A brief review on ISO 14644 Standard on Cleanroom Design and testings. With a look at PIC/S and WHO standards

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Tehran University of
Medical Sciences
School of Pharmacy
Cleanrooms
Classification, Design and Testing
Ahmadreza Barazesh
Under the supervision of Dr. Vatanara

2. Cleanrooms Slide 2 of 68
November 2014
Tehran University of
Medical Sciences
School of Pharmacy
References
 ISO 14644
– Part 1: Classification of air cleanliness
– Part 2: Continued compliance with
– Part 3: Metrology and test methods
– Part 4: Design, construction and start-up
 WHO Technical Report Series, No. 902, 2002
– Annex 6: Good manufacturing practices for sterile pharmaceutical products
 WHO Technical Report Series, No. 961, 2011
– Annex 5: WHO guidelines on good manufacturing practices for heating, ventilation and air-conditioning systems for
non-sterile pharmaceutical dosage forms
– Annex 6: WHO good manufacturing practices for sterile pharmaceutical products
 PIC/S GMP Guide (Part I: Basic Requirements For Medicinal Products)
 PIC/S Guide To Good Manufacturing Practice For Medicinal Products - Annexes
Disclaimer: The TUMS logo included, neither indicates that the lecturer is an official lecturer of TUMS nor the content
is approved by TUMS. It is just indicative of the department in which the lecture was prepared and presented.

3. Cleanrooms Slide 3 of 68
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Scope
 What will be covered during this presentation:
 A Brief review on cleanroom classification
 Introduction of Design Concepts and Considerations based on ISO
14644 Series Standards, PIC/S and WHO Guidelines.
 Testing Methods and Procedures According to

4. Cleanrooms Slide 4 of 68
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Introduction
 Cleanrooms provide for the control of airborne contamination
to levels appropriate for accomplishing contamination-sensitive
activities.
– Aerospace,
– Microelectronics,
– Pharmaceuticals,
– Medical devices,
– Healthcare (Hospitals)
– Food.

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Definitions
 Cleanroom: room in which:
– The concentration of airborne particles is controlled,
– Constructed and used in a manner to minimize the introduction, generation,
and retention of particles inside the room,
– Other parameters (temperature, humidity, and pressure) are controlled
 Installation: cleanroom or one or more clean zones, together with all
associated structures, air-treatment systems, services, and utilities.
 Classification: level of airborne particulate cleanliness,
represents maximum allowable concentrations (in particles per
cubic metre of air) for considered sizes of particles

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Definitions
 Particle: Solid or liquid object which, for purposes of classification of air
cleanliness, falls within a threshold size in the range from 0.1 to 5μm
 Occupancy states
 As-built: installation is complete, all services functioning, no
production equipment, materials, or personnel present
 At-rest: no personnel present
 Operational: the installation is functioning in the specified
manner, specified number of personnel present and working

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Classification
 The particulate cleanliness of air shall be defined in one or
more of three occupancy states, viz. “as-built”, “at-rest”, or
“operational”
 The maximum permitted concentration of particles, Cn, for
each considered particle size, D,
 In which, N is the ISO classification number, which shall not
exceed a value of 9. (ISO Class 1 to 9)

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Classification

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Classification
Graphical representation of ISO-class concentration limits for selected ISO classes

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Classification
 PIC/S Guide To GMP For Medicinal Products Annex 1

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Classification
 WHO Technical Report Series, No. 902, 2002 Annex 6

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Classification
 WHO Technical Report Series, No. 902, 2002 Annex 6
This comparison is defined based on at-rest limitations.

