Environmental Monitoring Handbook

for Pharmaceutical Manufacturers

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Table of Contents

Introduction...................................................................................................................................................... 1

Section 1. FUNDAMENTALS................................................................................................. 2

Fundamentals: Cleanrooms............................................................................................................................. 3

Fundamentals: Particle Counters..................................................................................................................... 5

Section 2. APPLICATIONS................................................................................................... 9

Applications: Cleanroom Standards.............................................................................................................. 10

Applications: EU GMP Annex 1........................................................................................................................ 18

Applications: Cleanroom Classification......................................................................................................... 25

Applications: Cleanroom Monitoring............................................................................................................. 32

Applications: Risk Assessment....................................................................................................................... 41

Section 3. SOLUTION........................................................................................................ 48

Solutions: Total Particle Portable Monitoring Best Practices....................................................................... 49

Solutions: Microbiological Portable Monitoring Best Practices.................................................................... 60

Solutions: FMS................................................................................................................................................ 67

Solutions: Alarm Rationale............................................................................................................................. 75

Solutions: Compressed Gas............................................................................................................................ 83

Particle Measuring Systems iii

List of Tables and Figures

Chapter 1. INTRODUCTION................................................................................................. 1

Chapter 2. FUNDAMENTALS: CLEANROOMS........................................................................... 3

Figure A-1  Turbulent Dilution of Contamination......................................................................................... 3
Figure A-2  Unidirectional Air wash............................................................................................................... 4
Figure A-4  Filling Mechanism diagrams........................................................................................................ 4

Chapter 3. FUNDAMENTALS: PARTICLE COUNTERS................................................................. 6

Figure B-1  lasair® pro AEROSOL PARTICLE COUNTER.................................................................................. 6
Figure B-2  particle counter schematic.......................................................................................................... 7

Chapter 4. APPLICATIONS: CLEANROOM STANDARDS............................................................12

Table C-1  Selected airborne particulate cleanliness classes..................................................................... 14
Table C-2  Selected airborne particulate cleanliness classes..................................................................... 15
Table C-3  Number of sample locations required........................................................................................ 16

Chapter 5. APPLICATIONS: EU GMP ANNEX 1........................................................................19

Figure D-1  European commission Logo...................................................................................................... 19
Figure D-2  Graphical Representation of Strategy 1.................................................................................... 21
Figure D-3  Graphical Representation of Strategy 2.................................................................................... 21
Figure D-4  EU GMP Annex 1 2022, Section 9.7............................................................................................ 23
Figure D-5  EU GMP annex 1 2002, Section 9.24........................................................................................... 23
Figure D-6  EU GMP Annex 1 2022, Section 10.10........................................................................................ 24

Chapter 6. APPLICATIONS: CLEANROOM CLASSIFICATION......................................................27

Table E-1  Airborne particulate cleanliness classes for cleanroom and clean zones ................................ 28
Figure E-1  Graphical representation of ISO class concentrations limits for selected classes.................. 29
Figure E-2  Diagram showing the locations for classification within the clean zone................................ 30
Figure E-3  Maximum Permitted Particle concentration calculation.......................................................... 30
Table E-2  Number of sample locations required........................................................................................ 30
Table E-3  Illustration of locations within the example clean zone and measurement results................. 31
Table E-4  EU GMP Annex 1:2008 room classification table........................................................................ 32

Particle Measuring Systems iv

 

Chapter 7. APPLICATIONS: CLEANROOM MONITORING..........................................................34

Table F-1  Monitoring Frequencies for In Operation Routine Particulate Sampling.................................. 38
Figure F-1  Graphical representation of strategy 1...................................................................................... 40
Figure F-2  Graphical representation of strategy 2..................................................................................... 40
Figure F-3  LASAIR® Pro AEROSOL PARTICLE COUNTER: CFR 21 PART 11.................................................. 41

Chapter 8. APPLICATIONS: RISK ASSESSMENT......................................................................43

Figure G-1  ICH (International Conference on Harmonisation) Logo......................................................... 43
Figure G-2  STEPS FOR RISK IDENTIFICATION AND ANALYSIS.................................................................... 45

Chapter 9. SOLUTIONS: TOTAL PARTICLE PORTABLE MONITORING BEST PRACTICES.................52

Figure H-1  Diagram of typical ISO rating in areas of cleanroom................................................................ 53
Figure H-2  PMS products comply with certification standards set for bio-contamination control......... 58
Figure H-3  Example Gemba walk paths...................................................................................................... 60
Figure H-4  Example sample locations......................................................................................................... 60

Chapter 10. SOLUTIONS: MICROBIOLOGICAL PORTABLE MONITORING BEST PRACTICES �����������63

Figure I-1  Example of active microbial air device: MiniCapt Mobile® Microbial Air Sampler................ 63
Figure I-2  Airflow model of an active air sampler....................................................................................... 65
Figure I-3  Active air sampler geometry....................................................................................................... 65
Table I-1  Technique Comparison................................................................................................................ 66

Chapter 11. SOLUTIONS: FMS............................................................................................69

Figure J-1  Diagram of typical ISO rating in areas of cleanroom................................................................. 70
Table J-1  EU GMP Annex 1 room classification table (Annex 1 2022)........................................................ 72
Figure J-2  Footnotes to above Table in Annex 1 2022 ............................................................................... 72
Figure J-3  STEPS FOR RISK IDENTIFICATION AND ANALYSIS..................................................................... 73
Figure J-4  Example Facility Monitoring System (FMS) Setup..................................................................... 73
Figure J-5  Main Page of Facility Monitoring Software................................................................................ 74
Figure J-6  Report Generator........................................................................................................................ 75
Figure J-7  Alarms page................................................................................................................................ 75

Chapter 12. SOLUTIONS: ALARM RATIONALE........................................................................77

Figure K-1  critical point in cleanroom environment.................................................................................. 77
Table K-1  Annex 1 (2003) particle cleanliness limits.................................................................................. 79
Figure K-2  Formula in ISO 14644-1:1999, Establishment of single sample volume per location............. 79
Figure K-3  EU GMP Annex 1 2003, Table 1, footnotes (a) and (e)............................................................... 79
Figure K-4  EU GMP Annex 1 2008, #4 and 5................................................................................................ 80
Figure K-5  EU GMP Annex 1 2008 text, #20................................................................................................. 80
Figure K-6  EU GMP Annex 1 2008 text, #13................................................................................................. 81
Figure K-7  2022 Annex 1, Table 1................................................................................................................ 81
Figure K-8  2022 Annex 1 text, Section 9.1.................................................................................................. 81
Figure K-9  2022 Annex 1 text, Section 9.9-9.10.......................................................................................... 82
Figure K-10  2022 Annex 1 text, Section 9.17 .............................................................................................. 82

Chapter 13. SOLUTIONS: COMPRESSED GAS.........................................................................86

Figure L-1  CRITICAL POINT OF A FILLING LINE........................................................................................... 86
Figure L-2  serratia marcescens.................................................................................................................... 88
Figure L-3  TYPICAL DECOMPRESSION IN MOST PHARMA ENVIRONMENTS: 2.5 TO 1.1 BAR..................... 88

Particle Measuring Systems v

 Without measurement there is no control

Introduction

Welcome to this first of our E-Books on life science applications. This first book will
Introduction
Introduction
focus on the overall topic of environmental monitoring; it presents new information and
also pulls from our extensive library of applications notes. As we head into a new era of
requirements with the release of the much anticipated EU GMP Annex 1 2022 along with
the adoption of a CCS, this book is a timely addition to any library.
Fundamentals
We will cover the fundamentals of particle counting and cleanroom designs, and this will
help establish the baseline of the technology being used to manufacture pharmaceutical
and life science products in controlled areas. It will also explain how to demonstrate that
control.
Applications
The second section will look at the standards applicable to production areas and the
expectation of regulatory bodies governing release to market of aseptic drugs, advanced
therapies, medical devices, and non-sterile products.

Solutions And finally, once the fundamental principles point the way, the standards and
requirements define what is necessary to fulfill quality attributes; we will look at the
instrumentation and techniques required to satisfy those needs.
If you have any questions regarding the content or have a desire to learn more about
these topics, please contact your local Particle Measuring Systems’ office, or us directly at
[email protected].

Frank Panofen
Director & General Manager Life Sciences Division

Particle Measuring Systems Page 1

 Without measurement there is no control

Fundamentals
Fundamentals

Cleanroom

Fundamentals
Fundamentals

Particle Counter
Fundamentals

Applications

Solutions

Particle Measuring Systems Page 2

 Without measurement there is no control

Fundamentals: Cleanrooms
Pharmaceutical Cleanroom Design Basics

To satisfy the assurances required by regulatory agencies, pharmaceutical products are

Fundamentals manufactured in a controlled environment. Cleanrooms are an example of a controlled
environment and are employed to reduce the contamination risk and variability of potential
production environment. As controlled environments, cleanrooms can be regulated to meet
Cleanroom
Cleanroom
specific standards. GMP regulations require that these environments are rigorously monitored
Fundamentals
Fundamentals to ensure that there is full and constant awareness of current environmental conditions for both
viable and nonviable contamination.
A cleanroom is the fundamental starting point for contamination control. A cleanroom is defined
Particle Counter as a room in which air filtration, air distribution, utilities, materials of construction, and equipment
Fundamentals are maintained in a controlled manner. Operational procedures are defined and regulated for
airborne particle concentrations to meet appropriate particulate cleanliness classifications.
There are essentially two basic types of cleanrooms:

Applications • Turbulent dilution of contamination

Turbulent airflow cleanrooms utilize the exchange rate of filtered air to dilute any
contamination down to an acceptable threshold. HEPA filtered air is delivered via a
central system through diffusion panels in the ceiling at a rate of between 10 to 40+
Solutions room volumes per hour (Air Exchange Rate).This value is a function of the operations and
number of personnel within each area. Return air is recirculated and uses a percentage of
fresh air to maintain comfort.

Figure A-1  TURBULENT DILUTION OF CONTAMINATION

• Unidirectional Air wash

Unidirectional air cleanroom utilizes the velocity of air to act as shroud to prevent
extraneous particles from a potential contaminating source to impact adjacent areas.

Particle Measuring Systems Page 3

 Where there are particles generated by the process or operators, the airflow also is
designed to sweep particles away from the critical zone; this can be affirmed by the
smoke airflow visualization test. Flowrates are optimally 0.36 – 0.54 m/s, however other
airflow velocities can be used.

Fundamentals

Cleanroom
Cleanroom
Fundamentals
Fundamentals

Figure A-2  UNIDIRECTIONAL AIR WASH

Particle Counter
Fundamentals
Within the cleanroom, operations will typically take place within HEPA filtered benches, allowing
for the manual manipulation of product, containers, and processing equipment. Flow direction
within these benches is either horizontal or vertical.
Horizontal flow goes directly toward the operator, sweeping
Applications
any operator-borne contaminants out of the critical zone.
If the flow is vertical, the air flows down over the process
ensuring a shroud is maintained of the critical areas.
The vertical flow also allows for fill and finish equipment to
Solutions
be automated and enclosed with a designed space.
The filling machine can be open to access from above and
utilize the unidirectional airflow within the room to ensure
isolation of the process to the outside activities within the
general room environment.
Restriction of access to the critical processing zone can be
limited using fixed or flexible curtains; this offers a certain
degree of isolation of the critical area verses the general room
environment where operators are able to intervene with the
process should it be required.
Alternatively, a dedicated filter can be employed to deliver
filtered air only to the processing equipment. This allows
for a lower grade of air for the background areas and a
higher degree of separation between the critical and the
background areas.
This design can be expanded and scaled up to create Figure A-4  FILLING MECHANISM DIAGRAMS

[email protected] Page 4
 restricted access barrier protection (RABS) where access to the critical zone can only be performed
using gloved aces ports. Full isolation can be achieved using fully closed isolator systems. These
also allow for enclosed sanitation and sterilization processing.
The nature of activities and choice of cleanroom used will also affect the type of environmental
monitoring required. The higher the access of operators to a process, the greater the risk of
contamination. This is because personnel are the single largest contributor of airborne
contaminants within a cleanroom.

Fundamentals Author
Mark Hallworth
Life Sciences Senior GMP Scientist
Life Science Division
Cleanroom
Cleanroom
Particle Measuring Systems
Fundamentals
Fundamentals
Mark Hallworth is the Life Sciences Regional Manager for Particle
Measuring Systems. He has lectured for pharmaceutical societies
throughout Europe, Asia, and the US on nonviable particle and facility
Particle Counter
Fundamentals
monitoring and the implications of validating those systems. He can be
reached at [email protected].

Editor: Noelle Boyton

Applications

Solutions

© 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the copyright owner is unlawful. Requests for permission or further
information should be addressed to Particle Measuring Systems, Inc. at +1 303-443-7100. Book Content. 10272022

[email protected] Page 5
 Without measurement there is no control

Fundamentals: Particle Counters

A Simple Guide to How Aerosol Particle Counters Work

Fundamentals
Overview
Aerosol particle counters are built to rapidly count and size contaminant particles in cleanroom air
and other controlled environments. Users tend to see this as a device where a button is pressed
and absolute results tumble out. However, when measuring anything small, it is important to be
Cleanroom aware of the technology within the instrument to understand the relevance of the generated data,
Fundamentals to put the operation of particle counters in context, and to be aware of the benefits and limitations
of the technology. No measurement is absolute―all measurements are relative to the measuring
technique employed. For example, if particles are measured with a scanning electron microscope,
it would not be a surprise to get slightly different
Particle
Particle
Counter
Counter
Fundamentals
Fundamentals results than from a different technology. The
instruments/technologies might even produce vastly
different results between these two at certain sizes
because each particular technology is producing a
Applications unique response.

Particle Types
Particles exist in a tremendous range of sizes, shapes,
Solutions and compositions; for example, inside a cleanroom
we could measure flakes of skin, small pieces of
silicon or metal, or fungal spores. The sources can be
very broad.
Figure B-1  LASAIR® PRO AEROSOL PARTICLE COUNTER

Particle Size
The size of particles is measured in micrometers (i.e., microns, µm), which is 10-6 meters (or a
millionth of a meter and a thousandth of a millimeter). State of the art semi-conductor facilities
measure in nanometers (nm), which is 10-9 meters (or a millionth of a millimeter); 1 micrometer
is 1000 nm. However, many readers may be more interested in the 0.5 and 5 micron particles
which are relevant to the pharmaceutical, health care, and medical device industries. To put size
into perspective, visible particles are around 50 microns (e.g., a human hair would be 50 to 150
microns), non-visible particles such as bacteria are somewhere between sub 1 and 15 microns,
and plant spores and pollens fall between the visible and non-visible at around 10 to 100 microns.
Most particles are non-uniform in structure, and this poses a question: How is the size qualified?
If you ask those working in industry, some would say it is based on the longest length, others the

Particle Measuring Systems Page 6

 volume, and some an equivalent hole size through which a particle could pass. There would be
many different answers, and it is probably fair to say that none of them would be wrong, provided
that they are qualified.
Particle counters determine the size of particles by matching a signal response generated by the
contaminant particle to an equivalent size of latex sphere. Users often look at the size and number
distribution reported by an instrument and treat this data as absolute without recognizing that
there are a number of operating variables, i.e., physical properties, refractive index, orientation,
etc. All these factors play a part in the size indicated by the counter, and therefore, they play a role
Fundamentals in the size channel in which the particle is counted.

Calibration Reference Standard using Latex Spheres

Cleanroom Aerosol particle counters are calibrated for size by sampling mono-dispersed (i.e., single size)
Fundamentals polystyrene latex spheres (PSLs) nebulized into the flow of HEPA/ULPA grade filtered air. The
instrument is adjusted for each test particle size used, and a calibration curve is generated within
the instrument.
Particle
Particle Counter
Counter The sizing response from environmental particles is referenced by the instrument as an equivalent
Fundamentals
Fundamentals to a perfectly spherical latex sphere and counted in one particular size range or channel. As a
result, any false sizing could affect not only the stated size but also the size channel into which the
particle is allotted, affecting the number distribution.

Applications
How Particle Counters Work
All of the commonly used cleanroom airborne particle counters, regardless of their manufacturer,
are based on the light scattering principle. Essentially, this means that they use a very bright light
source to illuminate the particles. Nowadays, this source is a laser diode, where previously gas
Solutions
lasers and ‘white light’ halogen bulbs were used.
This very bright light source shines through an optical block. Within the optical block are mirrors
and at least one photodetector. Sampled air is drawn through the laser beam by a small vacuum
pump. As the particles in the air pass through the laser beam, the laser light interacts with
particles and is scattered. The term ‘scattering’ means that the light undergoes a directional
change. This change occurs in all directions: Forwards, backwards and sideways. Below is a
schematic of how a sampled particle is read.

Figure B-2  PARTICLE COUNTER SCHEMATIC

[email protected] Page 7
 The elliptical clam shapes in the diagram are mirrors. In a particle counter, they are silvered so
that the reflecting surface is inside. As the light scatters, it is picked up by these mirrors, which
focus the scattered light onto one or more photodetectors.
The photodetector converts the burst of light energy from each particle into a pulse of electrical
energy. By measuring the height of the signal and referencing it to the calibration curve, we can
determine the size of the particle, and by counting the number of pulses, we can determine
quantity. It is relatively straightforward from that point to allocate particle numbers into size
channels.
Fundamentals
Light scattering is a general term and is composed of various different physical phenomena.
Scattering is made up of:

Cleanroom 1. REFLECTED LIGHT – when light hits a particle and is angularly deflected.
Fundamentals
2. REFRACTED LIGHT – when light goes through the particle and its direction of travel is
changed.

3. DIFFRACTED LIGHT – where the light comes close to the particle and is bent around it.
Particle
Particle Counter
Counter
Fundamentals
Fundamentals
There may also be a degree of absorption (when a percentage of the light energy is retained by the
particle), and in some instances, effects such as phosphorescence may occur from some particle
types. Therefore, scattering is a combination of many physical properties relating to light, and the
Applications interaction of light and particles. The interaction of light and particles depends on the particle
composition, its refractive index, and the difference between that particle and the background
medium, (i.e., air).
In operation, the instrument compares the response it is getting from the particle signal to the
Solutions calibration curve generated with latex spheres. What the instrument is actually doing is comparing
the response from the interaction of that particle and the laser light and then relating it to the
ideal latex sphere in a background of air. Users should be aware that particles with different
scattering responses will size either smaller or larger relative to the latex standard.
For example, a silicon particle has a high reflectivity relative to the latex standard and is going to
scatter a great deal of light. A particle of this material will size large compared to the standard. A
particle that absorbs light or doesn’t scatter very much light, such as a particle generated from
a heat source, is going to size small relative to the latex standard. Therefore we are not looking
at absolutes here. These sizing differences from the latex standard may assign the particles into
larger or smaller size channels.
The orientation that the particle takes when passing through the laser beam will also have an
effect on how it is sized. In an extreme example, if we sampled a rod shaped particle that exposes
its full length to the laser beam, the light would strike the largest surface area and scatter a
relatively large amount of light. If it only exposed one circular end, its relatively smaller cross
sectional area would cause it to size as a small particle.

[email protected] Page 8
 Conclusion
What particle counters do very well is allow users to take instantaneous samples and get a very
good real time indication of the load of particles in a room or around a critical process. Alternative
methods, such as using a filter, pump, or microscope, are time consuming, subjective, and labor-
intensive.
Aerosol particle counters have been, and continue to be, crucial and beneficial in the
development, operation, and advancement of cleanroom production environments. They are fast,
well defined, and non-subjective, and modern instruments are now extremely stable, robust, and
Fundamentals
simple to use. By using the scientific principles of light scattering and comparison to a calibrated
reference, these devices ensure repeatable, reliable data.

