National Agency of Health Surveillance | Anvisa
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Quality Guide for Air

Treatment Systems and
Environmental Monitoring in
Pharmaceutical Industry
 
 
 
 
National Agency of Health Surveillance | Anvisa
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Quality Guide for Air

Treatment Systems and
Environmental Monitoring in
Pharmaceutical Industry
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Brasília
2013
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Copyright © 2012. National Agency of Health Surveillance. Partial or complete reproduction of his work is allowed,
provided the source is acknowledged. National Agency of Health Surveillance (Anvisa)
 
 
1st edition, March 2013
 
President of the Republic
Dilma Rousseff
 
Ministry of Health
Alexandre Padilha
 
 
Director-president Director-President Assistant
Dirceu Brás Aparecido Barbano Luiz Roberto da Silva Klassmann
 
 
Directors Assistants
José Agenor Álvares da Silva Neilton Araújo de Oliveira
Jaime César de Moura Oliveira Luciana Shimizu Takara
 
Chief of Staff
Vera Maria Borralho Bacelar
 
General Manager of Inspection, Quality Monitoring, Control and Inspection of Raw Materials, Medicines and Products,
Advertising and Publicity
Bruno Gonçalves Araújo Rios
 
Manager of Inspection and Certification of Medicines, Pharmaceutical Ingredients and Health Products
Jacqueline Condack Barcelos
 
Redaction
Nélio Cézar de Aquino
 
Illustrations
João Dimas Ribeiro
Nélio Cézar de Aquino
 
Revision
Jacqueline Condack Barcelos
 
Cover, graphic design and layout
Camila Medeiros (Uncom/Ascec/Anvisa)
 
Comments and criticisms: guiasdaqualidade.gimep@anvisa.gov.br
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PREFACE
 
The basic technical information and guidelines presented in this Quality Guide were based in technical
articles, pharmacopoeias, foreign and international technical standards and also in the experience gained by
Anvisa during inspections conducted in domestic and foreign territories. They should be considered as
guidance to drug manufacturers for occasional needs for adjustments to current legislation and also to seek
continuous improvement.
Once recommendations are made for manufacturers of sterile and non-sterile medications, special attention
should be given to certain terms employed in order to avoid misinterpretations. The terms "clean area" and
"classified area" are reserved for installations subject to classification and monitoring as the count of viable
and non-viable particles. Most production areas of non-sterile drugs do not need this type of classification,
but should always be designed and maintained as "controlled area", i.e., areas that have conditions and
procedures defined, controlled and monitored to prevent degradation and products contamination.
Sometimes it is not appropriate to make general and categorical statements about the technical requirements
for full compliance with Good Manufacturing Practices, as operations and production facilities considerably
vary in size and complexity. In such situations, recommendations were made for the adoption of risk
assessments to determine the impact of procedures, control measures and any other action in the quality of
manufactured products.
The adoption of a broader approach based on the risk in replacement for the strict compliance with legal
requirements has been very stimulated by foreign regulatory authorities and is incorporated herein. This
approach, although not widespread in documents published by Anvisa, should be considered as a regulatory
trend and its adoption demonstrates the commitment of manufacturers with the quality, safety and efficacy of
their drugs and, consequently, with the health of the population.
Although targeted to manufacturers of medicines in general, many of the concepts presented in this Quality
Guide are also applied to manufacturers of pharmaceutical ingredients and health products.
 
 
 

SUMMARY
 
PART 1
Air treatment systems and manufacturing areas

1. INTRODUCTION .............................................................................................. 8
2. MAJOR TECHNICAL RULES ABOUT HVAC .................................................. 8
3. AIR TREATMENT SYSTEM............................................................................. 9
3.1 External Air Inlet (Fresh Air) ................................................................................. 11
3.2 Air Treatment Unit ................................................................................................ 11
3.2.1 Ventilation ........................................................................................................ 11
3.2.2 Dehumidification and Humidification ................................................................ 11
3.2.3 Filters ................................................................................................................12
3.2.4 Classification of Coarse, Medium and Fine Filters ............................................13
3.2.5 Classification of Absolute Filters .......................................................................13
3.2.6 Monitoring and Exchanges of Pre-Filters ..........................................................13
3.2.7 Monitoring and Exchanges of HEPA Filters ......................................................14
3.3 Ducts ................................................................................................................14
3.4 Manufacturing Areas .........................................................................................15
3.4.1 Finish Requirements .........................................................................................15
3.5 Air Renewal and Recirculation ..........................................................................16
3.6 Exhaust Systems and Extractors ......................................................................17
4. PARAMETERS AFFECTING THE SAFETY OF PRODUCTS AND
OPERATORS ................................................................................................... 18
4.1 Number of Particulates in the Air ......................................................................... 18
4.2 Distribution of ATU (Sharing) ............................................................................... 19
4.3 Number of Air Exchanges .....................................................................................20
4.4 Air Flow Standard .................................................................................................20
4.5 Requirement of Filtrating Elements (Types and Position) .....................................22
4.6 Temperature and Humidity....................................................................................24
4.7 Pressure Differential, Air Replacement and Physical Barriers ..............................24
4.8 Microorganisms in the Air and Surfaces ...............................................................27
4.9 Air Exhaustion .......................................................................................................27
4.10 Supervisory Systems ............................................................................................28
5. ENVIRONMENTAL PROTECTION .................................................................. 28
6. CLEANING AND MAINTENANCE OF COMPONENTS OF AIR TREATMENT
SYSTEMS ........................................................................................................ 29
 
 
7. CHOOSING THE APPROPRIATE AIR TREATMENT SYSTEM ..................... 30
7.1 Technologies of Air Conditioning ........................................................................ 31
7.2 Limitations of Use for Comfort Air Conditioning Systems .................................... 32
8. COMMISSIONING, QUALIFICATION AND REQUALIFICATION OF THE AIR
TREATMENT SYSTEM.................................................................................... 32
8.1 Commissioning .................................................................................................. 32
8.2 Qualification ....................................................................................................... 33
8.2.1 Clean Areas ....................................................................................................... 35
8.2.2 Differences Between ISO 14644 Ratings and GMP Guidelines for Clean Areas35
8.2.3 Microbial Classification of Clean Areas .............................................................. 37
8.3. Requalification ................................................................................................... 37

PART 2
Environmental Monitoring in Clean Areas

1. INTRODUCTION .............................................................................................. 39
2. MONITORING OF NON VIABLE PARTICLES ................................................ 40
2.1 Procures for Monitoring Non-Viable Particles .................................................... 40
2.2 Analysis of Routine Monitoring Data of Non-Viable Particles............................. 42
3. ENVIRONMENTAL MONITORING OF MICROORGANISMS DURING
OPERATIONS .................................................................................................. 43
3.1 Growth Promotion Testing of the Culture Media ................................................ 43
3.2 Volumetric Sampling of the Air ........................................................................... 44
3.3 Monitoring with Sedimentation Plates ................................................................ 45
3.4 Sampling of Microorganisms on Surfaces.......................................................... 46
3.4.1 Contact Plates ................................................................................................... 46
3.4.2 Swabs ................................................................................................................ 46
3.4.3 Fingerprinting of operators’ gloves ..................................................................... 47
4. ROUTINE MICROBIOLOGICAL MONITORING FREQUENCY ...................... 47
5. LABORATORY TESTING OF ENVIRONMENTAL SAMPLES ........................ 48
6. INVESTIGATIONS AND CORRECTIVE ACTIONS AND PREVENTIVE
ACTIONS (CAPA) ............................................................................................ 49
6.1 Alert and Action Limits for Environmental Monitoring ........................................ 50
6.2 Deviation Investigations ..................................................................................... 51
6.3 Corrective Actions and Preventive Actions ........................................................ 52
BIBLIOGRAPHIC REFERENCES ....................................................................... 53
 
PART 1
Air treatment systems and manufacturing areas
 
 
 
 
 
1. INTRODUCTION
 
The heating, ventilation and air conditioning (HVAC), often referred to by the acronym HVAC (heating,
ventilation and air-conditioning) or commonly as "air handling systems," play an important role in the
quality of pharmaceutical products. These systems, in addition to offering protection to the product during
manufacturing steps, also provide comfortable and safe conditions for operators and protect the environment
of contaminants from the manufacturing process.
Drawings of air handling systems significantly influence the architectural design of a production plant, since
these systems must be closely linked with the positions, locations and size of production areas, pre-chambers
and doors. Due to the diversity of the existing technical requirements for the different categories of drugs, it
is necessary that in the design of production areas and their respective HVAC systems also take into account
the criticality of the products that will be manufactured.
Therefore, planning is an essential step in the idealization of an architectural design, and post-construction
changes, besides costly, difficult and sometimes turns unfeasible the full compliance with Good
Manufacturing Practices (GMP).
The prevention of cross contamination, microbial contamination or from any other sources is an essential
consideration to be made during the preparation of a project for construction of a HVAC system. Important
parameters related to HVAC systems that can affect the quality of pharmaceutical products during all the
manufacturing or storage  steps, such as temperature, humidity, pressure differentials and air renewal and
cleaning, must be properly designed, controlled and monitored.
 
 
 
2. MAJOR TECHNICAL RULES ABOUT HVAC
 
Most countries have clear rules on the conditions under which HVAC systems must be designed, installed,
maintained and operated, as well which professionals are qualified for these activities. International technical
standards for HVAC systems are very often derived from the American Society of Heating, Refrigerating and
Air-conditioning Engineers, Inc. (ASHRAE), and the EN European standards. In Brazil, the technical
standards are prepared and published by the Brazilian Association of Technical Standards (ABNT).
 
 

  8
 Standards published by ABNT bring important concepts for strengthening national technological
development. Current ABNT standard that regulates the activities of air conditioning is NBR 16,401. This
standard, which has different parts, is applicable to the facilities of air conditioning systems in general, and
on non conflicting devices, in addition to special air conditioning systems, such as industrial and clean rooms
governed by other rules. As relevant specific rules, we must emphasize the NBR ISO 14,644, which covers
specific tests to be performed in the construction and maintenance of clean areas. Another specific technical
standard which content is relevant to the theme of this Quality Guide is ISO 14,698, which although not
prescribing specific recommendations for construction covers the requirements for control of
biocontamination in clean areas and other related controlled areas.
In addition to the standards published by ABNT, the Collegiate Board Resolutions (RDC) of the National
Health Surveillance Agency (ANVISA) are published, which in its intricacies address the required air quality
to supply areas used in the manufacture and storage of products subject to health surveillance. Anvisa
standards related to Good Manufacturing Practices are often derived from technical documents published by
the World Health Organization (WHO), which are called Technical Reports Series (TRS). Sometimes
technical documents published by other foreign authorities and international entities such as the
Pharmaceutical Inspection Convention (PIC) and the International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) are also used as
references.
In the normative published by Anvisa, criteria are established for the installation and maintenance of HVAC
systems, whose noncompliance sets up a health violation. Such criteria can sometimes be more
comprehensive and stringent than those set out in ABNT standards, since unlike many of these standards, the
RDCs of Anvisa are specific to certain production segments and aim primarily the manufacture of products
with quality, safety and efficacy for consumption by the population. Therefore, the ISO rules should be used
in addition to the Resolutions published by ANVISA, and should not be considered alone or even overlap the
RDCs when there are conflicting devices.
 
 
 
 
 
 
3. AIR TREATMENT SYSTEM
 
HVAC is a technology designed to provide comfort and also acceptable indoor air quality in closed
environments. The term "HVAC" refers to the three main functions of technology, which are heating,
ventilation and air conditioning.
The indoor air quality depends on the contamination from the outside air ("fresh"  air) entering into the
building, the efficiency of the HVAC system to remove contaminants from the air, and the activities in the
internal areas (pollution caused by building materials, equipment, persons etc.).
The design and specification of HVAC systems are usually in charge of specialist engineers, but due to
specific health rules, it is particularly important that in the design of building and laboratories other
professionals participate, especially those engaged in activities related to Quality Assurance and Production.

  9
 
 
 
To meet air quality requirements in productive areas, several functions are associated with air handling
systems such as heating, cooling, humidification, renewal, filtration, ventilation and dehumidification.
Systems can also include other features, such as pressurizing the air inside a given space.
In general, the air treatment systems used for supplying manufacturing plants of medicines can be divided
into subsystems. The main subsystems, from the viewpoint of GMP are shown in Figure 1 and will be
discussed in detail in the following subsections.