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Classification
Class
maximum particles/m3
FED STD
209E
equivalent
EU GMP
≥0.1 μm ≥0.2 μm ≥0.3 μm ≥0.5 μm ≥1 μm ≥5 μm Classification
ISO 1 10 2.37 1.02 0.35 0.083 0.0029
ISO 2 100 23.7 10.2 3.5 0.83 0.029
ISO 3 1,000 237 102 35 8.3 0.29 Class 1
ISO 4 10,000 2,370 1,020 352 83 2.9 Class 10
ISO 5 100,000 23,700 10,200 3,520 832 29 Class 100
Grade A and
Grade B
ISO 6 1.0×106 237,000 102,000 35,200 8,320 293 Class 1,000
ISO 7 1.0×107 2.37×106 1,020,000 352,000 83,200 2,930 Class 10,000 Grade C
ISO 8 1.0×108 2.37×107 1.02×107 3,520,000 832,000 29,300 Class 100,000 Grade D
ISO 9 1.0×109 2.37×108 1.02×108 35,200,000 8,320,000 293,000 Room air

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Classification
 PIC/S Guide To GMP For Medicinal Products Annex 1

15. Classification: Designation
 The designation of airborne particulate cleanliness for clean rooms and
clean zones shall include:
– the classification number, expressed as “ISO Class N”;
– the occupancy state
– the considered particle size(s), and the concentration(s), 0,1μm through 5 μm.
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 Example designation:
– ISO Class 4; operational state; considered sizes: 0,2μm (2 370 particles/m3), 1 μm
(83 particles/m3)

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 Airborne particle physical control:
– Filtration (HEPA)
– Dilution (Higher Airchange Rate)
– Isolation HEPA
class
retention (total) retention (local)
E10 > 85% ---
E11 > 95% ---
E12 > 99.5% ---
H13 > 99.95% > 99.75%
H14 > 99.995% > 99.975%
U15 > 99.9995% > 99.9975%
U16 > 99.99995% > 99.99975%
U17 > 99.999995% > 99.9999%

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Classification: PIC/S
 Grade A: The local zone for high risk operations:
– Filling zone, open ampoules and vials, making aseptic connections.
– Provided by a LAF work station with a homogeneous air speed in a
range of 0.36 – 0.54 m/s (guidance value)
– A unidirectional air flow and lower velocities may be used in closed
isolators and glove boxes.
 Grade B: For aseptic preparation and filling, this is the background
environment for the grade A zone.
 Grade C and D: Clean areas for carrying out less critical stages in the
manufacture of sterile products.

18. PIC/S General Paragraphs
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 Clean Areas:
– Entry through airlocks for personnel and/or for equipment and materials.
– Supplied with air has passed through filters of an appropriate efficiency.
 The various operations of component preparation, product preparation
and filling  in separate areas within the clean area
 Manufacturing operations:
– Product is terminally sterilized,
– Conducted aseptically at some or all stages.
 In order to meet “in operation” conditions, areas should be designed to
reach certain air-cleanliness levels in the “at rest” occupancy state.

19. PIC/S General Paragraphs
 “In operation” classification may be demonstrated during normal
operations, simulated operations or during media fills (worst-case)
 Clean rooms and clean air devices should be routinely monitored
– Monitoring locations based on risk analysis and the results of classification
– Grade A: full duration of critical processing
– Grade A: Such a frequency and sample size that all interventions, transient
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events is captured and alarms triggered
– Grade B: The same as grade A; the sample frequency may be decreased.
– Grade C and D: in accordance with the principles of quality risk management.

20. PIC/S General Paragraphs
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 Terminally Sterilized Products
– Preparation of components and most products should be done in at
least a grade D environment
• Where the product is at a high or unusual risk of microbial contamination 
Grade C
– Filling of products for terminal sterilization  Grade C
• Where the product is at unusual risk of contamination from the environment,
filling  Grade A with Grade C background.
– Preparation and filling of ointments, creams, suspensions and
emulsions should  grade C before terminal sterilization

21. PIC/S General Paragraphs
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 Aseptic Preparation
– Components after washing  Grade D
– Handling of sterile starting materials, unless subjected to
sterilization or filtration  Grade A with Grade B background.
– Otherwise  Grade C
– Handling and filling of aseptically prepared products  Grade A
– Transfer of partially closed containers, as used in freeze drying,
 either in a Grade A environment with grade B background or in
sealed transfer trays in a grade B environment

22. PIC/S Paragraphs on Premises
 All exposed surfaces should be smooth, impervious and unbroken
 To reduce accumulation of dust and to facilitate cleaning there should
be no uncleanable recesses and a minimum of projecting ledges,
shelves, cupboards and equipment.
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 False ceilings should be sealed.
 Sinks and drains should be prohibited in grade A/B areas
 Changing rooms should be designed as airlocks, The final stage of the
changing room should, in the at-rest state, be the same grade as the
area into which it leads.

