Contamination control

It is useful to clarify the different stages of the contamination control process. These definitions are taken from the forthcoming "Pharmaceutical Isolators and their Applications"¹ and wherever possible are based on ISO standards.

The above publication describes disinfection as "the process of reduction in the number of micro-organisms in or on an inanimate matrix by the action of an agent on their structure or metabolism to a level judged to be appropriate for a defined purpose". This may also be referred to as liquid sanitisation. A biodecontamination programme will usually consist of cleaning, disinfection and validation. Cleaning refers to removing contamination by physical means from a surface to render it visibly clean. Validation is the accumulation of documentary evidence to show that a system or process consistently performs as expected and to a pre-determined specification. Cleaning and disinfection may be carried out as separate processes or as a joint process. However, disinfection does not replace cleaning, as a contaminated surface may interfere with the effectiveness of a disinfectant. This is especially relevant for isolators that are being used to handle biological products. Cleaning should therefore usually be undertaken before disinfection, or combined with it. A separate cleaning stage is more likely to be needed if there has been a spillage of a product or sample. Even though isolators are designed to prevent contamination from entering them, they still require regular cleaning and disinfection to maintain the required microbial grade; for pharmaceutical isolators this would normally be Grade A as defined by EC GMP². Even though most isolators are fitted with HEPA filters and interlocked transfer devices as products are passed into the isolator, there is still potential for contamination. Work carried out looking at the different types of transfer disinfection procedures used to pass product into isolators showed the huge potential for starting materials to be contaminated. The results of the research found that 60% of consumables are contaminated with bacteria and 40% of consumables are contaminated with bacterial spores³. The study also showed that if the transfer disinfection procedure employed comprised of spraying solely with alcohol, only 27.6% of the spores would be removed. The results of the different methods tested are shown in Table 1, showing there is a potential for contamination to enter the isolator. Even if these figures suggest a move to gassing isolators, initial bioburden on consumables and surfaces still needs to be considered. This also highlights the importance of decontaminating all materials which are transferred into an isolator system.

Microbial contamination Microbial contamination can be broken down into four main categories – vegetative bacteria, fungi and moulds, viruses and bacterial spores. Bacterial endospores are the dormant state which some bacteria, e.g. Bacillus spp and Clostridium spp, are capable of assuming when environmental conditions place them under stress. They are extremely resistant to destruction by most chemical biocides, heat, UV light and radiation etc. They can remain viable and a potential source of contamination over a long period of time. When environmental conditions improve they will germinate to become vegetative bacteria.

Decontamination The actual process of decontamination is the same for both cleaning and disinfection. Due to the effect of biofilms, surface wiping is needed to assist in removing surface contamination. Wiping should never be carried out in a circular motion as this causes the wipe in its dirtiest state to be passed over an area which has just been cleaned. This point needs to be reinforced with operators, as a circular wiping pattern is the most comfortable and convenient, according to Siegerman⁴. The correct technique is to wipe towards you in straight horizontal lines, each time overlapping the previous one by 10-25%. A contaminated wipe should not be passed over an area that has just been wiped, unless it is folded and refolded to provide a clean surface. Quarterly folds are usually recommended, but must be validated with each operator concerned, as a quarterly fold can lead to confusion as to which surfaces of the wipe have been used. In this case wipes folded in half should be used. Surface wiping should be carried out from top to bottom, from back to front and from cleanest to dirtiest. The wipe itself should be constructed from a low particulate material. One specific factor that should be considered, when decontaminating isolator systems using a liquid sanitisation procedure, is that all surfaces must be reached. This is not always easy in large or half suit isolators, so consider investing in specially designed tools which aid reaching into awkward places. Any pads used on cleaning tools should be low particulate, sterile and disposable. Cleaning can be carried out with an isolator in its open state; however, it is recommended that disinfection or combined disinfection and cleaning should be carried out with the isolator closed¹. The aim of cleaning is to reduce the contamination level of the surface to a visibly clean state, ie remove dust and organic or inorganic soil. This will provide a greater likelihood that the disinfectant used will be effective. The cleaning programme should be carried out by the operators of the isolator, or suitably trained personnel to a validated SOP. The effectiveness of the cleaning procedure should be validated, documented and regularly monitored. The same principle also applies to the disinfection procedure. Cleaning should be carried out before and after every manufacturing, preparation or dispensing session and between activities that may result in cross contamination. Ideally, cleaning should also be carried out at a natural break, after meal times or the end of a working day. Gross soil should be removed with a detergent – a non-enzymatic variety is preferred for isolators. If a detergent is used, a rinsing cycle must be included to remove any residue. Any detergent residue remaining will have an adverse effect on any disinfectant used. Rinsing can be carried out with either Water for Injection (WFI), sterile purified or de-ionised water or sterile alcohol. It has been known for many years that alcohol is an effective disinfectant⁵: studies have shown that it is a better disinfectant when it is neither too weak nor too concentrated – disinfectant efficacy is optimal at concentrations between 50% and 80%⁶. Many institutions use 70% alcohol blends as standard.

