Methods
Room sanitization
Cleanrooms
and clean areas must be regularly cleaned and disinfected. This is normally
undertaken using a detergent step, followed by the application of a
disinfectant. It may be necessary to remove the residue of the disinfectant
using water.
When
cleaning rooms the equipment used (mops and buckets) should be of an
appropriate design for the grade of cleanroom. When undertaking cleaning a
strict cleaning regime should be followed. 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) and never in circular motions. 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.
Equipment sanitization
Effective
cleaning and sanitisation of equipment is important because equipment may not
be amenable to visual inspection and it 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 sterilisation, mechanical removal or chemicals, and is 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, with sodium hydroxide among the
most widely used. Such caustic alkalis can readily remove organic deposits
without affecting the equipment. It is important that equipment cleaning is
validated.
Glove sanitization
For
staff undertaking critical activities gloved hands should be sanitized on a
frequent basis using an effective hand sanitizer. Disinfected glove hands can
aid staff who need to carry out aseptic practices although the sanitizing agent
itself is not a replacement for poor aseptic technique.
There
are many commercially available hand sanitizers with the most commonly used
types being alcohol-based gels. To ensure that the hand sanitizer selected is
effective, within Europe there is a standard
describing the approach for their validation (EN 1499[i]
and EN 150025A[ii]). The
test determines if a hand sanitiser can reduce the number of transient
microflora under simulated practical conditions. The standard outlines the
approach for the evaluation of hygienic handrubs. Most hand sanitizers are
ethanol or iso-propanol alcohol based[iii].
There
are three key variables which affect the use of hand sanitizers. These are the
act of agitation and rubbing the hand sanitiser into the glove, the frequency
of application and the quantity applied[iv].
Types of sanitizing agents
A
disinfectant is one of a diverse group of chemicals which reduces the number of
micro-organisms present (normally 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). These standards involve challenging
disinfectants with high populations of a range of different microorganisms and
noting the log reduction over 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, where 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 grow. Other
disinfectants are bactericidal in that they destroy bacterial cells through
different mechanisms including causing structural damage to the cell;
autolysis; cell lysis and the leakage or coagulation of cytoplasm[v].
There
are many different types of disinfectants for use within the pharmaceutical
industry, with different spectrums of activity and modes of action. The
mechanisms of action are not always completely known and continue to be
investigated. A range of different factors needs to be considered as part of
the process of selection including the mode of action, and also efficacy,
compatibility, cost and with reference to current health and safety standards[vi].
Surface
disinfectants have varying modes of action against microbial cells due to their
chemical diversity. Different disinfectants and 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, then proceed to act on intracellular components.
There are different approaches to the categorisation and sub-division of
disinfectants, including grouping by chemical nature, mode of activity or by
bacteristatic and bactericidal effects on micro-organisms[vii].
There
are many different types of disinfectants and space does not permit a list of
all possible types. This guide describes some of the more commonly used types
of disinfectants. Surface disinfectants can be divided into:
Non-oxidising
disinfectants
a)
Alcohols
The
effectiveness of alcohols against vegetative bacteria and fungi increases with
their molecular weight (therefore ethanol is more effective than methanol and
in turn isopropyl alcohols are more effective than ethanol). Alcohols act on
the bacterial cell membrane by making it permeable and 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 odour and a quick evaporation[viii].
b)
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 some safety concerns about the use of some
aldehydes[ix].
c)
Amphoterics
Amphoterics
have both anionic and cationic character and possess a relative wide spectrum
of activity, but they 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.
d)
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.
e)
Quaternary ammonium compounds (QACs)
QACs
are cationic salts of organically substituted ammonium compounds and have a
fairly broad range of activity against micro-organisms. They are ineffective
against bacterial spores. QACs are possibly the most widely used of the
non-oxidising disinfectants within the pharmaceutical industry; examples
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[x].
Oxidising
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. Oxidising agents
have advantages in that they are clear and colourless, thereby avoiding surface
staining.
Sanitization regime
There
are a number of important steps involved with respect to cleaning and
disinfection. These are:
Cleaning
Cleaning,
in the context of pharmaceutical manufacturing, is the process of removing
residues and soil from surfaces to the extent that they are visually clean.
This involves defined methods of application and often the use of a detergent. Detergents
generally work by penetrating soiling and reducing the surface tension (which
fixes the soil to the surface) to allow its removal.
For
cleanrooms such cleaning steps are necessary prior to the application of a
disinfectant. It is essential that a surface or item of equipment has been
properly cleaned before the application of a disinfectant in order for the
disinfectant to work efficiently.
Disinfection
Disinfectants
are applied to surfaces which have been cleaned. When applying a disinfectant,
as previously discussed, the critical aspect 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 WFI) is employed between the change-over of
disinfectants. This is in order to remove traces of disinfectant and detergent
residue (such as anions) which may act to reduce the efficacy of the new
disinfectant.
Rotation of disinfectants
In
selecting disinfectants many pharmaceutical manufacturers will opt to have two
‘in-use’ disinfectants and sometimes to have a third disinfectant as a reserve
in case a major contamination incident arises, such as a bioburden
contamination build up, which appears resistant or difficult to eliminate using
the routinely used disinfectants. The reserve disinfectant will often be more
powerful and sporicidal, such as an oxidising agent, the routine use of which
is restricted because of likely damage to the equipment and premises. Typically
the two primary disinfectants are rotated. This is a requirement of regulatory
bodies with the EU GMP Guide stating that “where disinfectants are used, more
than one type should be employed” (Annex 1).
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 (which are 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
(including the procedure for sterilisation of disinfectants).
- Holding times for
detergents and disinfectants
[i] EN 1499. 1997; Chemical disinfectants and
antiseptics. Hygienic handwash. Test method and requirements (phase 2/step 2)
[ii] 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)
[iii] Best M and Kennedy ME. Effectiveness of handwashing
agents in eliminating Staphylococcus aureus from gloved hands, Journal of
Applied Bacteriology, 1992; 73: 63–66.
[iv] 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): 83–88.
[v] 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
[vi] Block S. 1977; Disinfection, Sterilisation and
Preservation, Third Edition, Lea and Febiger, Philadelphia
[vii] Denyer SP and Stewart GSAB. Mechanisms of action of
disinfectants, International Biodeteriroration and Biodegradation, 1998; 41:
261-268
[viii] McDonnell G and Russell A. Antiseptics and
Disinfectants: Activity, Action and Resistance, Clinical Microbiology Reviews,
Jan. 1999; 147–179
[ix] 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: 292–296
[x] Bergan T and Lystad A. Evaluation of Disinfectant
Inactivators, Acta Path Microbiol Scand Section B, 1972; 80: 507–510.
Posted by Dr. Tim Sandle,
Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)