Inlet of external air

(fresh) Air Treatment Unit
Return of the air
(recirculation) 

Production
Areas

Treatment of the air to be discarded

Figure 1 Pain subsystems of a HVAC system.

  10
 
 
 

3.1 External Air Inlet (Fresh Air)

The uptake of atmospheric air must be performed in a place well away from any sources of contamination or
heat such as unpaved streets, water cooling towers, chimneys, exhaust combustion engines and discharging
contaminated air points from other productive or laboratory areas. The neglect with this requirement creates
problems with the quality of the treated air, air treatment system damages (especially in the filter elements)
and possible increase in energy consumption. The entrance of the duct air inlet should be designed in order to
prevent, by using physical barriers, the entry of insects and large particles into the system.
Thus, during the project design of a production plant the positions of the fresh air inlet that will supply the air
treatment system should be considered.
 
 
 
3.2 Air treatment unit
An air treatment unit (ATU) is a device used for air conditioning and circulation, as a part of a HVAC
system. Occasionally, the air treatment units are also referred by the acronym AHU (air handling units) in
English.
The majority of the ATUs consist of a large metal box which contains a mechanical ventilator and heating
and cooling elements, filter elements, noise attenuators and inlet and outlet air grids. Usually, ATUs are
connected to air distribution, collection and return ducts. Several key functions of an HVAC system are
performed by the ATUs, highlighting the ventilation, heat exchange (heating and cooling), humidification,
and dehumidification and air filtration steps.
 
3.3 Ventilation
 
The main functions of ventilation are to provide clean air (“fresh”  air) and thus provide proper movement
and renewal of the air in order to meet the needs of occupants and dilute and remove contaminants generated
inside rooms, in addition to participate in the creation and maintenance of pressure differentials between
areas.
 
3.3.1 Dehumidification and Humidification
 
The functions of air dehumidification and cooling are often performed simultaneously in the ATU heat
exchangers, where dehumidification occurs through the condensation of the water present in the air.
Chemical desiccants containing silica or lithium chloride are acceptable to perform this function, provided
that they do not become sources of contamination.
Humidifiers should be avoided if possible, since they may become sources of contamination, such as for
example, promoting microbial growth. When required, the humidity should be provided by suitable means,
such as steam injection. However, a risk assessment of the possibility of contamination of products must be
made when steam is required for humidification purposes. In cases where it is essential to use humidifiers,
the steam must not contain hydrazine or other anticorrosion substances harmful to health or detrimental to
manufactured products. Air filters should not be installed immediately after humidifiers, since moisture can
promote bacterial growth on their surfaces. The humidification systems should be easily accessible for
maintenance and monitoring and only corrosion resistant materials must be used.

  11
 
 
 
It is not recommended to use heated-tray-type humidifiers, since in this system there is stagnant warm water,
which is a potential source for growth of micro-organisms. Other humidifiers such as evaporation systems,
atomizers and water sprays should not be used because they also offer potential for microbial contamination.
 
3.2.3 Filters
 
The air filtering is always present in HVAC systems, with the goal of providing air with acceptable levels of
particulate contaminants to within an installation. The degree of air purity can be obtained by proper use of
the ATU filters in supply and return ducts and also in the external air intake. Proper sizing of the filter
system is decisive for the establishment of cleaning standards of areas and to reduce particles in the air to
acceptable levels.
The technical standards of filters began appearing in the 1980s, but experienced major changes, such as
changes in the nomenclatures as coarse, fine and medium (Table 1) filters and HEPA filters (Table 2).
 
  EN 779:2002 ABNT NBR – EN 779:2012
NBR 6401:1980
16401:2008 ABNT NBR 16101:2012
Nomenclature Efficiency (%) Nomenclature Efficiency (%) Nomenclature Efficiency (%)
GO 30 - 59 G1 50 <Am< 65 G1 50 < Am < 65
G1 60 - 74 G2 65<Am<80 G2 65 < Am < 80
G2 75 - 84 G3 80<Am<90 G3 80 < Am < 90
G3 85 and above G4 90<Am G4 90 < Am
F1 40 - 69 F5 40<Em<60 M5 40 < Em < 60
F2 70 - 89 F6 60<Em<80 M6 60 < Em < 80
F3 90 and above F7 80<Em<90 F7 80 < Em < 90
- - F8 90<Em<95 F8 90 < Em < 95
- - F9 95<Em F9 95 < Em
 

Table 1. Comparison between nomenclatures used to classify filters in different standards and
versions.
Note: Table illustrative only, since differences between the tests performed for the purpose of assessing the
efficiency of the filters have not been taken into account.
 
EN 1822:2009 EN 1822:2002 ABNT NBR 6401:1980
Nomenclature Efficiency (%) Nomenclature Efficiency (%) Nomenclature Efficiency (%)
E10 > 85 H10 > 85 A1 85 ≤ Ef ≤ 94.9
E11 > 95 H11 > 95 A2 95 ≤ Ef ≤ 99.6
E12 > 99.5 H12 > 99.5 - -
H13 > 99.95 > 99.95 A3 (HEPA) Ef > 99.7
H14 > 99.995 H14 > 99.995
 

Table 2. Comparison between different versions of standards for the different nomenclatures and
efficiency values (Global Values).
Note: Table illustrative only, since differences between the tests performed for the purpose of assessing the
efficiency of the filters have not been taken into account.

  12
 
 
 
The nomenclatures used for filters are directly related to the methods used in efficiency tests. Thus, referring
to only the efficiency of the filters can lead to mistakes, for example, the use of different assay methods may
result in different efficiency values for the same filter. Thus, the nomenclatures of filters must always be
clearly described in formal documents and must preferably use classifications defined in current EN 779 and
EN 1822 versions or equivalent ABNT standards.
 
3.2.3.1 Classification of Coarse, Medium and Fine Filters
It is frequent the reference in standards of air filters testing that the results obtained in the laboratory cannot
be considered as predictions of filters behavior in the field. That is, they cannot be taken as true to predict the
efficiency or lifetime during normal use of a filter in a particular manufacturing facility. The main function
of these tests is to evaluate the performance of filters produced by different manufacturers (using sometimes
different technologies and materials) under standard conditions.
Tests to classify filters are described in EN 779 and ABNT NBR 16101 standards. The update of the EN 779
and publication of ABNT NBR 16101, both of which occurred in 2012, introduced the medium filters (Class
M), which have intermediate efficiency between coarse and fine filters.
It should be noted that the test reports of filters have several important information to users and, therefore,
they should be considered as fundamental tools for the selection and evaluation of air filters. In this
document information such as model, dimensions of the filter tested, filtering class and efficiency should be
included.
 
3.2.3.2 Classification of Absolute Filters
 
With the publication of the standard NBR 16,401 that became obsolete the NBR 6401, a Brazilian standard
for the Classification of Absolute Filters (HEPA) ceased to exist. Since then, it becomes frequent to use the
European standard EN 1822, which is in line with the recommendations made in TRS published by the
World Health Organization. The EN 1822 standard, updated in 2009, covers the method of testing and
classification EPA, HEPA and ULPA filters. The novelty of this standard from the previous version was the
change from the former category of HEPA filters (high efficiency particulate air filters), which was
subdivided into HEPA filters and a new class of filters, the EPA (efficient air filters) (Table 2). The
classification of ULPA filters (ultra low penetration air filters) remained unchanged.
Unlike coarse, medium and fine filters, the absolute/HEPA and ULPA filters shall be tested after installation
(field tests). These assays seek to detect leaks in the filter, in the sealing of the filter, in the sealing gasket and
support structure where the filter is mounted.
 
3.2.3.3 Monitoring and Exchanges of Pre-Filters
 
The air handling systems used in the supply of production areas normally have a sequence of filters installed.
The lower efficiency filters are commonly called "pre-filters" and filters with higher efficiency (e.g., HEPA
filters) called "final filters." The function of the pre-filters is to protect the higher efficiency filter against its
rapid saturation. Therefore, pre-filters are important for the correct functioning of the system and must be
part of a periodic monitoring program to ensure that they fulfill with their role.

  13
 
 
 
Monitoring of pre-filters is done with the aid of differential pressure gauges (analog, digital or supervisory
systems), which measure the “saturation pressure”. Saturation pressure is indicative of the amount of
particulate matter deposited on the filter which leads to increased resistance to air passage. Suppliers of
filters usually indicate an upper reference value for this parameter, and more restrictive  values can be
adopted. Once reached these values and not being possible to clean without compromising efficiency, filters
should be replaced.
To properly establish the monitoring periodicity of pre-filters historical data obtained over a period of more
intensive monitoring  should be considered. Whenever any deviation is detected in the filters, their impact
should be investigated and recorded.
 
3.2.3.4 Monitoring and Exchanges of HEPA Filters
 
The life cycle of the HEPA filters directly depends on environmental conditions such as the level of
cleanliness of the rooms supplied, the contamination of the outside air, the percentage of the air renewal by
the system, the installation conditions (e.g., efficiency of pre-filters and ducts sealing) and the maintenance
conditions of the installation (e.g. monitoring and exchange of pre-filters, cleaning and maintenance of
central of air handling).
The HEPA filters must be tested by the manufacturer of drugs to detect possible leaks. These tests are
performed with the filters installed and the periodicity to perform tests should be clearly defined in
procedures. The HEPA filters installed in clean areas must preferably be annually tested, and the frequency
of testing the HEPA filters used in other facilities must be properly grounded in a risk analysis.
Once rejected in the integrity test a HEPA filter can be repaired and retested. Repairs should be made as
recommended by EN 1822-4, respecting the maximum limits of repaired surface and also the maximum
surface of each individual repair. If alternative criterion is adopted, it should be agreed between purchaser
and manufacturer of filter, and duly justified by both.
 
 
 
3.3 Ducts
AHUs are linked to HVAC through ducts that both distribute the air conditioning throughout the building
and return extraction air to AHU. However, occasionally an AHU can inject and extract air to space to
ventilate directly, without passing through ducts.
Systems of supply and return ducts are often coated with thermal blankets, to prevent gain or loss of heat
from the treated air. The ducts, including those used for disposal of air, must be submitted to a periodic
program of maintenance and cleaning to ensure the absence of leakage and also that there is no accumulation
of contaminants in the system.

  14
 
 
 

3.4 Manufacturing Areas

The infiltration of non filtered air into a pharmaceutical plant is a source of contamination. Therefore,
ceilings, walls and sealed systems of doors and fixtures must limit the input and output of air. As the
efficiency of the system and the levels of air purity obtained are dependent on a correct design and materials
finishing of facilities, the following items should be considered:
• Pre-chambers, dressing rooms and other passages must be available and provide protected traffic between
areas with different cleaning conditions. These areas must have adequate systems for air supply and
extraction;
• Areas such as pre-chambers, dressing rooms and passageways shall be designed so that the necessary
pressure downstream is achieved and maintained;
• Detailed diagrams containing information about pressure  downstream, air flow directions and traffic
routes for operators and materials should be designed and kept updated;
• Whenever possible, people and materials should not move from one area of greater cleaning to another of
lesser cleaning and return again to areas of greater cleaning. The transit from a lesser cleaning area to an
area of higher cleaning must be performed following procedures to exchange uniforms and perform
decontamination, and
• The room used as a final step to dress operators must, in the "rest" state, have the same degree of
cleanliness of the area to which it leads.
Clean rooms are considered areas with environmental conditions defined in terms of contamination by viable
and non-viable particles. To achieve a good level of microbial quality in pharmaceutical products, it is
essential to know the sources and mechanisms that can cause contamination. Some factors such as the type of
installation, operation, maintenance, production processes, presence and activity of personnel are relevant
factors in the generation of contamination and dispersion of particles in clean rooms. Therefore, additional
requirements to guarantee the operating conditions, such as routine environmental monitoring of viable and
non-viable particles should be adopted by these areas.
 