Alcohol: cleans and disinfects Alcohol is suitable as a combined cleaning and disinfection agent provided no proteins are present, as alcohol will fix these by a process of protein denaturing to a surface. In this instance, a separate cleaning agent should be used. The alcohol can be diluted with either Water for Injection, purified or de-ionised water. Consideration should be given as to whether the product contact area requires a disinfectant which is not only sterile but also free from endotoxins, in which case alcohol diluted with Water for Injection should be used. The alcohol used can be either isopropyl alcohol (IPA) or denatured ethanol. There is very little difference in efficacy between the two alcohols as the results are almost identical⁷. The choice is down to other factors. IPA dries more readily but has a strong acrid smell and a lower occupational exposure limit than denatured ethanol. However, denatured ethanol has a sweeter smell that some users may find unpleasant.

Transfer disinfection Alcohol is almost exclusively used for transfer disinfection into isolators, as it is quick drying and leaves little or no residue. However, consideration should be given to the theoretical fire or explosion risk when spraying alcohol, as it is highly flammable and may need additional fans or ventilation to prevent the build up of vapour. The exposure limits should also be monitored when large quantities of alcohol are being sprayed and this can be simply checked by using chemical indicator tubes, such as Draeger tubes. Both of these factors can be minimised by the use of sterile impregnated wipes. These are available in a range of presentations including sachets, tubs or pouches. Care should be taken when using alcohol in an isolator as it can also have an adverse effect on some materials found in many isolator systems. Some isolator front panels are manufactured from acrylic which is attacked by alcohol. The crazing effect which can occur over time can be minimised or eliminated by ensuring that surfaces are dried after disinfection. Problems occur when the alcohol is in contact with a surface and cannot evaporate, so care should be taken not to let alcohol or any disinfectant accumulate in areas where it cannot be reached, for example, down the back of work-surfaces, doors or behind door seals.

Sporicidal disinfectants Many disinfectants, especially sporicidal ones, can have an adverse effect on the materials used in the construction of an isolator. Manufacturers should be able to provide information to support the validation of a particular agent. Shield commissioned some specialist test work⁸ by the Mechanical Engineering Department at the University of Wales utilising immersion tests to investigate the impact of our range of disinfectants on different materials used in isolators. As materials behave differently under stress, we looked at stressed and non-stressed examples. Sample results for IPA are shown in Table 2. If alcohol has so many cleaning benefits, why should we consider anything else? Firstly, alcohol is not sporicidal and secondly, GMP Annexe 1 states that "Where disinfectants are used, more than one type should be employed. Monitoring should be undertaken regularly in order to detect the development of resistant strains". Disinfectants have different modes of action; for example, alcohols disrupt the cell membrane, whereas a quaternary ammonium compound damages the cell wall. Disinfectants with differing modes of action should preferably be rotated. There are a wide range of branded disinfectants available, but they fall into a smaller group of active components. Disinfectants for bacteria and fungi include alcohols, quaternary ammonium compounds, phenols and amphoteric surfactants. None of these have a sporicidal effect. Sporicidal disinfectants tend to be more aggressive, as spores are difficult to kill. Many of these types of disinfectants have drawbacks, such as long contact times, health and safety issues or they are corrosive. Sporicidal disinfectants include aldehydes, hypochlorites, hydrogen peroxide/peracetic acid blends and unique formulations such as stabilised chlorine dioxide and quaternary ammonium blends. When deciding between one disinfectant and another it is important to take key factors into account right from the start. There are five main points to consider when validating a new disinfectant. 1. What microbiological testing is required? 2. What is the correct specification of the disinfectant and how will it be used? 3. What are the health and safety implications of introducing the new disinfectant? 4. What will be the impact on the environment? 5. What support can you expect from the manufacturer? 6. This particular aspect was considered in detail in Cleanroom Technology last year⁹.

Validation Cleaning validation is usually limited to a visual or white cloth test. Simply wipe a white cloth over the area that has been cleaned and check that it shows no visible recovery. Disinfectants should be validated using appropriate microbiological testing. There needs to be verification of the removal or inactivation of micro-organisms to the required level. This would normally be carried out using a combination of settle plates, contact plates and swabbing. Neutralising media should be used for any sampling that follows immediately after disinfection. All critical parameters should be controlled, monitored and documented as evidence to support the assurance of bioburden reduction. The contact time of the disinfectant should be determined in order to ensure the required level of biological decontamination. Cleaning agents and disinfectants should have a validation file which covers product specification, health and safety, compatibility, shelf life and validation data. If all these factors are taken into consideration, this should ensure a successful liquid decontamination programme can be implemented for an isolator system.