Cleaning and Disinfection: An Important Pillar of Contamination Control

Ratul Saha, PhD - Director, Contamination Control, Seres Therapeutics

Background Information:

Cleaning and disinfection of surfaces are not only the essential steps for maintaining the cleanliness and control of the manufacturing environment, but it is also one of the most important pillars of the Contamination Control framework. The overall regulatory expectations regarding this topic are well described:

US Code of Federal Regulation Sec. 211.56 Sanitation.

1. Any building used in the manufacture, processing, packing, or holding of a drug product shall be maintained in a clean and sanitary condition, any such building shall be free of infestation by rodents, birds, insects, and other vermin (other than laboratory animals). Trash and organic waste matter shall be held and disposed of in a timely and sanitary manner.
2. There shall be written procedures assigning responsibility for sanitation and describing in sufficient detail the cleaning schedules, methods, equipment, and materials to be used in cleaning the buildings and facilities; such written procedures shall be followed.
3. There shall be written procedures for use of suitable rodenticides, insecticides, fungicides, fumigating agents, and cleaning and sanitizing agents. Such written procedures shall be designed to prevent the contamination of equipment, components, drug product containers, closures, packaging, labeling materials, or drug products and shall be followed. Rodenticides, insecticides, and fungicides shall not be used unless registered and used in accordance with the Federal Insecticide, Fungicide, and Rodenticide Act (7 U.S.C. 135).
4. Sanitation procedures shall apply to work performed by contractors or temporary employees as well as work performed by full-time employees during the ordinary course of operations.

EudraLex Volume 4-Good Manufacturing Practice (GMP) Guidelines

Part 1 Basic Requirements for Medicinal Products

Principle

Premises and equipment must be located, designed, constructed, adapted and maintained to suit the operations to be carried out. Their layout and design must aim to minimize the risk of errors and permit effective cleaning and maintenance in order to avoid cross-contamination, build-up of dust or dirt and, in general, any adverse effect on the quality of products.

Revised Annex 1 Manufacture of Sterile Medicinal Products

Disinfection

 4.36 The disinfection of cleanrooms is particularly important. They should be cleaned and disinfected thoroughly in accordance with a written program. For disinfection to be effective, prior cleaning to remove surface contamination should be performed. More than one type of disinfecting agent should be employed to ensure that where they have different modes of action, and their combined usage is effective against all bacteria and fungi. Disinfection should include the periodic use of a sporicidal agent. Monitoring should be undertaken regularly in order to assess the effectiveness of the disinfection program and to detect changes in types of microbial flora (e.g., organisms resistant to the disinfection regime currently in use). Cleaning programs should effectively remove disinfectant residues.

 4.37 The disinfection process should be validated. Validation studies should demonstrate the suitability and effectiveness of disinfectants in the specific manner in which they are used and should support the in-use expiry periods of prepared solutions.

 4.38 Disinfectants and detergents used in Grade A zone and Grade B areas should be sterile prior to use (disinfectants used in Grade C and D may also be required to be sterile). Where the disinfectants and detergents are made up by the sterile product manufacturer, they should be monitored for microbial contamination. Dilutions should be kept in previously cleaned containers and should only be stored for defined periods. If the disinfectants and detergents are supplied “ready-made” then results from certificates of analysis or conformance can be accepted subject to successful completion of the appropriate vendor qualification.

 One of the more difficult tasks is the implementation of an effective Cleaning and Disinfection Program. The problem is multifactorial and therefore several aspects need to be considered to develop and implement a compliant and effective Cleaning and Disinfection Program.

1. What are we cleaning and disinfecting?
2. What are the sources of microbial contamination in a cleanroom?
3. Why should we care?
4. What are the definitions?
5. What are the different types of chemical agents and their modes of action?
6. What makes a chemical agent effective against microorganisms?
7. Does your Standard Operating Procedure (SOP) have the following?
8. What are some of the best practices of cleaning and disinfection?
9. What is the importance of training and periodic walkthrough?
10. Why the details matter?

A. What are We Cleaning and Disinfecting?

This is one of the key questions we need to understand and answer. Before answering the question, it is important to understand some of the basic definitions of the terminologies associated. Cleaning is the process of removing residues and soil from surfaces to the extent that they are visually clean. Cleaning involves the use of a detergent. Detergents are basically surfactants that work by reducing the surface tension, which allows the removal of the soil. Cleaning is key before the application of disinfectant (Sandle, 2012). Disinfectant is a substance, or mixture of substances, that destroys or irreversibly inactivates bacteria, fungi and viruses, but not necessarily bacterial spores, in the inanimate environment. (EPA, 2022). Disinfection describes a process that eliminates many or all pathogenic microorganisms, except bacterial spores, on inanimate objects (CDC, 2008).

