Pharmaceutical Facility Sanitization: Best Practices Considered

• Bio Products Laboratory Ltd

Introduction

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. Cleaning and disinfection should also extend to equipment. Furthermore, with personnel, sanitization is important in relation to glove hands.

This article reviews the key points to consider for the practical application of a cleaning and validation program within a pharmaceutical facility, with a focus on disinfectant selection.

Detergents

For cleanrooms, detergents are required to remove ‘soil’ (protein, grease and so on). Detergents penetrate soiling and reducing the surface tension (which fixes the soil to the surface) to allow its removal. This is necessary in order for the disinfectant to work effectively. Furthermore, microorganisms in suspension are easy to remove with rinsing or kill with the disinfectant.

For this task it is important to select an effective detergent. Here it is important that the detergent selected will:

• Work with different types of water (e.g. ‘hard’ and ‘soft’ water)
• Be compatible with the disinfectant
• Not damage the surfaces
• Non-foaming
• Be effective against different soils e.g. grease, dirt, oil, protein, rust, skin

In general, neutral detergents are ideal. Depending on the area of use, such as aseptic filling areas, detergents may need to be sterile.

Disinfection

Disinfection is either the inactivation or destruction of microorganisms. Some disinfectants are bacteriostatic, others are bactericidal. Importantly, disinfection is not the same as sterilization. Disinfection is about a standardized reduction of microorganisms. There are several types of disinfectants (or ‘sanitizers’) available, and they represent a diverse group of chemical agents.

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 cytoplasm¹.

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 standards² .

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, then 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-organisms³ .

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-Oxidizing Disinfectants

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 evaporation⁴.

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 aldehydes⁵ .

Amphoterics

Amphoterics have both anionic and cationic character and possess a relatively 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.

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)

QACs are cationic salts of organically substituted ammonium compounds and have a fairly broad range of activity against microorganisms. They are ineffective against bacterial spores. QACs are possibly the most widely used of the non-oxidizing 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 phospholipids⁶ .

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.

What Makes a Disinfectant Work Well or Badly?

There are a number of factors that affect whether a selected disinfectant works well or poorly⁷. These are briefly presented below:

1. Number of microorganisms: In general disinfectants are more effective against a low number of microorganisms than a higher number.
2. Types of microorganisms: Some microorganisms are more resistant than others. Here Gram-positive bacteria are generally easier to kill than Gram-negative bacteria; vegetative bacteria are easier to kill than fungi; and endospore forming bacteria are the hardest to kill (for these a disinfectant classed as a sporicide is required)⁸ .
3. Location of microorganisms: The key issue here is how likely are microorganisms to be fixed to surfaces? The degree of surface attachment can affect the removal and destruction of organisms.
4. Contact time: This is the time taken for a disinfectant to kill the microorganism and the time that the disinfectant must be left in contact with the surface. This will typically be 1 to 5 minutes, although the time can only be assessed through disinfectant efficacy testing⁹ .
5. Disinfection concentration: Disinfectants are manufactured or validated to be most effective at a set concentration range (the proportion of the chemical to water). Over- or under-dilution will lead to a loss of efficacy.
6. Temperature: The temperature at which the disinfectant is used at influences the rate of reaction. In general, lower temperatures, especially those below the threshold at which the disinfectant has been assessed; mean that the disinfectant may not work. This means that if disinfectants are used in cold storage areas, they should be assessed to see if they remain effective.
7. pH: Like temperature, extremes of pH can influence disinfectant efficacy.
8. Soil: As discussed above, if soil is not effectively removed this can either interfere with the disinfectant or prevent the disinfectant from making contact with the microbial cell wall.
9. Type of water: Hard water can be a problem. The water used to prepare disinfectants in the production facility should be incorporated into disinfectant efficacy studies.

How to Select the Appropriate Disinfectant?

Taking the above ‘influencing factors’ into account, t here are some key criteria that should be considered when selecting a disinfectant. It is good practice to use these as a check-list when making a selection¹⁰. The criteria are:

1. Wide spectrum of activity: This is the ability of the disinfectant to kill different types of microorganisms.
2. Possibly a ‘sporicide’: This will depend on the application and a review of the microorganisms recovered from the environmental monitoring program.
3. Rapid action: Ideally a contact time of
4. Compatible with a second disinfectant for rotation. 
5. Work across a wide pH and temperature range 
6. Detergent compatibility. 
7. Compatibility with different surface types: Disinfectants must not damage the material and problems of corrosion and discoloration can arise. For more aggressive disinfectants, a wipe down using water or an alcohol is recommended¹¹.
8. An assessment should be made to see if the disinfectant leaves surface residues. This could lead to surface damage or disinfectant/detergent computability issues. To avoid this, a surface rinse may be required (such as with Waterfor-Injections or alcohol).
9. Sterility: If the disinfectant is to be used in aseptic filling areas it must be capable of being rendered sterile without loss of efficacy. Methods of sterilization include gamma irradiation and filtration.
10. Format: The presentation of the disinfectant should be considered. Is a ready-to-use concentrate, trigger spray or saturated wipe format required?
11. Other considerations include operator health and safety and the environmental impact.

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.

Cleaning and Disinfection Techniques

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.

Rotation of Disinfectants

The scientific basis for disinfectant rotation, in terms of the buildup of resistant strains is questionable on the basis that no reliable evidence has been provided about the acquisition of resistance by microorganisms. A more plausible argument for rotation comes from the spectrum of activity that different biocides provide. Given, as indicated above, that different disinfectants have different modes of action and some are more effective against different microorganisms than others, then the use of two disinfectants to ensure a greater range of effectiveness against microorganisms is more logical. Whatever the merits of the argument, the use of two disinfectants in rotation is a common regulatory question and in Europe it is stated in Annex 1 of EU GMP. The guidance states that “where disinfectants are used, more than one type should be employed”.

Thus 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 oxidizing agent, the routine use of which is restricted because of likely damage to the equipment and premises. Typically the two primary disinfectants are rotated.

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 correctly¹². 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 sterilization of disinfectants).
• Holding times for detergents and disinfectants

Including these essential standard operating procedures will ensure that a cleaning and disinfection procedure can be applied consistently.

Equipment Sanitization

Effective cleaning and sanitization 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 sterilization, mechanical removal or chemicals, and is termed Clean-in-Place (CIP) or Steamin-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 1499¹³ and EN 150025A¹⁴). The test determines if a hand sanitizer 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 based¹⁵.

There are three key variables which affect the use of hand sanitizers. These are the act of agitation and rubbing the hand sanitizer into the glove, the frequency of application and the quantity applied¹⁶.

Summary

This article has considered sanitization in pharmaceutical facilities and has sought to present best practices. The article has not so much centered on the science of disinfection, but rather to offer practical advice for those who need to use and select disinfectants and to put them into effective use.

Ensuring that contamination is controlled in cleanrooms, on and in equipment, and in relation to personnel is an important step in maintaining microbial control of the production process and a focus on the fabric and structure in which pharmaceutical products are prepared is as important as bioburden reduction steps within the product itself.

References

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5. Angelillo, I.F., Bianco, A., Nobile, C. and Pavia, M. (1998) Evaluation of the efficacy of glutaraldehyde and peroxygen for disinfection of dental instruments, Letters in Applied Microbiology, 27: 292–296
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10. 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
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13. EN 1499. 2015; Chemical disinfectants and antiseptics. Hygienic handwash. Test method and requirements (phase 2/step 2)
14. EN 1500. 2015; 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)
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