Peer Reviewed: Facility Sanitation

Sanitation of Pharmaceutical Facilities

Tim Sandle

ABSTRACT

Maintaining environmental control

including microbiological contamination in a pharmaceutical manufacturing
environment is primarily dependent on
the facility sanitization program. Sanitization considerations are specific for facility rooms, equipment, and personnel.
Sanitization comprises cleaning and disinfection. Cleaning is necessary prior to
the application of disinfectant to enable
sufficient contact time of the disinfecting
agent with the surface. Disinfectants vary
in their spectrum of activity, modes of action, sites of action in microorganisms, and
efficacy. Disinfectants kill vegetative micro-organisms but do not necessarily kill
bacterial spores. There are many different
types and categorizations of disinfectants
such as non-oxidizing disinfectants and
oxydizing agents. Many pharmaceutical
manufacturers will have two in-use disinfectants and a third disinfectant for major
contamination incidents. Rotation of disinfectants is often implemented to satisfy
the requirements of regulators. Cleaning
and disinfection must be detailed in a Standard Operating Procedure (SOP) to ensure
consistency of practice. The effectiveness of
cleanroom sanitization is assessed through
the site environmental monitoring program. Viable monitoring is undertaken using microbiological growth medium. Regulatory agencies expect the pharmaceutical
manufacturer to have evaluated the efficacy
of disinfectants. While suspension testing
is useful for initial screening, comparative
surface (or carrier) testing is more relevant.

USP <1072> lists common materials used

in clean rooms that should be considered
when developing disinfectant surface testing. To demonstrate the effectiveness of a
disinfectant, it must be challenged using a
panel of organisms that is reflective of the
natural microflora of the facility. The biocidal activity of the disinfectant should be
taken into account when selecting the panel
of organisms. The use of microbial isolates
from the manufacturing facility is increasingly becoming a regulatory expectation.
Surface tests cannot demonstrate the effect
of a range of environmental factors in actual environmental conditions. Field trials are an important part of the qualification of a sanitizer to determine if cleaning
techniques are suitable and if the cleaning
frequencies of cleanrooms require modification.

INTRODUCTION

This article provides an introduction to

the sanitization and bio-decontamination
of pharmaceutical manufacturing facilities.
This topic is especially relevant for manufacturing of sterile products.
Pharmaceutical facilities used for manufacturing of sterile products are comprised
of a series of rooms called cleanrooms.
Cleanrooms and zones are typically classified according to their use or main activity
within each room or zone. Cleanrooms are
confirmed by the cleanliness of the air by
the measurement of particles. Pharmaceutical cleanrooms are classified by standards
in either EU and WHO GMP guidance
for aseptically filled products (alphabet-

ic notation) or by International Standard

ISO14644 (numeric classification). The
cleanliness of the air is controlled by the
HVAC system (Heating, Ventilation and
Air-Conditioning) in the facility.
Cleanrooms are designed to minimize
and to control contamination. There are
many sources of contamination. The atmosphere contains dust, microorganisms,
condensates, and gases. Manufacturing
processes will also produce a range of
contaminants. Wherever there is a process which grinds, corrodes, fumes, heats,
sprays, turns, etc., particles and fumes
are emitted and will contaminate the surroundings (1). The foremost concern in
pharmaceutical manufacturing of sterile
products is microbial contamination.
Maintaining environmental control in
a pharmaceutical manufacturing environment is primarily dependent on the facilitys cleaning and disinfection program.
The program requires the selection of the
appropriate disinfectants, their proper application, and validation of their capability
to inactivate vegetative cells.

TYPES OF SANITIZATION

Sanitization differs depending on the

specific area of concern. Three areas are
discussed: Rooms, equipment, and personnel.