Cleanroom
Fundamentals
Author
Allen Humphries

Particle
Particle Counter
Counter
Fundamentals
Fundamentals Editor: Noelle Boyton

Applications

Solutions

Lasair® is a registered trademark of Particle Measuring Systems, Inc. © 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the
copyright owner is unlawful. Requests for permission or further information should be addressed to Particle Measuring Systems, Inc. at 1-303-443-7100. App Note 59. 10272022

[email protected] Page 9
 Introducing the new

Helping you meet all contamination control regulations including:

• EU Annex 1 • 21 CFR Part 11 • ISO 14644-1/2

PARTICLE COUNTERS MICROBIAL MONITORS

Portable and Remote Portable and Remote

Advisors
PMS™

Contamination
er for m c e
Control
an
PMS™

Advisory Team
inin
Tra

g
™P

S
PM

DATA MANAGEMENT ADVISORY SERVICES

Processors and Software

For more information contact

www.pmeasuring.com
[email protected]
 Without measurement there is no control

Fundamentals

Applications
Applications

Cleanroom Standards
ISO14644-1
ISO14644-2
Applications
EU GMP Annex 1

Cleanroom
Classification

Cleanroom
Monitoring

Risk
Assessment

Solutions

Particle Measuring Systems Page 11

 Without measurement there is no control

Applications: Cleanroom Standards

ISO 14644 Revisions Summary

Fundamentals
Abstract
The recent revision of ISO 14644-1 and -2 has introduced several changes for cleanroom
classification and monitoring guidelines. This paper will highlight the major changes in the
new ISO 14644-1 compared to the previous version as well as the possible impact on the
Pharmaceutical EU GMP Annex 1 and FDA Aseptic Processing Guideline.
Applications

Introduction
Cleanroom
CleanroomStandards
Standards
Over the last five years, the ISO Technical Committee 209 has been working on the revision of the
ISO14644-1
ISO14644-1 basic airborne cleanliness classification, 14644-1 and -2.
ISO14644-2
ISO14644-2
The ISO community voted in favor of the revision to update and improve the standard specifically
to:

• Simplify the classification process and, if possible, remove the need to evaluate the 95%
EU GMP Annex 1 upper confidence limit (UCL) for low sample location numbers (currently required for 2/9
of cleanroom locations)

• Review the classification procedure and make it more applicable to cleanroom

Cleanroom
operation. In this situation, the contamination is not expected to be evenly distributed: an
Classification assumption the current statistical approach makes

• Generally, update the standard as required to current thinking and industry requirements

• Avoid any radical change to the principles of the current ISO cleanliness classes 1-9
Cleanroom
Monitoring The same technical committee has also been working on the revision of the ISO 14644-2:2000 in
conjunction with the revision of ISO 14644-1. The ISO community voted in favor of the revision to
improve the ISO 14644-2:2000 standard to:

Risk • Simplify and clarify requirement and guidance tables that specify frequency of testing
Assessment and monitoring of cleanrooms used to demonstrate continued compliance with the
cleanliness classification
• Refine how these intervals may be extended, provided that automated monitoring
systems show the cleanroom is under control
Solutions • Provide new guidance on aspects that should be considered when configuring a
monitoring system for a cleanroom

On October 29th 2015, during the last voting session, the revised 14644-1 and -2 Standards were
approved by a significant majority of the member nations participating in the ISO/TC 209 committee.

Particle Measuring Systems Page 12

 Definitions
To simplify ISO 14644-1:2015, summaries are provided below of sections relevant to the revisions [1].

ISO 14644-1:2015
Cleanrooms and Associated Environments
Part 1. Classification of air cleanliness by particle concentration

This section specifies classes of air cleanliness for the world’s cleanrooms and controlled
environments in terms of the number of particles expressed as a concentration in air volume. To
Fundamentals determine the class, a specified testing method is required, which includes selection of sampling
locations.

ISO 14644-1 Introduction

Applications
ISO 14644-1 represents the first chapter of a series of documents, which describe the method,
procedures, and limits to be applied in the cleanroom design, operation and controls. This standard
Cleanroom Standards is used in different industries including Microelectronics, Pharmaceuticals, Aerospace, Medical
Cleanroom
CleanroomStandards
Standards
ISO14644-1 Devices, Healthcare, and Food Production.
ISO14644-1
ISO14644-1
ISO14644-2
ISO14644-2
ISO14644-2 It specifies classes of air cleanliness for the world’s cleanrooms and controlled environments in
terms of the number of particles expressed as a concentration in air volume. To determine the class,
a specified testing method is required, which includes a strategic selection of sampling locations.

EU GMP Annex 1
ISO 14644-1 Scope

The scope of this International Standard is to provide guidelines, specifications, and rules to
be used for cleanroom certification in terms of airborne particle concentration. ISO 14644-1
Cleanroom addresses all consideration for classification purposes that have cumulative distributions based on
Classification threshold (lower limit) sizes ranging from 0.1 µm to 5 µm. Lower particle size concentration limits
(nanoparticles), are addressed in ISO 14644-12.

Cleanroom
Monitoring

Risk
Assessment

Solutions

[email protected] Page 13
 2015 Revision
ISO 14644-1:2015 – New maximum concentration limits
Table C-1  Selected airborne particulate cleanliness classes

ISO 14644- MAXIMUM CONCENTRATION LIMITS (PARTICLES/m3)

1:2015
Classification 0.1 µm 0.2 µm 0.3 µm 0.5 µm 1.0 µm 5.0 µm
Fundamentals Number (N)
ISO CLASS 1 10
ISO CLASS 2 100 24 10
ISO CLASS 3 1 000 237 102 35
Applications ISO CLASS 4 10 000 2 370 1 020 352 83
ISO CLASS 5 100 000 23 700 10 200 3 520 832
ISO CLASS 6 1 000 000 237 000 102 000 35 200 8 320 298
Cleanroom Standards
Cleanroom
CleanroomStandards
Standards
ISO14644-1 ISO CLASS 7 352 000 83 200 2 930
ISO14644-1
ISO14644-1
ISO14644-2 ISO CLASS 8 3 520 000 832 000 29 300
ISO14644-2
ISO14644-2
ISO CLASS 9 35 200 000 8 320 000 293 000
The ISO classification is based on a new table (Table C-1), which uses the current and well-known
formula for the intermediate decimal classes:
EU GMP Annex 1

Cleanroom
Classification where

is the maximum permitted concentration (particles per cubic meter) of airborne

particles that are equal to and greater than the considered particle size.
Cleanroom
Monitoring
is the ISO classification number, which shall not exceed a value of 9 or be less than 1

is the considered particle size, in micrometers, that is not listed in Table C-1

Risk
Assessment
is a constant, 0.1, expressed in micrometers

The foremost concern in the Life Science industry is the removal of the ≥ 5 µm particle
Solutions concentration in ISO Class 5 clean areas (for classification purposes) when compared to the ISO
14644-1:1999 version.

[email protected] Page 14
 In the 1999 version, the limit is 29 particles per cubic meter as reported on the table below
(Table C-2) [2]. This change to the ISO/DIS 14644 standard is a major concern for a number of
reviewers.

Table C-2  Selected airborne particulate cleanliness classes

ISO 14644- Maximum concentration limits (particles/m3)

1:1999
Fundamentals Classification 0.1 µm 0.2 µm 0.3 µm 0.5 µm 1.0 µm 5.0 µm
Number (N)
ISO CLASS 1 10
ISO CLASS 2 100 24 10
Applications ISO CLASS 3 1 000 237 102 35
ISO CLASS 4 10 000 2 370 1 020 352 83
ISO CLASS 5 100 000 23 700 10 200 3 520 832 29
Cleanroom Standards
Cleanroom
CleanroomStandards
Standards ISO CLASS 6 1 000 000 237 000 102 000 35 200 8 320 298
ISO14644-1
ISO14644-1
ISO14644-1
ISO14644-2 ISO CLASS 7 352 000 83 200 2 930
ISO14644-2
ISO14644-2 ISO CLASS 8 3 520 000 832 000 29 300
ISO CLASS 9 35 200 000 8 320 000 293 000

EU GMP Annex 1
The reasons for the de-emphasis on the ≥ 5 µm ISO Class 5 limit include:

• Sampling and statistical limitations for particles in low concentrations make this
classification inappropriate.
Cleanroom • Sample collection limitations for both particles in low concentrations and sizes greater
Classification
than 1 µm make classification at this particle size inappropriate, due to potential particle
losses in the sampling system.

Cleanroom
Monitoring
ISO 14644-1:2015 – Sample locations
In order to achieve the goals of the ISO community, the significant changes with ISO 14644-1 are
related to the revision of the classification method, summarized as follows.

Risk Number of sample locations

Assessment
• A new table has been developed for the determination of the number of sample locations,
replacing:

Solutions

from the ISO 14644-1:1999 version of the standard.

[email protected] Page 15
 • For all room sizes above 6 m2, the new table (Table C-3 below) results in an increase of
required sample locations.

• The ISO 14644-1:1999 standard required the UCL 95% (Upper Confidential Limit)
calculation for sample locations between 2 and 9.

• The new table has been pre-calculated to eliminate the need for this calculation. The new
method, when successfully applied, assures that at least 90% of the room is compliant at
a 95% confidence limit.

Fundamentals Table C-3  Number of sample locations required

AREA OF ZONE (m2) ISO 14644-1:1999 ISO 14644-1:2015

2 2 1
Applications 4 2 2
6 3 3
Cleanroom Standards 8 3 4
Cleanroom
CleanroomStandards
Standards
ISO14644-1 10 4 5
ISO14644-1
ISO14644-1
ISO14644-2
ISO14644-2
ISO14644-2 24 5 6
28 6 7
32 6 8
36 6 9
EU GMP Annex 1
52 8 10
56 8 11
64 8 12
Cleanroom 68 9 13
Classification
72 9 14
76 9 15
104 11 16
Cleanroom 108 11 17
Monitoring
116 11 18
148 13 19
156 13 20
Risk 192 14 21
Assessment
232 16 22
276 17 23
352 19 24
Solutions 436 21 25
636 24 26
1000 32 27
>1000 N/A SEE FORMULA A.1

[email protected] Page 16
 Formula A.1: Formula used to determine the number of sampling locations

Fundamentals The determination of each sampling location will be based on a semi-random sampling
technique, based on a “hypergeometric” distribution, which is the statistical model for
sampling without replacement.
This is a significant change from current practice, meaning that each time a zone is classified, the
Applications sample locations may be different. If a company has determined through a risk assessment that
certain locations need to be examined specifically, then these should be applied in addition to the
randomly selected locations.
Cleanroom Standards
Cleanroom
CleanroomStandards
Standards
ISO14644-1 Recognizing that the ≥ 5.0 micron class limit for ISO 5 has been removed in the revised standard,
ISO14644-1
ISO14644-1
ISO14644-2 parties wishing to use the standard for classifying the environments EU GMP Grade A and B “at
ISO14644-2
ISO14644-2
rest” will have to use the macro-particle limit table retained in the standard.

Instrument Calibration
EU GMP Annex 1
Another important change in the new revised Standard is represented by the need to use
ISO 21501-4 compliant particle counters.
The paragraph B.2.2 of the (previous) ISO 14644-1:1999 required using “calibrated” particle
Cleanroom counters, not mentioning any specific calibration technique.
Classification

Cleanroom
Monitoring
The newly released version of ISO 14644-1:2015 specifically requires the use of ISO 21501-4
compliant instruments, as stated in paragraph A.2.2.

Risk
Assessment

Solutions Not all instruments will be able to meet the requirements described in A.2.2, and the use of
non-compliant instruments will require an additional explanation for authorities.
More information about the ISO 21501-4 requirements can be found on a separate application
note, available on the Particle Measuring Systems website, Knowledge Page: Understanding ISO
21501-4.

[email protected] Page 17
 Conclusion
The new changes described here will impact cleanroom classifications, and any company that
needs to comply with this standard will be required to update their internal SOP in order to meet
the new ISO 14644 requirements.
ISO 14644-1:2015 was published on Nov. 1st, 2015 and will be effective starting from Jan. 1, 2016.
All users who want to be compliant with this standard will be required to take any necessary
action before the end of 2016.

Fundamentals All Particle Measuring Systems instruments will have updates applied in order to fully meet these
new ISO requirements.
For more information about the new ISO 14644-1:2015, instrument firmware upgrades, or expert
consultancies, contact your Particle Measuring Systems local representative or use our website to
Applications Contact Us.

Cleanroom Standards
References
Cleanroom
CleanroomStandards
Standards
ISO14644-1 1. International Standards Organization. Cleanrooms and associated controlled environments
ISO14644-1
ISO14644-1
ISO14644-2 — Part 1: Classification of air cleanliness by particle concentration, ISO Standard No. 14644-
ISO14644-2
ISO14644-2
1:2015 (2015).

2. International Standards Organization. Cleanrooms and associated controlled environments —

Part 1: Classification of air cleanliness, ISO Standard No. 14644-1:1999 (1999).
EU GMP Annex 1

Author
Daniele Pandolfi
Cleanroom
Classification Global Product Line Manager for Data Management/Aerosol
Life Science Division
Particle Measuring Systems
Cleanroom Daniele Pandolfi has had experience in particle counter
Monitoring instrumentation and cleanroom contamination control for over ten
years. While building strong customer relationships, he has helped
many people solve their cGMP issues. Outside of work, he is a semi-
Risk professional photographer, an enthusiast of electronics, a lover of new
Assessment technology, and a keen traveler. He can be reached at dpandolfi@
pmeasuring.com

Editor: Noelle Boyton

Solutions

© 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the copyright owner is unlawful. Requests for permission or further
information should be addressed to Particle Measuring Systems, Inc. at 1-303-443-7100. App Note 220b. 10272022

[email protected] Page 18
 Without measurement there is no control

Applications: EU GMP Annex 1

The New EU GMP Annex 1 Revision: Impact on Environmental Monitoring Programs

Fundamentals
Introduction
In August 2022, a new revision of the EU GMP Annex 1 regulatory standard of the EU Guideline
for Good Manufacturing Practice for sterile drug products and drug substances was released by
the European Commission, replacing the most recent draft from 2020 and the existing revision
from 2008 [1,2]. The deadline for operational use of the new standards is August 25, 2023: a year
Applications after the release. These requirements regulate the manufacturing of sterile drugs made in and
imported to the EU. Pharmaceutical manufacturing is performed in controlled environments
to reduce contamination, and changes recently announced by Annex 1 focus more on strategic
Cleanroom Standards control than on measurement of quality. This new revision better aligns the manufacturing
ISO14644-1 principles contained in the Annex 1 to those presented by the World Health Organization (WHO),
ISO14644-2
Pharmaceutical Inspection Cooperation Scheme (PIC/S), and US Food and Drug Administration
(FDA).
The new revision is a complete rewrite of the existing Annex 1 from 2008 [2] and almost
EU GMP
GMPAnnex 1 quadruples the length. It divides the document into 10 newly defined sections. One major
sectioning change is the separation and differentiation of Certification (Section 4) and Monitoring
(Section 9), which allows for expanded guidance and distinction between premise design/
qualification and ongoing routine monitoring. There is a new section that discusses the concept
Cleanroom
Classification
of contamination control strategy (CCS). This section shifts to a new paradigm of incorporating
CCS as a central holistic approach to how each aspect of contamination interacts with the facility
as a whole. There is also a new section that discusses and
identifies Quality Risk Management (QRM) as a central principle
Cleanroom to defining processes, operations, and limits, and it ties to
Monitoring
CCS to balance process against risk. Additionally, as laid out
in the new revision, regulations for Environmental Monitoring
is essentially the same with a few enhanced descriptions to
Risk better align with QRM.
Assessment Figure D-1  EUROPEAN COMMISSION LOGO

Annex 1 Structure
The new structure provides a comprehensive understanding of where to find relevant content.
While the former 2008 version was not well organized and became a patchwork of changes over
Solutions
time, the new authors compiled the content in a logical, easy-to-follow way.
Quality and Manufacturing Officers interested in environmental monitoring will readily find
relevant content primarily in Chapter 5 “Premises” and Chapter 9 “Viable and nonviable
environmental and process monitoring”.

Particle Measuring Systems Page 19

 Cleanroom Classification and Qualification
In the Annex 1 revision, most of the relevant principles of the ISO 14644-1:2015 standard are
included (Section 5.26) [3]. The initial number of sampling sites, their even distribution and related
sampling volume in critical zones (Grade A and B) rely on this standard.
However, it is clearly stated that these are only the minimum requirements sufficient to classify
a cleanroom. All further decisions must be based on process knowledge and risk assessment.
Consequently, it will become very difficult in the future to defend why lesser parameters for the
qualification were chosen, especially for inspection of manufacturing environment sampling. This
Fundamentals
ties deeply with the statement in Section 5.28 of the ISO 14644-1:2015 standards, where “Clean
room qualification (including classification) should be clearly differentiated from operational
process environmental monitoring”. A clear differentiation needs to be made between each phase
of a clean environment's lifetime.
Applications
Let’s bring this concept into a simple equation:

Initial classification ≠ Re-Qualification ≠ Process Monitoring

Cleanroom Standards
ISO14644-1
The classification of a cleanroom, covered in the ISO 14644-1:2015 standard, is based on particle
ISO14644-2
load. There are no microbial limits given for this part of the process, but there has been a major
change towards the 2008 version of the guideline:
In chapter 5.25, particles of the size equal to or bigger than 5 µm have been removed
EU GMP
GMPAnnex 1
from the classification and qualification limit table for Grade A, but kept in the
recommended limits for monitoring of the process environment.
The reasons for the de-emphasis on the 5 µm limit in Grade A class includes:
Cleanroom
Classification • Harmonization of the European Requirements with the recent release of ISO 14644-1,
where the 5 µm limit in ISO Class 5 has already been removed.
• Sampling and statistical limitations for particles in low concentrations make this
classification inappropriate.
Cleanroom
• Sample collection limitations for both particles in low concentrations and sizes greater
Monitoring
than 1 µm make classification at this particle size inappropriate, due to potential particle
losses in the sampling system.

De-emphasis of the 5 µm limit only refers to the cleanroom classification process. The 5
Risk
µm particles still represent an important indicator of possible contamination during the
Assessment
manufacturing process and, therefore, must be kept under control continuously during filling and
manufacturing.
Discrepancy in the treatment of 5 µm particles between classification and monitoring are of
Solutions foremost concern and may cause discussion on the possible risk of not considering certain
particle sizes during initial qualification. These sizes will need to be within certain limits during
monitoring.
The language pertaining to the responsibility of defining alert and action levels and limits has
been made stronger and clearly refers to the cleanroom user, who must define the appropriate

[email protected] Page 20
 values based on a formal risk assessment and data trending analysis. This change emphasizes the
expectation of regulators that manufacturers set their action and alert limits based on historical
data, process knowledge and a risk-based approach. In addition, it is important not only to define
particle limits, but also an appropriate alarm strategy which encourages the evaluation of ISO
14644-2 and its recommended practices (paragraph B.3.4) [4].
The following strategies consider the importance of evaluating an alert or alarm situation using a
series of events rather than a single spot value.

Fundamentals Strategy 1
Establish a trigger threshold value based on a series of consecutivey higher readings.
For example: 3 consecutive 1-minute reading all above a specified level.

Applications

Cleanroom Standards
ISO14644-1
ISO14644-2

EU GMP
GMPAnnex 1

Figure D-2  GRAPHICAL REPRESENTATION OF STRATEGY 1

Cleanroom Strategy 2
Classification
Establish a trigger threshold value based on a high frequency of elevated readings. This method is
commonly referred to as "x out of y, " where "x" is the number of events and "y" is the number of
minutes.
Cleanroom
Monitoring For example: 3 out of the last 10 readings/minutes are above the specified alarm threshold.

Risk
Assessment

Solutions

Figure D-3  GRAPHICAL REPRESENTATION OF STRATEGY 2

[email protected] Page 21
 More information about monitoring guidelines dictated by ISO 14644-2:2015 can be found in the
Cleanroom Monitoring Chapter of this book.

Re-qualification Frequency
Section 5.29 gives manufacturers a challenge: Bi-annual re-qualification of critical zones (Grade
A and B) are becoming a standard of the industry. It is already a widespread practice, but many
pharmaceutical companies have differing strategies that will need to be thoroughly explained in
upcoming inspections. Modern technologies, including real-time methods for viable counts, that
Fundamentals
minimize downtimes caused by the re-qualification process and increase productivity will become
more crucial to the success of pharmaceutical companies.
NOTE

Throughout the new revision, Grade A and B environments are

Applications considered almost equal in the way they are treated for cleanliness.

Annex 1 and Microbial Impurities

Cleanroom Standards Microbial impurities can be divided into “viable” and “nonviable” particles. “Nonviable” impurities
ISO14644-1
are inert and will not contain any microorganisms, and readers of the new regulation should be
ISO14644-2
educated on all technical terms used to prevent misunderstandings of the document.
Laser-based particle counting, typically used for determining “nonviable” levels in a critical
pharmaceutical environment, displays both “nonviable” and "viable" particles, which is
EU GMP
GMPAnnex 1
comprised of "viable and culturable" and “viable but not culturable” (VBNC) impurities. The
following equation represents what is seen by laser-based particle counting:

Cleanroom
Classification
inert
nonviable = + viable and culturable
+ viable but not culturable

Cleanroom
Monitoring This equation is nonsense but its components have widespread usage, and therefore difficult to
change. At a minimum, people using this terminology should be aware of the potential drawbacks
from the habitual use of this language.
Risk Cleanrooms for pharmaceutical use are not classified by microbiological parameters, but rather
Assessment on the nonviable/inert aspect. Therefore, microbiological considerations start when the rooms
are qualified for intended use. As in the 2008 version, this is called the “in operation” stage and
proposed action limits can be found in Table 2 of the document. Although the values in the table
look familiar, some major changes have been made:
Solutions
• The values for Grade A zones are now set to "no growth" and not <1.
• No averaging of results is allowed.

