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ISO 14644 - Revised Cleanroom Standard

Presentation February 2016
DOI: 10.13140/RG.2.1.5097.8323

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Revision to Parts 1 and 2: 2015

Tim Sandle

Welcome
Pharmig is a non-profit making professional organisation, established

in 1991, that represents the interests of individuals who work in, have
responsibility for, or work alongside microbiology within
pharmaceutical, healthcare, cosmetics & NHS Industries.

It provides a focus for continuing professional development and serves

as a unique network for the exchange of microbiological information

through training courses, conferences, publications and its website
forum.

Organising meetings, training courses, conferences and producing publications

that provide topical information and views on microbiologically related topics

Advancing the science of microbiology and its practical application

Influencing the development of regulations and guidelines

surrounding microbiology
Acting as a confidential forum for the dissemination of information
concerning all aspects of microbiology

Presenter
Dr. Tim Sandle
Pharmig committee
member
Pharmaceutical
Microbiology website:
http://www.pharmamic
roresources.com/

ISO 14644
ISO 14644
International cleanroom standard
Part 1 in 1999
12 part standard

Electronics, healthcare, biotechnology, pharmaceuticals

Replaced FS 209E in 2001

Accepted by EU GMP in 2003 for classification but not

monitoring
FDA aseptic filling guide in 2004.

ISO 14644 parts #1

ISO 14644-1:2015 - Part 1: Classification of air

cleanliness
ISO 14644-2:2015 - Part 2: Specifications for testing
and monitoring to prove continued compliance with
ISO 14644
ISO 14644-3:2005 - Part 3: Test methods
ISO 14644-4:2001 - Part 4: Design, construction and
start-up
ISO 14644-5:2004 - Part 5: Operations

ISO 14644 parts #2

ISO 14644-6: 2004 - Vocabulary
ISO 14644-7:2004 - Part 7: Separative devices (clean air hoods, gloveboxes,
isolators and mini-environments)
ISO 14644-8:2013 - Part 8: Classification of air cleanliness by chemical
concentration (ACC)
ISO 14644-9:2012 - Part 9: Classification of surface cleanliness by particle
concentration
ISO 14644-10:2013 - Part 10: Classification of surface cleanliness by chemical
concentration
No part 11 in draft
ISO 14644-12:draft - Part 12: Classification of air cleanliness by nanoscale
particle concentration
ISO 14644-13:draft - Part 13: Cleaning of surfaces to achieve defined levels of
cleanliness in terms of particle and chemical classifications
ISO 14644-14:draft - Part 14: Assessment of suitability for use of equipment by
airborne particle concentration

Cleanrooms
Cleanrooms
Design

Air
Filtration (HEPA)
Pressure differentials
Air changes
Clean-up times
UDAF: air velocity

Personnel
Gowning
Behaviours
Cleaning and disinfection

ISO 14644
Became live in December 2015:
ISO 14644-1 - Classification of air cleanliness
ISO 14644-2 - Specifications for testing and monitoring
to prove continued compliance by ACP.

Both parts deal with particles only

ISO 14644 Part 1 - purpose

Classification is the process of qualifying the

cleanroom environment by the number of particles

using a standard method.
Determine classification of cleanroom according to

standards e.g. Room x is ISO class y.

Distinct from routine environmental monitoring.
Distinct from process monitoring e.g. ongoing
assessment of aseptic filling.

Occupancy states
As built : condition where the installation is complete with all services
connected and functioning but with no production equipment, materials, or
personnel present
At rest : condition where the installation is complete with equipment installed
and operation in a manner agree upon by the customer and supplier, but with no
personnel present
Operational : condition where the installation is functioning in the specified
manner, with the specified number of personnel and working in the manner
agreed upon

9 classes

1999 version

In Operation:
Grade A
Grade B
Grade C
Grade D

=
=
=
=

ISO class 4.8

ISO class 7
ISO class 8
Not determined, class 8 normally applied

Particle sizes
Allows for one or more particle sizes to be assessed.
The standard requires the larger particle to be at least 1.5
times that of the smallest particle size measured.
But no longer features 5.0 m limit for particles for the
Grade A equivalent class for classification.
This does not replace EU GMP requirements. 0.5 and
5.0 m need to be assessed for monitoring.