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Tim Sandle
Room Sanitization

Cleanrooms and clean areas must be

regularly cleaned and disinfected. This is
normally undertaken by cleaning with detergent followed by the application of a disinfectant. Sometimes more than one disinfectant application is necessary such as
following a production shutdown. It may
be necessary to remove the residue of the
disinfectant using water.
Arguably the most effective cleaning and
disinfection process is undertaken manually by use of wiping techniques. Some facilities utilize fumigation or fogging methods.
These methods are effective when surfaces
are clean and the sanitizing agent can reach
all of the cleanroom surfaces.
When cleaning rooms manually, the
equipment used (mops and buckets)
should be of an appropriate design for the
grade of cleanroom. Cleaning procedures
require a strict cleaning regime. Cleaning and disinfection using cloths and mop
heads is ideally performed by saturating the
cleaning item and wiping the area using a
series of parallel overlapping strokes with
an approximate one quarter overlap. Circular motion should never be used. The
direction of the cleaning should be towards
the operator (from top to bottom, from
back to front). Only one application of the
disinfectant or detergent should be applied
to avoid over-concentration. Cleaning and
disinfection should begin with the visually
cleanest area first and towards the dirtiest
area last.
Cleaning is normally undertaken in
each process area before use. In general,
the frequency of cleaning should be established through risk assessment based on a
review of environmental monitoring data
before and after the disinfection process.
The review should consider the numbers
of microorganisms recovered and the types
of species. With species, a check should
be made on the frequency of recovery of
spore-forming microorganisms. A high
recovery of microbial contamination could
signal a concern with the disinfectants used
or with their frequency of rotation.

Equipment Sanitization

Effective cleaning and sanitization of

equipment is important because equipment
may not be amenable to visual inspection.
Also, equipment may be prone to biofilm
formation.
The main method for cleaning industrial equipment is by making the mechanism
for cleaning integral to the equipment itself.
This can be achieved by use of pressure,
heat, steam sterilization, mechanical removal, or chemical cleaning agents. Automated methods are termed Clean-in-Place
(CIP) or Steam-in-Place (SIP). Prior to
chemical or heat treatment, attempts must
be made to remove process residues and
particles using steam or high pressure water cleaning. Alkali-based disinfectants
and detergents are commonly used for CIP
systems; sodium hydroxide is among the
most widely used. Such caustic alkalis can
readily remove organic deposits without affecting the equipment. It is important that
the equipment cleaning process is validated.

Glove Sanitization

The gloved hands of staff undertaking

critical activities should be sanitized on a
frequent basis using an effective hand sanitizer. Aseptic techniques performed by
trained personnel are most important. The
sanitizing agent itself is not a replacement
for poor aseptic techniques.
There are many commercially available hand sanitizers. Most commonly
used types are alcohol-based gels containing either ethanol or isopropanol. An EU
standard (EN 1499 (2) and EN 150025A
(3) provides test methodology to validate
the efficacy of the hand sanitizer. Testing
determines if a hand sanitizer can reduce
the number of transient microflora under
simulated practical conditions. There are
three key variables which affect the use of
hand sanitizers. These are the agitation and
rubbing the hand sanitizer into the glove,
the frequency of application, and the quantity applied (5). Of these, consistency of
the rubbing technique in ensuring that all
surfaces of the hand come into contact with
the sanitizer is most important.

Journal of GXP Compliance Volume 18 Number 3

TYPES OF DISINFECTION
AGENTS

A disinfectant is one of a diverse group

of chemicals which reduces the number of
micro-organisms present on an inanimate
object. Disinfectants kill vegetative micro-organisms but do not necessarily kill
bacterial spores. To be effective, disinfectants must meet either European standards
(the CEN series) or US standards (the
AOAC standards) for disinfectant efficacy.
These standards involve challenging disinfectants with high populations of a range of
different microorganisms and noting the
log reduction after a period of time. Such
studies are undertaken for the disinfectant
solution (the suspension test), on surfaces,
and in the field to develop appropriate
cleaning frequencies.
Disinfectants vary in their spectrum
of activity, modes of action, and efficacy.
Some are bacteriostatic in which the ability
of the bacterial population to grow is halted. Here the disinfectant can cause selective
and reversible changes to cells by interacting with nucleic acids, inhibiting enzymes,
or permeating into the cell wall. Once the
disinfectant is removed from contact with
bacteria cells, the surviving bacterial population could potentially resume growth.
Other disinfectants are bactericidal in that
they destroy bacterial cells through different mechanisms including causing structural damage to the cell, lysis, and leakage
or coagulation of cytoplasm (6). The mechanisms of action are not always completely
known and continue to be investigated.
Surface disinfectants have varying
modes of action against microbial cells due
to their chemical diversity. Different disinfectants target different sites within the
microbial cell. These include the cell wall,
the cytoplasmic membrane (where the matrix of phospholipids and enzymes provide
various targets) and the cytoplasm. Some
disinfectants, on entering the cell either by
disruption of the membrane or through
diffusion, proceed to act on intracellular
components.