23. PIC/S Paragraphs on Premises
 Both airlock doors should not be opened simultaneously; interlocking
system or a visual and/or audible warning system should be operated.
 A filtered air supply should maintain a positive pressure and an air flow
relative to surrounding areas of a lower grade. a pressure differential of
10-15 pascals
 It should be demonstrated that air-flow patterns do not present a
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contamination risk.
 A warning system should be provided to indicate failure in the air
supply.

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Planning and Design
 A project plan shall be developed to define the requirements of the
products, the processes and the scope of the installation.
 A process equipment list shall be compiled, and shall include the critical
requirements for each piece of process equipment.
 Diversity factors shall be defined, considering peak and average
demand for each utility and environmental control system.
 A contamination control concept shall be developed for each zone of an
installation.

25. Design: Control and segregation concepts
 For economic, technical and operational reasons, clean zones are often
enclosed or surrounded by further zones of lower cleanliness.
 The zones with the highest cleanliness demands is reduced to the
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minimum size.
 Movement of material and personnel between adjacent clean zones
gives rise to the risk of contamination transfer,
 management of material and personnel flow

26. Design: Control and segregation concepts
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27. Design: Personnel flow and Material flow
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 Personnel flows considered:
– Manufacturing personnel
– Maintenance personnel
– Quality control personnel
 Material flows considered:
– Raw materials
– Finished goods
– Waste
– Product (In-process, Intermediate & Final)
– Equipment
• Clean and dirty components
• Portable equipment
• Product containers

28. Design: Personnel flow and Material flow
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29. Design: Personnel flow and Material flow
Desirable Layout Less Desirable Layout
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Design: Air Flow Patterns
 Air flow patterns:
– Cleanroom airflow patterns can be categorized as either
unidirectional or non-unidirectional (or mixed)
 Unidirectional airflow
– ISO Class 5 and cleaner
– may be either vertical or horizontal
– airflow rely upon a final filtered air supply and
– return inlets are nearly opposite air supplies to maintain the
airstream straight

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Design: Air Flow Patterns
 non-unidirectional airflow cleanrooms
– Air flow outlets located in multiple positions. Filter outlets may be
distributed at equal intervals or grouped over the core process.
– The final filter location may be remote, (avoid contamination ingress
between filters and cleanroom)
– Return air locations in non-unidirectional airflows are not as critical
– Distribute the returns to minimize dead zones within the cleanroom

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33. Disturbance of unidirectional airflow
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34. Contamination Control Concepts
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36. Design: Segregation Concepts
 In order to protect cleanrooms from contamination from adjacent less
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clean spaces
 Displacement concept (low pressure differential, high airflow)
– by means of a low turbulent "displacement" airflow, >0,2 m/s
 Pressure differential concept (high pressure differential, low airflow)
– The pressure differential in the range of
5 - 20 Pa, to allow doors to be opened
and to avoid unintended turbulence.
 Physical barrier concept
– Use of an impervious barrier to prevent contamination transfer to a
clean zone from a less clean zone.

37. Design: Layout of an installation
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 General Considerations:
 Size: of cleanroom should be minimum. if a large space is required, it
should be divided, with or without physical barriers.
 Workstation siting and organization: critical workstations away from,
major traffic pathways.
less clean operations site downstream of cleaner operations.
 Ancillary areas and adjacent cleanrooms:
– Pressure or flow differentials,
– Access and communication arrangements
(such as airlocks, speech panels and intercoms
cross-contamination from less
clean zones does not
compromise the cleaner zones.