3.4.1 Finish Requirements
 
Finishes are considered all materials intended for coatings, coverings, trimmings, internal partitioning,
ceiling lining and accessories in general. These finishes are also present in production areas, and in this case
should be specified meeting the rigid assumptions of use to which these environments determine.
In areas where the raw materials, primary packaging materials, intermediate or bulk products are exposed to
the environment, interior surfaces (walls, floor and ceiling) shall be lined with smooth, waterproof, washable
and resistant  materials, free of joints and cracks, easy to clean, allowing disinfection and not releasing
particles.
It is important and mandatory to keep these environments controlled with low concentration of dust and
particles. Thus, the finishing materials specified must meet the strict standards for particulate emissions, and
resist the frequent and necessary sanitation and hygiene actions, which require compatibility with chemical
and biocide agents. Walls and floors should have smooth, non-porous and sealed surfaces; they must be easy
to clean, with rounded corners and minimal protrusions; and have adequate resistance to impacts and
hygiene.

  15
 
 
 
The ceiling lining in turn must follow criteria like tightness, continuity, uniformity and not release particles.
There are several types of finishing materials commercially available, which should be selected considering
the needs of the process and also their resistance to manufacturing and cleaning processes. Some examples
are stainless steel to coat walls and countertops; painted galvanized steel for walls; melamine laminate for
walls and countertops; epoxy coating for walls and floor; vinyl for floor covering; aluminum anodized
coating for doors and corners; glass for windows and partitions; and silicone for sealing joints.
 
 
 
3.5 Air Renewal and Recirculation
The treated air can then be insulated in the productive area, be totally discarded or have a portion
recirculated. The choice between these two approaches should be based on aspects of GMP and also in
economical and ecological reasons.
In systems that air is recirculated, the percentage of fresh air used for renewal should not be arbitrarily
determined, but taking into account, for example, the amount of fresh air sufficient to compensate for
leakage through the installation and loss through exhaust systems; to comply with the legal regulations of
construction, and for odor control.
There should be no risk of contamination (including by fumes and volatiles) and cross-contamination due to
air recirculation. Multiproducts1 areas, depending on the type and amount of contaminants adduced in the
recirculation of the air, recirculation may be acceptable provided that HEPA filters are installed in the supply
system or in the return system to remove contaminants and thereby avoid cross-contamination. The HEPA
filters for this application should be at least H13. The HEPA filters, when installed in the air supply system
may be located in the air treatment unit or terminally installed (Figure 2).
HEPA filters may not be needed in manufacturing areas of non-sterile drugs when the air treatment system
supplies a dedicated2 installation and there is evidence that there is no possibility of cross contamination.
When the air recirculation is restricted to areas where there is no generation of powders, such as areas used
for secondary packaging steps, also there may be no need for HEPA filters in the system.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 Areas used to manufacture drugs containing different active pharmaceutical ingredients.
2 Areas used to manufacture a single drug and, therefore, in these areas only one active pharmaceutical
ingredient is handled.

  16
 
 
HEPA Filter

Production
Rooms

Air treatment unit containing the terminal filter

 
 
  HEPA Filter
 
 
 
 
 
  Production
  Rooms
 
 
System with final HEPA filter in terminal position

Figure 2 Position of terminal HEPA filters.

It is recommended that HEPA filters are installed terminally and connected to the air distribution ducts via a
flexible duct. However, when the flexible duct is used, it should be as short as possible and properly secured
to withstand pressure.
The air used to supply areas where highly toxic products are processed, as well as the air contaminated with
solvents or flammable vapors must not be recirculated by the air treatment system. In these cases, the air
treatment system must operate providing 100% fresh air for the production area.
If heat recovery wheels (or any other system) are used as a way of reusing energy in multipurpose areas, a
risk assessment to determine whether this component of air treatment system will not become a source of
cross contamination must be conducted.
 
3.6 Exhaust Systems and Extractors
The air coming from production areas, equipment (such as fluidized bed and tablet  coating equipment) or
extraction systems can carry large amount of dust. Thus, before being discarded, this air must be filtered to
prevent environmental contamination.
The required degree of the exhaust air filtration depends on the contaminants present in the air and regulatory
requirements. When the powders are not highly potent, terminal filters in the exhaust system shall be thin,
with F9 classification according to EN 779. On the other hand, in case of hazardous substances, it is
recommended to use at least one HEPA filter in the air exhaust system.
Dust, steam and smoke can be sources of contamination and must be removed from the inside of the
production facilities. Thus, careful must be taken when deciding the generation and extraction places.
Wherever possible, contaminants must be removed as close as possible to the source of generation.
Extraction systems in the workplace, such as points of ventilation and high speed fixed or articulated arms 
extractors must be employed. However, only points of extraction may not be sufficient to capture all the
contamination generated. In these cases, unidirectional flows must be used to help remove them.

  17
 

Powder extractors must be designed to have sufficient speed to ensure that the dust is removed from the
generation point and does not accumulate in the exhaust ducts. Additionally, periodic checks must be
performed to ensure that there is no accumulation of powder in the pipeline. The air speed is typically
defined according to the density of the powder to be extracted, i.e., the denser the powder, the greater will be
the speed of the air. The direction of air flow should be chosen and documented in procedures in such a way
that the operator is not exposed to powder, and also so that the operator does not jeopardize the quality and
safety of the product.
In a room operating with a pattern of turbulent air, the air introduced by diffusers located in the ceiling
should be removed from the back of the room, in a position close to the floor to help "clean up" the room.
The proper flow of this air can be verified by smoke testing.
When particularly dangerous products are handled, extra care to control the generation of contaminants may
be necessary, such as the use of "glove boxes" and insulators.
 
 
 
 
 
 
4. PARAMETERS AFFECTING THE SAFETY OF
PRODUCTS AND OPERATORS
 
The air treatment system is the main tool, but it is not only for protection of products and operators. To
ensure the required level of cleanliness in productive areas additional measures are needed, such as sanitation
and hygiene; staff training and proper dressing; adequate facilities and equipment; proper procedures for
materials and personnel  movement, and validation procedures for cleaning / sanitizing. However, because
they are not the scope of this Guide Quality, such measures will not be discussed. The following subsections
deal only parameters related to air handling systems that affect the safety of products and often the operators
themselves.
 
 
 
4.1 Number of Particulates in the Air
Many variables can affect operations in clean areas, but the concentration of particles in the air is one of the
most significant elements in the control of the risk to the quality of pharmaceutical products. This parameter
becomes even more significant in clean areas used for the production of sterile drugs and, thus, the
monitoring of the amount of particulate matter suspended in the air of these environments has to be carried
out.

  18
 
 
 

4.2 Distribution of ATUs (Sharing)

Multipurpose areas, is of special importance to carry out a risk assessment on the impacts of a possible cross-
contamination caused by the sharing of air handling units between different production areas. The
recirculated air between different production environments can carry particles of active pharmaceutical
ingredients from one to another and thus become a significant source of cross contamination.
In certain situations, GMP determine the use of segregated and dedicated facilities for the production of
certain drugs, such as certain biological preparations (e.g., live microorganisms) and highly sensitizing
materials (e.g., penicillin, cephalosporin, carbapenems and other beta-lactam derivatives), in order to
minimize the risk of serious damages to the health of medications users due to cross-contamination.
Although this requirement does not necessarily imply the need for separate buildings, facilities must provide
complete and total separation of all aspects of an operation, including independent air handling systems.
In order to demonstrate the correct distribution of air handling systems, documents containing schematic
drawings of production areas and their AHUs used in their supplies (Figure 3)  must be prepared and kept
updated. Information on the manufacturing process steps performed, handled products and their levels of
exposure to the environment in all areas should also be available.
 

AHU 01
AHU 02
AHU 03
AHU 04
AHU 05

Figure 3 Example of schematic drawn of AHUs distribution supplying a production facility.

Quality control laboratories must be separated from manufacturing areas, including the need for independent
air handling systems. If there is any sharing, it must be demonstrated through a risk assessment that there is
no negative impact on the quality and safety of products manufactured and the reliability of the results of the
analyses performed. However, the animal rooms and areas used for biological, microbiological (especially
those used for testing growth-promoting culture media) or radioisotopes testing must necessarily be
separated from production areas as well as each other.

  19
 
 
 

4.3 Number of Air Exchanges

In the manufacturing facilities, the number of air exchanges is usually assessed by factors such as:
• When a production area requires specific classification under rest and / or operation conditions, the rate of
air exchange must be defined according to the need of cleaning;
• Product characteristics (for example, release of smells, hygroscopicity, etc.);
• Quality and filtration of supply air ( “fresh” air);
• Amount of particles generated by the manufacturing process;
• Amount of particles generated by operators;
• The configuration of the room and the supply and exhaust air places;
• Amount of air sufficient to “clean” the air of the area;
• Amount of air sufficient to neutralize the heat load generated inside the room;
• Air enough to equilibrate the exhaustion rates;
• Air enough to keep the required pressure differentials between areas.
Often, the air exchange rates in manufacturing areas of non sterile products vary between 6 and 20 exchanges
per hour. However, the required number of exchanges may significantly vary depending on the use of the
facilities.
Due to more stringent criteria of cleaning, in clean areas the air exchanges rate is significantly higher than in
controlled areas only. The technical standards often adopt a value of at least 20 exchanges per minute, but the
higher the degree of cleanliness of the room, the greater the number of air exchanges required.
 
 
 
4.4 Air Flow Standard
Contaminants present in the productive environments must be controlled by dilution or by replacing the air.
These functions are performed through the treated air supply in production rooms, and the standard supply
can be unidirectional (e.g., existing pattern in the example B of Figure 4) or turbulent (e.g., existing pattern
in the examples A and C of Figure 4).

  20
 
 
 
AHU

Air Supply

Main Filter

Turbulent

Unidirectional Turbulent

Return of the air (recirculation)

 

Figure 4 Different Patterns of Air Supply.

Usually, the weighting or sampling cabin should provide a unidirectional standard of air flow to provide
protection to the operator and also to the product. They also must promote a slight influx of air in the room
where it is installed, in order to strengthen containment. The containment of powders dispersed must be
demonstrated by tests of air standards (smoke testing) or other appropriate tests. The unidirectional airflow
cabins can also be used in other steps of the production processes which generate large amounts of dust.
The speed of unidirectional air flow must be such as not to disturb the sensitivity of the scales in weighing
areas. If necessary, the speed can be reduced to avoid bias during the weighing, since the air flow is retained
in a pattern sufficient to provide containment.
Conventional systems of unidirectional air flow used when a grade A cleaning air is required have a
homogeneous speed of air flow of about 0.36 to 0.54 m/s at a distance of 15-30 cm below the filter or
distributor system. The air velocity in the same unidirectional flow system at working level must be not
lower than 0.36 m/s. However, in weighing and sampling cabins lower speeds can be adopted. For this type
of application, sometimes it is preferable to use the term “protection air flow cabin” instead of
"unidirectional air flow" to avoid confusion with the requirements performed for a grade A unidirectional
flow.
Typically, in an operating room with a pattern of turbulent air, the air must be introduced from ceiling
diffusers located near the entrance of the room and extracted at the back of the room, preferably in a low
position. These conditions provide the best "cleaning" of the air, and must be verified by visualization testing
of air standards (smoke tests).
The position at which the operator remains to the source of powder releasing and the air flow must be
determined to ensure that the operator does not be in the way of the air flow, becoming a source of product
contamination.

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4.5 Requirement of Filtrating Elements (Types and Position)

External contaminants must be removed by effective filtration of the supply air and also by appropriate
design of the production facilities. The degree of air filtration plays a major role in the prevention of
contamination and cross-contamination control.
The types of filters used for different applications depend on the quality of ambient air, return air (where
applicable) and also the rates of air exchange. For example, Table 1 provides information on the
recommendations of filters to be used for air filtration that supplies public environments.
 
Typical Application Class
Supermarket, malls, shopping centers, banks, post offices, commercial and service shops. G4
Offices, meeting rooms, CPD, typing rooms, call centers F5
Airport – lobby, departure lounges F5
Airport – control tower G3 + F6
Library, museum – public areas F5
Library, museum – exhibitions and dept of deposit sensitive works G3 + F8
3 star or more Hotels, apartments, lobby, living rooms, convention halls F5
Hotels - other, motels - apartments G4
Theater, cinema, auditoriums, places of worship, classroom F5
snack bar, cafe G4
Restaurant, bar, cocktail lounge, nightclub, disco, games room F5
Gym (public areas), fitness center, bowling, electronic games G4
Telephone exchanges computing G3 + F6
Homes G3
Control room - electronic environment sensitive G3 + F6
Printing - Lithographic, offset G3 + F7
Printing - film processing G3 + F8
 

Table 1 Recommended filtration in air systems supplying public environments (Extracted from NBR
16401-3).
Drug manufacturers, in turn, must determine and demonstrate the proper use of filters in their facilities. The
level of protection and cleanliness of air for different areas must be determined according to the product to be
manufactured, the manufacturing process and the product's susceptibility to degradation.
The areas used for performing the production steps of non-sterile drugs do not require classification
according to the count of viable and non-viable particles. However, although not being required for
classification tests, these areas must be designed in such a way that meet grade D under resting conditions
(at-rest condition). WHO, in its guide for HVAC systems for manufacturers of non-sterile dosage forms
(Annex 5 of TRS 961, published in 2011) lists different levels of protection that must be employed in
accordance with the exposure of the products to the environment. For each one of these different levels of
protection (Table 2) recommendations were made about the sequence and the efficiency of the filters to be
used (Table 3).

