Cleaning, Disinfection and the Problems Caused by Chemical Residues

Tim Sandle - Head of Compliance and Quality Risk Management, Bio Products Laboratory

Introduction

Cleaning and disinfection are necessary protective and reactive steps to address microbial deposition on surfaces within pharmaceutical cleanrooms. An appropriately designed cleaning and disinfection program plays a central role in a contamination control strategy. There is considerable literature on the importance of cleaning and with disinfectant selection. In addition, there is the regulatory expectation that a defined cleaning and disinfection program is in place including the requirement for rotation to occur between two or more (depending on whether the periodic use of a sporicide forms part of the duo-rotational pattern or is used at a different frequency) disinfectants. What receives less attention and yet is becoming an area cited by auditors is avoiding or addressing chemical residues remaining on a surface. Facilities should understand the impact of residues, particularly in relation to chemical interference or subsequent surface damage.

There are several concerns with residues remaining on cleanroom surfaces, including:

1. Surfaces that are sticky or slippery pose a safety risk.
2. The presence of a residue ensures that the odor of the chemical lingers for a prolonged period (which can cause operator discomfort or contribute to occupational exposure levels, especially in the case of oxidizing agents used as sporicidal disinfectants).
3. Residues can lead to discoloration, over time, of surfaces (for example, with phenolics staining vinyl).
4. The continuing action of some types of disinfectants can cause surface damage through continued reactivity (such as chlorine ions interacting and corroding with stainless steel).
5. The altered physiochemical properties of the surface due to the presence of the residue can aid bacterial attachment. Microorganisms attached to a surface (sessile state) are far more difficult to remove and inactivate compared with those that are not attached to surfaces (planktonic state).
6. The residue may aid biofilm development and subsequent protection of the microbial community from disinfection.
7. For certain applications, a disinfectant residue may present as an adulterant in terms of cross-contamination of the subsequently processed product. While this veers more into cleaning validation territory, the assessment of Maximum Residue Limits provides a related concern.
8. Residues could lead to contamination of the disinfectant, depending on the application technique (as with some bucket systems).
9. The development of resistant profiles can be promoted by disinfectant residues that persist in the environment at subinhibitory concentrations.
10. The presence of a residue from one disinfectant can interfere and partly inactivate another disinfectant when it is applied to the surface. This is a potential challenge when changing disinfectants as required by a disinfectant rotation regime.

Residues can also arise from some detergents used for cleanroom surface cleaning. The presence of cleaning product residues, which are often manifest as the surface remaining sticky, is due to the surfactant^.1 The likelihood of these depends upon the detergent selected, the concentration, and method of application. The best detergents will be of a neutral pH, low-foaming, and will not leave residues that are capable of interfering with the subsequent application of a disinfectant.

This article focuses foremost on the final item on the list of challenges and concerns: the likely presence of residues and the subsequent possibility for one disinfectant to inactivate another as the chemistries combine and interact. This issue of cleanroom surface residues leads into considerations of how to detect residues and strategies for residue removal.

Concern with Residues: Inactivation

Disinfectants vary in their efficacy and suitability for different uses within pharmaceutical facilities and an agent’s properties must be fully evaluated before adopting it for a particular purpose. This should include understanding the chemistries of the agents selected not only from the microcidal perspective but also in terms of their interactions with surfaces and, potentially as with the case of residues, in relation to each other.²

The point of concern addressed in this article is where residues from one disinfectant come into contact with the in-use disinfectant, affecting the chemicals microtidal or microstatic activity and hence microbial inactivating properties of the disinfectant. In cases where residues are present and where these are not removed, the extent to which inactivation happens is dependent upon the chemical nature of the residue and this determines the extent of inactivation. It may be that two in-use disinfectants are ‘compatible’ and the presence of surface residues is not significant. However, this is less likely given that one of the governing principles of disinfectant rotation is to use two disinfectants with different modes of activity and used in order to broaden the spectrum of microbial kill. Therefore, it is more likely, based on different types of disinfectants being used in the facility disinfection program, that the residues of one agent will interfere with the other.