38. Design: Layout of an installation
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 General Considerations (contd.)
 Utility services and ancillary equipment
– General: Utility services should be designed and installed such that
the cleanroom is not compromised by contamination.
– exposed piping, tubing and cable runs should be minimized,
– Vacuum-cleaning equipment
– Sprinkler systems
– Communication systems: to reduce personnel movement
– Glazing: Avoid heat loss and solar gain, non-opening double glaze

39. Design: Layout of an installation
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 Access:
 General: The number of openings should be minimized.
Normal (non-emergency) access should be through airlocks for both
personnel and material.
 Airlocks: In order to maintain pressure differential and integrity of
during entry and exit, airlocks or transfer hatches (pass-throughs) are
normally required.
 Emergency exits: Emergency exits should be provided with means
to show that they have been opened.

40. Design: Layout of an installation
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 Access (contd.)
 Changing rooms: Have three functional zones:
– Entry: access from ancillary. appropriate for removal, storage,
disposal and/or redonnning of garments not permitted within the
cleanroom;
– Transition zone: where garments or personal equipment dedicated
to the cleanroom are stored, donned or removed.
– Inspection/access zone: where inspection of the completed
gowning is accomplished and provides access to cleanroom.
 The three functional zones may be separated by a physical barrier (e.g.
a stepover bench or airlock)

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Changing Rooms

42. Design: Layout of an installation
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 The following requirements should be defined:
– number of people passing through the gowning procedure
– the gowning procedure (i.e. what garments are to be taken off and put on)
– the frequency of garment replacement.
 Consideration should be given to the following provisions:
– Storage and disposal of garments;
– Storage before use and disposal of consumable items
– Storage of personal items;
– Hand-washing and drying or other decontamination processes;
– Display or posting of gowning sequence, with clear instructions;
– full-length mirrors to check effective fit.

43. Design: Construction and materials
 The materials used should be selected to meet the requirements of the
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installation, and should take into account the following:
a) the cleanliness class;
b) effects of abrasion and impact;
c) cleaning and disinfection methods and frequencies;
d) chemical/microbiological attack and corrosion.
 Surface cleanliness and cleanability of materials of construction
 Fittings in airlocks: Minimum horizontal surfaces

44. Design: Construction and materials
 Ceilings: Ceilings should be sealed, penetration points should be kept
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minimum.
 Walls: Materials and surface finishes should meet all general requirements.
–Particular consideration to impact and abrasion. (rubbing strips, protective bars)
–Cover strips or seals between panels should be smooth, with rounded edges
–Use double glazing, with airtight seal, which can enable flush mounting
–Doors should present as few horizontal surfaces as possible, thresholds avoided.
–Consider use of push plates, automatic openings, or appropriate door-swing
 Floors: Floors or floor coverings should be non-porous, slip-resistant, abrasion-resistant,
conductive if necessary.

45. Design: Construction and materials
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46. Design: Control of air Cleanliness
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 Air filtration systems
– Air filtration systems including filter elements, mounting frames,
housings, gaskets, sealants and clamping systems should be
selected to suit both the cleanliness and using condition.
– Three basic stages of air filtration are recommended:
• prefiltering of the outside air to ensure adequate quality of air
supply
• secondary filtering in the air conditioning plant to protect the
final filters;
• final filtering before cleanroom supply.
– “Sacrificial" filters or temporary filters: considered to protect the air
cleanliness of air-handling systems during construction and
commissioning.

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HVAC Systems

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HVAC Systems

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HVAC Systems

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Tests Methods
 Cleanroom tests:
– Required Tests: An airborne particle count test shall be carried out
in order to classify an installation
– Optional Tests:
• Airborne particle count for ultrafine and/or Micro-particles
• Airflow test
• Air pressure difference tests
• Installed filter system leakage test
• Air flow direction tests and visualization
• Temperature, Humidity and Electrostatic tests
• Particle deposition tests
• Recovery tests
• Containment leak tests

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Tests Methods
Airborne particle count for classification and test measurement:
 Measurement of airborne particle concentrations with size 0.1 - 5 μm.
 A discrete-particle-counting, light-scattering instrument is used to
determine the concentration of airborne particles.
 Prior to testing, verify that all aspects of the cleanroom and functioning
in accordance with specifications.
– Airflow rate or velocity tests;
– Pressure difference test;
– Containment leakage test;
– Filter leakage test.