  22
 
 
 
Level Condition Examples of areas
1 General General Areas with normal organization and maintenance where there is no risk of
contamination for the product. E.g., warehouse.
2 Protected Protected area in which precautions are taken to protect raw materials or the product
from direct or indirect contamination or degradation. E.g., Secondary packaging, first
stage of changing of clothes.
3 Controlled Controlled area in which environmental conditions are defined, controlled and
monitored to prevent contamination or degradation of raw materials or products.
Environments where there is exposure of raw materials, components or product to the
environment, as well equipment washing areas and warehouse of equipment parts that
come in contact with the product.
 

Table 2 Levels of protection for non-sterile products in different areas.

 
Level Minimal filtration recommended
Level 1 (general) Only primary filters (E.g., filter EN 779 G4)
Level 2 (protected) Protected areas operating with 100% renewed air: primary and secondary filters (E.g.,
Filters EN 779 G4 + F8 or F9)
Level 3 (controlled) Plants operating with recirculated air, increasing the risk of cross-contamination:
primary, secondary and tertiary filters (E.g., filters EN 779 G4 + F8 + EN 1822 H13).
In areas operation with 100% renewed air – without recirculation
- only filters G4 + F8 or F9 are accepted
 

Table 3 Minimal requirements of filtrating elements for different levels of protection of non-sterile
products.
Clean areas require more efficient filtering elements, and usually the filters described in Table 4 are used. It
is noteworthy, however, that the sequence of filters described in Table 4 may not be suitable for all
installations, especially when the outside air captured has large amounts of contaminants. In such cases, the
use of more efficient filters must be considered. To demonstrate the choose appropriateness of the filter
systems, all the tests recommended for qualification of clean areas must be performed and subsequently tests
for the purposes of periodic requalification and also for routine environmental monitoring.
 
Area
Operating method of the areas Minimal filtration recommended
Classification
Grade D Production areas operating with 100% G4 e F8
renewed air;
Grade D Areas operating with recirculated air plus fresh G4, F8 and H13 (HEPA filter may be
air, and that there is risk of cross contamination; terminally located or at AHU)
Grade C Areas operating with recirculated air G4, F8 and H13 (HEPA filter may be
or 100% renewed; terminally located or at AHU)
Grade A or B Areas operating with recirculated air G4, F8 and H13 (HEPA filter must be
or 100% renewed; terminally installed)
 

Table 4 Minimal requirements of filtering elements for clean areas with different degrees of
cleanliness.

  23
 
 
 

4.6 Temperature and humidity

Where appropriate, temperature and relative humidity  must be controlled and monitored. Periodic records
must be made and, if applicable, alert and action  limits  must be defined. These measures are intended to
ensure the provision of necessary conditions for the quality of materials and products, the proper functioning
of equipment and, where applicable, to the comfort of the operators.
Maximum and minimum values of temperature and relative humidity must be appropriate and specified. The
operating or tolerance range between the minimum and maximum acceptable values should not be too
narrow since it may be difficult to be maintained and increases operating costs.
Rooms or facilities in which products requiring low relative humidity are processed must have walls and
ceilings well sealed and must be separated from adjacent areas with higher air relative humidity through
antechambers. Precautions must also be taken to prevent moisture migration that increases the load on the
HVAC system.
 
 
 
4.7 Pressure Differential, Air Replacement and Physical Barriers
When we talk about contamination of products by air, it is necessary to bear in mind different concepts,
especially concerning techniques for contamination control by differential pressure, by replacing air and by
physical barriers.
The concept of pressure differential (high pressure and low air flow between areas) is usually used in areas
where there is low or no generation of dust. This technique may be used alone or in combination with other
techniques of containment, as antechambers. When adopted, care must be taken to determine the magnitude
of the pressure differential, which must be sufficient to ensure containment of powders and preventing flow
inversion, but it should not be so high that would create problems of turbulence. A pressure differential of 15
Pa is typically used to achieve the contention between two different adjacent zones, but pressures from 5 to
20 Pa may be acceptable. Low pressure differentials may be accepted when the "sink"-type or "bubble"-type 
antechambers are used to segregate different areas.
To ensure that pressure differentials remain within the projected values for the area, properly calibrated
pressure gauges  should be used. Such instruments should preferably be kept fixed in places with easy
viewing by operators and must have a measuring graduation and range that allow the proper reading of
measured values. Routine operation ranges and alert and action limits must be established and be available at
the checkpoints. In these situations, a system of color coding (ribbons of different colors attached on the
differential pressure gauge indicating the lower and upper limits of pressure) can be quite useful. Another
common way to monitor the differential pressure is through central computerized systems such as BMS or
SCADA.
Antechambers may be important components in the setting and maintenance of pressure differentials and
also to limit contamination. They are designed and used for separation of areas of varying degrees of
cleanliness and traffic for both operators and materials. They are classified according to their patterns of air
flow, whereas there are basically three types: “downstream” (Figure 5) “bubble” (Figure 6) and “sink”
(Figure 7).

  24
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 5 Antechamber with “downstream” pattern. Figure 6 Antechamber with “bubble” pattern.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 7 Antechamber with “sink” pattern.
 
 
Antechamber doors must open toward the area of greatest pressure, so that when closed, the pressure
(together with the lock) assists to keep it closed. The doors that open toward the room with lower pressure
differential must have locks strong enough to keep them closed and prevent the pressure differential force its
opening. There must be a method for controlling both the antechamber doors are not opened simultaneously,
or alternatively, they have to be interlocked. Determining which doors should be interlocked shall be subject
to a risk  assessment. Passage boxes (pass-boxes) of materials can also be used to separate two areas with
different classifications. There are basically two types, those called dynamic pass-boxes (when there is air
supply or extraction in the proper box) and passive pass-boxes (when there is no air supply or extraction).
Dynamic pass boxes can function as bubble, sink or downstream antechambers.

  25
 
 
 
The concept of replacing air (low pressure and high speed of the air between different areas) is not a method
of choice to avoid dispersion of solid contaminants or smoke in a production area, due to the difficulty of
measuring the air flow between different areas. This concept is usually found in production processes that
generate large amounts of powder. This principle lies in supplying air to production  corridors, which
subsequently enters into production rooms through grids on doors (or even open doors) and is extracted from
the end of this room. The air speed must be high enough to avoid air turbulence. Although not recommended
and rarely used, this concept still exists in old production plants.
Where appropriate, impermeable barriers must be used (conceptually called physical barriers) to prevent
contamination between two different zones. Examples of this technology are the closed systems (isolators
and glove boxes) and transfer systems via peristaltic or vacuum pumps.
Manufacturing facilities must normally be maintained at a positive pressure relative to the outside
environment, to limit the entry of contaminants. When facilities need to be maintained under negative
pressure relative to ambient pressure, additional care must be taken, such as the choice of the location of the
production plant, which must be done carefully considering the surrounding area. Special attention must be
given to the building structure of such installations that must be well sealed. Negative pressure areas must, as
far as possible, be “encapsulated” in surrounding areas that have active air supply, so that only clean air can
infiltrate into the controlled area.
The choice of pressure differentials must be performed considering the product and/or manufacturing method
employed and, therefore, each installation must be individually evaluated according to the product handled
and the required level of protection. Examples of choices for pressure differentials are listed in Table 5.
 
Categories of products Requirements of pressure differentials
Solids Containment system of powders. The corridor must be maintained at a
pressure higher than the production room (cubicles), and the cubicles at a
pressure greater than atmospheric pressure.
Liquid and semisolid Less critical, however in certain steps may be required adequate flows to
products prevent cross contamination.
Sterile Products Product protection system, i.e., the production area must have higher
pressure than the corridors (higher pressure in cleaner areas).
Biological products - where Biocontainment system, i.e., corridors with pressure higher than working
there are microorganisms area.
exposed.
Highly potent products Powder containment system. The product must be produced in areas
negatively pressurized relative to atmospheric pressure.
Comments: There should be no direct ventilation air to the external
environment. HEPA filter should be used in the exhaust; Recirculation only
in the same areas (minimum H13 filter in the air return).
 

Table 5 Patterns of pressure differentials usually required to protect different categories of

manufactured products.

  26
 
 
 

4.8 Microorganisms in the Air and Surfaces

Microorganisms are present in virtually all nature. They are carried by air currents from the earth's surface to
the upper layers of the atmosphere. Even the typical microorganisms of the oceans can be found many
kilometers away, on the top of mountains.
Once the conditions that favor the survival and growth of many microorganisms are the same under which
human populations live, it is inevitable that we live among a large number of microorganisms. They are on
the surface of our body, in our digestive tract, and other natural orifices.
Thus, considering the risk of medicines contamination with micro-organisms offers to users, it is particularly
important the microbiological control of productive areas. Thus, facilities are needed to prevent the entry of
these microorganisms carried by the air and other appropriate measures, such as procedures for operators
dressing and decontamination of materials to control the amount of microorganisms introduced into
productive areas.
 
 
 
4.9 Air Exhaustion
It is preferable that the exhaust of the air insufflated into productive areas is made at a low level in the room
(example A of Figure 8). Otherwise, the system must provide a high number of air exchanges.
Points for exhaust air uptake located in the ceiling (Example B in Figure 8) can be used only in areas where
the particle generation is low. This exhaust position is not recommended for areas that require a higher
degree of cleanliness such as clean areas.

Air Handling Unit Pre-filter Main Filter

Air Insuflation

Air Return (Recirculation)

 
Figure 8 Positions of inlet points of exhaust air.

  27
 
 
 

4.10 Supervisory Systems

When automated systems are used for monitoring the air treatment system and production areas, they must
be able to indicate the out of specification values conditions, without delays, by means of alarms or similar
systems. If these systems are used to support critical decisions, they must be validated. Examples of
computerized systems are building management system (BMS), building automation system (BAS) or
system control and data acquisition (SCADA).
 
 
 
 
 
 
5. ENVIRONMENTAL PROTECTION
 
Details of the environmental protection requirements are not addressed in this document, since any material
disposal to the environment should be carried out according to the rules defined by the competent authorities
of environmental protection.
Powder, steam and smoke can be sources of contamination of products and must be removed from
production areas. Thus, care should be taken when deciding the location of the generation and extraction of
such contaminants.
The air collected in production areas and equipment, such as from fluidized beds and equipment for coating
tablets, or from extraction systems may carry large amount of powder. Soon, this air must be filtered to
prevent environmental contamination and the required degree of filtration depends on the contaminants and
regulatory requirements. When contaminants are not highly potent, terminal filters in the exhaust system
shall be thin, with F9 classification according to EN 779. The use of HEPA type filter elements in the
exhaust system is normally required only when hazardous materials are processed.

  28
 
 
 

6. CLEANING AND MAINTENANCE OF

COMPONENTS OF AIR TREATMENT SYSTEMS
 
There should be a formal program of preventive maintenance, and executions of the activities defined in this
program must be recorded. Operation and maintenance manuals, schematic drawings, protocols and reports
should be available and kept updated, containing all changes made to the system.
Maintenance personnel must have adequate training.
HEPA  filters, when necessary, should be replaced by experts or persons duly trained and tested for leaks
after installed. Any maintenance activity should be critically evaluated to determine its impact on the quality
of the product, including possible contaminations.
Maintenance activities should be scheduled to occur at times when there are no production activities, and as a
result of any system downtime an assessment of a possible need for requalification must be performed.
In Brazil there is no available technical documents published by regulatory authorities to define a specific
plan for maintenance, operation and control of air treatment systems in the pharmaceutical industry.
However, some documents are not specific and targeted to the maintenance of other air handling systems can
be adopted as guidelines for maintenance of air handling systems of pharmaceutical industries. They are the
Ordinance 3523/GM-1998 published by the Ministry of Health and Resolution RE 9/2003 published by
Anvisa. RE 9/2003, which sets Reference Standards of Quality Indoor Air at Environments Artificially
Conditioned of Public and Collective Use presents some frequencies of activities to be performed (Table 6).
 