Consequently, manufacturers are no longer allowed to “average out” non-welcome results by

looking at a scale of multiple measurements.

[email protected] Page 22
 Each single result should be considered and cause a deviation resulting in a full investigation.
However, this is only true for the qualification of the “in operation” stage and does not apply for
the monitoring of the process. True routine “in operation” monitoring limits can only be based
on historical data and locations, frequency, volume and duration of monitoring on a risk-based
approach and data generated during the qualification, as stated in Section 9.5. This may create
some confusion between the qualification stage's “in operation” and the routine monitoring
program's “in operation”.
Sections 9.7 and 9.27 follow previously established standards in terms of viable sampling which
Fundamentals can be found in other regulatory documents.

Applications
Figure D-4  EU GMP ANNEX 1 2022, SECTION 9.7

In essence, sampling should be done as close as possible to the critical area in Grade A
Cleanroom Standards environments, but without posing any risk to the process and sampling itself. To do both has been
ISO14644-1
a long-lasting dilemma, and often requires a specialized approach using technologies such as
ISO14644-2
single use.
The frequency of viable sampling has received an almost revolutionary renewal in the revision to
Annex 1, and it has become integrated with increasing control over the process by scientifically
EU GMP
GMPAnnex 1
sound rationale. One significant change is that viable sampling should be performed continuously
during routine process monitoring, as stated in Section 9.24. It will no longer be acceptable to
have only small, snapshot sampling that does not characterize the entire manufacturing process.
This concept was applied in the 2008 version for “nonviable” counts and has now been expanded
Cleanroom
Classification into “viable” counts, creating some short-term challenges for manufacturers. Continuous data
generation can only be achieved by either real-time methods or long-term, traditional viable
sampling that is quasi-continuous. The right combination of methods will become critical in
the decision-making process, and it will be an area of interest to see how inspectors push these
Cleanroom
requirements into the field and how manufacturers will respond. As always, the reasoning for all
Monitoring
decisions must be documented and based on risk assessment and historical/scientific data.

Risk
Assessment

Figure D-5  EU GMP ANNEX 1 2002, SECTION 9.24

Solutions Interestingly these strategies also apply to personnel monitoring (Section 9.25). At the moment,
manufacturers tend to avoid multiple samplings of operators in order to prevent contamination
build-up and the subsequent risk to the process and products. A possible solution could be the
implementation of more sampling techniques that are not susceptible for residuals, such as the
use of swabs instead of contact plates.

[email protected] Page 23
 A greater emphasis on the qualification and monitoring of personnel is seen in Chapter 7. The
stricter requirements may result in operators taking longer to contribute to the productivity of
the company (until they are fully qualified). Good personnel will become an even more precious
resource in the manufacturing of sterile drugs.
Grade A and B zones are now considered almost equivalent in how they are treated from a
monitoring perspective, and Sections 9.31 and 9.43 impose on manufacturers the need to identify
all microorganisms found in these environments down to the species level. This new requirement
emphasizes:
Fundamentals
1. The importance of Grade B in final product quality.

2. The need for investigations in both cleanrooms.

3. The need for understanding the instruments used in these zones and their capability to
Applications contribute to contamination.
Trending of environmental data, which has already been implemented on a worldwide scale, has
finally found its representation in Section 10.10.
Cleanroom Standards
ISO14644-1
ISO14644-2

EU GMP
GMPAnnex 1

Figure D-6  EU GMP ANNEX 1 2022, SECTION 10.10

Cleanroom
Classification Conclusion
The new Annex 1 revision provides insight into upcoming regulatory trends. In terms of
environmental monitoring content, there is significant emphasis placed on manufacturers basing
Cleanroom their decisions on scientifically sound and historical data while applying a risk-based approach.
Monitoring
From a microbiology and particle contamination perspective, this document may push modern,
relevant and scientifically sound monitoring methods into the pharmaceutical world, in addition
to a reasonable trending approach to data analysis.
Risk The overall quality of products is sure to increase with the released revision, and a stronger and
Assessment
deeper understanding of cleanroom performance inside each single company should be fostered
by allowing continuing discussion and evaluation of
the collected sampling data, and development of user
set alert and action levels. Monitoring plans should For a more detailed section
Solutions by section explanation of the
be proactively revised using growing knowledge of
the process and risk assessment tools. Rather than new Annex 1 revision check
considering each single monitoring session as isolated out this paper!
from the whole program, consider it part of a bigger Review of Annex 1 2022
picture.

[email protected] Page 24
 References
1. European Commission. The Rules Governing Medicinal Products in the European Union
Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human
and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products, Annex 1 (2022).
2. European Commission, EudraLex. The Rules Governing Medicinal Products in the European
Union Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for
Human and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products (corrected
version), Annex 1 (2008).
Fundamentals 3. International Standards Organization. Cleanrooms and associated controlled environments
— Part 1: Classification of air cleanliness by particle concentration, ISO Standard No. 14644-
1:2015 (2015).
4. International Standards Organization. Cleanrooms and associated controlled environments —
Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by
Applications particle concentration, ISO Standard No. 14644-2:2015 (2015).

Authors
Daniele Pandolfi
Cleanroom Standards
ISO14644-1 Global Product Line Manager for Data Management/Aerosol
ISO14644-2 Life Science Division
Particle Measuring Systems
Daniele Pandolfi has had experience in particle counter
EU GMP
GMPAnnex 1 instrumentation and cleanroom contamination control for over ten
years. While building strong customer relationships, he has helped
many people solve their cGMP issues. Outside of work, he is a semi-
professional photographer, an enthusiast of electronics, a lover of new
Cleanroom technology, and a keen traveler. He can be reached at dpandolfi@
Classification
pmeasuring.com

Frank Panofen, PhD

Cleanroom Director and General Manager Life Sciences
Monitoring
Life Science Division
Particle Measuring Systems
Dr. Panofen has a Diploma in Chemistry from the University of
Risk Bielefeld and a PhD in molecular and cell biology from the University
Assessment
of Osnabrück. He has expansive experience in the field of applied
pharmaceutical microbiology and serves as the Sterility Assurance/
Microbiology Product Line Manager at Particle Measuring Systems.
Frank has been an invited speaker at international conferences
Solutions
including ECA and PDA, with a strong regulatory background in
pharmaceuticals. He is a certified Microbiological Laboratory Manager
from ECA. He can be reached at [email protected].
Editor: Noelle Boyton

© 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the copyright owner is unlawful. Requests for permission or further
information should be addressed to Particle Measuring Systems, Inc. at 1-303-443-7100. App Note 268. 10272022

[email protected] Page 25
 Who defines your Contamination Control Strategy?
The experts you need to support you at every step of your process
including risk analysis and EM data interpretation.

Advisory Services
Helping you to achieve the highest level of
contamination control and sterility assurance

Contamination Performance
Training Control
Preparing your employees Advisory Team Optimization
o ensure success at every step Designing quality into your
operations using gold
standard PMS tools & processes

Environmental Cleaning and Aseptic Process Sterility Environmental

MonitoringRisk/ Disinfection Simulation Assurance Monitoring
Gap Aalysis Audits Trend Analysis

Gowning New Filling Crisis Utilities Process Risk

Line QbD Management Assessment

For more information contact

www.pmeasuring.com
[email protected]
 Without measurement there is no control

Applications: Cleanroom Classification

Cleanroom Classification for Pharmaceutical Applications

Pharmaceutical products are manufactured to meet exacting standards of both efficacy and
Fundamentals quality. All aspects of quality are reviewed considering the risks associated with the delivery
method (injected, ingested etc.) and the manner in which they were produced (aseptic,
terminally sterilized, or under lesser controlled conditions). This chapter looks at two parts
of that process: the quality of the environment in which the product is manufactured and the
Applications standards that surround the particle concentration limits that determine what a controlled
environment consists of.
This chapter examines the standards for physical testing (EN ISO 14644-1:2015 [1]) and those
Cleanroom Standards standards which apply in regulatory guidance (EU GMP Annex 1 [2]).
ISO14644-1
ISO14644-2
ISO 14644-1
In 1999, the new ISO 14644 room classification suite of standards became active, the first of
which was ISO 14644-1, which determined the method by which a room should be classified
EU GMP Annex 1
and the maximum allowable particles within a fixed volume of air. The reader should note that
although ISO 14644 has been adopted globally for cleanroom classification, there are differences
for routine monitoring, particularly between ISO 14644 and EU and WHO GMP.
Cleanroom
Cleanroom The certification state of the cleanroom must be defined in advance of testing; three states exist
Certification
Classification within the context of ISO 14644-1:
As Built: a completed room with all services connected and functional but without
production equipment or personnel within the facility.
Cleanroom
Monitoring At Rest: all the services are connected, all the equipment is installed and operating to
an agreed manner, but no personnel are present.
Operational: all equipment is installed and is functioning to an agreed format and a
specified number of personnel are present, working to an agreed procedure.
Risk
Assessment
The limits for the cleanroom concentration of particles greater than a prescribed size are defined
and presented in Table E-1 (below).

Solutions

Particle Measuring Systems Page 27

 Table E-1  Airborne particulate cleanliness classes for cleanroom and
clean zones (as indicated in ISO 14644)

ISO 14644-1:2015 MAXIMUM CONCENTRATION LIMITS (PARTICLES/m3)

Classification
Number (N) 0.1 µm 0.2 µm 0.3 µm 0.5 µm 1.0 µm 5.0 µm
ISO CLASS 1 10
ISO CLASS 2 100 24 10
Fundamentals ISO CLASS 3 1 000 237 102 35
ISO CLASS 4 10 000 2 370 1 020 352 83
ISO CLASS 5 100 000 23 700 10 200 3 520 832
ISO CLASS 6 1 000 000 237 000 102 000 35 200 8 320 298
Applications ISO CLASS 7 352 000 83 200 2 930
ISO CLASS 8 3 520 000 832 000 29 300
ISO CLASS 9 35 200 000 8 320 000 293 000
Cleanroom Standards These limits have been defined in accordance with the calculation from the standard using the
ISO14644-1 following formula for the intermediate decimal classes:
ISO14644-2

EU GMP Annex 1

where

is the maximum permitted concentration (particles per cubic meter) of airborne

Cleanroom
Cleanroom particles that are equal to and greater than the considered particle size.
Certification
Classification
is the ISO classification number, which shall not exceed a value of 9 or be less than 1

Cleanroom is the considered particle size, in micrometers, that is not listed in Table E-1
Monitoring

is a constant, 0.1, expressed in micrometers

Risk The relationship of particle size to its abundance within a population is therefore a function
Assessment of , and if the particle size is plotted against its concentration on a log/log scale, the slope
of the curve for each class is 2.08; this relationship is shown in the table above.

Solutions

[email protected] Page 28
Fundamentals

Applications

Figure E-1  GRAPHICAL REPRESENTATION OF ISO CLASS CONCENTRATIONS LIMITS FOR SELECTED CLASSES.
Cleanroom Standards
ISO14644-1
ISO14644-2 The designation for cleanroom or clean zone certification should also include the following
elements:

• The room classification number expressed as “ISO Class N”.

EU GMP Annex 1
• The occupancy state.

• The considered particle size. It is also possible to certify a cleanroom at multiple sizes; if
this is the case then the sample volume requirement for the largest particle size is used.
Cleanroom
Cleanroom
Certification
Classification
Example:
An example would be: unidirectional airflow device x is an ISO Class 5 clean zone at 0.5 µm
(3520 n/m3), operational state.
Cleanroom
Monitoring The clean zone now needs to be tested to prove the statement; the ISO 14644-1 standard identifies
each of the component steps required to prove compliance.
Assume we have a clean air device that we want to use for aseptic preparation area. This area
Risk needs to meet ISO Class 5 at 0.5 µm (≡ 10,000 /ft3) in the operational state, how do we go about
Assessment the process of determining the classification of this are?
The room is 12 m by 5 m (60 m2) and has a worktable in the center of the room.

Solutions

[email protected] Page 29
Fundamentals

Figure E-2  DIAGRAM SHOWING THE LOCATIONS FOR CLASSIFICATION WITHIN THE CLEAN ZONE
Applications

Step 1. Calculate the maximum permitted particle concentration

Cleanroom Standards
ISO14644-1
ISO14644-2
Figure E-3  MAXIMUM PERMITTED PARTICLE CONCENTRATION CALCULATION

3520 n/m3 is the maximum permitted concentration

EU GMP Annex 1
5 is the ISO classification number
0.5 µm is the considered particle size, in micrometers

Cleanroom
Cleanroom 0.1 µm is a constant, 0.1, expressed in micrometers
Certification
Classification

Step 2. Determine the number of sample locations from the Table below
Table E-2  Number of sample locations required
Cleanroom
Monitoring
Area of ISO 14644- ISO 14644- Area of ISO 14644- ISO 14644-
Zone (m2) 1:1999 1:2015 Zone (m2) 1:1999 1:2015
2 2 1 52 8 10
Risk 4 2 2 56 8 11
Assessment 6 3 3 64 8 12
8 3 4 68 9 13
10 4 5 72 9 14
24 5 6 76 9 15
Solutions
28 6 7 104 11 16
32 6 8 108 11 17
36 6 9

[email protected] Page 30
 Step 3. Calculate the sample volume

Fundamentals
From ISO 14644-1:2015, this formula finds a sample valume of 5.68 L, and for a standard particle
counter with a flowrate of 28.3 L/min, a 1-min sample would surpass the criteria of minimum
sample volume.
Applications
So, to meet specification we shall take a 1 minute sample at each of the 12 locations.
Step 4. Take measurements at each location (1 minute per sample) and record results
Cleanroom Standards Table E-3  Illustration of locations within the example clean zone and measurement results
ISO14644-1
ISO14644-2
LOCATION NUMBER /m3
1 708
2 885
EU GMP Annex 1 3 1522
4 2336
5 3363
6 2584
Cleanroom
Cleanroom
Certification
Classification 7 2301
8 2089
9 1344
Cleanroom
10 2013
Monitoring 11 1756
12 1897

Risk Step 5. Define report

Assessment

This area meets the specification for an ISO Class 5 clean zone at 0.5 µm and can now be used for
the purpose that it was designed for. Room classification will need to be repeated on a frequency
Solutions
defined by ISO 14644-2 [3].

[email protected] Page 31
 EU GMP Annex 1
The European Union GMP guidance for sterile manufacture was revised in 2003 to accommodate
the changes from various cleanroom standards to a single unified cleanroom standard [4],
ISO4644-1. The front page makes note of this:
“Annex 1 of the EC Guide to Good Manufacturing Practice (GMP) provides supplementary
guidance on the application of the principles and guidelines of GMP to sterile medicinal
products. The guidance includes recommendations on standards of environmental
Fundamentals cleanliness for clean rooms. The guidance has been reviewed in the light of the international
standard EN/ISO 14644-1 and amended in the interests of harmonisation but taking into
account specific concerns unique to the production of sterile medicinal products.”
Specifically, the means by which a cleanroom was certified needed to comply with the rules and
Applications format of the ISO14644-1 guidance, but, the ISO standard was modified with respect to sterile
medicinal products. To that end a table of cleanroom certification values that roughly translated
to ISO 14644-1 was defined.

Cleanroom Standards For clarity a series of notes appended the table, unfortunately the first of which, Note ‘a’, caused
ISO14644-1 certain confusion. This confusion was remedied in the 2008 release of the EU GMP Annex 1
ISO14644-2 which clearly outlines three phases that need to be performed [5]. Each cleanroom and clean
air device should first be classified; it should then be monitored to verify that conditions are
being maintained relative to product quality and that the data accrued from said monitoring be
reviewed in the light of risk to finished product quality.
EU GMP Annex 1
Table E-4  EU GMP Annex 1:2008 room classification table

Cleanroom
Cleanroom At Rest In Operation
Certification
Classification
EU GMP Grade Maximum number of particles permitted/m3 equal to or greater
than the tabulated size

Cleanroom 0.5 µm 5.0 µm 0.5 µm 5.0 µm

Monitoring A 3520 20 3520 20
B 3520 29 352,000 2900
C 352,000 2,900 3,520,000 29,000
Risk D 3,520,000 29,000 NOT DEFINED NOT DEFINED
Assessment
The reader will note, as indicated above, that the limits in the EU GMP table differ slightly to
those in the ISO 14644 standard. To perform the required certification, it is important to know the
workings of ISO 14644-1, how to certify a cleanroom in accordance with that standard, rules on
Solutions number of sample points, rules on sample point location, rules on volume of sample to be taken
at each location, and the rules on statistical analysis of cleanroom data that need to be followed.
However, rather than use the table for classification limits prescribed in ISO 14644-1, one should
be using the table shown above, as printed in the revised guidance document.

[email protected] Page 32
 Other expectations are also defined by the GMP. These can be expectations such as having a
sample volume for Grade A of 1m3 per sample location or using a minimum sample tubing length
due to the high precipitation of 5.0 µm particles in transport tubing (ideally no sample tubing
should be used). Also, recertification of the cleanroom should follow the guidance given in ISO
14644-2: once per year for ISO Grade 6 and greater and once per six months for ISO Grade 5.
Concessions are made for extending the ISO Grade 5 areas if a continuous monitoring system has
been implemented. Suitable times to perform certification are media fills or simulated filling runs.

Fundamentals References
1. International Standards Organization. Cleanrooms and associated controlled environments
— Part 1: Classification of air cleanliness by particle concentration, ISO Standard No. 14644-
1:2015 (2015).
Applications
2. European Commission. The Rules Governing Medicinal Products in the European Union
Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human
and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products, Annex 1 (2022).
Cleanroom Standards
3. International Standards Organization. Cleanrooms and associated controlled environments —
ISO14644-1
ISO14644-2 Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by
particle concentration, ISO Standard No. 14644-2:2015 (2015).

4. European Commission. EC Guide to Good Manufacturing Practice Revision to Annex 1:

Manufacture of Sterile Medicinal Products, Annex 1 (2003).
EU GMP Annex 1
5. European Commission, EudraLex. The Rules Governing Medicinal Products in the European
Union Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for
Human and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products (corrected
version), Annex 1 (2008).
Cleanroom
Cleanroom
Certification
Classification
Author
Mark Hallworth
Cleanroom Life Sciences Senior GMP Scientist
Monitoring Life Science Division
Particle Measuring Systems

Mark Hallworth is the Life Sciences Regional Manager for Particle

Risk Measuring Systems. He has lectured for pharmaceutical societies
Assessment throughout Europe, Asia, and the US on nonviable particle and facility
monitoring and the implications of validating those systems. He can be
reached at [email protected].

Solutions
Editor: Noelle Boyton

© 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the copyright owner is unlawful. Requests for permission or further
information should be addressed to Particle Measuring Systems, Inc. at +1 303-443-7100. Book Content. 10272022

[email protected] Page 33
 Without measurement there is no control

Applications: Cleanroom Monitoring

Understanding ISO Standards: ISO 14644-2:2015 Cleanroom Monitoring

Fundamentals
Abstract
This chapter underlines the major changes between the previous version of ISO 14644-2 and the
latest, second edition, dated December 12, 2015. This chapter discusses a specific approach to the
best practices to be adopted for an efficient and compliant cleanroom monitoring process.