Change A particle locations #1

New approach to selection of locations for particle counting
Previous approach:

NL =

NL is the minimum number of sampling locations (rounded up to a whole

number).

A is the area of the cleanroom or clean zone in square metres (m2) for

which the square root is taken.

Taking the surface of the room in square metres, assessing the square
root and using the obtained number (rounded up) to give the number
of locations, to be positioned equidistantly.

Change A particle locations #2

Change A particle locations #3

The new method is based on hypergeometric distribution.

Opposite to binomial distribution.

Samples are drawn randomly without replacement from a finite
population.
Each location can be treated independently with a 95% level of
confidence that at least 90% of the cleanroom will comply with
the maximum particle limit for the intended class.

Confidence an be increased if desired.

No calculations are required to determine the number of
locations - there is a look-up table (Table 1 - the only reference
for all sizes of particle from ISO 1 to ISO 9).

Look up tables #1
Where a room area is not

listed in the look-up

table, the next largest
size is selected.
This method has
generally led to an
increase in particle count
locations.

Look up tables #2
Cleanroom

Room size

A
B
C

200 m2
36 m2
8 m2

1999 version
location
numbers
15
6
3

Revised no.
of locations
23
9
4

Change B position of counters #1

Once the number of locations has been selected, the room

is divided up into equal sectors and a particle counter

placed in each sector.

Previous standard counter placed in approximate centre.

New standard - where the counter is placed within each sector
is determined by the user.

The standard allows counters always to be placed at the same

point within the sector; randomly placed within the sector; or
evenly distributed; or by risk.

Reason: counts no longer assumed to be homogenous within a

sector.

Addition locations can be added at the discretion of the

facility.

Change B position of counters #2

To align with GMP, the location should be orientated

to the point of greatest risk e.g. close to fixed

equipment. The standard recommends that the
following is accounted for:

Room layout;
Equipment layout;
Airflow patterns;
Position of air supply and return vents;
Air-change rates;
Consideration should be given to any unintended bias in the
sampling process.

Change C sample volumes #1

Volume of air to be sampled at

each location, the volume of air

must be sufficient to detect at
least 20 particles for the largest
particle size limit.
The operative figure is 5.0
microns
Volume to be sampled

(Vs) =

[20 x 1000 (constant)]

Class limit particles (largest size)

For example, Grade C

Volume to be sampled
=

20 x 1000 = 0.69 litres

29,000

Therefore, a minimum of 1 litre

would need to be taken at each

location.
However, ISO 14644 states that
the volume needs to be at least 2
litres, sampled over a one
minute period.
Therefore, a minimum of 2 litres
would need to be taken at each
location.

Change C sample volumes #2

For example, Grade B
Volume to be sampled =
20 x 1000
=
6.9 litres
2,900
Therefore, a minimum of 7 litres would need to be

taken at each location.

Grade A more complicated

Change D class limits #1

The Grade A issue
EU GMP Grade A does not equal ISO class 5, because of
the different 5.0 m limits

29 count limits for ISO 14644 class 5

20 count limit for EU GMP Grade A.

Where intermediate classes are required the standard no

longer permits increments of 0.1. So, to meet EU GMP, an

ISO class of 4.5 would need to be selected in theory.

Change D class limits #2

De-emphasis on the 5 m ISO Class 5 limit:
Sampling and statistical limitations for particles in low concentrations make classification

inappropriate; and
Sample collection limitations for both particles in low concentrations and sizes greater than
1 m make classification at this particle size inappropriate, due to potential particle losses
in the sampling system.

But Annex 1 of EU GMP requires 5.0 m particles to be

assessed
Options:

Just classify Grade A for 0.5 m and use 0.5 m / 5.0 m for operations,
Or continue with 20 or 29 as a limit as an additional option for 5.0 m.

Standard states: In some situations, typically those related to specific process requirements,
alternative levels of air cleanliness may be specified on the basis of particle populations that are not
within the size range applicable to classification.

This means continuing with one cubic metre per location.

BUT attempting this for 5.0 m size particle could be difficult due to potential particle loss from
tubing.