The consequences of inactivation mean that either the anti-microbial activity is diminished or that far more of the in-use disinfectant will be needed to produce the same antimicrobial effect (compared with a situation where there is no residue present).³ Inactivation is rarely a total ‘canceling out’ of disinfectant microcidal action. More probably, there will be a sublethal loss of biocidal activity meaning that the expected logarithmic reduction of the target microbial population is not achieved. Consequently, the numbers of survivor cells or spores remains at an unexpected level thereby posing a greater and continued product or process risk within the cleanroom.

Are Residues Likely?

To minimize the presence of residues, first an understanding of whether residues are present in the first place is required. This can be derived theoretically from an understanding of the disinfectants and their active ingredients. For example, with a chlorine compound the active residuals of concern may be free chlorine (along with various chloramines), combined chlorine, chlorine dioxide, chlorite ion, chlorate ion, and ozone.⁴ Other disinfectant products contain soluble active ingredients (such as sodium hypochlorite, quaternary ammonium salts, and so on) or other additives in the formulation are likely to form residues. Disinfectants can be divided into low- and high-residue formulations (as discussed below).

For disinfectant products considered more likely and capable of forming residues the rate of residue formation may not be consistent for there are factors that will influence the rate and level of residue formation. The rate of formation, for example, is influenced by the method of application (such as if liquid is allowed to gather in pools or puddles on a surface then residue build up is more likely) and the nature of the surface in terms of structural damage (where scratches, for example, can cause liquid to accumulate). The dilution of the disinfectant can also be a factor, especially when using concentrates that are under diluted.

Another dependency is time. A period of time exists before a problematic chemical residue starts to interact with the surface material. This is the time required to initiate and form a stable pit. The smoothness of the surface, quality of fabrication, and degree of polishing can decrease or increase the time required (crevices are the most common place for chemicals to gravitate towards and for a corrosive attack to begin). This makes crevices plus scratched or damaged surfaces more vulnerable (there are other reasons why a facility maintenance program should seek to minimize surface damage since crevices also provide more opportunities for bacterial surface attachment and they prove more challenging for cleaning and disinfection wiping application techniques to deliver the chemical to these regions and their microbial niches).

It also stands that some residues are stickier than others and the resultant physicochemical reactions can help to attract and stabilize other chemicals or microorganisms to the surface. For this reason, and due to the unknown variable of how different chemistries may interact, the level of any residue remaining is itself not always a useful measure for there are other properties that need to be considered.

To assess the presence of residues, the primary approach is through visual examination of the treated surface, although this is not always straightforward. Operators need to be trained in terms of what residues look like on different surfaces. It follows that some types of residues are more easy or difficult to detect on some types of surfaces than others and even with the same types of surface materials, the coloration of the surface makes detection more difficult (the use of darker colored vinyl for example). The quality of lighting is also of importance. The task of looking for patterns of non-uniformity on a surface is inevitably far easier to undertake within a laboratory and in a cleanroom. Residues that are visible will appear as smears, discrete particles, collections of particles, films, discoloration, and stains.⁵

Given the extent of stainless steel use in pharmaceutical manufacturing (and because it tends to be used to construct the most expensive items of equipment), the use of chlorine-based disinfectants on stainless-steel arguably presents the most troublesome residue concern. Chlorine is a very potent oxidizer and therefore high levels, or long-term exposure of chlorine may accelerate chloride corrosion of stainless steels. The level of any damage is dependent upon the grade of stainless-steel and concentration of the free chlorine (Cl₂ ). Stainless steel of grades 304 and 304L are less corrosion resistant and corrosion can occur at or above 2 ppm free chlorine; whereas 316 and 316L grades are more resistant and corrosion becomes a problem at or above 4 or 5 ppm (these conditions assume the surface is at 20°C (68°F) and of a neutral pH). There will be some variations based on the quality of the stainless-steel manufacturing such as the correct selection of alloys, application of good design principles and proper fabrication practices.