Peer Reviewed: Facility Sanitation

There are different approaches to the
categorization and sub-division of disinfectants, including grouping by chemical
nature, mode of activity, or by bacteristatic
and bactericidal effects on micro-organisms (7). These and other factors such as
efficacy, compatibility, cost, and current
health and safety standards (8) must be
considered when selecting disinfectants for
use in the facility. The following describes
the primary types of disinfectants currently
in use categorized as non-oxidizing and oxidizing agents.

Non-Oxidizing Disinfectants

The non-oxidizing disinfectants include

alcohols, aldehydes, amphoterics, phenolics, and quaternary ammonium compounds (QACs or quats).
Alcohols. The effectiveness of alcohols
against vegetative bacteria and fungi increases with their molecular weight (ethanol is more effective than methanol and
isopropyl alcohols are more effective than
ethanol). Alcohols act on the bacterial cell
membrane by making it permeable. Efficacy is increased with the presence of water
leading to cytoplasm leakage, denaturation
of protein, and eventual cell lysis. The advantages of employing alcohols include
a relatively low cost, little odor, and rapid
evaporation (9).
Aldehydes. Aldehydes include formaldehyde and glutaraldehyde. Aldehydes
have a non-specific effect in the denaturing of bacterial cell proteins and can cause
coagulation of cellular protein. There are
safety concerns about the use of some aldehydes (10).
Amphoterics. Amphoterics have both
anionic and cationic character and possess a relative wide spectrum of activity.
Amphoterics are limited by their inability
to damage endospores. Amphoterics are
frequently used as surface disinfectants.
Examples include the alkyl di(aminoethyl)
glycine group of compounds.
Phenolics. Synthetic phenols are widely
available such as the bis-phenols (triclosan)
and halophenols (chloroxylenol). Phenols
are bactericidal and antifungal, but are not

effective against spores. Some phenols

cause bacterial cell damage through disruption of proton motive force, while others attack the cell wall and cause leakage of cellular components and protein denaturation.
Quaternary ammonium compounds.
QACs or quats are cationic salts of organically substituted ammonium compounds
that have a fairly broad range of microbial
activity. They are ineffective against bacterial spores. QACs are possibly the most
widely used of the non-oxidizing disinfectants. Example QACs include cetrimide
and benzalkonium chloride. Their mode
of action is on the cell membrane leading
to cytoplasm leakage and cytoplasm coagulation through interaction with phospholipids (11).

Oxidizing disinfectants

This group includes oxygen-releasing

compounds (peroxygens) like peracetic
acid and hydrogen peroxide. They function by disrupting the cell wall, causing
cytoplasm leakage, and denature bacterial
cell enzymes through oxidation. Oxidizing agents have advantages in that they are
clear and colorless, thereby avoiding surface staining.

SANITIZATION REGIME

There are several important process

considerations involved with sanitization.
These include cleaning, disinfection, selection and rotation disinfecting agents, and
the specific procedures utilized.

Cleaning

Cleaning, in the context of pharmaceutical manufacturing, is the process of removing residues and soil (dirt, grease, protein
etc.) from surfaces to the extent that they
are visually clean. This involves clearly defined procedures that often require use of a
detergent or other cleaning agent. Detergents generally work by penetrating the soil
and reducing the surface tension (which
fixes the soil to the surface) to allow its
removal. Hence many of the products are
surfactants (surface active agents).
Cleaning as described above is necessary
for cleanrooms prior to the application of
disinfectant. It is essential that a surface

or item of equipment has been properly

cleaned before disinfectant application in
order for the disinfectant to work efficiently. This is particularly important for sporicidal disinfectants, many of which have
limited ability to effectively penetrate soil.

