This document provides information about cleanrooms, their classification, design, and testing. It defines cleanrooms and classifications based on maximum allowable particle concentrations. ISO classification ranges from 1 to 9, with lower numbers indicating cleaner rooms. Design considerations include personnel and material flows, air flow patterns to minimize contamination, construction materials for cleanability, and HVAC systems for air filtration and pressure differentials between zones. Parameters like particle levels, air changes, temperature and humidity are monitored regularly to maintain cleanroom quality.

1. School of Pharmacy
Cleanrooms
Classification, Design and Testing

2. 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.

3. 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

4. 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

5. 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)

6. Classification

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

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

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

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

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

12. 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.
 Example designation:
– ISO Class 4; operational state; considered sizes: 0,2µm (2 370 particles/m3), 1 µm
(83 particles/m3)

13.  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%

14. 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.

15. PIC/S General Paragraphs
 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

16. PIC/S General Paragraphs
 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

17. 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.
 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.

18. 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
contamination risk.
 A warning system should be provided to indicate failure in the air
supply.

20. Design: Control and segregation concepts

21. Design: Personnel flow and Material flow
 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

22. 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

23. 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

25. Disturbance of unidirectional airflow

26. Contamination Control Concepts

28. Design: Construction and materials
 The materials used should be selected to meet the requirements of the
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

29. Design: Control of air Cleanliness
 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.

30. HVAC Systems

31. FUNCTIONS OF HVAC
 To Control Temperature.
 To Control Humidity.
 To Develop Differential Pressure.
 To Prevent Cross Contamination.
 To Maintain proper Air Movement.

32. APPLICATION
S :
▶ Product protection:
protect from contamination , cross contamination,
prevent contamination by operatives, correct
conditions of humidity and temperature.
▶ Personnel protection:
prevent contact with dust, prevent contact with
fumes, good comfort condition
▶ Environment protection:
No dust discharge, no fumes discharge, no effluent
discharge
▶ Preservation of materials and equipment
Handling, holding and storage

33. TYPES OF HVAC
2. Split system
There are two types of Air-Conditioning system.
1. Ducted
2. Non Ducted
 Ducted
It includes,
 Chilled Water System
 Dx System (Direct Expansion)
 Non Ducted
It includes
1. Window System

34. TYPES OF HVAC
Ducted System:
Chilled water system.
In chilled water system the refrigeration system in chiller is used to first chill the
water, which is then used to chill the air used for cooling the rooms or spaces.
Technically speaking, water can be classified as a refrigerant
Chilled water system is indirect method of cooling the air.
A typical chiller uses the process of refrigeration to chill water in a chiller barrel.
This water is pumped through chilled water piping to the building AHU where it
will pass through a coil.
Air is passed over this coil and the heat exchange process takes place. The heat in
the air is absorbed into the coils and then into the water.
The water is pumped back to the chiller to have the heat removed. It then makes
the trip back to the building AHU and the coils.

35. TYPES OF HVAC
Chilled water system:
Chiller.
Many chillers have cooling towers where the heat removed in the chiller
barrel is transferred to another barrel.
It is the condenser barrel where the refrigerant is condensed and sent back to
the evaporator barrel to remove the heat. The process is in reverse in the
condenser barrel. The water absorbs heat from the refrigerant and allows it to
condense.
The water is then transferred to a cooling tower where the heat in this
water is removed to the atmosphere.
 Once the heat is removed from the water it is pumped back to the chiller
barrel to absorb more heat from the refrigerant.

36. TYPES OF HVAC
Ducted System:
Direct Expansion (DX Type).
In the DX system the air used for cooling the room or space is directly passed
over the cooling coil of the refrigeration plant.
DX is direct type of cooling air.
In the direct expansion or DX types of air conditioning plants the air used for cooling
space is directly chilled by the refrigerant in the cooling coil of the air handling unit
(AHU).
Since the air is cooled directly by the refrigerant, the cooling efficiency of the
DX plants is higher.
However, it is not always feasible to carry the refrigerant piping to the large distances
hence, direct expansion or the DX type of central air conditioning system is usually
used for cooling the small buildings or the rooms on the single floor.

37. TYPES OF HVAC
Non Ducted System:
Window system.
 Window system is the most commonly used air conditioner for single
rooms.
 In this air conditioner all the components, namely the compressor, condenser,
expansion valve or coil, evaporator and cooling coil are enclosed in a single box.
 This unit is fitted in a slot made in the wall of the room, or more
commonly a window.
Split System.
The split air conditioner comprises of two parts,
i. Outdoor unit
ii. Indoor unit
 The outdoor unit, fitted outside the room contain components like the
compressor, condenser and expansion valve.
 The indoor unit comprises the evaporator or cooling coil and the cooling fan.