Where a more detailed understanding of the level of residues is required, this is an area that can be added to disinfectant efficacy studies. This assessment could be placed after surface testing and prior to the commencement of the field trial. These studies should consider the concentration to be used in practice (which will have been established from laboratory studies) and the intended application technique (variables like pressure, number of wiping strokes, overlapping strokes, size of the area treated and so on). In addition, some wiper materials are more proficient at absorbing residues than others.

To consider residues, either a visual assessment can be used, or more specialized methods deployed. Many methods will require the use of external laboratory services due to the need for specialist equipment and the techniques for separating the residue from a surrounding matrix. Methods that can assess residues on surfaces include scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS); Fourier-transform infrared spectroscopy, surface enhanced Raman spectroscopy, and X-ray fluorescence spectrometry.⁶ A cruder method involves rinsing samples of a surface following disinfectant treatment and assessing the pH (an approach which can help to determine the number of likely rinses to be required prior to introducing a disinfectant into the facility for a field trial).

Low Residue Disinfectants

There are two possibilities for disinfectant selection in order to address the residue question: either select low residue disinfectants or have in place a post-application method to remove residues. This may be simplistic for there is a third consideration affecting low residue disinfectants as there may still be a requirement to target surfaces to remove residues at a lower frequency since a more gradual, cumulative build-up of residues could occur over a more prolonged period of time.

In opting for a low residue disinfectant there is no universally accepted definition of a ‘low residue’ and ‘low residue’ does not mean ‘zero residue’. There are two classes of common disinfectants that produce far lower levels of residues: hydrogen peroxide and alcohols, yet not all formulations are equivalent. With the low residue disinfectants, this is either based on their chemical nature (such as hydrogen peroxide which breaks down to water and oxygen on a surface) or as a more specific consequence of the formulation to render the disinfectant low residue (as with the development of some quaternary ammonium compounds).

On the subject of formulation, not all disinfectants are identical even when they contain a similar formulation. With hydrogen peroxide, for example, solutions may be in different states of equilibria or may contain certain stabilizers that are themselves residue forming. It should not be taken from this review that low residue disinfectants present the optimal choice since they may not be suitable for all applications. For instance, low residue disinfectants are generally those most prone to interference from the presence of any soil being present on a surface (such as organic material), which increases the importance of cleaning surfaces to be treated by these products prior to disinfection (there may be exceptions in low bioburden environments, and this is a topic that requires risk assessment).

In terms of defining ‘residues’ and ‘low residues’, the presence of residues on a surface is expressed in parts per million and classing a product as ‘low residue’ at <25 ppm is a good starting point. This is not intended to mean that all residues above this level are an immediate concern for the impact of a residue occurs when a new disinfectant type is used and further depends upon what happens in terms of the outcome of the chemical interaction. Nonetheless, it is sensible to either use of a low residue product or to practice periodic residue removal.

Removing Disinfectant Residues

With post-disinfection removal steps, these can either be targeted shortly after application, or after a longer period of time has elapsed. More immediate removal would be taken where:

a. There is concern about the disinfectant activity causing material surface damage. While surfaces need to be wet from the applied agent for the entire disinfection time to be actively working,⁷ under drier conditions subpotent residue activity may continue.⁸ The complete dry time for different disinfectants will vary and this can be assessed gravimetrically.

b. There is concern about the cumulative build-up of residues over time (which is a factor influenced by the amount of residue remaining, the frequency of disinfectant application and the frequency of cleaning).

c. There are concerns about the more immediate transfer of a chemical from one surface to another, such as through moving objects or directly via operators contacting with the surface. While the likelihood of transfer is dependent upon several factors (surface type, surface loading, contact motion, pressure, duration, and secondary surface condition), that chemical transfer that can occur is evidenced by fluorescence imaging studies.⁹

Alternatively, residue removal can be taken less frequently and after several disinfectant applications have occurred. Here, the residue removal is undertaken prior to disinfectant switch-over in order to avoid the two chemistries from the two different disinfectants from adversely interfering with each other. The severity of this will depend on the specific agents and the level of interaction and loss of potency can be quite high (as in the case of chlorine-based disinfectants interacting with phenolic-based disinfectants;¹⁰ in addition, anionic residues can neutralize quaternary ammonium compound disinfectants on a molar basis; and phenolic-type disinfectants are affected by many non-ionic chemical residues).^11,12 Where the formation of residues is likely and where the practice is to delay residue removal it must be understood that the number of repeated applications of the disinfectant and the progression of time will make any residue more challenging to remove.