52. Tests Methods
Airborne particle count for classification and test measurement:
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 Sampling
– Minimum number of sampling point locations:
– Sampling locations evenly distributed, at the height of the work activity.
– Sample a sufficient volume of air that a minimum of 20 particles would be
detected if the particle concentration for the largest considered
particle size were at the class limit for the designated ISO class.
– The volume sampled at each location shall be at least 2 litres, with a minimum
sampling time at each location of 1 min.
– Compute the overall mean of the averages, standard deviation, and 95% upper
confidence limit from the average particle concentrations for all locations.

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Tests Methods
 Airborne particle count for ultrafine particles
– Smaller than 0,1μm
– DPC, with a capability for accurate particle size definition up to at least 1μm.
– Condensation nucleus counter (CNC)
– Small sampling flow & long sampling tube  diffusion loss.
 Airborne particle count for macro-particles
– Larger than 5 μm.
– There are two general categories of macroparticle measurement methods.
• collection by filtration or inertial effects, followed by microscopic
measurement
• in situ measurement of the concentration and size of macroparticles with a
time-of-flight particle counter or DPC

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Tests Methods
 Two general categories of macroparticle measurement methods:
– collection by filtration or inertial effects, followed by microscopic measurement of
the number and size, or measurement of the mass of collected particles:
• filter collection and microscopic measurement will report macroparticles
using particle size based upon the agreed diameter;
• cascade impactor collection and microscopic measurement will report
macroparticles using particle size based upon the microscopist's choice of
reported particle diameter;
• cascade impactor collection and weight measurement will report
macroparticles using particle size based upon an aerodynamic diameter;
– In situ measurement of the concentration and size of macroparticles with a time-of-
flight particle counter or a DPC:
• DPC measurement of particle size based upon an equivalent optical
diameter;
• Time-of-flight particle size measurement based upon an aerodynamic
diameter.

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Tests Methods
Airflow Test
 To measure airflow velocity and uniformity, and supply airflow rate
 Measurement of velocity distribution is necessary in unidirectional airflow
cleanrooms, and supply airflow rate in non-unidirectional cleanrooms.
 Supply airflow rate (air volume supplied to the clean installation per unit
of time) can also be used to determine the air changes.
 Airflow rate is measured either downstream of final filters or in air
supply ducts; both methods rely upon measurement of velocity of air
passing through a known area.

56. Tests Methods: Air Flow Test
Procedure for unidirectional airflow installation test
 Supply airflow velocity
– Measured at approximately 150-300 mm from the filter face.
– Number of measuring points should be the square root of 10 times
of area in m2 but no less than 4. At least 1 point for each filter outlet
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 Uniformity of velocity within the cleanroom
– measured at approximately 150-300 mm from the filter face and the
subdivision into grid cells should be defined
 Supply airflow rate measured by filter face velocity
– The results of the airflow velocity test can be used to calculate the
total supply airflow rate.
 Supply airflow rate in air ducts
– by volumetric flowmeters (orifice meters, Venturi meters and anemometers)

57. Tests Methods: Air Flow Test
Procedure for non-unidirectional airflow installation test
 Air volume supply rate and air-change rate are the most important
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parameters.
 Supply airflow rate measured at the inlet
– Because of local turbulence, use of a flowhood that captures all of the air
issuing from each supply diffuser is recommended.
 Supply airflow rate calculated from filter face velocity
– Evaluation of the supply airflow rate without a flowhood may be done with
an anemometer downstream of each final filter. The supply airflow rate is
determined from the airflow velocity multiplied by the area of exit.