Component of the system Periodicity
External air intake and filter units (coarse filters) Monthly cleaning or disposal within up to three
months
Condensation tray Monthly
Humidifier and the heating and cooling coils Biannual decaling and quarterly cleaning
Ventilator Semiannual
Mixture Plenum / powerhouse Monthly
 

Table 6 Maintenance periodicity of components of the air treatment systems, according to RE 9/03.

  29
 
 
 

7. CHOOSING THE APPROPRIATE AIR TREATMENT

SYSTEM
 
As previously discussed, the three main aspects related to the role of an HVAC are the protection of
operators, the product and the environment (Figure 5).
 
GMP production 
environment 
 
 
  Protection of the   Protection of Protection of
   
  Product   Operators Environment
   
   
   
Contamination Prevent contact with Avoid disposal of
  (Product and Powders Powders
  Operators)
 
 
  Protection of the Prevent contact with Avoid disposal of
  product against Fumes  Fumes
  cross-contamination
 
  Correct Temperature Acceptable comfort Avoid disposal of
  and Humidity conditions  efluents
 
 
 
 
SISTEMS
 
 
 
VALIDATION OF
THE SYSTEM

Figure 9 Main aspects of an air handling system (adapted from WHO-TRS 961, 2011).
Thus, to assess the adequacy of a HVAC system is first necessary to know the production process, as well as
the characteristics of the manufactured products.
Sterile products, and also those who typically provide contamination risks to the environment, require
additional protection requirements when compared to other products (Figure 10). Therefore, the design of the
air handling system should always take into account the characteristics (therapeutic risks) of products.
Similarly, the air handling system should be designed to offer a higher level of protection to raw materials,
primary packaging materials, intermediate or bulk products when they are exposed to the environment.

  30
 
 
 

Product Protection Requirements

Aseptic process
Potentially Toxic
Non Sterile

Sterile
Therapeutic Risk
 

Figure 10 Protection requirements to the product versus therapeutic risk associated to products.
 
 
 
7.1 Technologies of Air Conditioning
The air conditioned systems may, in general, be classified into “comfort air conditioning” and “process air
conditioning”.
The comfort air conditioning systems are designed to provide an internal environment which conditions
remain relatively constant within the standards that provide comfort to people despite weather variations and
internal thermal loads. Their uses are most common in homes, shops, offices and cars. Most of the comfort
air conditioned systems use the principle of direct expansion, characterized by devices that have a coil where
a refrigerant fluid of high latent heat is expanded. During this change of state, the fluid absorbs heat and
promotes air refrigeration in contact with the coils. The standard ABNT NBR 6401/80 used the term “direct
evaporation” and defined as one in which “the refrigerant boils in the actual heat exchanger, which is directly
in contact with the air to be treated.” Examples of devices that use this principle are the window/wall and the
split type air conditioners0.
The process air conditioning systems, in turn, aim to ensure appropriate environmental conditions for a
particular process. Although these environmental conditions are often within the standards of human
comfort, the needs are that determine the process. They are used in operating rooms, pharmaceutical products
manufacturing rooms, clean rooms for the production of integrated circuits, among others. These systems
mostly use the principle of indirect expansion, i.e., the refrigerant does not enter into direct contact with the
coil located in a conditioned environment, but with an intermediate medium (secondary fluid), usually water.
The chilled water is produced in the evaporator of the liquid chiller (chiller) and sent, through pumps, to
another heat coil or exchanger (typically located in an AHU) where the cooling of the air used to supply the
internal environment will occur. The standard ABNT NBR 6401/80 used the term “indirect evaporation” and
defined it as one in which “there is an intermediate element, such as water or brine, supplied by a cold
producer central, which supplies the various air conditioners.”

  31
 
 
 

7.2 Limitations of Use for Comfort Air Conditioning Systems

The comfort air conditioning systems, such as split and wall types, have significant limitations to be used in
drug manufacturing areas. These devices typically use filters, such as polyurethane, glass wool, or synthetic
microfibers or steel mesh, which are not suitable for obtaining air quality (level of particulate and microbial
contamination) required by the processes of drugs. Furthermore, they favor the accumulation of
microorganisms and dust in the filter and do not promote adequate renewal of air, since the air renewed is
from external environments without proper filtration. In addition, with the use of this system, it is not
possible to create and control pressure differentials between areas and, despite reducing the humidity of the
air, is not effective for its control within preset limits. Moreover, such systems are not usually effective to
provide and maintain a homogenous distribution pattern of temperature throughout the environment where
they are installed.
Due to the inherent risks, comfort air-conditioning systems must not be used in areas where raw materials,
primary packaging materials, intermediates or bulk products are exposed to the environment. If duly justified
through a risk assessment that takes into account the conditions required by the product (temperature and
humidity) and its exposure time, these systems may be acceptable in areas where there is no exposure of
products to the environment, such as storage and secondary packaging areas.
 
 
 
 
 
 
8. COMMISSIONING, QUALIFICATION AND
REQUALIFICATION OF THE AIR TREATMENT
SYSTEM
 
 
8.1 Commissioning
Commissioning is the activity that aims to ensure that the systems and components of an HVAC system were
designed, installed and tested to operate and be maintained in accordance with the specifications of user
requirements (SUR). This activity is applicable both to new systems and systems already installed that are
found in process of expansion, modernization or changes. Commissioning must include the balancing,
adjustments and testing of all components of the air handling system.
The commissioning plan should begin to be established in the early stages of a project, so that it can be
integrated into the activities of qualifications and verifications. Before starting the physical installation of the
system, acceptance criteria must be defined for all system parameters and measurement results after
installation must meet them consistently. After the installation of the system, operators must be trained and
instructed for the activities of routine operations and maintenance also. In summary, the commissioning
should be a precursor for the system qualification and the validation process.
Commissioning activities of the system  must be recorded, in order to show that all measures of system
capacity have been performed.

  32
 

8.2 Qualification
Commissioning activities are, in essence, an assessment to ensure that the system or equipment has been
designed, installed and operates correctly from the point of view of engineering. The qualification term is
used to define the required tests to ensure that the system or equipment has been designed, constructed and
operates within the acceptance limits for the parameters identified as critical for the quality, safety and
efficacy of the product, i.e., the system operates according to the Good Manufacturing Practices guidelines.
Thus, a risk-based approach must be adopted to identify the minimum requirements for the qualification of
the HVAC system, in view of the manufactured products and processes developed in productive areas.
The qualification of the air treatment system must be described in a validation master plan (VMP). The
nature and extent of qualification, as well as their testing protocols to be followed, the results and
conclusions must be defined and documented. It must also be attached to the records of the qualification of
the AHU drawings, drawings of manufacturing areas, drawings containing information about the pressure
differentials between different areas, designs of production areas containing information about where the
particle count devices and other sampling points are allocated, etc.
The qualification steps for the air system must include the qualifications of the design (DQ), facilities (FQ),
operations (OQ) and performance (PD). The critical and non-critical parameters must be determined by a risk
assessment for all components of the air handling system, including its subsystems and its control devices.
Any parameters or even some system components, which may affect the quality of the product must be
considered as critical and included in the qualification process. Systems considered not critical and its
components do not necessarily require qualification, but must follow the Good Engineering Practices.
A precise and reasoned assessment to differentiate between critical and non-critical parameters is necessary
to prevent the qualification that become unnecessarily complex or does not comply with the minimum
requirements of the process and product. An example of this approach is described below.
Example: The cleaning condition of the production rooms is considered critical,
and so the air renewal rates and HEPA filters must be considered as critical
parameters and require qualification. Components such as the fan that generates
the air flow and the primary and secondary filters are not critical parameters, and
may not require operation qualification. In these cases, an adequate maintenance
and monitoring program is sufficient.
There must have a procedure describing how to assess the impact of changes on the air handling system, as
well as their components and controls that may affect critical parameters.
The condition of the project, normal operating ranges, alert and action limits must be defined and be realistic.
Out of specification limit results (e.g., deviations above the action limits) must be formally documented and
its impacts properly investigated.
For a pharmaceutical facility, based on a risk evaluation, some of the typical parameters of the HVAC
system to be classified may include temperature; relative humidity; supply of air quantities to all diffusers;
return of air quantities or air exhaustion; number of air exchanges in rooms; pressure differentials between
different areas; air flow patterns; unidirectional flow velocities; speed of containment systems; penetration
testing of HEPA filters; particle counting; cleaning rates of rooms (air renewal); microbial contaminants
counts in the air and on surfaces; dust removal operation and; alert or alarm  system. Table 7 provides a
number of tests that must be considered for qualification ends as well as references to be followed in its
conduction.

  33

Parameter tested
Particles counting test
(Assessment of the cleanliness and
clean rooms)
Pressure differential
(to assess the effectiveness for
avoiding cross contamination)
Air Volume
(to assess the number of air
exchanges)
Air flow speed
(To check the unidirectional air flow flows of protection must be measured.
or the contention conditions)
Leakage test of filters
(To check the integrity of the
filters installed)
Check of leakage/contention
(To check the absence of
cross contamination)
Recovery
(To check the cleaning
time of the room)
Visualizing the air flow
(To check the air flow patterns)
Table 7 Tests that may be required for qualification of an air handling system.
 
Procedure for the test
Results obtained by the particle counters must be printed and
attached to the records of qualification. The sample numbers of
collections must be in compliance with ISO 14644-1 Annex B5
and include collected samples in representing positions in the
process (at heights close to working positions, with 30 cm of
operations a value which can be considered as a reference.)
Pressure differential must be daily monitored, preferably
continuously. A pressure differential of 15 Pa is recommended
between different zones (changes from 5 to 20 Pa may be
acceptable). Test must be performed in accordance with ISO
14644-3 Annex B5.
Readings of air flows in the supply and exhaust grids must be
performed and rates of air exchange calculated.
The test must be performed according to ISO 14644-3, Annex
B13.
Speed of the air for containment systems and unidirectional air
The test must be performed according to ISO 14644-3, Annex
B4.
Penetration testing for HEPA filters must be conducted by
competent persons, in order to demonstrate that the filter, the
filter seal and the frame of the filters are intact.
The test must be performed according to ISO 14644-3, Annex
B6.
To demonstrate that the containment in a production room is
kept, using smoke tests to check the direction of air and pressure
tests in the rooms.
According to ISO 14644-3, Annex B4;
Test to assess the time that the clean area takes to recover from a
condition of contamination for a cleaning condition previously
specified. It should not take more than 15 min.
Testing is performed according to ISO 14644-3, Annex B13;
Tests to assess that the airflow:
• is in the direction from clean areas to dirty areas;
• is not a cause of cross-contamination;
• has uniform standard.
Test must be performed according to ISO 14644-3, Annex B7,
and recorded by video recording.
34
 
 
 
8.2.1 Clean Areas
 
Clean areas are areas with environmental control defined in terms of contamination by viable and non-viable
particulates, designed, constructed and used in order to reduce the introduction, generation and retention of
contaminants in its interior.
These areas must be designed, qualified and operated according to strict criteria of GMP, including drawings,
flow of personnel and materials, air handling systems, utilities, and qualifications of operators.
The clean areas are classified according to their environmental conditions for amount of viable particles and
sometimes also for non viable particles. There are different technical standards that address the clean areas
classifications, with the most used in the country being ISO 14644 and GMP standards.
 
8.2.2 Differences Between ISO 14644 Classifications and GMP Guidelines for
Clean Areas
 
The ISO standard defines different levels of cleanliness (ISO classes) according to the amount of particles of
different sizes suspended in a cubic meter of air in a given clean area (Table 3). However, these classes are
not specific for the condition of the test (“at rest” or “operating”) and also they don’t have any relationship
with limits of viable particles (microbial contamination).
 