The common methods to remove residues include surface rinsing with pharmaceutical grade water and the application of a close-to-zero residue disinfectant, such as alcohol.¹³ With the application of the water, the removal is primarily through a dilution and rinsing effect with the residues. In addition, hydrophilic, polar solutes, such as sugar and salt dissolve readily in water, and hence water performs as a good general-purpose solvent. Water is not suitable for general cleaning since hydrophobic dirt, such as grease, protein, and oil, is not water-soluble at normal temperatures.¹⁴ Some of the residue is also physically removed through the use of the double or triple bucket method or absorbed onto a wipe. Volatile alcohols are effective at removing many types of residue and here both of the commonly used alcohols used in cleanrooms – ethanol and isopropyl alcohol at around 70% concentration – are effective.¹⁵

Field Trials

Whichever approaches are taken, the primacy of the field trial remains. No level of laboratory testing can replicate facility disinfection. The field trial is important from both the assessment of survivor microorganisms through environmental monitoring and any lasting effect of chemical corrosion or chemical interactions. Laboratory tests are limited because the condition of the test microorganisms does not reflect that of environmental growth; biostasis cannot be accounted for; the effects of the cleaning phase are not taken into account; demonstrating the frequency of disinfectant rotation cannot be replicated; and microorganisms need to be removed from the surface to be enumerated, which leads to underestimations of kill.¹⁶ To fully capture both consistent microbial kill and any concerns surrounding interacting chemistries, field trials need to be challenging across cleanrooms of different grades, environmental conditions and surface types, and repeated for a statistically representative number of sessions.

Depending on the type of disinfectant, it is also possible to assess the presence of chemical residues on cleanroom surfaces in the field. This is an inexact measurement and not sufficiently advanced for universal acceptability criteria to be in place. Nonetheless, assessing residue levels can provide a baseline and periodic checks can be used to assess operator practices when it comes to residue removal or to refer back to when a new disinfectant is being evaluated through change control. For this purpose, instrumentation, such as a surface conductivity probe, can assess the surface for non-visible contaminants, particularly water-soluble salts. The optimal way to achieve measurement is through swabbing, using moistened swabs and a conductivity meter. This enables the possible detection of residual chlorides, sulphates and nitrates. Alternatively, with a point made earlier, visual assessment may be possible and prove to be just as effective.

Conclusion

This article has looked at the issue of residues from the cleaning or disinfection process on cleanroom surfaces, focusing on whether or not residues will be likely and the concern with disinfectant inactivation which can arise after one disinfectant has been changed for another. The consequence of this leads to the microcidal properties of a disinfectant being diminished, a higher microbial population remaining, and control potentially being lost. Another concern is with corrosion.

Given the complexity of assessing residues within the cleanroom (not all residues will be visible and analytical methods are not very practical in practice) the importance of considering a low[1]residue disinfectant or adopting a residue removal step is of more importance. Even with ‘low-residue’ disinfectants, periodic removal of accumulated residues may be required. Whatever approach is taken, there should be a developed rationale as part of the facility contamination control strategy.

References

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14. Pashley, R., Rzechowicz, M., Pashley, L. and Francis, M. De-Gassed Water Is a Better Cleaning Agent, J. Phys. Chem. B, 2005, 109 (3): 1231–1238
15. Bellanger, E. Evaporation of alcoholic solutions. What residues are left on equipment? Lavague, 2021, 70: https://www.a3p.org/en/evaporation-alcool-residus/
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