INTRODUCTION TO

PHARMACEUTICAL CLEAN ROOM

Introduction to Pharmaceutical Clean Room 1

 1 Introduction:
What is Clean Room & Clean Zones

2 Contamination Sources:
Principles, Types and Sources of Contamination

Contents 3 Contamination Control:

Contamination agents & Personnel Hygiene

4 Clean room Classification:

ISO, WHO, EU & Schedule M Classification
5 Particle Sampling:
Non-viable & Viable air particle sampling

Introduction to Pharmaceutical Clean Room 2

 6 Gowning Entry & Exit Procedure:
Change rooms, Entry & Exit, Recommended Frequency
for Garmenting

Contents 7 Air Handling Systems:

Airflow distribution & Requirements, HEPA,
ULPA, Clean room Design Criteria

8 Environmental Qualification:
Requirements, Risk Analysis, Sample Site Selection,
Sample Site Number, Clean room tests, Sampling plan,
Non-Viable Particle sampling, Clean room performance
testing, Sampling Frequency, Environmental Monitoring

Introduction to Pharmaceutical Clean Room 3

 INTRODUCTION
1
※ What are clean rooms

※ What are clean Zones

Introduction to Pharmaceutical Clean Room 4

 WHAT ARE CLEAN ROOM ?
BRITISH STANDARD -“A room which is defines a clean room with control of particulate
contamination, constructed and used in such a way as to minimize the introduction,
generation and retention of particles inside the room and in which the temperature,
humidity, airflow patterns, air motion and pressure are controlled”.

ISO 14644 -“A room which the concentration of airborne particles is controlled, and which
is constructed and used in a manner to minimize the introduction, generation and retention
of particles inside the room and in which other relevant parameters e.g. temperature,
humidity and pressure are controlled as necessary”.

FEDERAL STANDARD 209E-“defines a clean room as a room in which the concentration of

airborne particles is controlled to specified limits”.

Introduction to Pharmaceutical Clean Room 5

 WHAT ARE CLEAN ROOM ? Continuation…

A clean environment designed to reduce the contamination

of processes and materials. This is accomplished by removing
or reducing contamination sources.

Following two ISO Standards:

ISO 14644 -“Standards for Airborne Particulate Cleanliness Classes in Cleanrooms and
Clean Zones”.

ISO 14698 -“Cleanrooms and associated controlled environments – Biocontamination

control”.

Introduction to Pharmaceutical Clean Room 6

 WHAT ARE CLEAN ROOM ? Continuation…

Guidelines on Sterile Drug Products produced by Aseptic

Processing as per USFDA revised on September 2004.
CRITICAL AREA: One in which the sterilized dosage form, containers, and closures are
exposed to the environment, activities that are conducted in this area include manipulations
of these sterilized materials/product prior to and during filling/closing operations.

1. NMT 100 particles of 0.5µm per cubic foot.

2. Air must be supplied at the point of use as HEPA filtered laminar air flow.
3. Velocity 90± 20 feet per minute.
4. NMT 1 colony forming unit per 10 cubic feet.
5. A pressure differential of 0.05 inch of water is acceptable.

Introduction to Pharmaceutical Clean Room 7

 WHAT ARE CLEAN ROOM ? Continuation…

CONTROLLED AREA: An area in which it is important to control the environment, is the area
where un-sterilized product, in-process materials, and container /closures are prepared. This
includes areas where components are compounded, and where components, in-process
materials, drug product contact surfaces of equipment, containers, and closures, after final
rinse of such surfaces, are exposed to the plant environment.

1. NMT 10,000 particles of 0.5µm per cubic foot.

2. Sufficient air flow.
3. NMT 25 colony forming unit per 10 cubic feet.
4. Positive pressure differential relative to adjacent uncontrolled areas.
a) A pressure differential of 0.05 inch of water.
b) 20 air changes per hour.

Introduction to Pharmaceutical Clean Room 8

 WHAT ARE CLEAN ZONES ?

A- Zone (Local Zone): For operations that affords high risk for product quality. Eg. Filling,
closing, ampoule and vial opening zones. Laminar airflow used in this zone of similar velocity
of 0.36 – 0.54 m/s.