38. HVAC Systems

39. HVAC SYSTEM :
▶ Can be simply said to be a utility system used to provide air ventilation, heating,
cooling and air conditioning services to a building or a
pharmaceutical space for drug manufacturing.(1,2,3)
▶ COMPONENT OF HVAC :
▶ Ducting ( for delivery of controlled air)
▶ Fan component
▶ Vibration isolator (flex joint)
▶ Heating and /or cooling coil
▶ HEP
AFilters
▶ Damper ( fixed adjustment of volume of air)
▶ Dehumidifiers
▶ Flow rate controller
▶ Humidity, Temperature, Pressure sensors, alarms and audit log system
▶ Dust extractors

41. Air handling unit:

43. MATERIAL OF CONSTRUCTION:
1. Aluminum
2. Mild steel
3. Stainless steel
4. Plastic

45. HVAC Systems

46. Air filters
POSITIONING OF FILTERS
▶ The required cleanliness or purity of air can be achieved with
effective cleaning of the external air or recirculated air through
correctly designed and installed filters to meet the specification
Types of air filters :
 Ultra Low Particulate Air filter( ULPA)
 High Efficiency particulate air filter( HEPA)
 Packed towers
 Membrane filter cartridges
 Hydrophobic filters

47. HEPA FILTERS
▶ HEPAis an acronym for “High Efficiency ParticulateAir”
▶ This type of air filter can remove at least 99.97% of dust, pollen,
mold, bacteria and any airborne particles with a size of 0.3
micrometres (μm).

48. HEPA FILTERS

49. HVAC PARAMETER MONITORING
AND FREQUENCY
Sr no PARAMETER FREQUENCY
1 HEPA Filter
Integrity ( DOP
testing)
Annual /
Yearly ( 12
monthly)
2 Air Change rate Every 6
months(
biennially
3 Air pressure
differential
Daily
4 Microbial load (
settle
plate & Swabs)
Daily
5 Temperature Daily
6 Humidity (
Relative
Humidity)
Daily

50. ◾A laminar flow cabinet or laminar flow
closet or tissue culture hood is a carefully
enclosed bench
◾designed to prevent contamination of
semiconductor wafers, biological samples, or
any particle sensitive materials
◾air is passed through a HEPA (High Efficiency
ParticulatesAir) filter which removes all
airborne contamination to maintain sterile
conditions

51. ◾ A laminar flow hood consists of a filter pad, a fan
and a HEPA (High Efficiency ParticulatesAir) filter
◾ The fan sucks the air through the filter pad
where dust is trapped
◾ After that the prefiltered air has to pass the
HEPA filter where contaminating fungi,
bacteria, dust etc are removed
◾ sterile air flows into the working (flasking) area
where you can do all your flasking work without
risk of contamination.

52. HEPA fliter

53. s
◾ Laminar FlowCabinets are suitable for a
variety of applications
◾ where an individual clean air environment i
required for smaller items, e.g. particle
sensitive electronic devices.
◾ In the laboratory, Laminar FlowCabinets
are commonly used for specialised work.
◾ Laminar FlowCabinets can be tailor made
to the specific requirements of the
laboratory
◾ ideal for general lab work, especially in the
medical, pharmaceutical, electronic and
industrial sectors.

54. ◾ Laminar FlowCabinets, or laminar air flow
cabinets as they are also known, are normally
made of stainless steel with no gaps or joints
thereby preventing the build-up of bacteria
from collecting anywhere in the working zone.
◾ Laminar FlowCabinets are also known as clean
benches because the air for the working
environment is thoroughly cleaned by the
precision filtration process.

55. ◾Laminar FlowCabinets can be produced as
both horizontal and vertical cabinets
◾There are many different types of cabinets
with a variety of airflow patterns for different
purposes
 Vertical Laminar FlowCabinets
 Horizontal Laminar FlowCabinets
 Laminar FlowCabinets and Hoods
 Laminar Flow Benches and Booths

56. ◾direction of air flow
which comes from above
◾then changes direction
and is processed across
the work in a horizontal
direction.
◾ The constant flow of
filtered air provides
material and product
protection.

57. ◾ function equally well as
horizontal Laminar Flow
Cabinets
◾ laminar air directed
vertically downwards onto
the working area
◾ The air can leave the
working area via holes in
the base
◾ Vertical flow cabinets can
provide greater operator
protection.

58. ◾Important parameters to make sure that the
hood works efficiently:
 the HEPA filter has to remove all airborne
materials
 the air speed in the working area has to be about
0,5 m/s

60. ◾Before you start flasking in your laminar flow
hood you should do the following actions.
 Turn on the blower and wipe out the sterile area
with an alcohol soaked piece of kitchen paper.
 Let the blower run continuously for 30 minutes.
 When this time has passed, wipe out of the sterile
area with an alcohol soaked piece of kitchen paper.

