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Sanitization of Pharmaceutical Facilities

By Tim Sandle, Ph.D. Aug 29, 2014 10:00 am PDT

Peer Reviewed: Facility Sanitation

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 (alphabetic 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.
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.
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.
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 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.
Cleaning and disinfection procedures
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.
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)
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.
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 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 biocidal 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 (orin 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.
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