B- Zone (Circled A-Zone): It is used for an aseptic preparation.

C & D- Zone: These zones are used for less critical stages of manufacturing.

Introduction to Pharmaceutical Clean Room 9

CONTAMINATION SOURCES
2
※ Principles of Clean Environment
※ Types of Contamination

※ Potential sources of Contamination

Introduction to Pharmaceutical Clean Room 10

PRINCIPLES OF CLEAN ENVIRONMENT

 Air is highly (HEPA) filtered

(99.97% @ 0.3µm)

 Layout should minimize particles

sources in filtered air stream

 Air flow should remove most

particles generated by process

Introduction to Pharmaceutical Clean Room 11

 TYPES OF CONTAMINATION
Particulate: Dust, hair, skin foreign particles,
makeup items etc.,
Chemicals: Oil, grease, metal ions. etc.,
Biological contamination: Bacteria, fungi,
rodents, etc.,
Radiation: Ultra Violet light

Introduction to Pharmaceutical Clean Room 12

 MAIN SOURCES OF CONTAMINATION

People: Contribute 75% of contamination, are main source to control to

inhibit the contamination.

Ventilation: Contribute 15% of contamination

Equipment: Contribute 5% of contamination

Room Structure: Contribute 5% of contamination

Introduction to Pharmaceutical Clean Room 13

POTENTIAL SOURCES OF CONTAMINATION

Introduction to Pharmaceutical Clean Room 14

CONTAMINATION CONTROL
3
※ Contamination Agents
※ Personnel Hygiene

Introduction to Pharmaceutical Clean Room 15

 CONTAMINATION AGENTS

Personnel Control:

a) Dress code
b) Personnel hygiene
c) Gowning

Environmental Control:

a) Entry and Exit

b) Flow of materials and supplies
c) Atmospheric control (HVAC & Environmental monitoring (microbial
monitoring)
d) Cleaning and maintenance

Introduction to Pharmaceutical Clean Room 16

 PERSONNEL HYGIENE

Personnel Hygiene:

a) Everyday Shower before entering in the area

b) Leave all personal items in changing room
(wallets, keys, comb etc.)
c) Control Dermatitis & Dandruff
d) No chewing gum or tobacco
e) No Cosmetics , Jewellery or wrist watches
should be worn.
f) Do not move vigorously(Brisk movements
shed large particles from body movement)
g) Do not smoke before and after entry
h) Avoid coughing and sneezing if unavoidable
leave the clean room

Introduction to Pharmaceutical Clean Room 17

CLEAN ROOM CLASSIFICATION
4
※ ISO Standard
※ WHO Standard
※ EU cGMP Standard

※ Schedule M Standard
Introduction to Pharmaceutical Clean Room 18
 CLEAN ROOM CLASSIFICATION

ISO Standard 14644-1

for clean room
classification

EU cGMP
for Air Classification

WHO Guidelines
for Air Classification.

Schedule M
for Air Classification

Introduction to Pharmaceutical Clean Room 10

 Federal Standard 209

Class ½ Sized micro particles / ft2

1,000,000 1,000,000

100,000 100,000 Standard 209E denotes the number of

0.5 µm (or ½ - sized micron) particles
10,000 10,000
or larger permitted per cubic foot (ft2)
1,000 1,000 of air.
10 10

1 1

ISO 14644-1 replaced Federal Standard 209E on

the basis of clean room classification
Introduction to Pharmaceutical Clean Room 20
 ISO Standard 14644
Maximum Concentration Limits (particles/m 3 of air) for particles equal to larger than
the considered sizes shown below
ISO Classification
Number (N) 0.1 µm 0.2 µm 0.3 µm 0.5 µm 1 µm 5 µm

100 2 ISO 14644-1

ISO Class 1
taken account of
ISO Class 2 100 24 10 4
even smaller
ISO Class 3 1000 237 102 35 8 particles ranging
from 0.1µm to
ISO Class 4 10,000 2870 1020 352 83
0.5 µm per cubic
ISO Class 5 100,000 23700 10200 3520 832 29 meter of air
ISO Class 6 1000000 237000 102000 35200 8320 293