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Test Methods
Air pressure difference test
 Verify the capability of the complete installation to maintain the
specified pressure difference between separate spaces
 With all doors closed, the pressure difference between the cleanroom
and any surrounding should be measured and recorded.
 The following should be considered:
– installation of permanent measuring points;
– take measurements near the middle of the cleanroom
away from any supply air inlets or return air outlet.

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Test Methods
Installed filter system leakage test
 To confirm that the filter system is properly installed and that leaks have
not developed
 Introducing an aerosol challenge upstream of the filters and scanning
immediately downstream of the filters and support frame or by sampling
in a downstream duct.
 Applied to cleanrooms in “as-built” or in “at-rest” occupational states,
and when commissioning new cleanrooms, or existing installations
require re-testing, or after the final filters have been replaced
 Detection of leakage by Scanning / Stationary remeasuring

60. Test Methods: Filter Leakage Test
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Test Methods
 Apparatus and materials for installed filter system leakage tests
– Aerosol photometer
– Discrete-particle counter (DPC)
– Suitable pneumatic or thermal aerosol generator(s)
– Suitable aerosol dilution system.
– Suitable aerosol source substances

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Test Methods
Airflow direction test and visualization
 To confirm that the airflow direction and its uniformity conform to the
design and performance specifications
 can be performed by the following four methods:
1. Tracer thread method;
silk threads, single nylon fibers, flags or thin film tapes and effective lighting
2. Tracer injection method;
tracer particles illuminated by high intensity light sources (DI Water ,
alcohol/glycol)
3. Airflow visualization method by image processing techniques; (Quantitative)
4. Airflow visualization method by the measurement of velocity distribution.

63. Test Methods: Air Flow Visualization
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64. Cleanrooms Slide 64 of 68
November 2014
Tehran University of
Medical Sciences
School of Pharmacy
Test Methods
 Temperature test
– Capability to maintain the air temperature level within the control
– Measured at a minimum of one location for each temperature-controlled
zone.
– Measurement time should be at least 5 min with one value
recorded at least every minute.
– Comprehensive temperature test:
• At least 1 h after the air-conditioning system has been operated
• The number of measuring locations should be at least two.
• Probe should be positioned at work-level height and at a
distance of no less than 300 mm from the ceiling, walls, or floor
of the installation

65. Cleanrooms Slide 65 of 68
November 2014
Tehran University of
Medical Sciences
School of Pharmacy
Test Methods
 Humidity test
– Capability to maintain the air humidity level
– Expressed as relative humidity or dew point
– The sensor should be located at least at one location for each
humidity control zone, and sufficient time should be allowed for the
sensor to stabilize.
– The measurement time should be at least 5 min.

66. Cleanrooms Slide 66 of 68
November 2014
Tehran University of
Medical Sciences
School of Pharmacy
Test Methods
 Particle deposition test
– Sizing and counting particles that can be deposited from the air
onto product or work surfaces in the installation.
– Particles are collected on witness plates with surface
characteristics similar to those of the at-risk surface
– Are sized and counted using optical microscopes, electron
microscopes, or surface scanning apparatus.
– The witness plate should be placed in the same plane as the at-risk
surface. And at the same electrical potential as the test surface.

67. Cleanrooms Slide 67 of 68
November 2014
Tehran University of
Medical Sciences
School of Pharmacy
Test Methods
 Recovery test
– Ability of the installation to eliminate airborne particles.
– Only important and recommended for non-unidirectional airflow
systems
– This test is not recommended for ISO Classes 8 and 9.
– 100:1 recovery time is defined as the time required for decreasing
the initial concentration by a factor of 0,01

68. Cleanrooms Slide 68 of 68
November 2014
Tehran University of
Medical Sciences
School of Pharmacy
Test Methods
 Containment leak test
– Determine if there is intrusion of contaminated air into the clean
zones from non-controlled areas
– Particle concentration outside should be greater than the
cleanroom concentration by a factor of 103. If the concentration is
less, generate an aerosol.
– To check for leakage through construction joints, cracks or service
conduits, scan inside the enclosure at a distance of not more than 5
cm from the joint, at a scan rate of approximately 5 cm/s.