 
 
ISO Maximum limits of concentration (particles/m3 of air) for equal or higher particles
Classification than the sizes considered
Number
0.1 µm 0.2 µm 0.3 µm 0.5 µm 1 5
ISO Class 1 10 2  
ISO Class 2 100 24 10 4  
ISO Class 3 1 000 237 102 35 8  
ISO Class 4 10 000 2 370 1 020 352 83  
ISO Class 5 100 000 23 700 10 200 3 520 832 29
ISO Class 6 1 000 000 237 000 102 000 35 200 8 320 293
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
 

Table 3 Classification of areas according with air cleaning classes for particles in suspension
(Source: ISO 14644-1).
To comply with the requirements of Good Manufacturing Practices for the manufacturing of drugs, the
classification of a clean area must be assessed according to the amount of viable and non-viable particles in
two different conditions to perform the tests (“operating” and “at rest”). For this reason, the criteria for
classifying areas often imposed by Resolutions published by Anvisa use the classification of areas into
“grades” A, B, C and D. From these classifications, in addition to the conditions of test conduction
(operating and at rest), the criteria for non viable particles limits for two defined dimensions (Table 8) and
also limits for microbial contamination (Table 9) are inferred.

  35
 
 
 
  At rest (Stand by) Operating
 
Grade Maximum Number of particles allowed/m3
≥ 0.5 µm ≥ 5 µm ≥ 0.5 µm ≥ 5 µm
A 3,520 20 3,520 20
B 3,520 29 352,000 2,900
C 352,000 2,900 3,520,000 29,000
D 3,520,000 29,000 Not defined Not defined
 

Table 8 Limits of non viable particles in areas classified as Grade A, B, C e D.

 
 
Plates Contact plates 5 fingers glove
  Air Sample
Grade finger printing
(90 mm diameter) (55 mm diameter)
(cfu/m3) cfu/glove
(cfu/4 hours) cfu/plate
A <1 <1 <1 <1
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -
 

Table 9 Recommended limits for evaluation of microbiological contamination in areas classified as

Grade A, B, C e D.
The classification of clean areas in class 100, 10,000 and 100,000 was established by the U.S. Federal
Standard 209. This classification was directly related to the maximum allowable number of 0.5 mM particles
in suspension in a cubic foot of air. Subsequently, the FS-209-E was replaced by ISO 14644. Even with the
substitution, in 2004 the U.S. Agency responsible for regulating the pharmaceutical market (FDA) published
the document FDA Guidance for Industry - Sterile Drug Products Produced by Aseptic Processing - Current
Good Manufacturing Practice, which continued to use the terminology, established by FS 209, but had a
table with correlation with the ISO rating. According to the FDA’s document, the definition of the
classification of the clean room (100, 1,000, 10,000 or 10,000) should be carried out for particles of 0.5 mM
suspended in the air and were considered values of viable particles obtained from the active sampling of the
air and sedimentation plates.
Despite the trends of change in the U.S. usual classification, which tend to adopt the related ISO standards, it
is still usual the categorization of clean areas in Classes 100, 10,000 and 100,000. The equivalence between
these different types of classification at rest condition is described in Table 10.

  36
 
 
 
WHO - GMP US (usual) ABNT NBR ISO 14644-1 CE – GMP
Grade A Class 100 ISO 4.8* Grade A
Grade B Class 100 ISO 5 Grade B
Grade C Class 10,000 ISO 7 Grade C
Grade D Class 100,000 ISO 8 Grade D
 

Table 10 Comparison between the different systems of classification of clean areas (considering
particles of 0.5 µm or more) at rest (Source: EU Guidelines for Good Manufacturing Practice
Medicinal Products for Human and Veterinary Use, Annex 1 - Manufacture of Sterile Medicinal
Products. November 2008).
 
8.2.3 Microbial Classification of Clean Areas
 
Microorganism monitoring for this purpose should be accomplished in conjunction with the counting of non
viable particles at intervals not exceeding 12 months. 6-month intervals may be required for requalification
of grade A and B areas when frequent detection of microorganisms occurs.
Monitoring of microorganisms must be carried out both under rest conditions (static) and in operation
(dynamic) during the classification studies. These conditions must be described in the documents generated
during the qualification areas. Microorganisms detected during environmental monitoring should also be
considered as part of the classification of a clean room.
 
 
 
8.3 Requalification
The testing required and the frequency of such tests for the purposes of periodic requalification of production
areas (clean or not rooms) must be assessed by a risk assessment. The need for requalification must also be
provided in a procedure when any change that would affect the performance of the system is performed.
If energy saving procedures were adopted, such as reduction of air flow during hours when no production
activities, they must be accompanied by some precautionary measures in order to ensure that the systems
return working under appropriate environmental conditions necessary for the process and the product. These
measures should be based on a risk assessment to ensure that there is no possibility of any negative impact
on the quality of products manufactured.
As a reference for requalification of clean areas the periodicities provided in ISO 14644-2 (Tables 11 and 12) 
may be adopted. It should be noted that tests considered optional by ISO (Table 12), depending on the risk
related, may be required from the point of view of the Good Manufacturing Practices (e.g., integrity testing
of the HEPA filters installed). Milder alternative frequencies, if used, must be based on a risk analysis.

  37
 
 
 
Schedule for required tests to demonstrate continued conformity
Test Parameter Class Maximum Interval Test Procedure
Particles counting test A, B 6 months ISO 14644 -1 Annex B
≤ ISO 5
C, D 12 months ISO 14644 -1 Annex B
> ISO 5
Pressure Differential All Classes 12 months ISO 14644 -3 Annex B.5
Air flow volume or Air All Classes 12 months ISO 14644 -3 Annex B.4
speed
 

Table 11 Frequency of testing required for requalification of clean areas, according to ISO 14644-2.
 
Schedule of optional tests (Annex A – ISO 14644-2)
Test Parameter Class Maximum Interval Test Procedure
Leakage test of HEPA All Classes 24 Months ISO 14644-3 Annex B.6
filters
Leakages/Contention All Classes 24 Months ISO 14644-3 Annex B.14
Recovery All Classes 24 Months ISO 14644-3 Annex B.13
Viewing of air flow All Classes 24 Months ISO 14644-3 Annex B.7
(smoke test)
 

Table 12 Frequency of optional testing for requalification of clean areas, according to ISO 14644-2.

  38
 
 
 

PART 2
Environmental Monitoring in Clean Areas
 
 
 
1. INTRODUCTION
 
Although not considered in this Quality Guide, the need to conduct environmental monitoring of areas used
in the production of non-sterile drugs must be critically considered. The main objective must be to assess the
microbiological profile of these areas, essentially considering the susceptibility of contamination of
medicines manufactured in them. Therefore, based on a risk analysis the nature, timing and extent of
monitoring must be defined and documented.
Quality is built into a product when the process, facilities and equipment used are designed to minimize or
eliminate the potential risks of contamination. Production modern approaches also include a systematic
evaluation of potential vulnerabilities of the process and how the routine activities can affect each other.
An environmental monitoring (EM) carefully planned and executed provides greater assurance of quality of a
product, especially when it is manufactured by aseptic processing. However, the evaluation of data for
environmental control is just one of a number of measures used to indicate the state of the control of a
manufacturing process. Note that environmental monitoring is not a direct sterility measure of products due
to inherent variability of environmental monitoring methods and, more importantly, the lack of direct
correlation between the specific numerical levels of environmental monitoring and sterility of a lot.
The EM is an important tool for evaluating the effectiveness of contamination control measures to identify
specific threats for the quality and safety of products manufactured. Therefore, the results of environmental
monitoring must invariably be considered for deciding whether a batch can be released.
The MA provides microbial profile data existing in clean areas and data allowing identify new trends of
microbial counts and growth of a microflora inside clean rooms and controlled environments. The results
obtained with the EM provide information about the physical construction of the room, HVAC system
performance, procedures of dressing and cleaning operators, equipment and cleaning operations.
The monitoring of non viable total particle counts in clean environments, even with the use of electronic
instrumentation that generate continuous records (online monitoring), does not give information about the
microbiological contents of the environment. The microorganisms carried by the air are not freely in
suspension or individually as cells, but often are associated with particles from 10 to 20 µm. Particle counts
as well microbial counts in controlled environments vary according to the local sampling and the activities
that are being carried out during sampling.

  39
 
 
 
Therefore, monitoring of non-viable and viable particles is an important function of control because both are
important to achieve the requirements of particulates and sterility in injection products.
Microbiological monitoring programs in controlled environments must evaluate the effectiveness of the
cleaning and sanitizing procedures and people who perform them. Microbiological monitoring regardless of
the degree of sophistication is not able to identify and quantify all microbial contaminants present in these
controlled environments. However, routine monitoring must provide sufficient information to assess that the
controlled environment is operating in a proper control state.
 
 
 
 
 

2. MONITORING OF NON VIABLE PARTICLES

 
Procedures for sampling non-viable particles can be performed by members of the Department of Quality
Control, Quality Assurance, Production or others with training and expertise for such activity.
In clean environments, non-viable particles of defined diameters (0.5 and 5 mM) must be measured by
appropriate and duly calibrated instruments at defined intervals according to their type and use. In systems
with unidirectional flow pattern, the particle measuring equipment must always be equipped with isokinetic
probes in order to ensure accurate counting.
Samples must be collected in positions approximate to the height of working positions, with 30 cm from
operator being a value that may be considered as a reference.
When portable equipment for counting particles is transported between different areas the effectiveness of
measures taken to avoid cross contamination must be demonstrated. Particularly segregated areas must have
dedicated particle counters.
 
 
 
2.1 Procedure for Monitoring Non Viable Particles
For each clean room, an analysis of the layout, materials, equipment and the people present, types of
activities, as well as potential risks for the product must be performed. From this analysis, a routine sampling
plan based on risk must be established. A procedure must be developed detailing the frequency, sampling
points and volumes sampled. This procedure must also have a schematic drawing of the room showing the
sampling places, and a justification of their choices must be clearly documented. Risk assessments must be
kept updated. In new areas, facilities renovated or after interruptions of the plant, the additional sampling is
recommended to assess the possible locations where contamination is recurrent.
Sampling points must be representative of all areas of the clean room, but the places where the product is
placed at increased risk of contamination must be mandatorily included during routine monitoring. As an
example, in rooms where open operations are conducted in a unidirectional air flow cabin, the cabin must be
routinely sampled; but the surrounding area may be sampled at a lower frequency or at multiple locations on
a rotating basis.

  40
 
 
 
Areas of low risk (such as those distant of the product, material, or unidirectional air flows) must be sampled
occasionally to provide confidence that low levels of contamination are maintained in such areas. Sampling
plans in which a central point in a room is chosen and samples are taken only at this point are not an
appropriate approach for a correct environmental monitoring.
In a unidirectional air system grade A, in which multiple operators perform different activities in the product
exposed (where multiple sources of particulates or aerosols are present), multiple points and high sampling
frequency may be required. In biosafety cabins where operations obstruct the flow or create turbulence, if the
product is exposed, additional sampling may also be required. Regulate limits established for biosafety
cabins are equivalent to the unidirectional air flow systems.
Sampling probes must be allocated in positions that correspond to the height where the activities are
performed, so that the probability of particle detection is maximized. Whenever possible, the probes must be
allocated in positions that enable the collection of air that had passed through the product, and when it is not
possible, the probes must be allocated in the area around the product and not in places where clean air flows
directly from the HEPA filter.
If accepted that it is not always possible to demonstrate low levels of particles ≥ 5.0 mM at the filling point
when operations are ongoing due to the generation of particles or droplets from the product itself.
Sampling in static areas is not usually performed as a mean of routine environmental monitoring. However,
when carried out, the number of samples must be determined in accordance with risk analysis. In these cases
it is recommended to sample at least one point where the product is placed at risk and a point in the area that
surrounds it.
Samples in areas where operations are to be routinely collected and also in situations considered as “worst
case” for contamination. Therefore, it must be includes steps in which the products are in open containers in
direct contact with the environment, or when operators are prepared to open containers containing the
product. To monitor the “worst case”, the maximal number of persons usually involved in operations must be
present once the reduced quantity of personnel reduces the release of particles and microorganisms, and can
invalidate the data sampling.
Manufacturing areas often have maximum and minimum operating schedules depending on the number of
lots produced per time unit. Peak of operations may affect the performance of materials, significantly
increase the flow of personnel and material and affect the environmental control systems. When a facility
operates under different levels of demand, the environmental monitoring program must be established in
accordance with a risk assessment, taking into account the maximum and minimum levels of operation.
The sampling points must be close enough to the handling places of the product, so that the results obtained
are representative of the cleanness of the environment without unduly obstructing the operations. When the
sampling points chosen do not represent the “worst case” for monitoring due to obstruction of first choice
places, or when the sampling activity put products at greater risk of contamination, must be a documented
assessment describing the criteria used for defining the sampling plan adopted.
Isolators or other closed areas of work must be monitored according to the risks involved. The work areas
that do not remain closed (sealed) during the whole period of operations, such as for example, when panels
are opened for adjustment of equipment or materials must have a monitoring program that includes the
period of performing such incursions.