ISO Class 7 352000 83200 2930

ISO Class 8 3520000 832000 29300

ISO Class 9 35200000 8320000 293000

Classification based on amount of micron particles in one cubic meter of air
Introduction to Pharmaceutical Clean Room 21
ISO Standard Calculation 14644
ISO calculation is based on the following Equation:

0.1 2.08

Cn = 10N x [ D ]  
Where

Cn = is the maximum permitted concentration of air born particles that are equal to or larger than
the considered particle size. Cn is rounded to the nearest whole number, using no more that three
significant figures
N = is the ISO classification number, which shall not exceed the value of 9 Intermediate ISO classification
numbers may be specified, with 0.1 the smallest permitted increment of N.
D = is the considered particle size in µm
0.1 = is the constant with a dimension of µm

Introduction to Pharmaceutical Clean Room 22

WHO Guidelines Classification of Air and Micro-organism

Maximum number
Maximum Number of particles of viable micro-
Grade 3 organism per m3
permitted per m

0.5 – 5 µm >5 µm

A
3 500 None Less than 1
(Laminar Airflow Work
station)
B 3 500 None 5

C 350 000 2 000 100

DAir Classification system for manufacture

3 50 0000 20 of
000sterile products 500

Introduction to Pharmaceutical Clean Room 24

 Air Classification as per EU cGMP

Clean Area ISO ≥ 0.5 µm Microbiologic Microbiological

Classification Designationb particle/m3 al Active Air Plates Action
(0.5 µm Action Levelsc Levelsc,d
particles/ft3) (cfu/m3) (dia.90mm,
cfu/4hours)

100 5 3,520 1e 1e

1000 6 35,200 7 3

10,000 7 352,000 10 5

100,000 8 3,520,000 100 50

Air Classification system as per EU cGMP

Introduction to Pharmaceutical Clean Room 24

 Air Classification as per Schedule M
Maximum permitted number of particles/m3 equal or above

At rest In Operation
Grade
0.5 µm 5 µm 0.5 µm 5 µm

A 3,250 29 3,500 29
B 35,200 293 3,52,000 2930
C 3,52,000 2930 35,20,000 29,300
D 35,20,000 29,300 not defined not defined
Grade A and B corresponds class 100, M 3.5, ISO 5
Grade C correspond with class 10000, M 5.5, ISO 7
Grade D correspond with class 100000, M 6.5, ISO 8

Introduction to Pharmaceutical Clean Room 25

 PARTICLES SAMPLING
5
※ Non –Viable Air Sampling
※ Viable Air Sampling

Introduction to Pharmaceutical Clean Room 26

 Particle Sampling

Non-viable Particles :
A non-viable particles is a particle that does not contain
a living micro-organism but acts as a transportation for
viable particles.

Non-viable Air Sampling :

It is monitored using particle counters which do not
distinguish between viable and non-viable particles, but
are more technically advanced than air samplers.

Introduction to Pharmaceutical Clean Room 27

 Particle Sampling.. Continuation…
Viable Particles :
Viable particles are living microorganisms like bacteria, mold
spores, and yeast. Such microorganisms can cause allergic
reactions in certain people. For example, inhaling mold
spores can cause sneezing and flu-like symptoms in people
sensitive to molds.

Viable Air Sampling Using Air Sampler :

Viable air Samplers collect a pre-determined volume of air
and impact micro-organisms against agar based growth
medium. Once the sample collected and the medium is
incubated and the results are expressed in colony forming
units per cubic meter.
For Sampling viable particles in the air R2STM (Remote Slit
Samplers), SASTM (Surface air sampler) and other qualified
air samplers will be used.

Introduction to Pharmaceutical Clean Room 28

 Particle Sampling.. Continuation…

Viable Air Sampling Using Settle

Plates :
In this method, a petri dish containing
sterile growth media is kept in the open
air for about 30 to 60 minutes, but the
time can extend till 4 hours. Within this
period of time, viable microorganisms
settle on the plate which is then incubated
for germination and identification.

Introduction to Pharmaceutical Clean Room 29

 Calculation for Sample Air Volume

As per ISO 14644-1 :

Defines the minimum sample volume as “that volume whereby a minimum number of 20 particles
would be detected if the particle concentration for the largest particle size were at the class limit for
the designated ISO class”.

