Regulatory inspections of sterile facilities by Madhu Saghee,

Tim Sandle and

– the focal points. Palash Das

Part 2: Airflow visualisation

Introduction
Air is of fundamental importance to
Air flow visualisation is one of the less clear-cut activities
cleanrooms and clean air devices, either as a
required of aseptic manufacturers. Regulations refer to
contamination source (microorganisms
such studies, yet little in the way of guidance is
carried in the airstream) or as a control
provided. However, judging by warning letters and
measure to minimise contamination (through
other regulatory trends, many industries are running
the supply of clean air). Therefore,
behind in terms of compliance expectations. The
controlling a cleanroom requires careful
regulator findings suggest that evaluation, presentation
attention to air filtration and airflow.
and demonstration of airflow is not well understood. In
Assessing airflow is undertaken through
this article, we will discuss the main regulatory findings, airflow visualisation studies (or what are
as evidenced from recent USA Food and Drug colloquially termed ‘smoke studies’). The
Administration (FDA) 483s and assess these for the main purpose of flow visualisation is to confirm
areas for concern and offer best practices for addressing the smoothness, flow patterns and other
the regulatory observations. spatial and temporal characteristics of
airflow in an installation. For this, the airflow
General expectations is examined through airflow visualisation
Air pattern or ‘smoke studies’ are designed to mapping whereby smoke is generated, and
demonstrate unidirectional airflow (what used to be the behaviour of the smoke is studied and
called, inaccurately, ‘laminarity’) in relation to European then captured by a video camera1.
Union (EU)/World Health Organization (WHO) good
manufacturing practice (GMP) Grade A/International
● Videotape or digital recording mechanisms have
Organization for Standardization (ISO) 14644 Class 5
been found to be useful in assessing airflow
areas. The required airflow appears as a sweeping
initially as well as facilitating evaluation of
action over and away from exposed product
subsequent equipment configuration changes.
components (such as vials or stoppers) under dynamic
conditions. This requirement also extends to activities
It is important that all airflow studies are conducted in
such as loading lyophilisers. Such studies should be
operation (dynamic state). This is because even
periodically conducted2.
successfully qualified systems can be compromised by
Studies are also expected in relation to the areas
poor personnel, operational or maintenance practices.
housing EU/WHO GMP Grade A/ISO 14644 Class 5 areas,
Smoke studies and multi-location particulate data are
such as EU/WHO GMP Grade B/ISO 14644 Class 7 (in
vital when performing qualification studies to assess
operation) cleanrooms as per conventional aseptic
whether proper particulate control dynamics have been
processing (or lower specification area where isolators
achieved throughout the critical area.
are used). In these areas, the airflow is expected to be
turbulent, and the main concern is to ensure that air does
not move into the EU/WHO GMP Grade A/ISO 14644 Class
Best practices to be followed during
conducting study
5 area during activities like machine set-up or where an
When conducting airflow studies, there are several
intervention is required to be performed3. Other
factors to observe.
expectations with airflow studies include the following.
● All open-door interventions and manipulations
● Such studies should be well-documented with
through glove ports must be identified. Personnel
written conclusions.
should minimise interventions into the critical
● Major issues of turbulent airflow in EU/WHO GMP
zones. Such interventions can adversely disrupt
Grade A/ISO 14644 Class 5 areas need to be the unidirectional and should therefore be
addressed. designed to minimise both the extent and
● Data should be used for selecting appropriate frequency of occurrence. Once identified and
environmental monitoring locations, especially corrected, each intervention must be practiced as
with the positioning of settle plates. part of an airflow study.

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● Assess that the equipment does not obstruct the ● 211.63 Equipment used in the manufacture,
airflow, and, in critical areas, its design should not processing, packing or holding of a drug product
disturb unidirectional airflow. shall be of appropriate design, adequate size, and
● Rapid movements can create unacceptable suitably located to facilitate operations for its
turbulence in a critical area. Such movements intended use and for its cleaning and maintenance.
disrupt the unidirectional airflow, presenting a ● 21 CFR Part 211.113(b) is generally cited when the
challenge beyond intended cleanroom design and video shows operator activities are contributing
control parameters. Movements should be to turbulence, or the true air flow cannot be
assessed. demonstrated based on the poor placement of
● The principle of slow, careful movement should the smoke source and camera angle.
be followed throughout the cleanroom. ● 21 CFR Part 211.63 is generally cited when the

