment sterilization, and validation of aseptic processes. The main objectives are to prevent microbial contamination and maintain sterility throughout manufacturing.

1. By
SUNILBOREDDY
M.Pharmacy

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. Aseptic processing
 Objective is to maintain the sterility of a product,
assembled from sterile components
 Operating conditions so as to prevent microbial
contamination

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. 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. 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. 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.  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.  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. Environmental Monitoring
 Physical
◦ Particulate matter
◦ Differential pressures
◦ Air changes, airflow patterns
◦ Clean up time/recovery
◦ Temperature and relative humidity
◦ Airflow velocity

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. 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. 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. 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. 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. 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. 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. 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.  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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.  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.  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.  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. Environmental MonitoringEnvironmental Monitoring
ConsiderationsConsiderations

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.  Water monitoring:
◦ Total organic carbon
◦ Conductivity
◦ Microbial Contaminants
◦ Endotoxin
Environmental MonitoringEnvironmental Monitoring
ComponentsComponents

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.  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.  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.  CFR GMP regulations
 FDA Guidance Documents
 USP Informational Chapter
Regulatory Basis forRegulatory Basis for
Environmental MonitoringEnvironmental Monitoring
ProgramProgram

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.  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.  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.  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.  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.  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.  “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.  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.  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. 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.  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.  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.  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.  The area just preceding the sterile core should be
one classification higher than the core.
Critical AreaCritical Area

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.  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.  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.  Airborne viable contaminants
 Surface contaminants
◦ walls
◦ equipment surfaces
◦ countertops
◦ floors
 Personnel contaminants
Microbial MonitoringMicrobial Monitoring

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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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. 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.  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.  Obtained by a suitable process, usually one of the
following:
◦ deionization
◦ reverse osmosis
◦ combination
Purified WaterPurified Water

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.  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.  Purified Water Systems
◦ Weekly monitoring of system for:
 microbial quality
 TOC
 conductivity
Water System MonitoringWater System Monitoring

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.  Purified Water
◦ Preparation of terminally sterilized microbiological media
◦ Initial rinsing/cleaning
◦ Laboratory use
◦ Feed for WFI system
Water UseWater Use

82.  Potable Water
◦ Non-product contact uses
◦ Feed for purified water system
Water UseWater Use

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.  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.  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.  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.  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. Contamination Control

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. 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. Facility DesignFacility Design
 Can use localized clean areas
 Clean Benches: Horizontal and Vertical
Laminar Flow (HLF on left, VLF on right)

92. Facility designFacility design
 Isolators, Glove boxes provide better protection
from outside contamination

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

95. 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

96. 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

97. 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.

98. UDF More Important forUDF More Important for
Cleaner AreasCleaner Areas

99. 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

100. 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

101. Airborne ContaminationAirborne Contamination
From Cleanrooms
Magazine, 2000
Invisible to naked
eye below ~50um
without special
illumination

102. 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.

103. 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.

104. 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

105. 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.

106. 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

107. Contamination Control andContamination Control and
Its RelationshipsIts Relationships
 All sources of contamination and control are
interrelated

108. 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

109. 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.

110. 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

111. 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

112. Tacky rollersTacky rollers
 Efficiency depends of tackiness of roller,
cleanliness of tacky surface and softness of
roller are also very important

113. 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.

114. 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!!

115. 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