  41
 
 
 

The sampling frequency must be based on a risk analysis and be properly established in procedures.
Operations in which the product is more subject to contamination require a higher sampling frequency. The
monitoring of the areas in which the defined values are exceeded in technical regulations must be more
frequent than the monitoring of areas in which results regularly meet the limits. The recommended
frequencies of monitoring for non viable particles in different areas of operation are described in Table 4.
 
Routine sampling of non viable particles (in
Classification
operation)
Grade A (aseptic filling operations) Throughout the duration of operation
Grade B Days when the operations are performed
Grade C Weekly
Grade D Not required
Unidirectional air flow stations in areas grade B Days when the operations are performed
Unidirectional air flow stations in areas grade C Weekly
Unidirectional air flow stations in areas grade D Monthly
Unidirectional air flow stations in unclassified areas Periodic requalification are sufficient
 

Table 4 Frequency of particles monitoring during routine operations.

The particle monitoring in areas grade A must be performed throughout the duration of the critical steps of
the process, including equipment assembly, except when justified due to the risk of damages to the particle
counter or due to contaminants generated in the process representing risk, as for example, living organisms
and radiological risks. In such cases, monitoring must be conducted during routine operation of equipment
adjustments, before exposure to the risk.
 
 
 
2.2 Analysis of routine monitoring data of non viable particles
Each result must be separately  analyzed, and the average must not be made by combining results from
multiple sampling points, or one single location sampled at different times. Any value above the regulate
limit should be seen as an excursion and an investigation must be conducted.
Events that generate particles for a limited time, such as brief procedures, equipment failures or spill
materials affect some units on a lot, but not all. In these situations, the results obtained from environmental
monitoring samples collected close to the workspace may be more relevant than the collections in more
distant points. Similarly, samples collected during certain activities may be more relevant than those
collected when these activities are not in progress. Therefore, the results must be analyzed taking into
account such factors, and samples that reflect the higher risk for purity, quality, safety and efficacy of the
product must be weighted more than others.

  42
 3. ENVIRONMENTAL MONITORING OF
MICROORGANISMS DURING OPERATIONS
 
GMP guides published by Anvisa often carry a set of four techniques employed for monitoring
microorganisms in clean rooms, namely: volumetric air sampling, sedimentation plates, contact plates and
samples for the evaluation of contaminants present in the surface of the glove fingers of operators (Table 7).
However, it is not possible that all tests are performed during all monitoring activities in each type of clean
room and operation room.
In Grade A area, volumetric sampling, the exposure of sedimentation plates and fingerprint collection from
operators’ gloves must be performed on each shift of operations. According to the criteria set by the
company, multiple samples can be collected, for example, at the beginning, middle and end of operations.
These additional data assist in investigations when some result is found outside the specified limits.
Environmental monitoring must be conducted in accordance with a formal program, prepared by risk
analysis. In the sampling plan must be included the places where the product is exposed and times when
there are product handling activities by operators. Points close to the places where products are exposed and
where materials are handled or surfaces that will come in contact with the product. It must also be performed
a risk analysis in order to prove the right choice of locations and frequency of monitoring. It is acceptable a
program that defines the performance of routine sampling of areas with high working demand (i.e.,
unidirectional) and sampling only in rotation of those little used.
Permission to access the high-risk areas for implementation the environmental monitoring and related
procedures must be governed by a risk-benefit ratio. Forms of sampling must be developed in these areas that
minimize the risk for the product, such as the use of sampling ports during aseptic operations instead of
placing sampling devices in an area of unidirectional air flow. When the activities performed turn impossible
the sampling activity or if the risks for the product are considered unacceptable, the decision to not perform
the sampling should be based on evidence, which must be properly documented and approved by the
responsible persons.
 
 
3.1 Growth Promotion Testing of the Culture Media
Bacteria and fungi are present in almost all types of environments and surfaces, and growth is influenced by
environmental conditions. However, unless these microorganisms can be collected survive and grow into
visible colonies, they will not be detected. Particularly in clean rooms, where the contamination regulatory
limits are low, the use of insensitive detection methods or that recovers only a small percentage of
contaminating microorganisms can make environmental monitoring procedures ineffective. Therefore,
testing for fertility evaluation of culture media, also called tests of growth promotion, is of particular
importance in a program of environmental monitoring of microbial contaminants.
There should be a formal program describing the tests of growth promotion. This program must include the
evaluation of the culture media used for quality control testing for release of the product and also for
environmental monitoring. Tests for growth promotion must be carried out with predefined microorganism
strains, from sources considered reliable. Microorganisms to be used in fertility tests of different culture
media are defined in the official compendium, but the test must not be restricted to these microorganisms.

  43
 
Microorganism strains recurrently identified in environmental monitoring (assuming they are present) must
be included. This list must represent a reasonable range of microorganisms that may be encountered in
production environments (such as rods and Gram-positive  cocci, filamentous fungi or yeasts and gram-
negative rods).
Agar plates used for environmental monitoring must be tested for their fertility. The growth promoting tests
must be performed on all batches prepared (individually by sterilization cycle). The plates must be tested for
its ability to promote the growth of low numbers of bacteria and fungi (e.g., <100 CFU of Staphylococcus
aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans and Aspergillus brasiliensis) when
incubated at standard temperature during predetermined times. The proper recovery of small numbers of
microorganisms must be considered a prerequisite for the success of the procedure for environmental
monitoring. When amounts smaller than 50% of microorganisms are detected for each species tested, the
procedures must be carefully examined and reviewed.
In addition to the reference strains of bacteria and fungi used for growth promotion tests and sterility testing
methods, further studies must demonstrate that strains of bacteria and fungi encountered in the environment
production (such as environmental isolates that may become resistant to disinfection processes and
production strains) are detectable by the method used.
Expiration dates must be assigned to culture media batches and culture media used for monitoring of surfaces
must include, if necessary, neutralizing agents of disinfection agents. In certain situations, such as in the
production of antibiotics may be required neutralizing agents with activity of the formulation components of
the product.
In areas with high air flow with strong turbulence with increased temperature or low humidity conditions,
sedimentation plates used may dry or otherwise modify its properties (for example, changes in the dissolved
gases, pH, or deterioration of certain components of culture media) so that the fertility of the medium is
compromised. Validation studies must be performed to determine how long a sedimentation plate can be left
under the specific conditions of use and still retain full growth promotion for the microorganisms of interest.
Care should be taken during the validation studies for the dry plates are not rehydrated with inoculum
volume (≤ 0.1 ml is a maximum recommended).
 
 
 
3.2 Volumetric Sampling of the Air
Volumetric samples can quantify bacteria and fungi in suspension in the air surrounding the exposed product.
Active sampling can detect active homogeneous suspensions of microorganisms in the air, but it is not a
reliable measure of sporadic contamination that occurs during operation.
Commercially, many types of air samplers for quantification of microorganisms are  available. The rules
issued by ANVISA not specify which equipment should be used, but determines, even indirectly, that the
chosen instrument demonstrably meets the current standards of detection and sensitivity.
The sampling points and the sampling frequency must be chosen based on a risk analysis. Although the
sampling procedures can, by themselves, represent a threat to the process, the process closer samples are
collected and longer the sampling time, the sample will be more representative of the production
environment.

  44
 
 
 
The volume of the sample, and consequently the time spent on sampling requires a balance between the need
of a representative sample of the process (where large samples must be collected over long periods of time)
and sensitivity (suction forces required for large volumes of air can dry plates containing culture medium or
decrease the viability of microorganisms) and can become a threat to the process itself. In general, the
sampling instrument manufacturer recommends sample sizes, and these recommendations should be
considered in the design of sampling strategies. Typically, sample sizes greater than 1 m3 must be collected
for each measurement. However, if this sample size results in a number of countless colonies small volumes
can be used to monitor Grade C areas unless duly justified.
The design, including among other factors the sampling size and speed of the air inlet, and the validation of
sampling should cover the volumetric efficiency of the device to capture the reference microorganisms, as
well as those of the microbial flora of interest, such as production  strains, isolated from environmental
monitoring, or endemic microorganisms that may infect operators.
The drying effect of the capture plate during sampling and transport to the microbiology laboratory must be
determined by a validation study. The times for transport and storage of the plates must be established to
ensure that the microorganisms remain viable until transfer to a suitable environment for growth promotion
(incubators).
The risks involved in the transport of potentially pathogenic environmental or production microorganisms of
the production area from other areas, and their growth and analysis in the microbiology laboratory must be
evaluated. Whenever possible, you should give preference to the use of instruments that can be sterilized or
disinfected.
 
 
 
3.3 Monitoring with Sedimentation Plates
Sedimentation plates can detect bacteria and fungi which solidify in the air column above the plate. Although
the sensitivity of the technique depends on the size of the plate, the deposition rate of microorganisms, and
growth-promoting properties of the chosen plate, the sedimentation plates are the only method that provides
continuous monitoring of microorganisms in a productive area.
Sedimentation plates must be allocated in high risk of contamination areas of the product. They must be
placed as close as possible to where the activities are performed, but without causing obstruction of the
activities or contamination by plates themselves.
Measurements with sedimentation plates must be made during periods of increased activity, or when
material’s aerosol may be generated. When drying the plates can occur, the plates must be replaced at
intervals of less than 4 hours. The exposure time of individual plates must be assessed according to data
obtained in the validation studies.
In the case where sporadic operations or with short duration are performed (such as the combination of
sterile solutions or assembly of parts of equipment connections in a system of unidirectional airflow), the
total exposure time of the plates can be reduced to correspond to the total time of operations.

  45
 
 
 

3.4 Sampling of Microorganisms on Surfaces

 
3.4.1 Contact Plates
 
The contact plates for surfaces of productive areas, equipment, and fingers of gloves operators are used to
detect contamination by microorganisms in the vicinity of the working area. The sampling of operator
becomes even more relevant when manual operations are performed, and in these cases, additional tests for
the detection of contaminations in other surfaces of the uniform may be needed.
Contact plates must be used for detecting microorganisms on surfaces that may lead to contamination of the
product. Therefore, it should be designed a sampling system based on a risk assessment considering the type
of activity performed to monitor relevant surfaces where contaminant microorganisms can be found. These
surfaces can include working surfaces, equipment, walls and ceilings of the unidirectional air flow systems.
When there is the possibility of spilling materials that may contaminate the floor, specific points of the floor
must also be included in the sampling program. When operators work in close proximity to the product
exposed, such as an open flow cabin, surface samples of uniforms, gloves, masks or other representative
positions of the operator must be collected. Since duly justified, it is not necessary that all sampling points
are always tested and a program of random or rotating sampling may be defined.
Surface samples must be collected after the completion of production activities or so that there is no
contamination of the clean areas by monitoring activity. Samples must be collected before cleaning the area.
When frequent sanitizing is performed (for example, by spraying with ethanol solutions), the samples must
be collected before sanitizing procedures in order to maximize the probability that the microorganisms are
detected. Samples collected on still wet surfaces with cleaning solutions must not be considered valid.
Recovery of microorganisms on contact plates must be validated, with more than 50% of the tested
microorganisms must be retrieved during these studies.
 
3.4.2 Swabs
 
Swabs or other adsorbent materials moistened with sterile water or other diluents can be used for sampling
uneven or difficult to access surfaces, such as equipment, filling needles, pipes and corners. They are also
useful for the sampling of large areas after cleaning or disinfection  procedures. The recovery of
microorganisms from smears must be validated, including the sampling method chosen, the suitability of the
wetting liquid from the swab, and the transfer of microorganisms to the growth medium. The validation
studies must demonstrate a recovery exceeding 50% of each of the microorganisms strains used.

  46
 
 
 
It is noteworthy that there is no regulatory limits established for swab samples, but any detection of
microorganisms using this method must be investigated as part of the batch release procedure. The liquids
used for recovery of microorganisms must contain neutralizing disinfectants when required.
 