● When undertaking activities in Grade A/ISO Class video shows the line design/equipment
5 areas, personnel should keep their entire body placements are contributing factors to turbulence.
out of the path of unidirectional airflow.
Unidirectional airflow design is used to protect FDA requirements are also outlined in the FDA guidance
sterile equipment surfaces, container closures and document “Sterile Drug Products Produced by Aseptic
product. Disruption of the path of unidirectional Processing – Current Good Manufacturing Practice”
flow air in the critical area can pose a risk to (September 2004)4. Here the text states the following.
product sterility.
● Proper design and control prevents turbulence
● Manufacturers should be aware of a device's air
and stagnant air in the critical area. Once relevant
monitoring capabilities, and the air sampler
parameters are established, it is crucial that
should be evaluated for its suitability for use in
airflow patterns be evaluated for turbulence or
an aseptic environment based on potential
eddy currents that can act as a channel or
disruption of unidirectional airflow. In addition, it
reservoir for air contaminants (e.g. from an
is best practice that air samplers are assessed for
adjoining lower classified area). In situ air pattern
collection efficiency, cleanability, and ability to be analysis should be conducted at the critical area to
sterilised. Furthermore, exposure conditions demonstrate unidirectional airflow and sweeping
should preclude desiccation (e.g. caused by action over and away from the product under
lengthy sampling periods and/or high airflows), dynamic conditions. The studies should be well
which inhibits recovery of microorganisms. documented with written conclusions, and include
evaluation of the impact of aseptic manipulations
Regulatory requirements (e.g. interventions) and equipment design.
This next section assesses the regulatory expectations Videotape or other recording mechanisms have
for conducting airflow studies. Each of these should be been found to be useful aides in assessing airflow
assessed and included, as appropriate, as part of best initially as well as facilitating evaluation of
practices. subsequent equipment configuration changes. It is
important to note that even successfully qualified
USA FDA expectation systems can be compromised by poor operational,
The USA FDA describes the following requirements for maintenance or personnel practices4.
airflow control and airflow patterns in the Code of
Federal Regulations (CFRs). WHO GMP For Sterile Pharmaceutical Products
Working Document QAS/09.295 Rev.1
● 211.113(b) Appropriate written procedures,
The requirement in the WHO GMP Working Document5
designed to prevent microbiological is as follows.
contamination of drug products purporting to be
sterile, shall be established and followed. Such ● “Grade A: The uniformity and effectiveness of the
procedures shall include validation of any unidirectional flow shall be demonstrated by
sterilization process. undertaking airflow visualization tests”5

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EU Guidelines to GMP Annex 1 ISO 14644

Airflow patterns are referenced in Annex 1 of the EU FDA’s Inspectional Observations (483s) on Air
Guidelines to GMP6, where the text states the following. Flow Pattern Visualization
As indicated above, there is some description in the
● “It should be demonstrated that air flow patterns various regulations (albeit sometimes lacking in detail).
do not present a contamination risk, e.g. care Despite this guidance, regulatory observations about
should be taken to ensure that air flows do not airflow control and the importance of conducting
distribute particles from a particle generating airflow studies features regularly in terms of regulatory
person, operation or machine to a zone of higher citations. This section provides some recent examples,
product risk”6 drawn from FDA warning letters. Alongside each of the
seven observations, we have provided some
The importance of airflow control and need to study
commentary.
airflow is afforded greater detail in the revised Annex 1,
where the current draft reads as follows. Observation 1: Smoke studies in ISO 5 hoods were not
conducted under dynamic conditions.
● “Grade A: The local zone for high risk operations,
e.g. filling zone, stopper bowls, open ampoules and Author comments: With this observation the company
vials, making aseptic connections. Normally, such have simply not adhered to the regulatory requirement
conditions are provided by a localised air flow for conducting airflow studies in a critical area. Not
protection, such as laminar air flow work stations or conducting studies places the operation at risk.
isolators. Unidirectional air flow systems should
provide a homogeneous air speed in a range of Observation 2: There has been no air flow pattern (i.e.
0.36–0.54m/s (guidance value), the point at which the smoke study) evaluation study performed to determine
air speed measurement is taken should be clearly the acceptability of the horizontal air flow, that is, the
justified in the protocol. During initial qualification air flow is not compromised (i.e. air turbulence/air
and requalification air speeds may be measured eddies) during the aseptic operations that are
either close to the terminal air filter face or at the performed in the ISO 5 area.
working height, Wherever the measurement is taken
it is important to note that the key objective is to Author comments: With this observation the regulatory
ensure that air visualization studies should correlate comments are more specific. This relates to a horizontal
with the airspeed measurement to demonstrate air airflow (which the regulator may have more questions
movement that supports protection of the product about compared with a vertical airflow); and the lack of
and open components with unidirectional air at the data showing what happens when operations are
working height, where high risk operations and undertaken in terms of the activity triggering
product and components are exposed. The turbulence.
maintenance of unidirectional airflow should be
demonstrated and validated across the whole of the Observation 3: There has been no air flow pattern
Grade A area. Entry into the Grade A area by evaluation to determine that the personnel activities
operators should be minimized by facility, process and manual transfer of materials between the ISO 8 and
and procedural design.”7 ISO 7 areas negatively affect the air movement and air
cascade.
Pharmaceutical Inspection Convention (PIC/S)
GMP Annex 1 Revision 2008 Interpretation of Author comments: With observation 3, the focus is on the
Most Important Changes for the Manufacture of cleanroom and how this may or may not be disrupted by
Sterile Medicinal Products – Recommendation the movement of personnel and how they undertake
January 2010 their tasks, and also on the transfer of materials. It would
appear that some kind of airlock or transfer site falls
● “Non-viable particles should be measured and are between two cleanrooms of a different classification. The
expected to meet Grade A requirements. Smoke interface between cleanrooms of different grades is
studies should be performed.”8 frequently an area of regulatory concern.