Cleanroom

Room size

X
Y
Z

200 m2
36 m2
8 m2

1999 sample
time per
location
3
6
12

Revised
sample time
per location
1
1
1

1999 sample
time (entire
room)
45 minutes
36 minutes
36 minutes

Revised
sample time
(entire room)
23 minutes
9 minutes
4 minutes

Assuming one available particle counter:

Cleanroom

Room size

X
Y
Z

200 m2
36 m2
8 m2

Assessment of results #1
Record the results for each sample location.
Convert the results to one cubic metre for the room:
Result per room

No. particles @each location (or average) x (conversion factor to make one cubic
metre)
Volume of air sampled @each location
For example:
Using a particle count that counts at 28.3 litres per minute (or one cubic foot
per minute), each result would need to be multiplied by 35.3
Using a particle counter counting at 50 litres per minute, each result would be
multiplied by 20.
Individual results must be within limits per sector (unless more than one
sample per sector)

Assessment of results #2
Example: Grade B cleanroom,
assessed for 0.5 m particles using
a 1-minute counter

There is no longer a

grand total for the

cleanroom, each
individual sector must
pass.
The room is determined
to have met the ISO class
provided that the
obtained result does not
exceed the desired class.

Sample
locatio
n

Sample
(count
s per
28.3
litres)

Counts
per
cubic
metre
(x 35.3)

Limit
for 0.5
m

Pass /
Fail

52

1836

352,000

Pass

12

424

352,000

Pass

91

3201

352,000

Pass

97

3424

352,000

Pass

19

682

352,000

Pass

271

352,000

Pass

Change E probe positions

The counter probe must be orientated into the airflow (for

unidirectional air) or pointed upwards for turbulent flow

air.
There are no changes to occupancy states, the ideal
position is that cleanrooms should be classified when
occupied (at the normal occupancy level).

Clarification of test certification

Test certificates must state:

Name and address of the testing organization.

Date of testing.
No. and year of the publication of the relevant part of ISO 14644 e.g.
ISO 14644: 1 2015.
Location of cleanroom (or clean zone).
Specific representation of locations e.g. diagram.
Designation of cleanroom:
ISO class (plus EU GMP)
Occupancy.
Particle count sizes considered.
Test method used (and any departures or deviations).
Identification of test instrument and calibration certificate.
Test results.

ISO 14644 Part 2

Very little change:
Reclassification is a minimum of annual (change).

EU GMP states aseptic filling to be six-monthly, unless justified.

Requirement for a monitoring strategy in addition to

cleanroom classification. This should be by risk

assessment.

Levels are likely to be higher during processing.

Other changes
Recommendation that particle counts that meet ISO

21501 are used (error rate at each particle size of no

more than 20%). Counters must be certified.
ACP - initialism for Airborne Cleanliness Particles.
This is to differentiate airborne particles from surface

particles.
So ISO class 7 becomes ISO-ACP class 7.

ACV - Airborne Viable Concentration.

Viable contamination is not addressed further in the
standard.

Actions and implications

Rooms require reassessment for number of particle

count locations.
Location of particle counters within a sector to be
assigned, accounting for risk.
Sample sizes to be re-calculated.
Decision on 5.0 m particles for Grade A.
Contract test costs may alter:
More locations but,
Shorter sample run times.

Summary
The number of measuring points is no longer calculated as

the square root of the surface but given in a table.

5 m particles for ISO 5 has been dropped from the limit
value table. But remains for EU GMP.
No more statistical UCL calculation: there is no need to
perform an observation of all measuring points in the room
any longer. Each single measuring point is considered
individually and has to meet the limit value.
The tubing length to the particle counter should be less
than 1 m.
The classification number, the sample volumes/ measuring
period remain unchanged compared to the version of 1999.

Pharmig publications
Current perspectives on Environmental Monitoring
Review # 1
Guide to Disinfectants and their use in the
Pharmaceutical Industry
Microbiological Control for Non-Sterile
Pharmaceuticals

See:
https://www.pharmig.org.uk/en/products/publications/
or email: info@pharmig.co.uk

Dr. Tim Sandle

If you have any questions please

email:
info@pharmig.org.uk