Disinfection

The critical aspect for disinfectant efficacy is the contact time. The disinfectant
is only effective when left in contact with
the surface for the validated time. This
can be achieved more easily when the disinfectant is applied in overlapping strokes.
When rotation of disinfectants is required,
a water rinse normally employing Water for
Injection (WFI) is employed between the
change-over of disinfectants. This rinse removes traces of disinfectant and detergent
residue such as anions which may reduce
the efficacy of the subsequent disinfectant.
A disinfectant will achieve the desired
effectiveness if it remains on the targeted
surface for an appropriate length of time.
Determining the optimal contact time often means striking a balance between what
is necessary to achieve the desired microbial reduction and what is practical for real-time use in the facility. At minimum, the
manufacturers recommended contact time
should be tested. Additional contact times
may also be evaluated if the manufacturers
recommended time is demonstrated to be
ineffective or if a shorter contact time is desired (12).

Rotation of Disinfectants

Many pharmaceutical manufacturers

will routinely use two in-use disinfectants in a specified rotational sequence
for the site disinfection program. A third
disinfectant will be available in reserve in
case a major contamination incident arises.
For example, a bioburden contamination
increase which appears resistant or difficult to eliminate using the routinely used
disinfectants may necessitate use of an additional disinfectant. The reserve disinfectant such as an oxidizing agent will often be
more powerful and sporicidal, the routine
use of which is restricted because of likely damage to the equipment and premises.
The rotation of two primary disinfectants
is a requirement of regulatory bodies. The

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Tim Sandle
EU GMP Guide states that where disinfectants are used, more than one type should
be employed (Annex 1). Nevertheless, the
case for rotation has not been scientifically proven in that there are very few studies
providing empirical evidence. However, it
remains that rotation is often implemented
to satisfy the requirements of regulators.

Viable monitoring is designed to detect

levels of bacteria and fungi present in defined locations during a particular stage in
the processing and filling of product. Viable monitoring is designed to detect micro-organisms and answer questions such
as how many, how frequent, when do they
occur and why do they occur? (14)

Cleaning and disinfection procedures

Viable monitoring is undertaken using

microbiological growth medium (agar or
other substances) in different presentations. It is important that the culture media
used for environmental monitoring contains a neutralizer to eliminate any residues
of the disinfectant.

Cleaning and disinfection must be detailed in a Standard Operating Procedure

(SOP) to ensure consistency of practice.
Furthermore, sufficient detail in SOPs is
important because detergents and disinfectants are only partially effective if they
are not applied correctly. An SOP should
describe:
The type of detergents and disinfectants to be used. These agents must be
compatible
The frequency of rotation of disinfectants
A list of suitable cleaning materials
Cleaning techniques
Contact times
Rinsing
Frequency of cleaning and disinfection
Procedure for the transfer of cleaning
agents and disinfectants into and out
of clean areas
Procedure for sterilization of disinfectants
Holding times for detergents and disinfectants.

ASSESSING SANITIZATION
EFFECTIVENESS

The effectiveness of cleanroom sanitization is assessed through environmental

monitoring. Environmental monitoring
is a program which examines the numbers
and occurrences of viable micro-organisms and non-viable particles such as dust
or skin cells. Environmental monitoring is
ideally targeted to those areas of the production process where the risk cannot be
adequately controlled. Trend analysis of
environmental monitoring data provides
an indication if the cleanroom disinfection
program is moving out-of-control (13).
Viable monitoring of surfaces is the most
relevant approach for assessing the effectiveness of surface sanitization.

The environmental monitoring program

is normally controlled by the site microbiology department who establish the appropriate frequencies and durations for monitoring based on a risk assessment approach.
The sampling plan takes into account the
cleanliness level required at each site to be
sampled.

QUALIFICATION OF
DISINFECTANTS

Regulatory agencies expect the pharmaceutical manufacturer to have evaluated

the efficacy of disinfectants. While suspension testing is useful for initial screening, it
is surface (or carrier) testing that is more
relevant. Qualification of a disinfectant is
demonstrated through performance testing
to show that the disinfectant is capable of
reducing the microbial bioburden found on
manufacturing area surfaces. Representative manufacturing surface samples are inoculated with a selection of microbial challenge organisms. A disinfectant is applied
to the inoculated surfaces and exposed for
a predetermined contact time after which
surviving organisms are recovered using a
qualified disinfectant-neutralizing broth
and test method (surface rinse, contact
plate, or swab). The number of challenge
organisms recovered from the test samples
(exposed to a disinfectant) is compared
to the number of challenge organisms recovered from the corresponding control
sample (not exposed to a disinfectant) to
determine the ability of the disinfectant to
reduce the microbial bioburden. Successful completion of the validation qualifies
the disinfectant evaluated for use. The disinfectant efficacy validation should provide