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Observation 4: Smoke studies have not been properly Further observations from warning letters issued in the
documented for the air flow patterns of the ISO 6 rooms past 2 years include the following.
or ISO 5 laminar air flow hoods used in the processing of
injectable products. 1. Dynamic conditions, or not reflective of the firm’s
processes.
Author comments: With observation 4 is would
appear that airflows have been conducted; however, – Smoke studies were not performed under
dynamic conditions to verify that operators and
the airflows have not been written up. While airflows
processing equipment do not alter or impede the
can be recorded, a report is still required so that a
unidirectional cascade of air from the HEPA filters
firm conclusion can be made as to the airflow
to the ISO 5 laminar flow benches where sterile
acceptability.
drug products are opened and manipulated, and
to the rest of the ISO 7 clean room.
Observation 5: The air flow pattern video does not
– No smoke study procedure was used to evaluate
present data to adequately assess the requested
your ISO 5 IV hood under dynamic conditions and
“downward sweeping air flow pattern” for the ISO 5
no additional smoke studies have been conducted
aseptic fill zone. The firm failed to evaluate the
since the IV Hood was installed in 2009 [Note
potential product impact of the turbulence air eddies
date of the inspection – August 2014].
observed in the middle of the ISO 5 hoods during
dynamic operations. – Smoke studies were not conducted for the ISO 5
hoods; they were only conducted in the ISO 7
Author comments: In observation 5, the regulator seems cleanroom with [redacted] present, which is not
to be disagreeing with the company about the air representative of routine aseptic operations with
movement. The company has determined that the [redacted] operators.
airflow is moving downwards, but the regulator – A review of The Critical Manufacturing Area
disagrees and then notes that the inadequate airflow Smoke Test, completed 05/21/2014, found that the
has not been adequately risk assessed. ISO 5 classified critical zones in each clean room
were not evaluated under dynamic conditions
Observation 6: Smoke study did not include an with compounding equipment and components in
evaluation of the personnel activities performed in the place.
adjacent ISO 5 hoods to determine that the personnel – Smoke studies are conducted during [redacted];
activities do not negatively affect air flow patterns however, these studies are handled in static
within ISO 5 hoods. conditions and do not show adequate coverage
of the ISO 5 area or the ISO 7/8 entryway and
Author comments: In observation 6, an airflow has been
pass through(s).
conducted but it is not suitably comprehensive, not
taking into account the full extent of personnel – The firm’s in situ air pattern analysis (smoke
activities on the air patterns. studies) was not conducted under dynamic
conditions, simulating routine production (i.e.
Observation 7: The smoke study does not demonstrate compounding equipment in place and operations
critical aseptic connections performed during the ongoing).
assembly of ISO 5 hoods used to fill sterile – Clean room certification and smoke studies for
pharmaceuticals. the ISO 7 Cleanroom were not performed during
the operation of the [redacted]. Smoke studies
Author comments: With this observation, an airflow were not performed under dynamic conditions to
study has been conducted but again it appears not to verify that operators, processing equipment, or
be suitably comprehensive, missing out one of the key activities of the ISO 7 clean room do not alter or
risk factors with aseptic processing – the sterile impede the unidirectionality of air from the HEPA
connection. Understanding whether air movement filters to the [redacted] ISO 5 laminar flow
presents a risk to the fluid path is critical for benches where products are aseptically processes.
maintaining contamination control. Smoke studies are not performed under dynamic