Applications
Introduction
In combination with the revision to Part 1: Classification of air cleanliness by particle concentration
of ISO 14644-1:2015 [1], the ISO Technical Committee TC 209 has been working on the update to
Cleanroom Standards
ISO14644-1 the basic airborne cleanliness monitoring guidelines contained in ISO 14644-2 [2].
ISO14644-2 The TC 209 community voted in favor of the revision to update and improve the standard
specifically to:

• Simplify and clarify requirements and guidance tables that specify frequency of testing
EU GMP Annex 1 and monitoring of cleanrooms used to demonstrate continued compliance with the
cleanliness classification
• Refine how these intervals may be extended, provided that automated monitoring
systems show the cleanroom is under control
Cleanroom
• Provide new guidance on aspects that should be considered when configuring a
Classification
monitoring system for a cleanroom

The new standard's introduction also lists the goals of the ISO 14644-2, when successfully applied:
Cleanroom
Cleanroom • Emphasize the needs and advantages of a planned cleanroom contamination monitoring
Monitoring
Monitoring
• Provides the method for a correct particles contamination alarm and warning limits
setting, based on a careful evaluation of data trends
• Define the differences between a simple periodic cleanroom control and a more
Risk intensive/complex monitoring strategy
Assessment
• Enhance the installation and process knowledge, as to improve the risk assessment
evaluation and a faster reaction to any unexpected cleanroom performance
• Concretely reduce the operation cost by preventing production loss

Solutions

Particle Measuring Systems Page 34

 Terminology
Before we get into the specific requirements, the understanding of ISO Standard terminology is
essential. Below is a list of the most important terms and their definitions:

Monitoring
Monitoring is an observation of the process made in accordance with a specific
method, able to provide clear evidence of cleanroom performance.
Monitoring can be continuous, sequential, or periodic.
Fundamentals

Action Level Alert Level

User set level that, when User set level that is defined to
Applications exceeded, will require provide early warning of a drift
immediate intervention, from normal conditions. This level
root cause investigation, and should be used to prevent action
Cleanroom Standards corrective actions. level conditions.
ISO14644-1
ISO14644-2
Risk Assessment
The appropriate creation of risk assessment documentation is a basic requirement for
implementing a monitoring plan.
EU GMP Annex 1
The new ISO 14644-2:2015 requires a well-developed risk assessment document as a tool to
correctly understand the process, the critical areas/locations, possible sources of contamination,
and any element/event that may compromise or negatively impact the cleanroom performance,
Cleanroom product quality, or operation cost.
Classification
ICH Q9 – QUALITY RISK ASSESSMENT
The 2005 International Conference on Harmonisation
Cleanroom
Cleanroom of Technical Requirements for Registration of
Monitoring
Monitoring
Pharmaceuticals for Human Use is one of the best
guidelines available for proper risk assessment
development, review, and application. It is well
Risk integrated with good manufacturing practices
Assessment required by the Pharmaceutical Industry Standards.

PDA - PARENTAL DRUG ASSOCIATION

Solutions
Fundamentals of an Environmental Monitoring Program, Technical
Report No.13 is also a useful document when approaching monitoring
plan development, and it addresses the necessity of a meaningful,
manageable, and defensible monitoring program.

[email protected] Page 35
 Other useful tools to be considered for reliable risk assessment development are HACCP, FMEA/
FMECA, PHA, FTA, and/or HAZOP.
A responsible understanding of the production process and installation performance aids in the
prevention of unexpected out of specification conditions and also assists in achieving energy
saving targets.

Monitoring Plan
Fundamentals Particle concentration control is mandatory for cleanroom classification (ISO 14644-1:2015) and
monitoring (ISO 14644-2:2015). It provides clear evidence of cleanliness level and the capability of
cleanrooms or clean zones to accomplish required production performance.

CLEANROOM MONITORING CAN BE:

Applications
SEQUENTIAL
Sequential monitoring is normally performed by using
Cleanroom Standards
multiplexing manifold systems. This practice is well accepted
ISO14644-1
ISO14644-2 in semiconductor industries where small particle size (no
greater than 1 micron) are monitored.
The sequential monitoring method typically utilizes long
EU GMP Annex 1
transport tubes, which is unacceptable in Pharmaceutical
Manufacturing cleanrooms where larger particles are
considered and the risk of particle loss in tubing isn’t
negligible.
Cleanroom Read more about Particle Loss in Tubing.
Classification
CONTINUOUS
The continuous method uses multiple particle counters, one
Cleanroom
Cleanroom per individual location.
Monitoring
Monitoring
This monitoring approach provides a continuous flow of data
over time and is normally active during the entire production
phase.
Risk
The correct use of this control method allows for the immediate
Assessment
evaluation of unexpected contamination events, thereby
allowing for swift performance of corrective actions.
Pharmaceutical aseptic production is required to perform
Solutions continuous particle monitoring during the entire production
time. This methodology provides trend information that is
useful for alarm and action level evaluation.

[email protected] Page 36
 PERIODIC
The periodic method consists of performing particle monitoring
at a scheduled frequency (i.e., once per week) to demonstrate
continuous compliance of the cleanroom from one classification
test to the next.
ISO 14644-2:2015 allows periodic monitoring to be performed,
provided the user clearly specifies the test frequency.

Fundamentals Considerations
The ISO 14644-2:2015 standard lists the main items to be taken
into account when developing an airborne particle monitoring
system:
Applications
• Understand the contamination sources and their impact
on the activity in the cleanroom
• Locate particle counter probes as close as possible
Cleanroom Standards to critical zones
ISO14644-1
ISO14644-2 • Potential adverse impact of the sampling system on the process or the process
environment (e.g. possible effects of the rate of the extraction of the sample volume in
small volume environments)
• Airborne particle collection efficiency, suitability to monitor the selected particle size(s),
EU GMP Annex 1 and accessibility for maintenance, calibration, and repair
• Air sample flow rate and volume
• Frequency and duration of the collection of each air sample (determined by the sampling
rate)
Cleanroom
Classification • Sample probe configuration and orientation with respect to airflow (e.g. isokinetic or
anisokinetic)

HOW LONG CAN MY TUBING BE?

Cleanroom
Cleanroom
Monitoring
Monitoring • The use of long sample transport tubes must be avoided when intending to evaluate
particle concentrations at sizes greater than 1 µm. ISO requires the adherence to
maximum tubing lengths as specified by the particle counter manufacturer, which is
typically between 1.5 and 2 meters.
Risk • In case longer sample tubing is needed, the particle loss rate should be evaluated by
Assessment
measuring the number of particles that remain trapped in the transportation tubes.
• Some method examples can be found in How to Conduct Particle Transport Tests.

Solutions

[email protected] Page 37
 WHAT’S THE RIGHT ISOKINETIC PROBE POSITION?
• The sampling probe (i.e. the Isokinetic Probe or ISP) provides the advantage of
harmonizing the sampling flow speed with the typical laminar flow patch speed (90 ft/min
or 0.45 m/s) for the purpose of not creating any potential adverse impact of the sampling
system on the process with respect to the ISO requirements.
• The position of these probes must be evaluated in the risk assessment
and should not be located right under the filtration system (unless
specifically required).
Fundamentals • The position of the sampling probe must be representative of the
cleanroom performance and should be placed as close as possible to
where the production occurs and where risk to the product is highest.
Also, it must be clearly determined and included in monitoring SOPs to
allow for reproducible sampling operations.
Applications WHICH MONITORING METHOD SHOULD I USE?
ISO 14644-2:2015 does not specifically provide a link between the ISO class of the cleanroom and
the recommended monitoring method. That means that it is up to the user to choose the most
Cleanroom Standards appropriate method based on their specific manufacturing requirements and risk assessment.
ISO14644-1
ISO14644-2 Life science industries may also consider other standards to correctly and reasonably set up their
monitoring method and frequency. For example, the World Health Organization(WHO) issued
a document titled Environmental Monitoring of Clean Rooms in Vaccine Manufacturing Facilities
in November 2012 which includes instruction on determining the best monitoring frequency
EU GMP Annex 1 approach based on cleanroom cleanliness grade class (see Table F-1 below).

Table F-1  Monitoring Frequencies for In Operation Routine Particulate Sampling

Cleanroom
Classification Classification In Operation (Dynamic) Routine Particulate Sampling

Grade A
(filling operation)
For the full duration of operation
Cleanroom
Cleanroom
Monitoring
Monitoring Grade B Daily (working days)

Grade C Weekly
Risk
Assessment Grade D Not required

UDAF work stations in B Daily (working days)

Solutions UDAF work stations in C Weekly

UDAF work stations in D Monthly

UDAF in UNC areas Runtime re-qualification of UDAF is sufficient.

Table taken from Environmental Monitoring of Cleanrooms in Vaccine Manufacturing Facilities. WHO, 2012.

[email protected] Page 38
 How to Set Action and Alert Limits
The determination of action and alert limits is extremely important and must be supported by the
risk assessment as well as a consistent quantity of historical sampling data. In addition, they must
be supported by a consistent amount of data collected during previous monitoring controls.
ISO 14644-2:2015 states the importance of a long term evaluation as well as a yearly assessment
of limits, methods, and frequency. While not necessarily requiring a change, the assessment is
an important exercise in the critical evaluation of a monitoring plan. For old plans especially,
it should be questioned whether they are still applicable and consistent with the actual
Fundamentals
performance, activities, and needs of the cleanroom.
The standard provides some important recommendations, as well as applicable strategy to
keep in mind when setting alert and action limits. One with high significance is provided in the
paragraph B.3.1.3, quoted below:
Applications
B.3.1.3 When setting alert and action levels, it is important to be sensitive
to the high variability of airborne particle concentrations with time and at
different locations. In particular, special care shall be taken when considering
Cleanroom Standards
ISO14644-1 alert and action levels for cleanliness classes ISO Class 5 and cleaner with low
ISO14644-2 concentrations of particles. In these circumstances, the occurrence of “nuisance
alarms” due to false counts and/or natural variability of particle concentration is
more likely and should be avoided by careful selection of alert and action levels.
Frequent “nuisance” alarms should be avoided as they can lead to alarms being
EU GMP Annex 1
ignored by users.
This concept is frequently ignored when setting alert or action alarms for large particle sizes in
areas of high cleanliness, and this neglect may result in extensive and useless investigation to
Cleanroom determine the reason of alarms.
Classification
Good methods to deal with large particle sizes (i.e. 5 µm) in ISO 5 cleanrooms are described in
paragraph B.3.4 of the ISO 14644-2:2015 standards.
The following strategies consider the importance of evaluating an alert or alarm situation by
Cleanroom
Cleanroom taking into consideration a series of events rather than a single spot value.
Monitoring
Monitoring

Risk
Assessment

Solutions

[email protected] Page 39
 Strategy 1:
Establish a trigger threshold value based on a series of consecutively high readings. For example: 3
consecutive, 1 minute readings all above a specified level.

Fundamentals

Applications

Cleanroom Standards Figure F-1  GRAPHICAL REPRESENTATION OF STRATEGY 1

ISO14644-1
ISO14644-2 Strategy 2:
Establish a trigger threshold value based on a high frequency of elevated readings. This method
is commonly referred as “x out of y”, where “x” is the number of events and “y” is the number of
minutes.
EU GMP Annex 1
For example, 3 out of the last 10 readings/minutes are above the specified alarm threshold.

Cleanroom
Classification

Cleanroom
Cleanroom
Monitoring
Monitoring

Risk
Assessment
Figure F-2  GRAPHICAL REPRESENTATION OF STRATEGY 2

Solutions

[email protected] Page 40
 ISO 14644-2:2015 COMPLIANT – LEARN FROM FDA
483 AND TECHNICAL REPORTS
Being compliant with any standard requires experience,
knowledge, and a critical approach, enabling the
harmonization of regulation requirements with specific
production environments. Jumping on this task can be
done well in advance by studying the observations of FDA
inspectors made to users in similar situations.
Fundamentals
Several 483 warning letters have been redacted over the last years, and many of them are strictly
linked with monitoring plans' lack of compliance. Some of them are summarized in Scott Sutton's
The Environmental Monitoring Program in a GMP Environment.

Applications Some issued letters may refer to basic concepts when read by cleanroom experts, which remain
a useful component when preparing a demonstration of an efficient, compliant, and defensible
monitoring program.

Cleanroom Standards The following warning letter states the need of having a monitoring plan in place, a description
ISO14644-1 of the location to be tested, and a specific sampling method. Monitoring results are considered
ISO14644-2 insufficient if they don't support and link to a clear and approved plan.
…Regarding the increased non-routine surveillance monitoring performed to
further evaluate the Building 37 Flu manufacturing facility, there was no plan in
EU GMP Annex 1 place specifying the locations to be tested, method of sampling, and actions to
be taken when microbial contamination was noted…
The FDA emphasizes the need to develop a monitoring plan based on risk analysis. Compliance
with the reference standards before implementation must be verified. The following is an excerpt
Cleanroom
Classification
of the warning letter:
…The [redacted] method used for increased
surveillance monitoring of the environment has not
been qualified…
Cleanroom
Cleanroom
Monitoring
Monitoring One warning letter, dated 2001, requires the cleanroom user to
proactively and critically review the sampling historical data,
as it must be referenced to correctly set up the appropriate
alert and action levels. This requirement was likely difficult
Risk
Assessment to accomplish in 2001, but is now easily achievable using the
appropriate software platform (CFR 21 Part 11 compliant),
capable with a particle counter's data storage. Here is an
excerpt of the letter:
Solutions …the alert and action limits established for
Figure F-3  LASAIR® PRO AEROSOL
PARTICLE COUNTER: CFR 21 PART 11
the manufacturing areas are not based on
historical data taken from the EM Program…

[email protected] Page 41
 Conclusion
ISO 14644-2:2015 is not only a new standard to be compliant with but is also a beneficial tool to
use in achieving mature cleanroom environmental control.
The standard's main goal is to cultivate and promote a strong knowledge of cleanroom
performance inside every company. This goal is accomplished by the enforcement of continuing
discussion and evaluation of collected sampling data, the development of user-set alert and
action levels, and the proactive revision of monitoring plans and risk assessments based on the
ongoing developments of monitoring activities rather than considering each single monitoring
Fundamentals
session as isolated from the entire program.
ISO 14644-2:2015 was published on December 15, 2015, and all users who want to be compliant
with this standard are required to take necessary action immediately. For more information
about ISO 14644-2:2015 or other specific questions concerning cleanroom contamination control,
Applications
contact your Particle Measuring Systems local representative or navigate to our Contacts web
page.

Cleanroom Standards
ISO14644-1 References
ISO14644-2
1. International Standards Organization. Cleanrooms and associated controlled environments
— Part 1: Classification of air cleanliness by particle concentration, ISO Standard No. 14644-
1:2015 (2015).
EU GMP Annex 1 2. International Standards Organization. Cleanrooms and associated controlled environments —
Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by
particle concentration, ISO Standard No. 14644-2:2015 (2015).

Cleanroom
Classification
Author
Daniele Pandolfi

Daniele Pandolfi is the Global Product Line Manager, Aerosol in

Cleanroom
Cleanroom
Particle Measuring Systems’ Life Science Division. He has over ten
Monitoring
Monitoring
years’ experience in particle counter instrumentation and cleanroom
contamination control, built building close customer relationships.
He helps customers to solve their cGMP issues. He can be reached at
Risk [email protected].
Assessment

Solutions

© 2022 Particle Measuring Systems. All rights reserved. Lasair® is a registered trademark of Particle Measuring Systems. Reproduction or translation of any part of this work without the permission of the
copyright owner is unlawful. Requests for permission or further information should be addressed to Particle Measuring Systems, Inc. at +1-303-443-7100. App Note 246. 10272022

[email protected] Page 42
 Without measurement there is no control

Applications: Risk Assessment

Risk Assessment as a Process Quality Assurance Tool

Fundamentals
Abstract
Environmental contamination control is a critical component of sterile pharmaceutical
manufacturing, and Risk Management is necessary to ensure that the correct control practices
are in place. The Risk Management process is a series of steps, including risk assessment, that
allows for a deeper understanding of the manufacturing environment. Removing, reducing, or
Applications monitoring activities associated with a product or process to mitigate risk may be the result of
such an assessment. This qualitative risk assessment can be transformed into a quantitative
evaluation using modern risk analysis tools and procedures; these tools deliver the fully
Cleanroom Standards documented rationale behind the path chosen.
ISO14644-1
ISO14644-2
The Concept of Risk in Pharmaceutical Production
In the released revision of EU GMP Annex 1, the importance of risk management (Risk
Management) is highlighted as an appropriate tool for ensuring the quality of a process. The
EU GMP Annex 1
revision is explicit about the necessity of risk management for sterile drug manufacturers, and
it also widely recommends risk management for other product types, especially where control
of microbial, particle, and pyrogen contamination is required (e.g., certain liquids, creams,
Cleanroom ointments, and low bacterial intermediates) [1].
Classification The production and use of a drug (medicine) and its components necessarily involves a certain
degree of risk. It is important to understand that product quality must be maintained throughout
the product life cycle. This ensures that the attributes important to the quality of the drug (Critical
Cleanroom Quality Attributes) remain consistent throughout the development and production phases of the
Monitoring drug. According to ICH (International Conference on Harmonisation) Q6A, drug quality refers to
the suitability of a drug substance or drug product for its intended use [2].
In general, a risk management procedure focuses on analyzing each process in a product life cycle
Risk
Risk with the intent to perform an assessment, mitigation, and review of the associated risks over time.
Assessment
Assessment As defined in ICH Q9, risk assessment is a systematic process of organizing information to support
risk decisions that are made as part of a risk management process [3].
When talking about pharmaceutical quality, the
term “process” can take on different meanings.
Solutions It can refer to any of the stages of development,
production, testing, inspection, distribution, up
to and including drug delivery.
Figure G-1  ICH (INTERNATIONAL___
CONFERENCE ON HARMONISATION) LOGO

Particle Measuring Systems Page 43

 Additionally, it can include the design, qualification, and validation of equipment, instruments,
and facilities. Thus, a process is any activity that may directly or indirectly affect the quality
of the final product. It is immediately apparent that the scope of pharmaceutical quality risk
management is very broad.
Before delving into risk management procedures, it is good to reflect on some key distinctions,
starting with the difference between hazards and problems. Problems are related to the
perception or implementation of a process [4], whereas a hazard is understood as an intrinsic
property or quality that has the potential to cause damage to the process and, thus, to the end
Fundamentals customer.
Therefore, risk is a probabilistic concept; it is the combination of the probability of a certain event
occurring and the ability of this event to cause damage. This is further complicated by the inability
to detect this risk at the time of occurrence. From a technical point of view, risk is the product of
Applications probability and severity [3], where detectability - if introduced - should consider the risk that the
detection system will fail. The notion of risk thus implies the existence of a source of danger and
the possibility of it turning into harm [5].
Cleanroom Standards It is from this moment in the analysis of Risk Management that the risk is effectively actualized
ISO14644-1
and translated into something scientific and documented. From this point forward, the tests and
ISO14644-2
evaluations of the risk assessment must be transformed into something visual and quantitative so
that both the capacity to manage the risk and the source of the risk can be understood externally.
The final risk assessment tool is the identification of hazards and the scientific analysis and
EU GMP Annex 1
evaluation of the risks associated with exposure to them. This includes severity of damage
or harm to health, including logistical damage that may result from loss of product quality or
availability.
Cleanroom It is essential to have both a robust quality management system and good manufacturing
Classification practices in place to reduce the risks to product quality, patient safety, and company reputation to
an acceptable level.
There are two primary principles of quality risk management:
Cleanroom
Monitoring • Risk assessments should be based on scientific knowledge and driven by patient
protection.
• The level of commitment, formality, and documentation of the quality risk management
process should be commensurate with the level of risk.
Risk
Risk Assessment
Assessment
Risk Assessment
The risk assessment process consists of different steps ranging from the identification of hazards
to the analysis and evaluation of the risks associated with exposure to these hazards. Figure G-2
Solutions shows the steps developed during the analysis.

[email protected] Page 44
Fundamentals

Applications

Figure G-2  STEPS FOR RISK IDENTIFICATION AND ANALYSIS

Cleanroom Standards
ISO14644-1
ISO14644-2 The risk identification step is composed of the systematic use of information to identify potential
sources of harm (hazards) and possible consequences (impact/effect). The identification of these
harms and consequences is based on historical data, theoretical analysis, informed opinions,
stakeholder concerns, brainstorming sessions, etc. This information is necessary to deepen the
EU GMP Annex 1
knowledge of processes.
These three basic questions are often useful as an aid to clearly defining risk:

1. What could go differently than expected?

Cleanroom
Classification 2. What is the probability (likelihood) of it going differently from the expectation?

3. What are the consequences (severity)?

Risk analysis is the estimation of the risk associated with identified hazards. It is a qualitative or
Cleanroom
quantitative process of linking the probability and severity of harm (severity being a measure of
Monitoring
the possible consequences of a hazard) by evaluating the design/measures that have control over
their occurrence and detection [3]. Analyzing the degree of risk leads to defining the appropriate
tools or actions for its management over time. With some risk management tools, the ability
Risk to detect damage (detectability) may also be considered as a factor influencing the overall risk
Risk Assessment
Assessment
estimate.
Risk assessment then leads to the comparison of the estimated risk with certain risk criteria.
This is done through a quantitative or qualitative scale that determines its significance and,
Solutions subsequently, defines a threshold of acceptability. When risk is expressed quantitatively, a
numerical probability is used. Alternatively, risk can be expressed using qualitative descriptors,
such as "high," "medium," or "low," which should be defined in as much detail as possible. The
purpose of risk control is to reduce the risk to an acceptable, defined level.

