As isolator systems are being used more frequently in pharmaceutical manufacturing, it is worth considering the effect of using these types of systems with contamination control programs. The cleaning and disinfection of an isolator system using liquid sanitation techniques presents a variety of challenges to the cleaning and disinfection of cleanrooms. In isolator cleaning, one must also consider the particular application, whether it be pharmaceutical, biotechnology, medical device manufacturing, or hospitals.

Definitions

It is useful to clarify the different stages of the contamination control process. These definitions are taken from the Cleaning and Disinfection chapter of the soon-to-be-published Pharmaceutical Isolators and their Applications1 and wherever possible are based on ISO standards. The reference describes disinfection as the process of reduction in the number of microorganisms 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 sanitization.

A biodecontamination program will usually consist of cleaning, disinfection, and validation. Cleaning refers to the removal of 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 performed 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 be undertaken prior to 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.

Why do we need to consider contamination control?

Even though isolators are designed to prevent contamination from entering, 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 GMP.2

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 to enter. Work carried out looking at the different types of transfer disinfection procedure used to pass product into isolators showed the huge potential for starting materials to be contaminated. Their research found that:

•60% of consumables are contaminated with bacteria

•40% of consumables are contaminated with bacterial spores.3

The rest of the study 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. Therefore 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 can be broken down into four main categories; vegetative bacteria, fungi and molds, 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, 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.

Cleaning and Disinfection Methods

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 the removal of 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 Siegerman.4

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. Usually quarterly folds are 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 sanitization procedure is that all surfaces must be reached. This is not always easy in large or half-suit isolators, so consideration should be given to 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 closed.1

Cleaning

The aim of cleaning is to reduce the contamination level of the surface to a visibly clean state, i.e. remove dust and organic or inorganic soil. This will give a greater likelihood that the disinfectant used will be effective. The cleaning program 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. It is preferable that cleaning is carried out at a natural break, meal times or the end of the 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 deionised water or sterile alcohol.

Combined Cleaning and Disinfection

It has been known for many years that alcohol is an effective disinfectant;5 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 is suitable as a combined cleaning and disinfection agent provided no proteins are present, as alcohol will fix these to a surface by a process of protein denaturing. In this instance a separate cleaning agent should be used.

The alcohol can be diluted with either WFI, purified, or deionized water. Consideration should be given 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 IPA or denatured ethanol. There is very little difference in efficacy between the two alcohols – the results are almost identical.7 The choice is reduced 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.

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: this can be simply checked by using chemical indicator tubes such as Draeger tubes. Both of these factors can be minimized 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 have an adverse effect on some materials found in many isolator systems. Many isolator front panels are isolator systems. Many isolator front panels are manufactured from acrylic which is attacked by alcohol. The crazing effect which can occur over time can be minimized 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, e.g. down the back of work-surfaces, doors, or behind door seals.

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. Specialist test work was commissioned8 by the Mechanical Engineering Dept. at the University of Wales to investigate the impact of our range of disinfectants on different materials used in isolators, utilizing immersion tests. As materials behave differently under stress, we looked at stressed and non-stressed samples. Sample results for IPA are shown in Table 2.

Why use anything else?

If alcohol has so many benefits why should we consider anything else? One, alcohol is not sporicidal and two, EC 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, e.g. 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 the sporicidal 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 stabilized 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 ?

Validation

Cleaning validation is usually limited to a visual or white cloth test. Simply wipe a white cloth over the area which 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 microorganisms to the required level. This would normally be carried out using a combination of settle plates, contact plates, and swabbing. Neutralizing 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.

Consideration of all these factors should ensure a successful liquid decontamination program can be implemented for an isolator system.

References

1 B. Midcalf, W. M. Phillips, J. Neiger, T. Coles. “Pharmaceutical Isolators and Their Applications (provisional title),” Pharmaceutical Isolators Working Party. To be published late 2003.

2 Euraldex, Good Manufacturing Practice, Vol.4, Annexe 1, Manufacture of Sterile Medicinal Products.

3 M. G. Cockcroft, D. Hepworth, J. C. Rhodes, P. Addison, A. M. Beaney. “Validation of Liquid Transfer Disinfection Techniques for Transfer of Components Into Hospital Pharmacy Cleanrooms,” Hospital Pharmacist (September, 2001).

4 H. Siegerman. “Wiping Surfaces Clean,” A2C2 Magazine (April, 2003).

5 Beck WC. “Benefits of Alcohol Rediscovered,” AORN J. Vol. 40 (1984).

6 Larson, Morton. “Disinfectants and Antiseptics.”

7 “Comparison of Denatured Ethanol and IPA,” Technical Report No TR0301R, Shield Medicare Ltd. (March, 2003).

8 Work carried out by Dr J C Arnold, University of Wales. “An Investigation Into the Effect of Klercide™ Products on Commonly Encountered Materials, Technical Report No. TR0011R (January, 2001).