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conditions. The smoke study was not performed – The smoke studies performed in the ISO 5
under dynamic conditions to verify that the [redacted] area of SVP Line 1 are inadequate in
operator or activities in the ISO 7 cleanroom do that there is insufficient smoke to clearly show
not affect the unidirectional airflow from the unidirectional air flow, the dynamic portion of
HEPA filters in the ISO 5 hood where drug the smoke study at the [redacted] included an
products are produced. operator who did not move, and the study did
not include smoke near the window on the back
Author comments: As can be seen from the above, side of holes that were open to the ISO 8 room in
failure to perform airflow studies when the equipment order to perform [redacted].
is being used and with personnel present is a common
finding. Such dynamic conditions need to be simulations Author comments: Another common observation is with
(so that product is protected) are essential as airflow studies deemed to be inadequate. The reasons
undertaking airflow studies in the static state (‘at rest’) given here are more varied. Examples include failure to
is not reflective of the greatest level of potential air video record the activity; failure to film all elements of
disruption and hence risk to the product. the operation; not scanning the camera to show where
all of the air is moving to; and failure to capture every
2. Inadequate studies. type of intervention.
– The smoke studies were not recorded to Other areas that were not correct included practices
demonstrate laminar air flow during dynamic in airflow studies not being reflective of what is
conditions. happening in production (where the inspector may have
observed a process activity and then watched the
– No dynamic airflow pattern studies (smoke
airflow study video). With this point, it is very important
studies) have been performed in the [redacted]
to ensure that the airflow study is representative of
ISO 5 hoods inside your ISO 7 room where sterile
normal practice.
drug products are formulated and filled.
– Limited smoke was used and did not completely 3. Turbulent air flow.
confirm unidirectional flow inside the filling line
– Smoke studies conducted show turbulent and
and all of its components.
stagnant air within ISO 5 areas used to sterilize
– Not all operators’ interventions were included and fill drug product unit containers.
such as multiple interventions conducted at the
– Video of the dynamic smoke studies conducted to
same time or line set-up activities.
demonstrate unidirectional airflow [redacted]
– Operators were observed standing directly in located in clean room [redacted] showed
front of wall air returns but video failed to follow considerable turbulent airflow when [redacted] was
the smoke pattern at this area. placed within the work zones of the [redacted].
– Operators were observed performing very slow – The above observations should form part of best
movements while adding stoppers and opening practices and the areas referenced as not being
[redacted] doors – unlike current practice included or not being adequately covered should
observed during routine operations at the same be assessed and added to airflow study shooting
line on 11/17/14. scripts, as required.
– The smoke studies are not reviewed by your
Author comments: Perhaps the most serious
personnel and there is no final report regarding
observations relate to the failure to show unidirectional
the adequacy of the airflow.
airflow, and where turbulent airflow has been recorded
– The firm has no documentation of smoke studies (where none is expected) and the company has failed to
conducted under dynamic conditions to indicate act on this.
adequate unidirectional air control during sterile
compounding. Evaluation methodology
– Smoke studies do not indicate vertical laminarity As well as ensuring that airflow studies are performed
of air flow from your ISO 5 vertical flow hood. and that essential activities are included, an evaluation

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Table 1. Example assessment of airflow observations.

Evaluation of smoke study in ISO Class 5 laminar airflow unit/isolator/restricted access barrier system
Intervention type Assembling filling pump
Study demonstrates laminarity Yes/No
Sweeping action over and away from product Yes/No
Turbulence observer recovers smoothly after intervention Yes/No
Air flow shows unidirectional path Yes/No
Video clip demonstrates complete intervention Yes/No
Smoke quality and study visualisation evaluation
Intervention type Corrective/inherent/new intervention
Adequate smoke density +++ /++ /+
Continuous smoke flow +++ /++ /+
Visibility of intervention in smoke +++ /++ /+
Complete smoke flow path is visible in the video clip Yes/No