[email protected] Page 45
 Therefore, the assessment should lead to either an acceptance of the risk itself (risk control), if the
level is acceptable, or to a reduction of the risk, if it is not an acceptable level.
The risk control process might include actions to:

• decrease the probability of occurrence of the identified hazards and risks

• decrease the severity of the identified hazards and risks
• increase the detectability of the identified hazards and risks

Fundamentals It must always be kept in mind that the implementation of risk reduction measures may
introduce new risks into the system (induced risk) or increase the significance of other already
existing risks (correlated risk). Therefore, it may be appropriate to review the assessment after
the implementation of a risk reduction process to identify and evaluate any possible changes.
The frequency of any review should be based on the level of risk. The risk review may include
Applications reconsideration of risk acceptance decisions [3, 4].
Acceptance is only possible when it is scientifically proven that the final quality of the process is
not critically impacted by the identified or residual risk. For some types of damage, even the best
Cleanroom Standards
quality risk management practices may not eliminate all of the risk. In these circumstances, the
ISO14644-1
ISO14644-2 application of an appropriate risk management strategy reduces the risk to quality to a specified
(acceptable) level. This acceptable level will depend on many parameters and should be decided
on a case-by-case basis. Various processes, including a cost-benefit analysis, can be used to
understand the optimal level of risk control, while always in compliance with regulatory and
EU GMP Annex 1 normative requirements [3].

Risk Management and Possible Approaches

Cleanroom Several scientific methods are currently available for use in risk analysis. These can be used
Classification independently or in conjunction with each other to achieve the most constructive outcomes. For
example, using a combination of the HACCP method with FMEA could lead to the production of
more comprehensive documents than either would on its own. We cover three of these below.
Cleanroom
Monitoring HACCP
The HACCP approach is a systematic, proactive, and preventive tool for ensuring product
quality, reliability, and safety [7]. HACCP stands for Hazard, Analysis, Critical, Control, Points,
Risk and it is based on the development of a "Decision Tree." Through this "decision Tree", the
Risk Assessment
Assessment HACCP approach facilitates the identification of critical and non-critical areas of the process
under analysis.

FMEA
Solutions
The FMEA approach is a systematic analysis of potential failure modes aimed at preventing
failures. It is a process of preventive action implemented before introducing new products,
modifications, or processes. FMEA stands for Failure, Modes, Effect, Analysis, and ideally,
FMEA analyses are conducted in the process/product design or development phases,
however, it can also be very useful when applied to existing products and processes. This

[email protected] Page 46
 approach is applicable in a variety of areas including the pharmaceutical manufacturing and
assembly. It consists of several steps that include reviewing the process, identifying potential
error modes, listing the potential effects of each error mode, assigning severity/occurrence
and detection to each effect, and calculating Risk Priority Number (RPN) to prioritize
mitigating actions that eliminate or reduce the risk.

Risk Priority Number (RPN)

The calculation of the Risk Priority Number represents the result obtained from the
Fundamentals evaluation of three parameters: "Severity" (the severity of the risk), "Detection" (the ability to
be able to detect the risk), and "Occurrence" (the probability that risk may occur and recur
within the process under analysis). For each of these parameters, variables are evaluated
and then multiplied to obtain the final evaluation. (RPN = Severity x Detection x Occurrence)
Applications [6]. Once the variables for each individual parameter have been established, the maximum
and minimum scores can be calculated to define the ranges. A risk value of "low" would be
assigned if the score value falls within the lowest range, "medium" if the score value falls
Cleanroom Standards within the intermediate range, and "high" if the RPN value falls within the highest range. The
ISO14644-1 division into different risk ranges is necessary for the completion of the next risk control step.
ISO14644-2 Assigning a risk value to one range rather than another contributes to the decision-making
process of risk reduction or acceptance. If the total risk identified is medium or high, then
corrective or preventive risk control actions must be applied to the process to lower its value
(i.e., risk of impact on quality). Additionally, where possible, the risk value should be reduced
EU GMP Annex 1
to an acceptable threshold value. Being in the "low" range determines the acceptance of
the risk as being below an established acceptable threshold and not requiring corrective or
preventive action because it is already under control.
Cleanroom If we take the fill and finishing of a product as an example, a good strategy for preventing
Classification environmental contamination of the sterile production process becomes essential in ensuring the
quality of the finished product. The risk assessment is therefore a necessary and fundamental tool
for pharmaceutical companies to utilize to strengthen the quality assurance process, combined
Cleanroom with the instrumentation used to practically perform what is theoretically assessed. Good data
Monitoring historicization and adequate data representation can be parameters to be considered to facilitate
the risk control and review process.
The appropriate use of quality risk management can facilitate (but does not obviate) the industry's
Risk obligation to comply with regulatory requirements [3].
Risk Assessment
Assessment

Conclusions
An effective approach to risk management can further ensure delivery of a high quality drug or
Solutions medicine to the patient by providing a proactive means to identify and control potential quality
issues during drug development and manufacturing. The main goal of any risk management
should be the protection of the end-user of the product, and product quality is the definitive
measure of the success of a quality risk strategy that identifies and maintains the safety of the

[email protected] Page 47
 end customer. A fully documented Risk Assessment enables pharmaceutical companies to obtain
high-quality products and comply with regulatory guidelines and requirements.
Ongoing product quality management is ensured through microbiological and particle control of
air and surfaces according to a defined monitoring plan. It is also important to keep in mind the
interdependence of the risk management and risk assessment approaches.
It can be useful to utilize external organizations for guidance. These experts can help the
company define and manage their risks by sharing
information gathered in clear and systematic ways. Let Particle Measuring Systems
Fundamentals industry experts support your risk
The communication can take place at any point in the
analysis and assessment needs.
process and the information shared could concern the Learn more:
existence, nature, form, likelihood, severity, acceptability, pmeasuring.com/consultancy-and-
control, treatment, detectability, or other aspects of the training-services/
Applications quality risks as required.

References
Cleanroom Standards 1. European Commission. The Rules Governing Medicinal Products in the European Union
ISO14644-1 Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human
ISO14644-2
and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products, Annex 1 (2022).

2. EMEA: European Medicines Agency, International Conference on Harmonisation of Technical

Requirements for Registration of Pharmaceuticals for Human Use. Q6A Specifications:
EU GMP Annex 1 Test Procedures And Acceptance Criteria For New Drug Substances And New Drug Products:
Chemical Substances, ICH Q6A (1999).

3. EMEA: European Medicines Agency, International Conference on Harmonisation of Technical

Requirements for Registration of Pharmaceuticals for Human Use. Quality Risk Management,
Cleanroom ICH Q9 (2005).
Classification
4. International Standards Organization, International Electrotechnical Commission. Safety
aspects — Guidelines for their inclusion in standards, ISO/IEC Guide No. 51:1999 (1999).

Cleanroom 5. Italy Government Agencies. Article 2, Legislative Decree No. 81 (2008)

Monitoring
6. Robin McDermott, Raymond J. Mikulak, Michael R. Beauregard. The Basics of FMEA, ISBN
0527763209 (1996).

7. World Health Organization. WHO Application of Hazard Analysis and Critical Control Point
Risk (HACCP) Methodology in Pharmaceuticals, WHO Technical Report Series No 908, Annex 7
Risk Assessment
Assessment (2003).

Solutions

[email protected] Page 48
 Author
Marco Castaldo
Head of Advisory Team
Particle Measuring Systems
Marco has a diverse background in the pharmaceutical field,
including expertise in the areas of Sterility Assurance, Quality
Assurance and Compliance. He gained extensive experience in
Fundamentals large, multinational companies before becoming a Consultant
and a Project Manager. Marco helps pharmaceutical companies
with projects including: validation of new technologies,
reduction of microbiological contamination, and internal audits
for aseptic behaviors. As Head of the Advisory Team for Particle
Applications
Measuring System, he is focused on supporting pharmaceutical
companies worldwide to improve their sterility assurance
approach and strategy. He can be reached at mcastaldo@
Cleanroom Standards pmeasuring.com.
ISO14644-1
ISO14644-2
Editor: Noelle Boyton

EU GMP Annex 1

Cleanroom
Classification

Cleanroom
Monitoring

Risk
Risk Assessment
Assessment

Solutions

© 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the copyright owner is unlawful. Requests for permission or further
information should be addressed to Particle Measuring Systems, Inc. at +1 303-443-7100. App Note 317. 10272022

[email protected] Page 49
Complete Cleanroom Contamination Monitoring
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• Microbial Samplers

Services Environmental
Monitoring Systems
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• Project Management ASSURANCE • Sampling Automation
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 Without measurement there is no control

Fundamentals

Applications

Solutions
Solutions Solutions
Total Particle Portable
Monitoring Best
Practices

Microbiological Portable
Monitoring Best
Practices

Facility Monitoring
Systems

Alarm Rationale

Compressed Gas

Particle Measuring Systems Page 51

 Without measurement there is no control

Solutions: Total Particle Portable

Monitoring Best Practices
Fundamentals Pharmaceutical Requirements for Portable Monitoring in Cleanrooms

All pharmaceutical and biotech products must be manufactured in accordance with the
current Good Manufacturing Practice (cGMP) regulations. Environmental monitoring of these
pharmaceutical manufacturing areas meets the requirement for contamination control of an
Applications environment, which is essential in defining clean manufacturing and demonstrating the necessary
controls are working.
A pharmaceutical company must have a quality department that is responsible for the routine
quality assurances that:
Solutions
Establishes documented evidence which provides a high degree of assurance that a specific
process will consistently produce a product meeting its predetermined specifications and
quality attributes. [1]
Total Particle Portable
Total Particle Portable
Monitoring Best
Monitoring Best Practices To satisfy these requirements, the products are manufactured in controlled environments/
Practices
cleanrooms. Cleanrooms are employed to reduce the variability of production environments,
and as controlled environments they can be tested and proven to meet specific standards. GMP
Microbiological Portable require that these environments be rigorously tested and monitored to ensure that there is full
Monitoring Best and constant awareness of current environmental conditions for both viable and nonviable
Practices
contamination.
A cleanroom is the fundamental starting point for contamination control; a cleanroom is defined as
Facility Monitoring A room in which air filtration, air distribution, utilities, materials of construction, and
Systems equipment are maintained in a controlled manner. [2]

Operational procedures are defined and regulated for airborne particle concentrations to meet
appropriate particulate cleanliness classifications. The International Standard, ISO 14644-1 [3] is
Alarm Rationale
the current international standard of defining cleanroom contamination levels.
Pharmaceutical cleanrooms are classified according to required particle concentrations of
the air that meet the cleanliness criteria for the manufacturing process being performed. The
determination of the cleanroom class is a process based on statistically valid measurements,
Compressed Gas
and its a function of the filtration and operations status of the room; it is, in essence, a calibration
of the room to ensure it meets its intended classification. It is not, primarily, a function of risk of
application.

Particle Measuring Systems Page 52

 There are three measurement phases involving particle counting in cleanrooms:
As Built: a completed room with all services connected and functional but without
production equipment or personnel within the facility.
At Rest: all the services are connected, all the equipment is installed and operating to an
agreed manner, but no personnel are present.
Operational: all equipment is installed and is functioning to an agreed format and a
specified number of personnel are present, working to an agreed procedure.
Fundamentals
The airborne particle count test is performed by counting particles at defined grid locations within
the cleanroom. The test points should be equally spaced throughout the room and at work height
to demonstrate the quality of the air cleanliness at the work area.
Applications Pharmaceutical cleanrooms typically operate at Class 5 (most aseptic areas), Class 7 (surrounding
areas), or Class 8 (support areas).

Solutions

Total Particle Portable

Total Particle Portable
Monitoring Best
Monitoring Best Practices
Practices

Microbiological Portable
Monitoring Best
Practices

Facility Monitoring
Systems

Figure H-1  DIAGRAM OF TYPICAL ISO RATING IN AREAS OF CLEANROOM

Alarm Rationale Pharmaceutical Cleanroom Utilization

Once a cleanroom has been tested to compliance to cleanroom classification (typically using
a Light Scattering Aerosol Particle Counter (LSAPC)), the classification achieved dictates which
production activities can be performed in that cleanroom or clean air device. The FDA defines two
Compressed Gas areas.

1. Critical Areas

2. Supporting Clean Areas

[email protected] Page 53
 Critical area: This area is defined as critical because it contains products that are vulnerable to
contamination if exposed. To maintain product assurance, it is essential that the environment in
which aseptic operations are conducted be controlled and maintained at an appropriate quality.
One aspect of environmental quality is the particle content in the air. Particles are significant
because they can enter a product as an extraneous contaminant and can also contaminate it
biologically by acting as a vehicle for microorganisms.
Air in the immediate proximity of exposed sterilized containers/closures and filling/
closing operations would be of appropriate particle quality when it has a per-cubic-meter
Fundamentals particle count of no more than 3,520 in a size range of 0.5 µm and larger when counted at
representative locations normally not more than 1 foot away from the work site, within the
airflow, and during filling/closing operations. This level of air cleanliness is also known as
Class 100 (ISO Class 5).
Applications We recommend that measurements to confirm air cleanliness in critical areas be taken at
sites where there is the most potential risk to the exposed sterilized product, containers, and
closures. [4]

Solutions Supporting Clean Areas: Classification of a supporting clean area is explained by the FDA as
follows:
The nature of the activities conducted in a supporting clean area determines its
Total Particle Portable classification. It is recommended that the area immediately adjacent to the aseptic
Total Particle Portable
Monitoring Best
Monitoring Best Practices processing line meet, at a minimum, Class ISO 7 standards under dynamic (operational)
Practices
conditions. Manufacturers can also classify this area as Class ISO 6 or maintain the entire
aseptic filling room at Class ISO 5. An area classified at a Class ISO 8 air cleanliness level is
Microbiological Portable appropriate for less critical activities (e.g., equipment cleaning). [4]
Monitoring Best
Practices
Environmental Monitoring
Once a cleanroom or clean air device has been proven to meet the requirements for cleanliness
from a certification perspective, it must also demonstrate that this control can be maintained
Facility Monitoring
Systems throughout production periods. The environment needs to be rigorously monitored to ensure
that there is full and constant awareness of current conditions, including the detection of periodic
events which could be catastrophic if gone unnoticed. Constant monitoring creates a continuous
flow of information, resulting in a large quantity of data which can be used to watch for trends.
Alarm Rationale The manufacturing facility should therefore have a comprehensive environmental monitoring
program, which includes monitoring for nonviable and viable airborne particulates, surface
viable contamination, and, in the aseptic areas, personnel. These procedures should address
frequencies and locations for the monitoring sample points, warning and alarm limits for each
Compressed Gas area, and corrective actions which need to be undertaken if any of the areas show a deviation
from expected results. Actions taken when limits are exceeded should include investigation into
the source of the problem, the potential impact on the product, and any measures required to
prevent a recurrence.

[email protected] Page 54
 In general, less frequent monitoring is required in areas of a lower classification (ISO 7, ISO 8, or
unclassified rooms). This reduced frequency monitoring performed in "controlled" environments
(ones with some level of particulate controls) should be of the same integrity as that sampled in
the highest classification.
For both critical areas and supporting clean areas, a portable particle counter can be used.
Portable particle counters are chosen based on several influencing factors for ergonomics and
suitability for the areas to be tested.

Fundamentals • Size channels required. There is a traditional requirement for two primary channels
for monitoring, 0.5 µm and 5.0 µm. However these sizes can be complimented with
additional sizes both smaller and larger than the given range. The expansion of sizes
allows for further investigation for out of tolerance trends, as events may generate
Applications particles within a specific band indicative of a failure mode.

• Sample flow rate of 1 CFM (1 cubic-foot/minute = 28.3 liters/min) is the most suitable
for the majority of cleanroom activities, including certification. This flowrate has suitable
particle transport qualities that allow for up to 2 m of tubing to be used without significant
Solutions losses of the larger sized particle counts. Higher flowrates are available (up to 100 LPM);
these very high flowrates allow for rapid qualification testing of clean environments where
low populations of the 0.5 µm particles exist. The higher flowrate gives a statistically
Total Particle Portable significant sample volume in a shorter period.
Total Particle Portable
Monitoring Best
Monitoring Best Practices
Practices • Mains or battery-operated functionality allow for ease of portability and ergonomics
of the instrument chosen. Batteries should last for the duration of required testing or be
available to be live exchanged without compromising data quality.
Microbiological Portable
Monitoring Best
Practices • Mobility, either by hand or cart: the technician may require several instruments in order
to perform environmental monitoring. If small spaces are required to be tested, then the
instrument can be placed in the clean air device, or tubing can be used to transport the
Facility Monitoring sample from within an environment to the particle counter.
Systems
• Accessories such as a local display for controls and alarming and a built-in printer for
sample point generated reports may also be required. Electronic transfer of data to a
central software or LIMS may be required as thermal paper has limitations for durability
Alarm Rationale and transposition requirements for reporting.

A portable instrument is typically used throughout the facility by moving it from location to
location after each measurement. The data record for each sample point should consist of the
following information:
Compressed Gas

[email protected] Page 55
 Primary Data

• Sample Date and time

• Sample location
• Channel data and results and units

• Instrument status (hardware alarms)

Additional information to support the primary data includes.

Fundamentals • Operator taking sample
• Instrument identification (serial number)
• Alarm threshold excursions
• Sampling parameters
Applications
• Occupancy state

This data is then either printed locally or exported to a software management application for
further review.
Solutions
Reviews of data should be performed in three phases. Short term, medium term, and long term
trending.

• Short term trends look at sample to sample relationship and stability of a process; it also
Total Particle Portable
Total Particle Portable allows for ‘n’ out of ‘m’ nuisance controls where samples might reflect the process and not
Monitoring Best
Monitoring Best Practices first air.
Practices
• Medium term trends allow for a day-to-day or batch-to-batch review. Trend data reviews
at this time scale show if a particular process or occupancy influences particle burden
Microbiological Portable during production
Monitoring Best
• Long term trends are every 3, 6, or 12 months and may identify a gradual decline in the
Practices
effectiveness of installed controls.

Facility Monitoring The reporting and trending requirements feed into the Contamination Control Strategy (CCS) of a
Systems site and should be performed and documented routinely.

Alarm Rationale

Compressed Gas

[email protected] Page 56
 Choosing the Most Suitable Particle Sample Point Locations in the Cleanroom

Introduction
As environmental system designers, we are often asked where to place sample points for particle
monitoring whether it be performed in a pharmaceutical cleanroom or clean device (RABS,
isolator, etc.).
The answer is not always straightforward. There are several guidance documents that offer advice
Fundamentals on what processes need to be monitored and also advice on suitable distances from the process
being monitored. The goal of this chapter is to identify the considerations, establish the most
suitable location for monitoring a process, and build a scientific rationale for that decision.
Particle counting in pharmaceutical applications can be clearly segregated into one of three
Applications categories: certification, qualification, and monitoring. Each category requires a different
approach.
Certification: Measuring a cleanroom to a standard. The only standard recognized worldwide is
ISO 14644-1, which defines how a cleanroom performs and its ability to show uniformity across
Solutions the entire space. This is done irrespective of the activities performed in it.
Qualification: The process of analyzing risk assessment for the activities in the room.
Qualification follows grid methodology testing methods. Particle counts are measured in both
Total Particle Portable
Total Particle Portable
operational and at-rest states; however, the operational data is the most valid.
Monitoring Best
Monitoring Best Practices Monitoring: The ongoing sampling of the cleanroom on a frequency relative to the degree
Practices
of control required to prove management over risk to the finished product. The number of
sample points and their locations are determined by risk assessment and the qualification and
Microbiological Portable certification processes.
Monitoring Best
Practices
Certification
As mentioned above, cleanroom certification is based on ISO 14644-1 standards. The specifics
Facility Monitoring of the assessment may vary slightly between FDA and EU GMP regulations, but the underlying
Systems methodology is standard.
Certification demonstrates that the entire area meets a specific ISO classification by
particle concentration. That is, irrespective of the final use of the room, only the design and
Alarm Rationale implementation of the filtration system are considered. The international standard means that a
cleanroom tested to meet compliance for ISO 5 standards will meet that standard independent
of geography and regulatory aspects (i.e., FDA or EU GMP). This provides a universal standard
to show that a cleanroom level has been established. Particle Measuring Systems’ products,
including the Airnet® II and IsoAir® Pro-E Aerosol Particle Sensors, comply with new ISO standards
Compressed Gas
set in 2015. The interactive software of the Lasair® Pro Aerosol Particle Counter can even step the
user through the certification process.
There are many different resources to prove ISO compliance and this paper will not cover these
in depth. However, using the example of a classic filling machine (Grade A/ISO 5) within a Grade B
(ISO 7) background area, the basic rules of testing can be demonstrated.