Journal of GXP Compliance Volume 18 Number 3

documented evidence that the disinfectant

demonstrates bactericidal, fungicidal, and/
or sporicidal activity necessary to control
microbial contamination in the facility
(15).
The selection of surfaces to be assessed
for disinfectant efficacy is an important
consideration. Given the multitude of
available surfaces in a facility, a pragmatic
view should be taken. Where the surface
is considered critical in terms of cleaning
and disinfection, i.e., contact with product
and personnel, it should be considered for
disinfectant surface testing. USP chapter <1072> lists common materials used
in clean rooms that should be considered
when developing disinfectant surface testing. Stainless steel and other surfaces
within the manufacturing facility should
be tested such as different grades of vinyl
and stainless steel, different types of plastic,
glass from windows and vessels, and other
materials as appropriate.
The test involves examining preparations of micro-organisms dried onto
surfaces. A prepared sample of the disinfectant is added to the dried surface containing and microbial suspension. The
surface is then transferred to a previously
validated neutralization medium and testing performed to measure the reduction
in viable counts. One variation of the test
involves drying 0.05 ml suspensions of
the micro-organisms with interfering substances such as bovine serum albumin onto
different surfaces. The micro-organisms
should have a population range of 1.5 - 5.0
x 108 for bacteria and 1.5 - 5.0 x 107 for
fungi and are equilibrated to 25oC before
use. Once applied to the surface, the drying
of the micro-organisms maybe accelerated
using an incubator operating at 36-38oC.
Disinfectant solutions (where disinfectants
are made with Water of Standard Hardness) are added to the surfaces. After the
specified contact time (five minutes is the
target), the surfaces are transferred to the
validated neutralization medium and then
pour plates are prepared for incubation and
counting. An alternative method is available using a soaked swab step (16).
To demonstrate the effectiveness of a
disinfectant, it must be challenged using a
panel of organisms that is reflective of the
natural microflora of the facility. The bio-

Peer Reviewed: Facility Sanitation

cidal activity of the disinfectant should be
taken into account when selecting the panel
of organisms. Some organizations use type
cultures, some use environmental isolates,
and others a combination of the two. The
use of isolates from the manufacturing facility is increasingly becoming a regulatory
expectation.
The surface test described above cannot
demonstrate the effect of a range of environmental factors like temperature, pH, detergent residues, mechanical stress, and attachment in the facility. For these reasons,
a disinfectant which appears effective for
the surface test can show marked variability
when applied to practical conditions. This
is due to problems in drying and differences between surfaces. In terms of drying
microbial suspensions, there is a marked
loss in the viability of a population when
dried onto a surface. Attempts to speed the
drying process do not significantly reduce
the variability of the actual number of micro-organisms challenged. Surfaces introduce another variation because surfaces.
Surfaces of the same grade of material are
not truly identical. There have been marked
problems in achieving reproducibility and
repeatability for the surface test between
laboratories particular in estimating the
concentration of disinfectant required to be
effective. Some of these limitations can be
addressed through field trials.
Field trials (or in situ studies) are an important part of the qualification of a sanitizer. These trials determine if cleaning
techniques are suitable and if the cleaning
frequencies of cleanrooms require modification. Filed trials involve a considerable
amount of environmental monitoring for
the counts before and after disinfection
and the types of microorganisms recovered
must each be evaluated (17).

SUMMARY

This paper has presented an overview

of the application of sanitization of pharmaceutical facilities. Sanitization is a
key part of contamination control within
cleanrooms. It has examined some of the
techniques and control required, and has
compared different types of disinfectants
available for use. It has also emphasized
the importance of qualifying disinfectant
in order to demonstrate their effectiveness,

and for undertaking a vigorous monitoring

regime in order to show that the cleanroom
environment remains in control. The emphasis has been upon the assessment of
surfaces and the evaluation of disinfectants
in the field. While the article has focused
on microbiological assessments, there are
other variables at play including wiping
technique, expiry times, and chemical stability that must also be considered. This
acknowledged, it is hoped that this article
has provided information and advice useful
to compliance practitioners that can be applicable within the manufacturing facility.