One of the factors that affect efficacy of disinfection is the prior cleaning of the object: - level of organic and inorganic load present. Disinfectants are poor at penetrating soil and microorganisms hidden within the soil cannot be easily reached by the disinfectants without effective cleaning with a surfactant. Now as the purpose of disinfection is to eliminate microorganisms it will be important to define microorganisms.

An organism that can be seen only through a microscope. Microorganisms include bacteria, protozoa, algae, and fungi (NIH). What about viruses? According to the National Cancer Institute, although viruses are not considered living organisms, they are sometimes classified as microorganisms. Within viruses there are enveloped and non-enveloped viruses.

When it comes to microorganisms five factors are key from a cleaning and disinfection standpoint: a) they are invisible (due to the sizes) to the naked eye; b) their number; c) their diversity; d) their persistence on inanimate surfaces (varies both at species and at strain level) and e) innate resistance of microorganisms (this also varies at species as well as at strain level). When discussing microorganisms, it would be prudent to talk about microbial structures such as bacterial endospores and fungal spores. It is important to keep into perspective that the behavior of the vegetative form of the microbial (bacteria and fungi) cells are very different than the spores (bacterial endospores and fungal spores) towards disinfectants.

To summarize, cleaning is required to remove soil to expose the microorganisms so that the disinfectants could reach and eliminate them. Nowadays most commercially available disinfectant solutions have a detergent within the formulation and therefore the activity of cleaning and disinfection could occur simultaneously, basically the formulations act as a one-step cleaner.

Additional definitions related to cleaning and disinfection will be discussed under the question around definitions.

B. What are the Sources of Microbial Contamination in a Cleanroom?

Now that we understand what we are cleaning and disinfecting it would be important to understand the sources of microbial contamination within a cleanroom keeping into perspective that microorganisms are ubiquitous. According to literature, there are five main sources of microorganisms in a cleanroom environment (Sandle, 2011):

• Personnel
• Facility
• Utilities
• Materials
• Equipment

Out of the above five key sources, personnel and material-equipment transfer are the major contributors for surface contamination and therefore, personnel and material flow, gowning, cleanroom behavior and robust material/equipment transfer processes play a key role in controlling the number and diversity of microorganisms in a cleanroom environment. In addition to personnel, the material transfer process plays an important role in contamination control within the cleanroom environment and therefore, it is key to understand the sources of contaminants and their route of ingress. For example, wheels of carts, cardboard boxes, wooden pallets, fibers, shoes etc. are important sources of microbial spores.

C. Why Should We Care?

We should care about a robust and effective cleaning and disinfection program not because it is required by the law, but it is the right thing to do from a product quality and patient safety standpoint. Microorganisms could cause harm in several ways:

• Mere presence of them in a product (parenterals)
• Number and types (non-sterile products)
• Producing metabolites and toxins (both parenterals and non-sterile products)
• Causing degradation or spoilage (non-sterile products)

D. What are the Definitions?

In addition to the definition of cleaning, disinfection and microorganisms it is also important to understand other definitions associated with cleaning and disinfection. In this respect, it is important to understand spores and sporicidal agents, and more importantly the difference between bacterial and fungal spores. Broadly there are two types of spores: - a) bacterial spores (endospores) and b) fungal spores (fruiting bodies).

1. Bacterial spores: Microorganisms respond to the lack of nutrients and other environmental fluctuations by undertaking different survival strategies, one of the strategies is to form endospores. Bacterial endospores are simplified forms of the bacteria, consisting of the DNA genome, some small amount of cytoplasm, and a specialized coating that confers resistance to heat, radiation, and other harsh external conditions. Endospores are virtually immortal, and can be re-activated, under favorable growth conditions, after lying dormant for hundreds or perhaps millions of years (Berman, 2012).
2. Fungal spores: Are microscopic biological particles that allow fungi to be reproduced, serving a similar purpose to that of seeds in the plant world. Fungal spores are adapted for airborne dispersal and might also withstand unfavorable conditions.

Therefore, a sporicide is an agent that destroys bacterial and mold spores. Because spores are more difficult to kill than vegetative cells, a sporicide can also acts as a sterilizing agent. In this regard, a sterilant is used to destroy or eliminate all forms of microbial life including spores. Whereas a sanitizer lowers the number of bacteria on surfaces to levels that are considered safe by public health organizations. Sanitizers tend to be faster and safer than disinfectants, but disinfectants usually have broader kill and log reduction claims.

E. What are the Different Types of Chemical Agents and Their Modes of Action?

As it is evident from the definitions above that an agent can be used for different applications (sanitizer, disinfectant, sporicide, sterilant) depending on its claims therefore, indicating that there are different types of chemical agents along with different mode of actions.

Broadly, there are two types of chemical agents: - a) non-oxidizing (e.g., alcohols, aldehydes, amphoterics, phenolics, quaternary ammonium compounds) and b) oxidizing (e.g., peracetic acid, hydrogen peroxides, sodium hypochlorite).