[email protected] Page 57
 Fundamentals

Applications

Solutions Figure H-2  BIOCAPT® SINGLE-USE AND STAINLESS STEEL MICROBIAL IMPACTORS, AND THE MINICAPT® MOBILE MICROBIAL AIR
SAMPLER COMPLY WITH ISO 14698-1, CERTIFICATION STANDARDS SET FOR BIO-CONTAMINATION CONTROL

1. The number of sample points is based on a statistical function of the area. Calculate the area
Total Particle Portable
Total Particle Portable of Grade A/ISO5. Find the number of required sample locations in the table.
Monitoring Best
Monitoring Best Practices
Practices • Calculate the area of Grade A/ISO5. Find number of required sample locations in the table.
• Calculate the area of Grade B/ISO7. Find number of required sample locations in the table.
Microbiological Portable 2. Sample point placement for the Grade A (ISO5) area:
Monitoring Best
Practices • The sample points must all be equidistant and at work height, irrespective of the activity at
the location of their placement.
• Samples are taken in a grid pattern at the identified locations. Derive the minimum
Facility Monitoring number of sampling locations, NL, from ISO 14644-1 Table A.1. This table provides the
Systems number of sampling locations related to the area of each cleanroom or clean zone to be
classified and provides at least 95% confidence that at least 90% of the cleanroom or clean
zone area does not exceed the class limits.
• PASS/FAIL criteria are calculated for ISO and EU GMP Annex 1. It is recommended to have
Alarm Rationale both sets of data, as the FDA requires ISO14644-1 and the EU requires Annex 1 data points
(although the EU data would suffice for the FDA).
3. Sample point placement for the Grade B (ISO7) area:

• Repeat the steps used for the Grade A (ISO5) area.

Compressed Gas • It may be more difficult to determine the locations of the sample points due to the unusual
shape of a room. Derive the minimum number of sampling locations, NL, from ISO 14644-1
Table A.1. This table provides the number of sampling locations related to the area of each
cleanroom or clean zone to be classified and provides at least 95% confidence that at least
90% of the cleanroom or clean zone area does not exceed the class limits.
4. A final report is created and marks the end of the certification phase.

[email protected] Page 58
 Qualification
The qualification phase considers the risks to the quality of the finished product. Each activity
must be considered and measured. Continuing with the example of the filling line, let us consider
the accumulator table at the exit of the sterilizer tunnel. The risk is that glassware (vials/bottles)
are exposed to the open environment and operator. Therefore, contamination can fall into clean
vials/bottles prior to filling. Operator intervention and the shifting of glassware causes turbulent
air movement on the table, impacting contamination risk to the exposed vials/bottles. Therefore,
it is an area of contamination risk and the following actions should be taken:
Fundamentals

Applications

Solutions

Total Particle Portable

Total Particle Portable
Monitoring Best
Monitoring Best Practices
Practices
1. Divide the area of risk into a 3 x 3 or a 4 x 4 grid. If the activity can occur at several levels, then
each level (working height, +150 mm from work height, and +300 mm from work height)
Microbiological Portable must be considered.
Monitoring Best
Practices 2. Take a particle sample at the center of each of the grid squares at each level.

• Samples are taken during ‘At Rest’ and ‘Operational’ states. It may be required to work
around an activity or operator to gain suitable data.
Facility Monitoring • Slight movement of sample points within the grid square is acceptable. A location is
Systems invalid if found to impede normal activities.

3. When all samples are taken this will provide a particle map of the pharmaceutical activity.
Each of the key functions within the cleanroom (filling point, stoppering, general background
Alarm Rationale
activities, etc.) should be analyzed accordingly.

Monitoring
Compressed Gas The location of the monitoring points must be based upon a formal risk assessment using tools
such as but not limited to Failure Mode and Effects Analysis (FMEA) or Failure Mode, Effects,
& Criticality Analysis (FMECA), with data from the certification and qualification testing. Other
factors, such as equipment interference, mounting points, operator impedance, and operator
intervention, contribute to selecting the final location for the sample probe. In the current
regulatory environment, a risk assessment is absolutely required. Without a risk assessment, poor

[email protected] Page 59
 or incorrect sampling methodology can lead to data unreliably associated to the process. It could
also lead to potential impact to finished product quality. Without the option of correlating events,
the lack of connection between location and sample frequency can lead to long investigations for
out of tolerance events.
There are several steps to defining a risk-based environmental monitoring plan:

1. Process understanding: You must study personnel and material flows within the assessed
area in addition to the production operations. This will give an understanding as to how the
system is used and what evidence there is to support its state of control, such as:
Fundamentals
• Current monitoring practices
• Historical data
• Smoke studies
Applications
This Gemba walk of the process and
rooms is necessary to define the
scope of monitoring required and to
aid in applying a process that fits with
Solutions an organization's internal practices.
Figure H-3 is an example.
Figure H-3  EXAMPLE GEMBA WALK PATHS

Total Particle Portable

Total Particle Portable 2. Definition of critical areas: Identification using Hazard Analysis Critical Control Point
Monitoring Best
Monitoring Best Practices
Practices
(HACCP) helps which critical areas require environmental monitoring, and identifies areas
which meet the needs of a critical sample location.

3. Evaluation of sampling methods: You need to make a determination between traditional

Microbiological Portable methods such as volumetric air samplers, newer technologies such as Rapid Microbiological
Monitoring Best Methods, or manual collection techniques such as swabbing and contact plates. Also,
Practices determine if the chosen method needs to be portable, continuous, remote, etc.

4. Definition of potential sample locations: Determine a single sample location within each
critical area, following this criteria (as shown in the adjacent figure):
Facility Monitoring
Systems • Check the available space around
the critical area.
• Measure the size of probes and
plate holders.
Alarm Rationale
• Determine the accessibility to the
location for operator maintenance.
• Assess the interaction between the
process operation with personnel
Compressed Gas and material flows.
• Calculate the probability of
potential contamination events.
Figure H-4  EXAMPLE SAMPLE LOCATIONS

[email protected] Page 60
 5. Definition of critical control points (CCP): Each individually considered location is
evaluated according to the FEMA method to rank and identify critical sample locations.

6. Define sampling parameters: The sample frequency is found based on the criticality of
operations along with any additional criteria, such as incubation parameters and mitigating
measures that might be put into place prior to establishing a monitoring plan.

Sampling practicalities include elements such as:

• The isokinetic sample probe should face into the air stream.
Fundamentals • The minimum length of tubing should be used.
• Although different manufacturers claim specific lengths of tubing can be used with
their particle counter, this is typically a function of vacuum pump dynamics and not
of particle transportation. Particles that are 0.5 µm move freely in long lengths of
Applications tubing. However, 5.0 µm particles do not have this same mobility. As 5.0 µm particles
are a greater concern, the tubing should be maintained at its shortest recommended
lengths (For more information on tubing, read, “Particle Loss in Transport Tubing” by
Particle Measuring Systems).
• Particle Measuring Systems quotes maximum tubing lengths based upon the same
Solutions conditions of airflow and has a recommended maximum length of 3 m. However, for
pharmaceutical particle systems we advise a reduced recommended length of 2 m
to ensure transportation of the larger particles.
Total Particle Portable From the FDA’s Aseptic Processing cGMP Guideline:
Total Particle Portable
Monitoring Best
Monitoring Best Practices
Practices

“filling/closing
Air in the immediate proximity of exposed sterilized containers/closures and
operations would be of appropriate particle quality when it has a
Microbiological Portable
Monitoring Best per-cubic-meter particle count of no more than 3520 in a size range of 0.5 µm and
Practices
larger when counted at representative locations normally not more than 1 foot
away from the work site, within the airflow, and during filling/closing operations.
Facility Monitoring
Systems
This level of air cleanliness is also known as Class 100 (ISO 5).
”
The frequency of sampling should reflect the risks and follow from the FDA guidelines on
sterile manufacturing and the EU GMP Annex 1. Particle monitoring should be automated and
Alarm Rationale maintained in a continuous state when glassware and products are exposed.

Compressed Gas

[email protected] Page 61
 References
1. Federal Drug Administration. General Principles of Validation, (1987).

2. Institute of Environmental Sciences. Federal Standard Airborne Particulate Cleanliness

Classes in Cleanrooms and Clean Zones, Federal Standard 209E (1999).

3. International Standards Organization. Cleanrooms and associated controlled environments

— Part 1: Classification of air cleanliness by particle concentration, ISO Standard No. 14644-
1:2015 (2015).
Fundamentals 4. U.S. Department of Health and Human Services, Food and Drug Administration, Center for
Drug Evaluation and Research (CDER,) Center for Biologics Evaluation and Research (CBER),
Office of Regulatory Affairs (ORA). Guidance for Industry Sterile Drug Products Produced by
Aseptic Processing — Current Good Manufacturing Practice, (2004).
Applications
Author
Mark Hallworth
Life Sciences Senior GMP Scientist
Solutions Life Science Division
Particle Measuring Systems

Mark Hallworth is the Life Sciences Regional Manager for Particle

Total Particle Portable Measuring Systems. He has lectured for pharmaceutical societies
Total Particle Portable
Monitoring Best throughout Europe, Asia, and the US on nonviable particle and facility
Monitoring Best Practices
Practices monitoring and the implications of validating those systems. He can be
reached at [email protected].

Microbiological Portable
Monitoring Best Editor: Noelle Boyton
Practices

Facility Monitoring
Systems

Alarm Rationale

Compressed Gas

Airnet®, BioCapt®, IsoAir®, Lasair®, and MiniCapt® are registered trademarks of Particle Measuring Systems, Inc. © 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the
copyright owner is unlawful. Requests for permission or further information should be addressed to Particle Measuring Systems, Inc. at +1 303-443-7100. App Note 79. 10272022

[email protected] Page 62
 Without measurement there is no control

Solutions: Microbiological Portable

Monitoring Best Practices
Fundamentals Comparing Microbiological Air Monitoring Techniques for Critical Environments

Abstract
This paper discusses the use of active air and passive air settle plate monitoring for routine and
continuous pharmaceutical manufacturing. Scientific literature supporting settle plates and active
Applications
air sampler monitoring effectiveness is evaluated to support the transition from passive to active
air monitoring.

Solutions Introduction
Contamination is monitored continuously with the latest in cleanroom technologies. However,
traditional growth-based solutions, limited to 4-hour sampling periods, are still the most common
method used for microbial monitoring. To provide evidence that a continuous sampling of
Total Particle Portable
Monitoring Best
cleanroom air is performed, pharmaceutical manufacturers widely use settle plates even if
Practices scientific and regulatory experts agree that they are a non-quantitative and non-validatable
method. Compare this to active microbial air samplers, validated to run for a prolonged period in
continuous mode to sample one cubic meter of air.
Microbiological Portable
Microbiological Portable
Monitoring Best
Monitoring Best Practices
Practices

Facility Monitoring
Systems

Alarm Rationale

Compressed Gas

Figure I-1  EXAMPLE OF ACTIVE MICROBIAL AIR DEVICE: MINICAPT MOBILE® MICROBIAL AIR SAMPLER

Particle Measuring Systems Page 63

 Method Efficiency
ISO 14698:2003's Annex B describes a technique for determining collection efficiency of microbial
air samplers, broken into two separate types [1]:

• Physical efficiency is the ability of the sample to collect various sizes of particles.
• Biological efficiency is the efficiency of the sample in collecting microbe-carrying
particles.

Physical efficiency is the same for inanimate particles, particles carrying a microorganism, or
Fundamentals
particles that are microorganisms. Biological efficiency is expected to be lower than physical
efficiency because it depends on the survival of the collected microorganisms and the growth
medium. Annex B is mainly concerned with physical efficiency.
In a 2005 publication, a highly accredited author, Dr. William Whyte, concluded settle plates were
Applications
a “fundamental method of measuring the number of microbe-carrying particles that will deposit
onto a given area in a given time. There is therefore no need to determine its collection efficiency”
[2]. In 2016, the European Journal of Parenteral & Pharmaceutical Science in partnership with
Whyte and T. Eaton reassessed and suggested improvements to EU cGMP’s Annex 1, specifically
Solutions for how airborne concentration and settle plate counts of MCPs contribute to the grade of a
pharmaceutical cleanroom [3]. Using more accurate deposition velocities, the EU cGMP maximum
concentrations can be revised to provide more accurate settle plate counts.
Total Particle Portable ISO 14698:2003's Annex A specifies the selection of the microbial air sampling device to be
Monitoring Best
Practices
dependent on the purpose of sampling. In addition, the device should have an impact velocity
(speed of the air hitting the culture medium) that is a compromise between:

Microbiological Portable 1. A high enough velocity to allow the entrapment of viable particles down to
Microbiological Portable
Monitoring Best approximately 1 µm, and
Monitoring Best Practices
Practices
2. A low enough velocity to ensure viability of particles by avoiding mechanical damage
or the break-up of clumps of bacteria or micromycetes.
Facility Monitoring
Systems The ISO standard's recommendation has generally been a sampler at or near 50 percent physical
recovery at 1 µm (a D50 of 1 µm). From a microbiological stance, 1 µm is the size of most common
species of individual bacteria. Fungal particles are usually 2 to 5 µm, and Bacillus anthracis spores
have a size range from 0.65 to 2.0 µm.
Alarm Rationale

Compressed Gas

[email protected] Page 64
 The Importance of Design
If a stream of gas undergoes a sharp change in direction, the particles it transports will tend to
continue in their original direction. Particles having different dimensions and densities will follow
different trajectories and may be collected separately. When a jet of air is accelerated through a
nozzle, the particles it transports are carried at the same speed as the fluid and follow its flow line.
If the fluid flow lines rapidly change direction at the nozzle output, the particle trajectories will
appreciably depart from the airflow lines, depending on the inertia associated with the particles.
In other words, the particles will tend to run in a straight line, and if they find a surface in their
Fundamentals
path, they can adhere to it.

Applications

Solutions

Total Particle Portable

Monitoring Best
Practices

Figure I-2  AIRFLOW MODEL OF AN ACTIVE AIR SAMPLER

Microbiological Portable
Microbiological Portable Active air impactors are designed to sample particles in the air or other gas through a collision
Monitoring Best
Monitoring Best Practices
Practices with a solid surface. The impactor’s geometry is optimized to allow laminar flow into the nozzle
(e.g., Re < 2300), with a velocity as high as possible and a D50 as low as possible.

Facility Monitoring
Systems

Alarm Rationale

Compressed Gas

Figure I-3  ACTIVE AIR SAMPLER GEOMETRY

[email protected] Page 65
 Comparing Monitoring Methods
Due to modern cleanroom clothing features, the microbial-carrying particles detected in aseptic
areas with operators are mainly in the size range of 0.5 µm to 5 µm. For these reasons, continuous
microbial active air sampling in Grade A is recommended, replacing the combination of settle
plates and single or intermittently used active air sampling activities. The following table
summarizes the differences between techniques.
Table I-1  Technique Comparison

Fundamentals
CONTINUOUS MICROBIAL
COMPONENT SETTLE PLATES
ACTIVE AIR SAMPLING

Continuous
BOTH CAN BE USED TO MONITOR ALL PHASES OF PRODUCTION.
Applications monitoring

MEASURES THE NUMBER OF MEASURES THE

Measuring the
MICROORGANISMS SETTLING FROM TOTAL NUMBER OF
concentration of
THE AIR ONTO A KNOWN SURFACE MICROORGANISMS IN A
microorganisms in
Solutions the air
AREA WITHIN A KNOWN TIME IN A QUANTIFIABLE VOLUME
TURBULENT ENVIRONMENT. OF AIR.

NOT A QUANTITATIVE METHOD.

Total Particle Portable RESULTS ARE OFTEN THE NUMBER A QUANTITATIVE METHOD.
Monitoring Best Quantitative OF CFUS PER SETTLE PLATE, THE RESULTS CAN BE
Practices method WITH THE SIZE OF THE PLATE AND ANALYZED IN TERMS OF
THE TIME EXPOSED OFTEN NOT TIME AND AIR QUALITY.
REPORTED.
Microbiological Portable
Microbiological Portable
Monitoring Best DOES NOT DETECT LOW
Monitoring Best Practices
Practices Detect low CONCENTRATIONS OF DETECTS LOW
concentrations MICROORGANISMS AND PROVIDES CONCENTRATIONS OF
of microorganisms VERY LOW SENSITIVITY IN GRADE A MICROORGANISMS.
Facility Monitoring DUE TO HIGH AIRFLOW.
Systems
BOTH CAN BE PLACED CLOSER THAN TRADITIONAL VOLUMETRIC
Position in the SAMPLERS TO CRITICAL AREAS WHERE THE PRODUCT IS EXPOSED
filling machine TO AIR. BOTH CAN BE PROVIDED IN STERILE FORM AND ARE
SMALLER THAN TRADITIONAL VOLUMETRIC SAMPLERS.
Alarm Rationale
Comparison of CORRELATION OF DATA
THE CORRELATION OF DATA IS
microbiological IS POSSIBLE BECAUSE
NOT DEFINABLE DUE TO THE TWO
and particulate AIRFLOW SPEED OF THE
METHODS’ DIFFERENCES.
Compressed Gas data TWO SYSTEMS IS SIMILAR.

VALIDATED ACCORDING
Validation NOT VALIDATED.
TO ISO 14698-1.

Table 1 highlights that active air sampling is an improved approach compared to both settle
plates and traditional volumetric air samplers in Grade A continuous monitoring applications.

[email protected] Page 66
 Monitoring in Different Cleanroom Areas
When patient safety is key, the qualification of pharmaceutical cleanrooms is a necessary step.
Particularly, microbiological qualification verifies the cleanliness level of rooms where medicines
are manufactured. Following qualification, companies must design a monitoring plan that
demonstrates air quality is in accordance with specifications established during qualification.
Through monitoring, microbiological contamination can be controlled and minimized.
Grade A (ISO 5) areas include the product and materials in contact with the product, including
the surrounding environment (i.e., air). For this reason, they are considered extremely critical and
Fundamentals
subject to continuous monitoring with high air frequencies during all production phases. Grade B
(ISO 7) areas are used to protect Grade A areas and include the presence of operators in a variable
number depending on the production process. Here, the purpose of microbiological monitoring is
to verify the level of microbiological contamination is within specifications. The microbial trend of
Applications these areas must always be constant or slightly decreasing.
Settle plates are not recommended in Grade A areas because they do not detect low
concentrations of microorganisms and offer low sensitivity with their high airflow rate. Settle
plates are only acceptable in Grade B, C, and D areas where less turbulent air movement allows for
Solutions microbe-carrying particles to be deposited at a higher rate.
Continuous microbiological monitoring of Grade A air is already required by cGMP and
implemented for total particle monitoring. It provides key information on the amount and size of
Total Particle Portable total particles present in the air at a given sampling point. It is therefore extremely important to
Monitoring Best
have a strategy for both particle and biological monitoring that utilize validated methodologies
Practices
quantifying both the microorganisms and particles present in a sampling area. Doing so will aid in
determining potential correlation between events and provide the data necessary for root cause
Microbiological Portable investigations.
Microbiological Portable
Monitoring Best
Monitoring Best Practices
Practices

Conclusion
The settle plate method is a non-validated, non-quantifiable method that does not take into
Facility Monitoring
Systems account a microorganism's recovery rate. This method should not be used in Grade A (ISO 5)
where high air speeds are common and air changes are frequent. These conditions make it
difficult or impossible for microorganisms and particles containing microorganisms to settle.
Settle plates have more viability in static environments.
Alarm Rationale Continuous microbiological monitoring of air in critical areas should be performed with validated
methodologies with a recovery rate as high as possible. Regulations will keep pushing standards
higher, and this strategy promotes better process knowledge while substantially increasing sterility
assurance of the released product.
Compressed Gas

[email protected] Page 67
 References
1. International Standards Organization. Cleanrooms and associated controlled environments
— Biocontamination control — Part 1: General principles and methods, ISO Standard No.
14698:2003 (2003).