20
Formula: Vs = x 1000
C n,m

Where

Vs = The minimum single sample volume per location in litres

Cn,m = The class limit (no. of particles per cubic meter for the largest considered particle size (n)

specified for the class limit (m)

Introduction to Pharmaceutical Clean Room 30

Microbial Limits for Contamination in Operation

Air Sampler Settle plates Contact plates Glove print,

Grade (cfu/m3) (diameter 90mm) (diameter 55mm) 5 fingers
cfu/4 hours (a) cfu/plate cfu/glove

A <1 <1 <1 <1

B 10 5 5 5

C 100 50 25 -

D 200 100 50 -

(a) Individual settle plates may be exposed for less than 4 hours

Introduction to Pharmaceutical Clean Room 31

 6
GOWNING , ENTRY & EXIT PROCEDURE

※ Change Rooms
※ Primary Change Rooms
※ Gowning

※ Entry & Exit procedure

※ Recommended Garmenting and Frequency of Change

Introduction to Pharmaceutical Clean Room 32
 Change Rooms

Change Rooms :
Suitable changing facilities or lockers provided at
appropriate location for the storage of employees
clothing and personnel belongings.

Employees must be trained on procedure for

changing street clothing and foot wear.

And other procedures like applying disinfectant

solution, crossover bench to primary change
room.

Introduction to Pharmaceutical Clean Room 33

 Primary Change rooms

Primary Change rooms :

After crossing through cross over bench, person will
enter in primary clothing area and wear appropriate
lint free trousers, shirts and cap from the hanger
/locker.

Wear clean footwear (company designed footwear) or

shoe covers placed in rack.

Wear nose mask before entering into the operational

area.

Apply disinfectant and enter into respective working

area.

Introduction to Pharmaceutical Clean Room 34

 Gowning

:
Gowning Order (lint free polyester clothing)
- Hair Cover
- Hood
- Shoe Covers
- Coverall
- Gloves
- Face mask
- Safety glasses

Introduction to Pharmaceutical Clean Room 35

 Entry and Exit

- Entry and Exit in respective area as per SOP/training

provided to the employees
- Only one person should enter at one time
- Each user must use individual user access cards
(Wherever applicable)
- Pass through gowning area to operational/working
area slowly to avoid /reduce migration of particles.
- Restricted the number of person into the aseptic
area.
- Wear appropriate gowning before entering into
aseptic area.

Introduction to Pharmaceutical Clean Room 36

 Recommended Garmenting and Frequency of Change

ISO 8 ISO 7 ISO 6 ISO 5 ISO 4

Apparel (M6.5 or Class (M5.5 or Class (M4.5 or Class (M3.5 or Class (M2.5 or Class 10
Type 100,000) 10,000) 1,000) 100) or Aseptic area)
Hair Cover ○ ○ ○ ○ ○

Gloves ○ ○ ○ ○ ○

Facial Cover ○ ○ ○ ○ ○

Hood ○ ○ ○

Lab coat/Frock ○ ○
Coverall ○ ○ ○ ○

Shoe cover ○ ○ ○ ○ ○

Boot Cover ○ ○ ○ ○

Typical Frequency 2x / Week 2x / Week 3x / Week Daily Per Entry

of Change
○ RECOMMENDED, ○ APPLICATION SPECIFIC

Introduction to Pharmaceutical Clean Room 37

 7
AIR HANDLING SYSTEM

※ Airflow Distribution Control

※ Air Handling Systems requirements
※ Schematic diagram of AHU and HVAC
※ HEPA and ULPA
※ Clean room design criteria
Introduction to Pharmaceutical Clean Room 38
 Air Flow Distribution and Control

Unidirectional :
It is an airflow pattern in which essentially the
entire body of air within a confident area moves
with uniform velocity and in single direction with
parallel airstreams. Clean rooms: Class 100 and
below have unidirectional airflow pattern.
Laminar Airflow is → 120 FPM

Introduction to Pharmaceutical Clean Room 39

 Air Flow Distribution and Control

Non-unidirectional :
Airflow is not unidirectional by having a varying
velocity, multiple pass circulation or non-parallel flow
direction. Conventional flow clean rooms (Class 1000
& Class 10,000) have non-unidirectional or mixed air
flow patterns.