of how the smoke study is executed is equally "+++", Good "++", Improvement required "+" and
important. The study should be assessed against the Poor "-". An example is shown in Table 1.
following criteria.
Summary
● Studies should demonstrate unidirectional flow This article has examined the importance of airflow
over the product path. studies as a means to visually understand the airflow
● Sweeping action should be away from the within a clean air device, and has indicated that
product path. understanding the air pattern is important in order to
know the level of contamination risk and to identify
● Activity/intervention should to be performed as
any potential weak areas within clean air design and
per procedure.
operations. This article has contextualised the
● Any turbulence observed in smoke flow shall be importance by examining recent trends from FDA
addressed. warning letters and has drawn this review together by
● Air flow should demonstrate a unidirectional providing some best practice tips for conducting
path. meaningful air flow studies that will satisfy the
● Air flow should to be from supply and evacuate regulatory considerations.
through return.
● Video clips should be used to demonstrate the
References
complete interventions. 1 Klinberg S. Smoke studies: clearing the mystery of air flow
● Adequate density of smoke is preferred. visualisation. Journal of Validation Technology
2010;Summer:11–17.
● Continues smoke flow during intervention is 2 Sandle T. Airflow visualisation in an aseptic facility. Cleanroom
preferred. Technology 2012;20(5):13–17.
3 Sandle T, Budini M and Rajesh T. Airflow studies and airflow
● Visibility of intervention should to be part of the
mapping. In: Sandle T and Saghee MR, Eds. Cleanroom
evaluation. Management in Pharmaceuticals and Healthcare. Passfield, UK:
Euromed Communications; 2013.
In the case of poor visibility, the accompanying should 4 Food and Drug Administration. Guideline on Sterile Drug
mention the reasons (such as reflection/less Products Produced by Aseptic Processing Rockville, MD, USA:
FDA; 2004.
lighting/denser smoke). In putting reports together, 5 World Health Organization. WHO GMP for Sterile
some users assess the results of the above as either Pharmaceutical Products, Working Document QAS/09.295 Rev.1.
‘Yes’ or ‘No’ or provide a rating, such as: Very good Geneva, Switzerland: WHO; 2009.

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6 European Commission. Eudralex The Rules Governing Medicinal Convention: http://www.ema.europa.eu/docs/en_GB/

Products in the European Union, Volume 4 EU Guidelines to document_library/Scientific_guideline/2015/02/WC500181863.pdf
Good Manufacturing Practice Medicinal Products for Human 8 Pharmaceutical Inspection Convention. GMP Annex 1 Revision
and Veterinary Use. Annex 1 Manufacture of Sterile Medicinal 2008 Interpretation of Most Important Changes for the
Products. Brussels, Belgium: European Commission; 2009:. Manufacture of Sterile Medicinal Products – Recommendation.
Available at: https://ec.europa.eu/health/sites/health/files/files/ Geneva, Switzerland: PIC/S; January 2010.
eudralex/vol-4/2008_11_25_gmp-an1_en.pdf 9 International Organisation of Standardisation. Cleanrooms and
7 European Medicines Agency. Concept paper on the revision of Associated Controlled Environments. I. Classification of air
annex 1 of the guidelines on good manufacturing practice – cleanliness; II. Specifications for testing and monitoring to
manufacture of sterile medicinal products, probe continued compliance with ISO 14644-1; III. Metrology
EMA/INS/GMP/735037/2014, issued jointly by the European and test methods. BS EN ISO 14644 Parts 1,2 and 3. 1999 (Part
Medicines Agency and the Pharmaceutical Inspection 3: revised 2005).

Madhu Saghee is a Sterile Manufacturing Specialist working in Corporate Quality at Aurobindo Pharma, India. His areas of expertise
include all aspects of quality and compliance for systems, processes, facilities and operations, particularly for sterile products. He is the
Director of the Indian Region for the PHSS, and sits on the international review board of Cleanroom and Containment Journal and GMP
Review magazine. He has been the recipient of the IDMA’s Young Analyst Award. Madhu has edited four books in subjects dealing with
quality and compliance, handling of FDA inspections, cleanroom management and sterility assurance. He has a Masters in Microbiology
from Andhra University and a Masters in Chemistry from Nagarjuna University.

Dr. Tim Sandle ([email protected]) is Head of Microbiology at the UK Bio Products Laboratory. In addition, Tim is a visiting tutor with
the School of Pharmacy and Pharmaceutical Sciences, University of Manchester. Tim serves on several national and international
committees relating to pharmaceutical microbiology and cleanroom contamination control. Outside of work, Tim runs an on-line
microbiology blog (www.pharmamicroresources.com) and he has written extensively on the subject of pharmaceutical microbiology and
contamination control.

Palash Das is a recognised expert in the field of aseptic manufacturing of parenteral products and is a frequent presenter at several web
conferences and technical trainings for sterile drug manufacturing. He earned his Master’s for Pharmaceutical Chemistry from the West
Bengal University of Technology in India. Since 2011, Palash has been associated with installation, qualification and operation of
production-lines in cleanrooms as well as in isolators at a major pharmaceutical firm in Vizag, India.

NB: Can’t find reference 9 in text!

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