2. Whyte, W. (2005) 'Collection efficiency of microbial methods used to monitor cleanrooms'.

European Journal of Parenteral and Pharmaceutical Sciences, 10 (2). pp. 3-7. ISSN 0964-4679

3. W Whyte (School of Engineering, University of Glasgow, Glasgow G12 8QQ) and T Eaton
Fundamentals (AstraZeneca, Macclesfield, Cheshire, SK10 2NA) 'Deposition velocities of airborne microbe-
carrying particles' - European Journal of Parenteral & Pharmaceutical Sciences 2016; 21(2):
45-49

Applications Authors
Gilberto Dalmaso, PhD.
Anna Campanella, PhD.
Paola Lazzeri
Solutions
Editor: Noelle Boyton

Total Particle Portable

Monitoring Best
Practices

Microbiological Portable
Microbiological Portable
Monitoring Best
Monitoring Best Practices
Practices

Facility Monitoring
Systems

Alarm Rationale

Compressed Gas

Lasair® and MiniCapt® are registered trademarks of Particle Measuring Systems, Inc. All trademarks are the property of their respective owners. © 2022 Particle Measuring Systems. All rights reserved.
Reproduction or translation of any part of this work without the permission of the copyright owner is unlawful. Requests for permission or further information should be addressed to Particle Measuring
Systems, Inc. at 1-303-443-7100. App Note 282 10272022

[email protected] Page 68
 Without measurement there is no control

Solutions: FMS
Designing an Environmental Monitoring Solution for GMP applications

Fundamentals
Introduction
With the release of the new EU GMP Annex 1revision, a review of current practices is required to
ensure that the installed monitoring system, chosen to meet the Annex 1 needs, complies with the
requirements. This chapter will review the needs of Annex 1 with systems designs currently being
installed [1].
Applications

Cleanroom Classification
Pharmaceutical cleanrooms are classified according to the particle concentration of the air
Solutions that is required to meet the cleanliness criteria for the manufacturing process being performed.
The determination of the cleanroom class is a process based on actual statistically valid
measurements, and a function of the filtration and operations status of the room, it is in essence a
calibration of the room to ensure it meets its intended classification, it is not, primarily, a function
Total Particle Portable
of risk of application [2].
Monitoring Best
Practices There are three measurement phases involving particle counting in cleanrooms:
As Built: a completed room with all services connected and functional but without
Microbiological Portable production equipment or personnel within the facility.
Monitoring Best At Rest: all the services are connected, all the equipment is installed and operating to an
Practices
agreed manner, but no personnel are present.
Operational: all equipment is installed and is functioning to an agreed format and a
Facility
Facility
Monitoring
Monitoring specified number of personnel are present, working to an agreed procedure.
Systems
Systems(FMS)
The airborne particle count test is performed by counting particles at defined grid locations within
the cleanroom. The test points should be equally spaced throughout the room and at work height
to demonstrate the quality of the air cleanliness at the work area.
Alarm Rationale
Pharmaceutical cleanrooms typically operate at Class 5 (most aseptic areas), Class 7 (surrounding
areas), or Class 8 (support areas).

Compressed Gas

Particle Measuring Systems Page 69

 Fundamentals

Applications

Solutions
Figure J-1  DIAGRAM OF TYPICAL ISO RATING IN AREAS OF CLEANROOM

Total Particle Portable Pharmaceutical Cleanroom Utilization

Monitoring Best Once a cleanroom has been tested to compliance to cleanroom classification typically using
Practices a Light Scattering Aerosol Particle Counter (LSAPC), the classification achieved dictates which
production activities can be performed in that cleanroom or clean air device. The FDA defines two
areas.
Microbiological Portable
Monitoring Best 1. Critical Areas contains products that, if exposed, are vulnerable to contamination,
Practices
these areas are designated Grade A (ISO5), within the Annex 1 document. To maintain
product assurance, it is essential that the environment in which aseptic operations are
conducted be controlled and maintained at an appropriate quality.
Facility
Facility
Monitoring
Monitoring
Systems
Systems(FMS) 2. Supporting Clean Areas are used for all other activities outside the critical core, these
are designated as grade B/C/D within the Annex 1 and are typically a lower risk to
finished product contamination.

Once a cleanroom or clean air device has been proven to meet the requirements for cleanliness
Alarm Rationale from a certification perspective, it must also demonstrate that this control can be maintained
throughout production periods. The environment needs to be rigorously monitored to ensure
that there is full and constant awareness of current conditions, including the detection of periodic
events which could be catastrophic if gone unnoticed. Constant monitoring creates a continuous
Compressed Gas flow of information, resulting in a large quantity of data which can be used to watch for trends.
The manufacturing facility should therefore have a comprehensive environmental monitoring
program, which includes monitoring for nonviable and viable airborne particulates, surface
viable contamination and, in the aseptic areas, and personnel. These procedures should address
frequencies and locations for the monitoring sample points, warning and alarm limits for each

[email protected] Page 70
 area, and corrective actions which need to be undertaken if any of the areas show a deviation
from expected results. Actions taken when limits are exceeded should include an investigation
into the source of the problem, the potential impact on the product, and any measures required
to prevent a recurrence.

Contamination Control Strategy

A Contamination Control Strategy (CCS) will include the environmental monitoring program and
should be implemented across the facility. The CCS should define critical control points as part of
Fundamentals
a risk assessment and assess the effectiveness of the controls and monitoring measures used to
manage risks associated with contamination. The CCS should be reviewed frequently, especially
during the early phases of implementation, and it should be updated to drive continuous
improvement of the monitoring and control methods, ultimately improving overall quality of
Applications process.
Elements that should be considered as part of a Contamination Control Strategy will include:

i. Design of the plant and processes.

Solutions ii. Premises and equipment.

iii. Personnel.

Total Particle Portable iv. Utilities.

Monitoring Best
Practices v. Raw material controls.

vi. Product containers and closures.

Microbiological Portable vii. Vendor approval –key suppliers.

Monitoring Best
Practices viii. Outsourced services, such as sterilization, ensure the process is operating correctly.

ix. Process risk assessment.

Facility
Facility
Monitoring
Monitoring x. Process validation.
Systems
Systems(FMS)
xi. Preventative maintenance.

xii. Cleaning and disinfection.

xiii. Monitoring systems - the introduction of scientifically sound, modern methods that
Alarm Rationale optimize the detection of environmental contamination.

xiv. Prevention – trending, investigation, corrective, and preventive actions (CAPA).

The scope of a Facility Monitoring System should encompass those identified in the list above (i, ii, iii, ix,
Compressed Gas
x, xii, xiii and xiv), many of the CCS considerations should be included in the Environment Monitoring (EM)
program.

[email protected] Page 71
 Environmental Monitoring Requirements

Table J-1  EU GMP Annex 1 room classification table (Annex 1 2022)

At Rest In Operation
EU GMP Grade Maximum number of particles permitted/m3 equal to or greater
than the tabulated size
Fundamentals
0.5 µm 5.0 µm 0.5 µm 5.0 µm
A 3520 NOT 3520 NOT
SPECIFIED(a) SPECIFIED(a)
B 3520 NOT 352,000 2930
Applications SPECIFIED(a)
C 352,000 2,930 3,520,000 29,300
D 3,520,000 29,300 NOT NOT
PREDETERMINED(b) PREDETERMINED(b)

Solutions

Total Particle Portable Figure J-2  FOOTNOTES TO ABOVE TABLE IN ANNEX 1 2022

Monitoring Best
Practices Monitoring should be performed using suitable techniques that meet the needs of the Risk
Assessment; for many of the monitoring requirements of lower classification areas, a portable
instrument can be deployed and used. However, the grade A area should be continuously
Microbiological Portable
Monitoring Best monitored (for particles ≥0.5 and ≥5 µm) with a suitable sample flow rate (at least 28.3 LPM /
Practices 1CFM) so that all interventions, transient events, and system deterioration is captured. The system
should frequently correlate each individual sample result with alert levels and action limits at such
a frequency that any potential excursion can be identified and responded to in a timely manner.
Facility
Facility
Monitoring
Monitoring Alarms should be triggered if alert levels are exceeded. Procedures should define the actions to be
Systems
Systems(FMS) taken in response to alarms, including the consideration of additional microbial monitoring.
The requirement for continuous monitoring within the Grade A is satisfied by using point of use
dedicated sensors; these are connected to a central monitoring software application that can
send alarm outputs to operators within the cleanroom or messages to concern groups. These
Alarm Rationale
alert and alarm excursions are also permanently recorded in the audit trail of the system.
One aspect of the system that needs to be determined is the location of the sample point(s);
these should be determined following a documented Environmental Monitoring Risk Assessment
(EMRA) and include the following information:
Compressed Gas
• Sampling locations
• Frequency of monitoring
• Monitoring method used and
• Incubation conditions (e.g. time, temperature(s), aerobic and/or anaerobic conditions).

[email protected] Page 72
 and be based on the following inputs from site:

• Detailed knowledge of; the process inputs and final product, the
• Facility, equipment,
• Specific processes,
• The operations involved,
• Historical monitoring data,
• Monitoring data obtained during
Fundamentals qualification and
• Knowledge of typical microbial flora
isolated from the environment.
• Air visualization studies should also be
Applications included

Suitable sample point locations are also

impeded by:

Solutions • Physical installation of sample probe

• Physical installation of instrument
• Tubing length, bends and bends radii
Total Particle Portable between the two
Monitoring Best Figure J-3  STEPS FOR RISK IDENTIFICATION AND ANALYSIS
Practices

Typical Automated Continuous Monitoring System

Microbiological Portable
Monitoring Best
Practices

Facility
Facility
Monitoring
Monitoring
Systems
Systems(FMS)

Alarm Rationale

Compressed Gas

Figure J-4  EXAMPLE FACILITY MONITORING SYSTEM (FMS) SETUP

[email protected] Page 73
 Instrumentation used in constructing an integrated solution will typically include:
Particle Counting – The need for continuous data requires a dedicated sensor at each location
that samples continuously during the set-up and production phases of manufacturing. The
sensor(s) send data back to a central processing component that is used to manage response
processing, data buffering, and sensor controls. The sensor can have an internal pump or a
remote vacuum source; both are controlled using the central interface within the software
application.
Microbial Sampling – Where a risk has identified the need for total particle counting, there
Fundamentals
is an associated requirement for microbial sampling. The sample head only is placed within
the environment, ensuring that any exhaust is managed by the system and not emitted locally
within the critical space. Microbial samplers are fixed flowrate devices (typically 25 LPM), and
this flow control is performed either locally (using a dedicated device) or centrally (using the
Applications same central vacuum source as the particle counter sensor sub-system). Start and stop controls
are performed via the software interface.
Alarm Beacons – These additional devices allow for local annunciation (visible and audible)
and can alert operators within the controlled space if a system is out of tolerance. Additional
Solutions information can be achieved by situating a remote interface within the viewing space of the
operators; these can also be interactive if they are within the clean core of the facility.
Central Software System - The system is designed with Industrial Automation architecture,
Total Particle Portable which consists of a central processing system that collects data from field sensors and controls
Monitoring Best
Practices remote devices while communicating with a SCADA (Supervisory Control and Data Acquisition)
software package. The following features
should be available to interact effectively
Microbiological Portable with the system and data reports.
Monitoring Best
Practices Data and Status Information
Displays - The main page is used for
visualization of the facility layout with
Facility
Facility
Monitoring
Monitoring current data and status information for
Systems
Systems(FMS) each sample point.
Data, status, and sampling information
can also be viewed for each dedicated
area on a single screen.
Alarm Rationale
Figure J-5  MAIN PAGE OF FACILITY MONITORING SOFTWARE

User and Area segregation - According to CFR 21 Part 11 and Annex 11, single user access
shall be controlled and managed to ensure each individual operated as described in the
user Standard Operating Procedures and, according to the role, responsibility and training
Compressed Gas
received.
The SCADA software should also ensure proper segregation of data whenever multiple
departments are connected and controlled by the same supervising system.

[email protected] Page 74
 Report Generator - The SCADA software requires a data report generator capable of
providing human readable reports
for all recorded data such as audit
trails (events), data/statistical
summaries, and trend charts.
The system should be capable
of retrieving data historically as
defined in the site User Requirement
Fundamentals Specification for the associated
system. Using filters for data, time,
location, and batch, data should
be readily accessed and, where
required, exported or printed to
Applications
support the release of product.
Figure J-6  REPORT GENERATOR

Alarms - The alarms display provides date, time, area, description, value, etc. for alarms and
provides an alarm acknowledgment function. The alarms display also offers the capability
Solutions to sort alarms by different criteria. Defining the alarm set point within the software is based
upon the limits table in Annex 1 and based on historical data for each sample location.

Total Particle Portable

Monitoring Best
Practices

Microbiological Portable
Monitoring Best
Practices

Figure J-7  ALARMS PAGE

Facility
Facility
Monitoring
Monitoring
Systems
Systems(FMS)
Annex 1 (2022) also notes in Section 9, that
“The occasional indication of macro particle counts, especially ≥ 5 µm, within Grade A
may be considered to be false counts due to electronic noise, stray light, coincidence loss
Alarm Rationale etc. However, consecutive or regular counting of low levels may be indicative of a possible
contamination event and should be investigated. Such events may indicate early failure of
the room air supply filtration system, equipment failure, or may also be diagnostic of poor
practices during machine set-up and routine operation”
Compressed Gas Therefore, when considering alarm rationale, it should also take into account the frequency of
event and not singularly the magnitude; these factors should be considered within the CCS.
Additional information and alarming strategies can also be found in ISO 14644-2:2015, paragraph
B.3.1; Environmental Monitoring Systems should allow for seamless and validated configuration
of an “N of M” strategy to ensure sequences of out of specification events are promoted to alarms
when the conditions are met.

[email protected] Page 75
 As with all integrated systems, especially those using a central software package, the validation
is a significant element on the timeline of any installed project. The review and circulation of
documents can take several weeks where multiple departments are involved, and the start to
finish time of a project should be discussed with the installation project team to ensure it meets
the site requirements for shutdown accessibility.

Conclusion
Monitoring of pharmaceutical aseptic production environments is well established and the
Fundamentals
changes presented in the revision of Annex 1 (2022) do not change many aspects of the
requirements for monitoring. The formal risk assessment and inclusion of data within a CCS create
a more comprehensive addition to the continuous systems installed under past regulations. More
emphasis is given to establishing the correct sample locations and techniques based on risk and
Applications reviewing data to support product release. This emphasis is however an enhancement to the
documentation requirements more than the traditional expectations of a continuous system.

References
Solutions
1. European Commission. The Rules Governing Medicinal Products in the European Union
Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human
and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products, Annex 1 (2022).
Total Particle Portable
Monitoring Best 2. International Standards Organization. Cleanrooms and associated controlled environments
Practices — Part 1: Classification of air cleanliness by particle concentration, ISO Standard No. 14644-
1:2015 (2015).

Microbiological Portable
Monitoring Best
Author
Practices Mark Hallworth
Life Sciences Senior GMP Scientist
Life Science Division
Facility
Facility
Monitoring
Monitoring Particle Measuring Systems
Systems
Systems(FMS)
Mark Hallworth is the Life Sciences Regional Manager for Particle
Measuring Systems. He has lectured for pharmaceutical societies
throughout Europe, Asia, and the US on nonviable particle and facility
monitoring and the implications of validating those systems. He can be
Alarm Rationale reached at [email protected].

Editor: Noelle Boyton

Compressed Gas

© 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the copyright owner is unlawful. Requests for permission or further
information should be addressed to Particle Measuring Systems, Inc. at +1 303-443-7100. Book Content. 10272022

[email protected] Page 76
 Without measurement there is no control

Solutions: Alarm Rationale

Alarm Rationale for Continuous Particle Counting Systems

Fundamentals
Introduction
Many of us have grown accustomed to performing routine environmental monitoring with a cart
of equipment that takes samples at predetermined cleanroom locations. We collect these particle
counts and microbial burdens with portable particle
counters, dynamic air-samplers, and settle plates,
Applications and we take surface samples to adhere to gowning
protocols. This process occurs regularly based on
guidance from industry [1] with frequencies ranging
from each shift up to monthly, depending on the
Solutions associated risk to the final product.
We approve conditions within controlled spaces such
as cleanrooms by affirming the results taken from
Total Particle Portable these ‘spot reading’ campaigns, where we aim for
Monitoring Best each individual sample to be:
Practices
• As accurate and representative as possible
• As statistically significant as the conditions of the
Microbiological Portable
environment allow
Monitoring Best
Practices • Appropriate so as to confirm controls are being
maintained

Figure K-1  CRITICAL POINT IN CLEANROOM ENVIRONMENT

Facility Monitoring
However, regulations require continuous particle monitoring in the most critical zones during
Systems
production and prior to manufacturing, as this reflects a better demonstration of environmental
control.
This shift to continuous monitoring can yield an ocean of data. Rather than using a portable
Alarm
Alarm
Rationale
Rationale instrument to, for example, collect three one-minute samples at each selected and defined
location, data is now a continuous stream flowing from the sensor to a centralized monitoring
system. This, in turn, continuously allows the state of control to be quantified and shows any shift
between “normal” and “anomalous” conditions. Where previously we needed to prove control
Compressed Gas through discrete sample data, control is now the assumed state and out of tolerance (OOT) events
become the discrete event requiring investigation.

Particle Measuring Systems Page 77

 This chapter is a description of current good manufacturing practices (cGMPs) and how they
can be applied to a continuous monitoring system installed within a facility. This process
should enable Quality and Production teams to establish new monitoring understandings and
harmonization with standards. This review will look at the configuration of alert and action levels,
potential reactions, and the reporting requirements of such systems.

Current Requirements in Industry

Forming a stance on what approach to take requires a look at the historical changes to GMP
Fundamentals
that define the current understanding and expectations of environmental monitoring (EM) in
controlled areas.

Applications “ The purpose of the EM program is to document the state of control of

the facility, not to determine the quality of the finished product. ”
SCOTT SUTTON, PH.D. - AUTHOR AND SPEAKER FOR THE PHARMACEUTICAL INDUSTRY

Solutions
Cleanrooms are classified to a common standard. In 1999, the standard changed from the Federal
Standard 209E:1992 (FS209E) [2] to a new metric standard, ISO 14644-1:1999 [3]. Specifically, the
USA replaced FS209E in favor of the ISO 14644-1 on November 29, 2001. At this point, cleanrooms
Total Particle Portable were now considered by their metric classification number (ISO 5, ISO 7, etc.) and not as the
Monitoring Best FS209E number (Class 100, Class 10000, etc.), and the monitoring requirements also became
Practices
metrified. Data to meet the new standard must be normalized and displayed as counts per cubic
meter (n/m3), regardless of actual sample volume taken.
Microbiological Portable With the advent of a new ISO 14644 classification requirement, the EU GMP Annex 1 [4] was also
Monitoring Best
updated. In 2003, a new version of the Annex 1 was released to reflect the ISO 14644 changes. The
Practices
following statement/note was made on the front cover:

The guidance has been reviewed in the light of the international standard
Facility Monitoring EN/ISO 14644-1 and amended in the interests of harmonization but taking
Systems
into account specific concerns unique to the production of sterile medicinal
products.

A few points to note: Annex 1 has been amended to reflect the industrial certification standard ISO
Alarm
Alarm
Rationale
Rationale 14644-1, sterile medicinal products, and additional concerns that apply to more critical, sterile
environments. A new table of required limits was also imposed:

Compressed Gas

[email protected] Page 78
 Table K-1  Annex 1 (2003) particle cleanliness limits

At Rest In Operation
EU GMP Grade Maximum permitted number of particles permitted/m3 equal to or
above

0.5 µm 5.0 µm 0.5 µm 5.0 µm

A 3500 1 3500 1
Fundamentals
B 3500 1 350,000 2000
C 350,000 2,000 3,500,000 20,000
D 3,500,000 20,000 NOT DEFINED NOT DEFINED
Applications
The limit for 5.0 µm, prior to the 2003 release, was zero due to the correlation between macro
particles (> 5.0 µm) and the potential for microbial activity to sustain to colony forming units.
Individual microbes either use moisture, dust, or biomass as an aid to offset desiccation. They
can also slough off as larger components from operators. However, the original zero is technically
Solutions impossible to achieve while following the ISO 14644-1 rules for sample volume, so the number
needed to be the lowest possible integer to allow the formula to operate.