61. Environmental monitoring methods
• Daily Monitoring: (Micro Area-LAF/UV Passbox /Room)
• Monthly Monitoring: (MFG Area/Equipments/Drain Points)
• Quarterly Monitoring: (Compressed Air)
• Half Yearly Monitoring: (Operators/Personnel Hygiene monitoring)
• Yearly Monitoring: (HVAC/AHU System Validations)
•Occasional Monitoring: (As and When required-During
Technical issues with filters/ on maintenance/Filter change)

62. Environmental monitoring 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

63. 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.

65. 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.

66. 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

67. 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.

68. 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

69. 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.

70. 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.

71. 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

72. 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.

73. PERSONNELL
THE TRAINING •
The people who produce sterile products are usually non-professional
persons, supervised by those with professional training. To be effective
operators, they must be inherently neat, orderly, reliable, and alert,
and have good manual dexterity. • All employees should be in good
health and should be subjected to periodic physical examinations. They
should understand their responsibility to report the developing
symptoms of a head cold, sore throat, or other infectious diseases so
that they can be assigned to a less—critical area until they have fully
recovered.

74. FOLLOW SOPS •
Personnel entering the aseptic areas should be required to follow a
definite preparatory procedure. This should include removing at least
outside street clothing, scrubbing the hands and arms thoroughly with a
disinfectant soap, and donning the prescribed uniform.
UNIFORMS •
The attire worn by personnel in the aseptic areas usually consists of
sterile coveralls, hoods, face masks, and shoe covers. Sterile rubber
gloves also may be required.

75. CONTAINERS
1. PLASTIC CONTAINERS •
The principal ingredient of the various plastic material used for containers is the thermoplastic
polymer i.e. polyethylene low density, polypropylene, polyvinylchloride, polycarbonate and
polystyrene etc.
ADDED SUBSTANCES IN PLASTICS •
Although most of the plastic materials used in the medical field have a relatively low amount of
added ingredients, some contain a substantial amount of: - Plasticizers - Fillers - Antistatic agents -
Antioxidants
TOXICITY WITH PLASTIC CONTAINERS •
Tissue toxicity can occur from certain polymers, but additives are a more common cause. Reactivity
due to sorption (absorption and/or adsorption) has been found to occur most frequently with the
polyamide polymers, but additives leached from any of the plastic materials may interact with
ingredients of the product.

76. AUTOCLAVING •
All of the polymeric materials except low-density polyethylene and polystyrene can be autoclaved if
they have been formulated with a low amount of plasticizers, although most of them soften at
autoclaving temperatures and care must be exercised to avoid fusing adjacent surfaces or
otherwise deforming them.
TOXICITY TESTING •
The USP has provided test procedures for evaluating the toxicity of plastic materials. Essentially the
tests consist of three phases: - Implanting small pieces of the plastic material intramuscularly in
rabbits. - Injecting eluates using sodium chloride injection, with and without alcohol, intravenously
in mice, and injecting eluates using polyethylene glycol 400 and sesame oil intraperitoneally in
mice. - Injecting all four eluates subcutaneously in rabbits.
The reaction from the test samples must not be significantly greater than nonreactive control
samples.

77. 2. GLASS CONTAINERS •
Glass is still the preferred material for containers for Injectable products. The two general types of
glass are soda-lime and borosilicate. TYPE 1 (borosilicate glass) is preferred for most sterile
products.
TYPE I: BOROSILICATE GLASS
• It is least reactive and highly resistant glass. It is more chemically inert than soda lime glass. A
substantial amount of alkali or earth cations are replaced by boric oxide.
• This type of glass has higher ingredient like aluminum and zinc and higher processing costs and is
therefore used primarily for more sensitive pharmaceuticals such as parenteral or blood products
e.g. Ampoules and vials.
• Although the glass is considered to be a virtually inert material and is used to contain strong acids
and alkalis as well as all types of solvents, it has a definite and measurable chemical reaction with
some substances, notably water.

78. CHEMICAL RESISTANCE •
The USP provides the Powdered Glass and the Water Attack tests for evaluating chemical resistance of
glass. The test results are measures of the amours of alkaline constituents leached from the glass by
purified water under controlled elevated temperature conditions.
• The Powdered Glass test is per formed on ground, sized glass particles. Water Attack test is performed
on whole containers. The conditions of the test must be rigidly controlled to obtain reproducible since
the quantity of alkaline constituents leached is small.
PHYSICAL CHARACTERISTICS •
Ultraviolet rays can be completely filtered out by the use of amber glass.
• If the product contains ingredients subject to iron catalyzed chemical reactions, amber glass cannot be
used. The product must then be protected from ultraviolet rays by means of an opaque carton
surrounding a flint (colorless) glass container.
• In addition to other physical characteristics, glass containers should have sufficient physical strength to
withstand the high-pressure differentials that develop during autoclaving and the abuse that occurs
during processing, shipping, and storage.
• A low coefficient of thermal expansion to withstand the thermal shocks that occur during washing and
sterilization procedures.

79. IPQC and finished product Quality
control tests for Parenterals