:
Mixed Patterns Combine some of each flow type

Introduction to Pharmaceutical Clean Room 40

Air Handling System Requirements

Air Handling System Requirements :

- Temperature should be 15 - 20°C
- Relative Humidity 45 – 55%
- Pressure differential between two areas should be
0.05 – 0.1 inch water gauge
- At least 20 air changes per hour
- Cleanliness required for class 100

Introduction to Pharmaceutical Clean Room 41

Schematic Diagram of AHU

Introduction to Pharmaceutical Clean Room 42

Schematic Diagram of HVAC

Introduction to Pharmaceutical Clean Room 43

 HEPA Filters

High Efficiency Particulate Air Filters (HEPA) :

- Box type depth filters used for air filtration
- Made of glass filters
- Efficiency of HEPA filters 99.97% against 0.3µm
particles.

HEPA filters are tested for:

- Hot DOP test (efficiency test), Cold DOP test (Integrity
test), air flow resistance test

Introduction to Pharmaceutical Clean Room 44

 ULPA Filters

Ultra Low Penetration Air Filters (ULPA) :

- Most of ULPA filters are replaceable extended media
dry filters that have a minimum particle collection
efficiency of 99.9997% efficient particles greater than
or equal to 0.12 µm particles in size.

ULAP filters are tested for:

- Hot DOP test (efficiency test), Cold DOP test (Integrity
test), air flow resistance test

Introduction to Pharmaceutical Clean Room 45

 Clean Room Design Criteria
(Mechanical Practices)

Class Limits
Criteria
10 100 1000 10,000 100,000

Air changes per hour 600 300 - 480 150 – 250 60 – 120 10 - 40

HEPA Filter Coverage 100 70 -100% 30-60% 10-30% 5 – 10%

CFM per sq.ft 90 65 – 36 32- 18 16 – 9 8 -5

Typical Filter Efficiency 99.9997 99.997 99.997 99.997 99.97

60 – 110
Typical Filter Velocity 50 -90 FPM 40 -90 FPM 25 -40 FPM 10 -30 FPM
FPM
Uni- Uni-
Air Flow Type Mixed Mixed Mixed
directional directional

Introduction to Pharmaceutical Clean Room 46

 ENVIRONMENTAL QUALIFICATION
8
※ Qualification Requirements ※ Risk Analysis

※ Sample Site Selection & Sample Site Number

※ Clean Room Test ※ Sampling Plan

※ Non-viable particle sampling

※ Clean room performance testing ※ Sampling Frequency
Introduction to Pharmaceutical Clean Room ※ Environmental Monitoring requirements
※ Sampling Frequency for routine monitoring 47
Environmental Qualification ?

Environmental Qualification :
It is the process by which classified rooms/zones are
verified to meet established microbial and total
particulate environmental standards requirements upon
sampling testing protocols.

Introduction to Pharmaceutical Clean Room 48

Qualification Requirements

Qualification Requirements :
• Risk assessment documentation containing rationale
for sampling sites, sampling frequency and use of
settle plates.
• Program must provide data to make effective decisions
about the level of environmental control necessary to
maintain the required levels of microbial and total
particulate cleanliness.
• Contamination control procedures

Introduction to Pharmaceutical Clean Room 49

 Risk Analysis

Risk Analysis :
A documented risk analysis is suggested to
select the monitoring sites during
characterization and it must document the
rationale for site selection and shall be based
on the needs of the process and classified area.

Introduction to Pharmaceutical Clean Room 50

 Sample Site Selection

Sample Site Selection :

• Sites have greater opportunity for contributing bioburden
to the product should be sampled more frequently.
• Sampling locations must include those locations which
may have an impact on the product and must be sampled
more often.
• In addition to walls and floors surfaces, representative
surface samples must be taken where human activity
occurred.
• Sampling must not be intrusive to the process to avoid
the probability of product contamination.

Introduction to Pharmaceutical Clean Room 51

 Sample Site Number

Sample Site Number :

Sample sites for Total Particulate monitoring by room can be determined using the following equation.