Total Particle Portable

Monitoring Best
Practices

Figure K-2  FORMULA IN ISO 14644-1:1999, SECTION

Microbiological Portable
B.4.2 ESTABLISHMENT OF SINGLE SAMPLE VOLUME PER LOCATION
Monitoring Best
Practices This leads to a sample volume of 20 m3. Such a result is impractical for the classification of
cleanrooms because significant time is required to fulfill the requirement. The Annex 1 clarified
this in the footnotes listed below the table (see Figure K-4).
Facility Monitoring
Systems

Alarm
Alarm
Rationale
Rationale

Figure K-3  EU GMP ANNEX 1 2003, TABLE 1, FOOTNOTES (A) AND (E)

Compressed Gas
Note (a) can be broken down into the following directives:

1. Use a particle counter.

2. Use a continuous measuring system for monitoring Grade A.

3. Take a 1 m3 sample for routine classification (testing) purposes.

[email protected] Page 79
 In real terms, the zero had to be dropped to meet ISO 14644-1 statistical requirements, but there
should be essentially no particles present greater than 5.0 µm. The notes were misinterpreted
to read “use a particle counter and sample 1 m3 for routine monitoring,” which it does not. This
caused confusion and was clarified with a new classification table with the release of EU GMP
Annex 1, coming into effect in 2009 [5]:

Fundamentals

Applications

Solutions

Total Particle Portable

Monitoring Best Figure K-4  EU GMP ANNEX 1 2008, #4 AND 5
Practices
This revision allowed for the formula result to meet the 1 m3 sample for classification. It also
clarified its application as ONLY for classification, resulting in the adoption of an ISO Class 4.8 for
Microbiological Portable
Monitoring Best Grade A. Paragraph 4 states that classification should be clearly differentiated from operational
Practices process environmental monitoring.
So, what to do for the alert and action limits? There is guidance (hidden) in the 2008 revision. If
we look at paragraph 20, it reflects current risk assessment practices that state process conditions
Facility Monitoring should define the limits:
Systems

Alarm
Alarm
Rationale
Rationale Figure K-5  EU GMP ANNEX 1 2008 TEXT, #20

Therefore, limits set by room classification are those “appropriate” to determining control over
potential risk, based upon room dynamics such as number of personnel, product type, activity
Compressed Gas type, etc. These limits are frequency based due to small-number statistics, in line with USP <1>
and USP <1116> where trend analysis is a better reflection of control than absolute numbers with
questionable statistical significance. Paragraph 13 in the Annex 1 gives the following directive:

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 Figure K-6  EU GMP ANNEX 1 2008 TEXT, #13

When reviewing an alert and action philosophy and its application to particle counting and
Fundamentals environmental control, the RISK should define what constitutes “occasional” vs “consecutive or
regular”. In all cases, the limit is not prescribed as 1 count/m3 or 20 counts/m3 but as a “low level”,
which can only be defined by a specific site, room, or process operation.

Applications 2022 Revision to EU GMP Annex 1

A new revision of EU GMP Annex 1 was published in 2022 [6]. It echoes the revision to ISO 14644-
1:2015 [3] where the 5.0 µm limit for ISO 5 classified spaces was dropped due to statistical
significance of particles in the larger sizes.
Solutions

Total Particle Portable

Monitoring Best
Practices

Microbiological Portable Figure K-7  2022 ANNEX 1, TABLE 1

Monitoring Best
Practices
Classification is section 4 and monitoring for both total particle and microbial contaminants has
been relocated to section 9, separating the approach required for each exercise.
Facility Monitoring Monitoring now requires a Contamination Control Strategy (CCS), which looks more holistically at
Systems the process of creating controlled spaces, HVAC, gowning, data review and monitoring then using
required techniques. It states:

Alarm
Alarm
Rationale
Rationale

Figure K-8  2022 ANNEX 1 TEXT, SECTION 9.1

Compressed Gas

Alert and action limit requirements are carried over from the 2008 version and clarified. They
do not mention what number determines periodic events, nor do they define what number
constitutes an actionable threshold. That is instead documented with the Risk Assessment and
CCS.

[email protected] Page 81
 Figure K-9  2022 ANNEX 1 TEXT, SECTION 9.9-9.10

Fundamentals

Applications

Figure K-10  2022 ANNEX 1 TEXT, SECTION 9.17

Solutions Monitoring procedures should define the approach to trending. Trends can include, but are not
limited to:

• Increasing numbers of action or alert level breaches

Total Particle Portable • Consecutive breaches of alert levels
Monitoring Best
Practices • Frequent or regular isolated breaches of action limits that may have a common cause

Reporting
Microbiological Portable
System reports should be considered as part of the CCS. As noted earlier, a room monitored using
Monitoring Best
Practices a portable device produces data that needs to reflect that control was maintained. As such, the
data needs to be ‘absolute’ and shown in all reports. However, the transition to a continuous
monitoring system now continuously demonstrates adherence to control parameters (defined by
Facility Monitoring alert and action levels), and only instances where there is a loss of control require investigation.
Systems Overall trend reports and audit trails show when the system was out of control and any remedial
actions taken as part of the SOP.
System reports should focus on:

Alarm
Alarm
Rationale
Rationale 1. The report boundary, what sensors are included, what time frame the report covers,
and any additional batch information that uniquely identifies this period.

2. The audit trail, alert and action threshold excursions, remedial actions, user log in, start
end time data, system changes, etc.
Compressed Gas
3. The trend report as a way to show data as an easy to read trend plot (graph) image,
demonstrating no loss of data.

4. The location of the out of tolerance event that caused an action level to be exceeded.
This period can be covered by an exception report to demonstrate that, prior to the
event, all was running typically and that, post-event, normal conditions were restored.

[email protected] Page 82
 Application of Strategy
Alert and action limits can be applied to reflect a trending condition referred to as an N:M, where
the number of events (N) is within a population of events (M). For example, an N:M of 2:6 would
refer to a rolling set of numbers where any 2 samples out of a total of any 6 samples would require
an action, with the initial trigger setting the clock to review the next 6 samples for a second
triggering event. If none are present, the counter is reset until the next activation event.
Using this application, the system alerts are converted to non-actionable events, where the trigger
is only used for data review. To ensure the system does not trend adversely over longer periods,
Fundamentals
the non-actionable events allow for comparison to similar time periods (e.g., weeks, months,
quarters) or to similar events (e.g., filling, batch, lot). This ensures the limits prescribed in the
system are reliable triggers of adverse conditions, and the limits reflect a loss of control over the
environment as it pertains to product quality.
Applications
The particle levels are set with each of the following:

1. Alert – If any value goes above the ‘normal’ operating condition values when
normalized to cubic meters (x35), an alert event is recorded. If alert levels are exceeded,
operating procedures should prescribe assessment and follow up, which includes
Solutions consideration of an investigation and/or corrective actions to avoid any further
deterioration of the environment. This is essentially a non-actionable event.

• Data is reviewed periodically for adverse trending and can be correlated against the
Total Particle Portable room production events to ensure that where potential contamination may occur;
Monitoring Best the future revisions can reflect the required changes.
Practices
2. Alarm – The frequency (N:M) is set such that a single location must be in ALERT for
a determined frequency before triggering an ALARM event, beacon, siren, audit trail
Microbiological Portable etc. When these events occur, operating procedures should prescribe a root cause
Monitoring Best
investigation, an assessment of the potential impact to product, and requirements for
Practices
corrective and preventive actions. This is an out of control condition.

Facility Monitoring Alert and Action Level Setting

Systems It follows then that a determination of the level to be used to catalyze needs should be
determined.
Guidance from the PDA Technical report 13 gives the following approaches:
Alarm
Alarm
Rationale
Rationale • Cutoff Value – A 95% or 99% control limit based on data previously gathered from a
single site or collection of similar sites.
• Normal Distribution – The mean and standard deviation of a set of data, where high
counts exist. Where a predominance of zeros prevail, skewed data sets may occur and
Compressed Gas
non-parametric rules apply.
• Non-parametric Tolerance – The statistical determination that a room meets
compliance with at least 95% confidence.

A data review is required per sample location or group of sample locations which exhibit similar
properties. The practice of normalization is more to put the data into an SI unit mentioned in

[email protected] Page 83
 regulations than understanding process controls. For example, pressure can be used as either
inches of water gauge (in H2O), pascals (Pa), or millibar (mBar), but each has a different number
value due to scaling. Data per minute gives a balance between measuring at a short enough
interval for response rate, while providing enough sample volume (assuming a 28.3 LPM flowrate),
and, therefore, associated potential counts and statistical confidence in the data.
We can also consider the extremes of each time period. Long sample periods (35.3 minutes) will
give great statistical confidence in the data, but poor response to control conditions and very
short sample periods (5 second intervals) will give great dynamic responses. Low confidence data
Fundamentals is where the few counts yielded requires multiple records to gain statistical insights and leads
back to low responses. As such, the raw data from the instrument can be used, given as counts per
minute or counts per cubic foot. However, industry has
found it more accommodating to reflect the standard in Let Particle Measuring Systems
Applications counts per cubic meter (n/m3). industry experts support your risk
assessment and contamination
Regardless of units chosen to reflect the level at which control strategy needs. Learn more:
an out of tolerance event occurs, the number of pmeasuring.com/consultancy-and-
training-services/
particles which trigger the event and the frequency with
Solutions which an event is escalated to actionable needs to be
determined.

Total Particle Portable References

Monitoring Best
Practices 1. Parenteral Drug Association. Fundamentals of an Environmental Monitoring Program, PDA
Technical Report No. 13 (2016).

2. Institute of Environmental Sciences. Federal Standard Airborne Particulate Cleanliness

Microbiological Portable
Classes in Cleanrooms and Clean Zones, Federal Standard 209E (1999).
Monitoring Best
Practices 3. International Standards Organization. Cleanrooms and associated controlled environments
— Part 1: Classification of air cleanliness by particle concentration, ISO Standard No. 14644-
1:2015 (2015).
Facility Monitoring
4. European Commission. EC Guide to Good Manufacturing Practice Revision to Annex 1:
Systems
Manufacture of Sterile Medicinal Products, Annex 1 (2003).

5. European Commission, EudraLex. The Rules Governing Medicinal Products in the European
Union Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for
Alarm
Alarm
Rationale
Rationale Human and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products (corrected
version), Annex 1 (2008).

6. European Commission. The Rules Governing Medicinal Products in the European Union
Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human
and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products, Annex 1 (2022).
Compressed Gas

[email protected] Page 84
 Author
Mark Hallworth
Life Sciences Senior GMP Scientist
Life Science Division
Particle Measuring Systems

Mark Hallworth is the Life Sciences Regional Manager for Particle

Measuring Systems. He has lectured for pharmaceutical societies
throughout Europe, Asia, and the US on nonviable particle and facility
Fundamentals monitoring and the implications of validating those systems. He can be
reached at [email protected].

Editor: Noelle Boyton

Applications

Solutions

Total Particle Portable

Monitoring Best
Practices

Microbiological Portable
Monitoring Best
Practices

Facility Monitoring
Systems

Alarm
Alarm
Rationale
Rationale

Compressed Gas

Lasair® is a registered trademark of Particle Measuring Systems, Inc. © 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of the
copyright owner is unlawful. Requests for permission or further information should be addressed to Particle Measuring Systems, Inc. at 1-303-443-7100. App Note 304. 10272022

[email protected] Page 85
 Without measurement there is no control

Solutions: Compressed Gas

Compressed Gas Monitoring of Microbes: The contamination Control Strategy in
Pharmaceutical Manufacturing Environments

Fundamentals Abstract
Compressed gases, such as nitrogen, carbon dioxide, and oxygen, are used for a variety of
applications in pharmaceutical manufacturing. These applications, such as aseptic packaging,
purging, and filtration, are critical processes monitored for their efficacy. To avoid sampling
Applications compressed gases, it has been speculated by some manufacturers that the rapid decompression
of a gas when exiting its container kills any microbial contamination. However, it has been
shown by multiple studies that microbial survival is not impacted by the typical compression or
decompression seen in pharmaceutical process gases.
Solutions Many GMP standards recommend sampling process gases for contamination before use in critical
areas of manufacturing. This step, along with many others that make up a contamination control
strategy, contributes to the purity and quality of the final product.
Total Particle Portable
Monitoring Best
Practices Introduction
The quality attributes of manufactured pharmaceutical products include the physical, chemical,
and microbiological characteristics of the raw materials, excipients, active pharmaceutical
Microbiological Portable ingredients (APIs), and final drug products. Here, absence of microbiological contamination
Monitoring Best
Practices
is critical because it can dramatically impact a drug's safety. As a result, the cleanliness of
compressed gases, which often come into contact with pharmaceutical products, is also critical.
For the variety of gases used in manufacturing, their compressed state refers to how they are
Facility Monitoring contained. Compressed gases are
Systems typically sampled by taking a small
amount from a gas line and drawing it
into a smaller space (i.e., a sampler).
The decrease in volume that the
Alarm Rationale gas occupies increases its pressure.
Gases are decompressed when exiting
whatever is containing them for use
in a manufacturing line. Process
Compressed
Compressed
Gas
Gas gases are therefore more likely to be
decompressed before coming into
contact with the product.
Figure L-1  CRITICAL POINT OF A FILLING LINE

Particle Measuring Systems Page 86

 Regulatory Requirements
The latest revision of Annex 1, Manufacture of Sterile Medicinal Products, introduced the concept
of a "contamination control strategy" which limits particulate and microbial contamination and
promotes process understanding [1]. A manufacturer's contamination control strategy must
demonstrate:

• A thorough understanding of potential sources of contamination.

• Regular trend analysis is performed, ensuring appropriate critical quality attributes of
Fundamentals high-risk utilities.
• Gases and other high-risk utilities that come in direct contact with the product or primary
container are of appropriate chemical, particulate, and microbial quality.

Specifically, actions should be taken to ensure the sterility of process gases, including filtration
Applications through a sterilizing filter at the point where the gas is used in production and sterilization of
any subsequent piping or tubing. Filtration should be part of batch standards with certification
guaranteed before release. Integrity testing should be performed for both critical and non-critical
gas filters. Care should be taken to avoid introducing moisture to filtration systems, as this
Solutions promotes the growth of microbes. In lines 715 - 716, Annex 1 states:

"When gases are used in the process, microbial monitoring of the gas should be
performed periodically at the point of use."
Total Particle Portable
Monitoring Best
Practices In a similar vein, the FDA's Guidance for Industry: Sterile Drug Products Produced by Aseptic
Processing makes specific mention of the need for purity in a compressed gas. Its microbiological
and particle quality after filtration should also be equal to or better than where it's going [2]. ISO
Microbiological Portable 8573 consists of nine separate parts pertaining to the quality of compressed air, with the first
Monitoring Best
specifying the quality requirements and parts two through nine concerning the methods of testing
Practices
for a range of contaminants. The test method for microbial monitoring of compressed gases is
provided in ISO 8573-7.

Facility Monitoring All the data generated from testing should be recorded to show proof of conditions for any
Systems generated product. Auditors will want to see the end results of testing in addition to the systems in
place to verify claims.

Alarm Rationale
Microbial Survival
The microbial component and, more particularly, the sampling methodology of compressed
gases has been the subject of extensive discussion. It was assumed that the sudden
decompression of a compressed gas before sampling was considered to have a deleterious effect
on microbial survival, thereby voiding sampling results. This claim has been proven false with
Compressed
CompressedGas
Gas
extensive study.
In an FDA study from 2014, bacterial cultures in food products were found to require 2500 to 3000
bar of pressure to inactivate. Cells subjected to pressures less than 1000 bar had no significant
loss in viability [3]. In another study, Serratia and Carnobacterium strains were found to survive

[email protected] Page 87
 conditions similar to those found on Mars [4].
Lessened microbial growth and metabolism in vivo
was only seen in pressures higher than 1000 bar.
In fact, microbial cells have been found to survive
volatile external pressures found in the harshest
environments Earth has to offer, including those in
deep-sea environments. In these extremes, microbes
conserve their ability to sustain life and reproduce.
Figure L-2  SERRATIA MARCESCENS
Fundamentals
In a study of decompression, Escherichia coli and Corynebacterium xerosis were shown to survive
rapid decompressions from 300 to 0 bar [5]. In a typical
compressed gas sampler, decompressions are on a
much smaller scale (i.e., from around 10 bar to 1 at
Applications
most). Only the viable counts of gram-negative, gas
vacuolate bacteria types, such as Marmoricola aquaticus,
Prosthecomicrobium pneumaticum and Meniscus
glaucopis were shown to be affected by minimum
Solutions decompression pressures from 25 to 50 bar [5].
Figure L-3  TYPICAL DECOMPRESSION IN MOST PHARMA
ENVIRONMENTS: 2.5 TO 1.1 BAR

Total Particle Portable

Monitoring Best Instrument Selection
Practices The effects of compressed gas sampling on microbes are insignificant and should not affect
instrument selection. Helpful parameters include ease of cleaning and disinfection, associated data
management tools, and simplicity. Cost is also one to consider when the monitoring compressed
Microbiological Portable
Monitoring Best gases is infrequent6. Different monitoring uses can occur at various frequencies:
Practices
• Classification frequency: Performed monthly or quarterly.
• Routine testing frequency: In Europe, twice a year. The US requires it to be once a year.

Facility Monitoring Investment into dedicated monitoring equipment can be hard to justify. Alternatives include
Systems hiring a service provider for scheduled sampling or using equipment that can be purchased with
an accessory for compressed gas sampling (i.e., the MiniCapt® Mobile Microbial Air Sampler with
compressed gas kit). Deciding on the most feasible solution will require a comparison of these
solutions.
Alarm Rationale

Conclusion
Regulations have maintained their stance on the importance of contamination control and continue
Compressed
CompressedGas
Gas to be a necessary tool to ensure products meet key standards for safety. GMPs are regularly updated
and reflect the modernization and improvements made to manufacturing systems made in recent
years. Knowing these guidelines and requirements will expand their reach; it is practical and
responsible for companies to seek forward-thinking solutions to better their own processes. In the

[email protected] Page 88
 case of microbial monitoring, taking steps to ensure sterility of equipment and process gases is
a vital part of the contamination control strategy. The methods for monitoring contamination
levels should not be determined with speculation and antiquated reasoning, but with constructive
comparison and validated study.

Author
Frank Panofen, PhD
Fundamentals Director and General Manager Life Sciences
Life Science Division
Particle Measuring Systems
Dr. Panofen has a Diploma in Chemistry from the University of
Applications Bielefeld and a PhD in molecular and cell biology from the University
of Osnabrück. He has expansive experience in the field of applied
pharmaceutical microbiology and serves as the Sterility Assurance/
Microbiology Product Line Manager at Particle Measuring Systems.
Solutions Frank has been an invited speaker at international conferences
including ECA and PDA, with a strong regulatory background in
pharmaceuticals. He is a certified Microbiological Laboratory Manager
from ECA. He can be reached at [email protected].
Total Particle Portable
Monitoring Best
Practices Editors: Noelle Boyton and Briana Krueger

References
Microbiological Portable 1. European Commission. The Rules Governing Medicinal Products in the European Union
Monitoring Best
Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human
Practices
and Veterinary Use: Annex 1, Manufacture of Sterile Medicinal Products, Annex 1 (2022).

2. U.S. Department of Health and Human Services, Food and Drug Administration, Center for
Facility Monitoring Drug Evaluation and Research (CDER,) Center for Biologics Evaluation and Research (CBER),
Systems Office of Regulatory Affairs (ORA). Guidance for Industry Sterile Drug Products Produced by
Aseptic Processing — Current Good Manufacturing Practice, (2004).

3. Federal Drug Administration. Kinetics of Microbial Inactivation for Alternative Food Processing
Technologies - High Pressure Processing (HPP): A report of the Institute of Food Technologists
Alarm Rationale for the Food and Drug Administration of the U.S. Department of Health and Human Services,
Journal of Food Science, Volume 65, Issue Supplement s8 (2000).

4. Nicholson, W. (2013). Growth of Carnobacterium spp. from permafrost under low pressure,
temperature, and anoxic atmosphere has implications for Earth microbes on Mars. pnas
Compressed
CompressedGas
Gas 110(2):666- 671.

5. Hemmingsen, B. B. & Hemmingsen, E. A. (1980). Rupture of the Cell Envelope by Induced

Intracellular Gas Phase Expansion in Gas Vacuolate Bacteria. Journal of Bacteriology. 143 no.
2:841-846.

6. Based on author's personal communications with customer

MiniCapt® is a registered trademark of Particle Measuring Systems, Inc. © 2022 Particle Measuring Systems. All rights reserved. Reproduction or translation of any part of this work without the permission of
the copyright owner is unlawful. Requests for permission or further information should be addressed to Particle Measuring Systems, Inc. at 1-303-443-7100. App Note 289. 10272022

[email protected] Page 89
 Contamination Control Advisory Solutions
Our Contamination Control Advisory Team can support you at every step, whether it be
non-compliance issues, setting up a new process, GAP analysis, or training employees.

Environmental Cleaning and Aseptic Process Sterility Environmental

MonitoringRisk/ Disinfection Simulation Assurance Monitoring
Gap Aalysis Audits Trend Analysis

Gowning New Filling Crisis Utilities Process Risk

Line QbD Management Assessment

Advisory Services
Helping you achieve the highest level of contamination
control and sterility assurance.

Performance Optimization
Designing quality into your operations using gold standard
PMS® tools and processes.

BioCapt® Single Use

Microbial Impactor

Training
Preparing your employees to ensure success at every step.

Basic and Advanced Coaching

For more information contact

www.pmeasuring.com
[email protected]