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.
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.
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:
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].
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].
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.
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].
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.
There are a number of important steps involved with respect to cleaning and disinfection. These are:
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.
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.
- 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.