NL = √ A

Where,
NL = represents the minimum number of samples

A is the surface area of the room in m2

Area ISO Class L (m) W (m) Area (m^2) √Area (m^2) # Locations

Assembly 5 8.5 4 34 5.83 6

Gowning 7 4 4 16 4.00 4

Calculation of number of locations as per ISO 14644

Introduction to Pharmaceutical Clean Room 52

 Clean Room Tests
Clean Room Tests:

1. Airflow velocity
2. HEPA/ULPA filter installation leak tests
3. Air generated aerosol challenge & aerosol photometer
filter scan test method
4. Alternative source aerosol particle challenge & discrete
particle counter filter scan test method
5. Airborne particle count test
6. Room pressurization count test
7. Airflow parallelism test
8. Temperature/RH test
9. Lighting level test
10.Sound pressure (Noise) level test
11.Flooring resistance test
12.Point to point test
13.Point to ground test
14.Testing swing/balance voltage and decay time of ionizers

Introduction to Pharmaceutical Clean Room 53

 Sampling Plan
Sampling Plan:
1. Room classification
2. Room Configuration
3. Criticality of operations in the room
4. Process and material flow
5. Sample sites with greatest potential for particulate
contamination
6. Gowning requirements
7. Cleaning and sanitization procedures
8. Personnel quantity for room
9. Personnel traffic patterns
10. Equipment
11. HVAC system (airflow patterns and design)
12. Duration of campaign / manufacturing batches
13. Process/engineering controls
14. In-process monitoring

Introduction to Pharmaceutical Clean Room 54

 Non-Viable Particle Sampling

Non-Viable Particle Sampling:

1. Take three readings of one minute one-CFM (28.3
liters) samples per location for statistical reliability
2. Test Laminar flow work stations and barrier isolators
3. While sampling sample for viable organism at the same
time.

Introduction to Pharmaceutical Clean Room 55

 Clean Room Performance Testing

Clean Room Performance Testing:

1. “As built” a test used to establish that the
cleanroom contractor has met his contractual
obligations.

2. “At rest” (Static) a test taken with the room

fully equipped ready for production but with no
equipment running and absence of personnel.
Three samplings are performed after 24 hrs of
cleaning process.

3. “In-use” (Dynamic) a test taken with the room

fully operational. Three samplings are performed
after 24 hrs of cleaning process.

Introduction to Pharmaceutical Clean Room 56

 Sampling Frequency

Sampling Frequency: It may vary plant to plant:

1. Sample site selection

2. Type of facility
3. Seasonal variations
4. Room design
5. Manufacturing process
6. Human activity and interventions
7. Cleaning and sanitization procedures
8. Gowning requirements
9. Historical data

Introduction to Pharmaceutical Clean Room 57

Environmental Monitoring (EM) requirements

Environmental Monitoring Requirements:

1. Sampling and testing methods
2. Monitoring frequency
3. Classification and identification of the areas to be
monitored and sites to be sampled
4. Alert and action levels
5. Excursion response/investigation, including re-
sampling requirements
6. Organism identification

Introduction to Pharmaceutical Clean Room 58

 Sampling Frequency for routing monitoring for
Class C and Class D

Test Minimum Frequency

Air Particle Monitoring Every 3 months

HEPA Filter Integrity Every one Yearly

Air change rate Every 6 months

Air Pressure differentials Daily

Temperature and Humidity Daily

Microbial Monitoring Once in every month / during manufacturing process

Introduction to Pharmaceutical Clean Room 59

 References
 FDA guidance for Industry-sterile drug products
produced by Aseptic processing – Current Good
manufacturing process
 ISO 13408 – Aseptic processing of health care
products
 ISO 14644-1 Cleanrooms & associated controlled
environments – Part 1: Classification of air
cleanliness
 ISO 14698, Cleanrooms & associated controlled
environments – Biocontamination controls
 PIC/S Recommendations on the Validation of
Aseptic Processes
 USP <1116> Microbiological Evaluation of Clean
Room and other controlled Environments
 USP <797> Pharmaceutical Compounding – Sterile
preparations
 http:// www.particlecounters.org/

Introduction to Pharmaceutical Clean Room 60

Thank you for your attention

Introduction to Pharmaceutical Clean Room 61