3.4.3 Fingerprinting of operators’ gloves
 
The fingertips of the operators are the most likely areas to come in contact with microbial contamination
existing on work surfaces, materials, or even the operator. This contamination can be transferred to the
products and therefore surface samples of the five fingers of operators’ gloves must be collected to control
this possibility. Sampling must be performed before the routine sanitization of gloves with alcohol or
exchange of outer gloves (in cases where they are worn by two operators).
 
 
 
 
 
 
4. FREQUENCY OF ROUTINE MONITORING OF
MICROORGANISMS
 
When an area is not used for short periods of time, such as on weekends, environmental monitoring may be
suspended. However, before resuming work in the area after long break (weeks or months); intensive
sampling should be carried out to ensure that the area still meets the cleaning requirements previously
established.
After periodic plant shutdowns, maintenance of the HVAC system, or significant changes in function of the
equipment or procedures, a short series of repeated sampling should be performed to ensure that the area still
corresponds fully to the previously defined classification.
Frequencies for routine monitoring of microbial contaminants must be established according to the
recommendations found in Table 5. Companies can use higher or lower frequencies, when warranted by the
results of monitoring, except in classes A and B. The static routine monitoring (in areas without operations)
is recommended to ensure that cleanliness levels are maintained when the area is not in use for short periods
of time or to check the effectiveness of cleaning procedures before operations.

  47
 
 
 
   
  Plates Contact plates (55
   
  Air Sample (90 mm diameter) mm diameter) 5 fingers glove
Classification
cfu/m3 cfu/4 hours fingerprint
cfu/plate
cfu/glove
Grade A (aseptic Once per shift Throughout the Once per shift Once per shift
filling operations) time that
production
activity is
performed
Grade B Daily Daily Daily Daily
Grade C Weekly Weekly Weekly N/A
Grade D Monthly Monthly N/A N/A
Unidirectional air Once per shift Once per shift Once per shift Once per shift
flow stations in
areas grade B
Unidirectional air Weekly Weekly Weekly Weekly
flow stations in
areas grade C
Unidirectional air Monthly Monthly Monthly N/A
flow stations in
areas grade D
 

Table 5 Routine monitoring frequencies of microorganisms (in operation).

The practice of volumetric air sampling at the beginning, middle and end of the filling operations gives better
environmental monitoring and facilitates investigations related to the batch release. Therefore, depending on
the type of activities, this strategy should be adopted.
When grades A and B areas used for aseptic filling are kept constantly in operation, and the sampling
operation is performed per shift, and there are data showing that the area operates consistently in a state of
control, the manufacturer may at its discretion change the frequency of static control (at rest areas) reducing
it to monthly or even eliminate it.
 
 
 
5. LABORATORY TESTING OF ENVIRONMENTAL
SAMPLES
 
Microorganisms of interest (e.g., production strains; strains previously identified during environmental
monitoring; resistant or sensitive  strains, or infections predictable in operators due to local endemics) may
require special conditions for collection and growth.
The environmental control samples must be incubated for at least two temperatures in order to detect bacteria
and fungi. In practice, the time of 3 to 5 days of incubation at 20 to 25°C, followed by incubation at 30 to
35°C for an additional 2 to 3 days is enough for the detection of most bacteria and fungi. The method chosen
by each manufacturer must be carefully validated and standardized.

  48
 
 
 
Alternative methods are acceptable when high recoveries (> 90%) of microorganisms of interest can be
demonstrated consistently.
When mycobacteria, mycoplasmas, anaerobic bacteria and thermophilic bacteria and bacteria or other
microorganisms which require growth specific conditions that are of interest, they are likely to be present in
the areas, specific methods must be developed to detect such microorganisms during the environmental
monitoring  program. The strategy for sampling and detection must take into account the need for specific
methods (e.g., nutrients, specific growth temperature, balance of gases, humidity, anaerobic conditions,
longer incubation times etc.).
When bacteria or fungi are detected in critical areas in values above the level of alert or action limits, your
identity must be verified to the species level. When this is impossible, a justification must be documented.
Recurring detections of the same microorganism indicates that a constant source of contamination is present.
When the detection of in-house flora becomes recurring due to a population of strains resistant to antibiotics
or cleaning agents, corrective actions should be taken. Sporicidal agents must be used to eliminate spore
forming species.
Unexpected or exotic species of microorganisms (non-native in local country) may come from contaminated
raw materials, indicating a change of supplier or supplies, or operators recently exposed to a not endemic
disease to the manufacturing plant. In such cases, corrective actions should be taken usually more
immediately.
Average of the results achieved in environmental monitoring at different times or locations must be avoided.
When the procedure is not identical over time and events occur that can produce aerosol particles of
microorganisms (vortex or shaking liquids, opening of pressurized containers, transfer of liquids, machinery
faults, material spills, breakage of glass bottles or syringes, incursions of operators in clean areas, coughing
or sneezing operators etc.), “dilution” of the true value with other values is unacceptable. Likewise, if
multiple locations within a working area are selected, but only one is likely to be representative of the risk
contamination (for example, near the hands of operators, the flow of air around places of performing
activities, etc.), the use of averages “dilute” the relevant result and EM does not provide any information
about the potential risk to the product.
 
 
 
 
 
 
6. INVESTIGATIONS AND CORRECTIVE ACTIONS
AND PREVENTIVE ACTIONS (CAPA)
 
Pharmaceutical companies must have adequate procedures to investigate deviations. They must also have
implemented a program for the adoption of Corrective Actions and Preventive Actions (CAPA) resulting
from Deviation Investigations of nonconformities, rejection of product lots, complaints and recalls, non-
conformities detected in audits and regulatory inspections and also activities related to product quality
monitoring.
The correct functioning of the investigations and a CAPA system covering also environmental monitoring
activities is a critical component of a quality system.

  49
 
 
 
Values above the environmental monitoring limits of viable and non-viable particles must be treated as
excursions, peaks, deviations, or out of specification results. When values that exceed the regulatory limit
occurs, the environmental impact of production and batch release must be considered, especially for critical
areas classified as grade A and B.
Conducting thorough and impartial investigations to evaluate any possible impact on product quality, purity
or safety is an important indicator of the robustness of a quality system. In poor systems, pressures on
operators and managers for batch release can substitute any reasonable scientific concern about possible
contamination of a batch.
 
 
 
6.1 Alert and Action Limits for Environmental Monitoring
The maximum limits for viable and non-viable particles for clean rooms are set in GMP guides. On the other
hand, alert and action limits must be set by the manufacturer. Such limits should be set so that, when
exceeded, trigger activities that promote the system return to normal. The monitoring of clean areas have
variations in the non viable particles and microorganisms, which makes the historical changes over time the
best indicator of the cleanliness level. When there is a good working of the clean area, the variation will be
low, and out of specification values of GMP standards are often indicative of a new problem in the same
room or even problems in sampling method.
The alert limits must be defined by the manufacturer for its own benefit. Values of alert limits for particles or
microorganisms are values below the regulatory maximum, but must be sufficiently above the normal range
of contaminants results historically found. The answer to a value above the alert limit is often only a record
of the event that will serve as a basis for an analysis of potential trends, i.e., to check that the event is not part
of a set of values abnormally high.
The action limits are established values below the maximum regulatory limits, and are usually defined as
values above the alarm limits. Occasionally, alert and action limits can be set as the same value. When the
sensitivity of the methods used to detect viable and non-viable contaminants is high, and the clean room has
demonstrated to operate consistently in a state of control, action limits can be the same set in technical
regulations as maximum contamination limits. Once exceeded the limits of action, actions should be
triggered. Therefore, it is important that the values are carefully chosen to avoid that the number of actions
required is excessive. Depending on the situation, these actions triggered should include items that may be
responsible for high levels of environmental contamination, such as:
a. Investigation of possible changes in procedures or equipment (including HVAC systems, water and other
utilities), which may be responsible for high contamination levels;
b. Review of the operations performed and the behavior of operators in the affected area;
c. Trend analysis of counts of viable and non-viable contaminants over the time, mainly due to the
seasonality of water supply or raw materials and endemic diseases that can infect operators;
d. Repeat or increase the frequency of monitoring;
e. Increase the number of monitoring points;

  50
 
 
f. Functionality verifications and maintenance history of equipment, including an assessment if the
processes are still operating within the limits defined in the project and used in validation studies;
g. Identification of detected microorganisms, with follow-up of the investigation on their possible origin
(e.g., the emergence of resistant strains, or infection of operators with a transmissible microorganism);
h. Alert operators for the problem and, when necessary, conduct refresher trainings.
i. Requalification of equipment or validation of processes using relevant process parameters.
 
 
6.2 Deviation Investigations
A procedure should be developed that details how to investigate and react to an excursion above the limits
for environmental control. Events identified should be investigated and the conclusions of the investigation
recorded. The conclusion of the investigation must be impartial and scientifically based and final disposal of
the lot or lots involved must be made considering the possible impact on the purity, quality, safety and
efficacy of the product.
The batch release by the Quality Assurance Department should be suspended until all investigations on
excursions in environmental monitoring are successfully completed. These lots only must be released when
the investigation results indicate unequivocally that there is no unacceptable risk to the patient.
The impact of an excursion in environmental monitoring should be evaluated for all lots produced in the area
during the time period in which such deviation may have persisted. A common error in environmental
monitoring programs is the adoption of a very low frequency of sampling, and when excursions are detected,
it is ignored the fact that the area could have been out of the minimum cleaning conditions for weeks or even
months. In practice, if failures are detected in the filters of the HVAC system or the contamination of the
area by a pathogenic microorganism, all batches produced in this area since the last result (or results) proved
suitable must be considered suspicious. The need to gather lots released in the period must be especially
considered in the investigation. Failure to meet these requirements is a serious failure of a Quality Assurance 
System.
Excursions in environmental monitoring and significant deviations in operating parameters of clean areas
must generally lead to rejection(s) of the lot(s) affected. Similarly, a piece of the equipment that does not
meet specifications (e.g., HVAC systems, autoclaves, fermenters, or freeze-dryers); can cause excursions in
environmental monitoring, which usually lead to rejection of lot(s) affected.
In the absence of other aggravating circumstances, trends in results are an important tool to determine if an
event is indicative of a serious problem or not. When the area is under continuous control for a long period of
time, and a single and unexpected episode of low level of contamination occurs, this can be used for the
decision to release the batch. Conversely, when environmental excursions have occurred consistently over
time, or when a set of events is under investigation, the concern with the acceptance of the lot must be
higher. For the investigation of microbial contaminants, identification of the microorganism may similarly be
used to determine if the event is a result of a continuous contamination of the installation by one or more
species or it represents the introduction of a new and potentially more dangerous species through unknown
mechanisms. Trends over time must be documented and presented in such a way that “normal” and
“abnormal” values are readily identified during the analysis.

  51
 
 
 

6.3 Corrective Actions and Preventive Actions

Corrective Actions and Preventive Actions (CAPA) are the logical outcome of investigations that occur after
excursions above the action and alert limits  adopted. Forcefully, effective programs of corrective and
preventive actions significantly reduce the recurrence of excursions, resulting in a higher efficiency of the
production process.
Departments that have interfaces in clean areas such as Engineering, Production, Quality Control and Quality
Assurance, must participate in the design and implementation of CAPA.
If large amounts of particles are generated by a given operation in the production process, such an operation
can be modified or secreted to minimize the problem. If microorganisms are detected in the workspace of a
particular operator, hygiene, work practices and training records of the operator must be evaluated.
When Deviation Investigations are inconclusive (root causes are not identified), greater efforts should be
made to improve the operation of the clean room and prevent the occurrence of new events. The
environmental monitoring should then be increased to cover more areas and have higher sampling frequency,
in order to try to find the source of contamination and to obtain data which can confirm that the clean area
actually operates according to specifications.
Independent analyzes of operations, operator behavior, material flows and operators must be considered. The
effectiveness of the cleaning area procedures and other procedures can be reviewed and reassessed.
Analyses after the implementation of corrective or preventive actions adopted are necessary to ensure their
effectiveness. This activity must be clearly defined in procedures and records must be documented.
After approval of the implementation of corrective and preventive actions, deadlines must be established for
completion of the required improvements, and the persons or departments nominally responsible must be
identified. The reasons for delays or adoption of excessively long periods of time in implementing the
required improvements must be documented.

  52
 
 
 

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