References
1.

Halls, N. (2004): Effects and causes of contamination in sterile manufacturing in Hall, N. (ed.):
Microbiological Contamination Control in
Pharmaceutical Clean Rooms, CRC Press, Boca
Raton, pp1-22
2. EN 1499. 1997; Chemical disinfectants and antiseptics. Hygienic handwash. Test method and
requirements (phase 2/step 2)
3. EN 1500. 1997; Chemical Disinfectants Quantitative Carrier Test to Evaluate the Bactericidial
Activity of a Hygenic Handrub Solution (Phase
2/2). Chemical disinfectants and antiseptics. Hygienic handrub. Test method and requirements
(phase 2/step 2)
4. Best M and Kennedy ME. Effectiveness of handwashing agents in eliminating Staphylococcus
aureus from gloved hands, Journal of Applied
Bacteriology, 1992; 73: 6366.
5. Larson E, Mayur K and Laughon B. Influence of
two handwashing frequencies on reduction in
colonizing flora with three handwashing products used by health care personnel, American
Journal of Infection Control, 1989; 17(2): 8388.
6. Sandle, T.: Selection and use of cleaning and
disinfection agents in pharmaceutical manufacturing in Hodges, N and Hanlon, G. (2003):
Industrial Pharmaceutical Microbiology Standards and Controls, Euromed Communications,
England
7. Block S. 1977; Disinfection, Sterilisation and
Preservation, Third Edition, Lea and Febiger,
Philadelphia
8. Denyer SP and Stewart GSAB. Mechanisms of
action of disinfectants, International Biodeterioration and Biodegradation, 1998; 41: 261-268
9. McDonnell G and Russell A. Antiseptics and
Disinfectants: Activity, Action and Resistance,
Clinical Microbiology Reviews, Jan. 1999; 147
179
10. Angelillo IF, Bianco A, Nobile CGA and Pavia
M. Evaluation of the efficacy of glutaraldehyde
and peroxygen for disinfection of dental instruments, Letters in Applied Microbiology, 1998;

27: 292296
11. Bergan T and Lystad A. Evaluation of Disinfectant Inactivators, Acta Path Microbiol Scand
Section B, 1972; 80: 507510.
12. Bessems, E.: The effect of practical conditions
on the efficacy of disinfectants, International
Biodeterioration and Biodegradation, 41, 1998,
pp177-183
13. Johnson, S. M. (2004): Microbiological Environmental Monitoring in Hodges, N. and Hanlon,
G. Microbiological Standards and Controls, Euromed, London
14. Vincent, D.(2002): Validating, Establishing and
Maintaining A Routine Environmental Monitoring Program for Cleanroom Environments:
Part 1, Journal of Validation Technology, August
2002, Vol. 8, No.4
15. Vina, P., Rubio, S. and Sandle, T. (2011): Selection and Validation of Disinfectants, in Saghee,
M.R., Sandle, T. and Tidswell, E.C. (Eds.) (2011):
Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices, New Delhi:
Business Horizons, pp219-236
16. Bloomfield, S.F., Arthur, M., Van Klingeren, B.,
Pullen, W., Holah, J.T. and Elton, R.: An evaluation of the repeatability and reproducibility of a
surface test for the activity of disinfectants, Journal of Applied Bacteriology, 1994, 76, pp86-94
17. Sandle, T. (2012). Application of Disinfectants
and Detergents in the Pharmaceutical Sector.
In Sandle, T. (2012). The CDC Handbook: A
Guide to Cleaning and Disinfecting Cleanrooms, Grosvenor House Publishing: Surrey,
UK, pp168-197

About the Author

Tim Sandle, Ph.D., is the head of the microbiology

department at Bio Products Laboratory Limited,
a pharmaceutical organization owned by the UK
Department of Health. Dr. Sandle is, additionally, a
visiting tutor at the School of Pharmacy, Manchester
University. E-mail: Tim.Sandle@bpl.co.uk

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