Based on this broad classification of whether an agent is non-oxidizing or oxidizing there could be different mode of actions (Figure 1) leading to growth inhibition of the microorganism or lethal action.

(https://www.pharmawareness.com/what-is-disinfectants-and-mode-of-action-of-disinfection-solution/)

Possible mode of actions:

• Action on the external membrane of the cell
• Action on the cell wall
• Action on the cytoplasmic membrane
• Action on the cytoplasm and nucleus
• Action on spores

To learn in more detail please visit, Center for Disease Control and Prevention (CDC) Guideline for Disinfection and Sterilization in Healthcare Facilities (2008).

https://www.cdc.gov/infectioncontrol/guidelines/disinfection/ disinfection-methods/chemical.html

F. What Makes a Chemical Agent Effective Against Microorganisms?

There are several factors that could affect the efficacy of disinfection:

• Number and location of microorganisms: All conditions remaining constant the larger the number of microbes, the more time a chemical agent would need to eliminate them all. Therefore, controlling and reducing the number of microorganisms potentially increases the efficacy of a chemical agent. The location of microbes must also be considered. Surfaces that have crevices, joints and angles are more difficult than flat surface. In addition, surfaces that are in direct contact with the chemical agent would get disinfected so it would be important to avoid air pockets. Therefore, adequate coverage of surfaces as well as strictly adhering to the vendors application method would be key.
• Types of microorganisms: Microbes vary greatly in their resistance to chemical agents. The variation could be due to cellular composition, structure, and other structures (e.g., spores).
• Concentration of disinfectants: It is important to pay attention and adhere strictly to the concentration of the chemical agent recommended by the vendor or supported by disinfectant efficacy testing (Saha, 2019) to achieve expected efficacy. In this regard, it is important to follow the procedure and document the preparation of the in-use solutions where in-house dilution of concentrated chemicals might be required before application for disinfection. Similarly, considering the length of the disinfection time, which depends on the potency of the disinfectant is also important during selection of chemical agents for surface application.
• Physical and chemical factors: Temperature, pH, and hardness of water plays an important role in the efficacy of the chemical agent. According to the CDC, the activity of most disinfectants increases as the temperature increases, but some exceptions exist. Furthermore, too great an increase in temperature causes the disinfectant to degrade and weakens its germicidal activity and thus might produce a potential health hazard. An increase in pH improves the antimicrobial activity of some disinfectants (e.g., glutaraldehyde, quaternary ammonium compounds) but decreases the antimicrobial activity of others (e.g., phenols, hypochlorites, and iodine). The pH influences the antimicrobial activity by altering the disinfectant molecule or the cell surface. Water hardness (i.e., high concentration of divalent cations) reduces the rate of kill of certain disinfectants because divalent cations (e.g., magnesium, calcium) in the hard water interact with the disinfectant to form insoluble precipitates. Taking into considerations these aspects and challenging them in the disinfectant efficacy testing is prudent to have an efficacious chemical agent. Similarly, adhering to these factors during application is equally important.

Relative humidity is the single most important factor influencing the activity of gaseous disinfectants/sterilants, such as EtO, chlorine dioxide, and formaldehyde.

• Soiling Matter: The type and amount of soil can have an impact on the efficacy of the chemical agent. Organic soil could interact with the chemical agent making it ineffective or less effective. Microorganisms could also hide within soiling agents and inaccessible to the chemical agent.
• Wet Contact Time: It is one of the critical parameters that directly has an impact on the efficacy of a disinfectant. It is also known as “dwell time” and “contact time”. The wet contact time is determined during disinfectant efficacy testing using coupons representative of different facility surfaces on which the disinfectants will be applied. It is important to note that disinfectant manufacturers register their disinfectants with specific wet contact time based on studies performed on limited surface types to meet relevant authority’s regulations, but pharmaceutical manufacturers must then demonstrate disinfectant efficacy on their broad cleanroom’s surfaces (USP<1072>) to qualify their chemical agents. As mentioned within the article (Azab, 2020) that the disinfectant efficacy testing performed within a laboratory in general only takes into account microbial kill, disinfectant formulation, disinfectant concentration and cleanroom surface but in real world setting wet contact would be impacted by temperature and humidity in the cleanroom, air exchanges in the cleanroom, application techniques, surface cleanliness and actual time for the disinfectant to dry. Therefore, there are two approaches to have an efficacious disinfection from a wet contact time standpoint: - a) if the surface dries out before achieving the wet contact time, then reapply the chemical agent and b) perform the disinfectant efficacy testing with the actual wet contact time (time the surface remains wet within a cleanroom with a single application of the chemical agent) demonstrating efficacy of the disinfectant. The criticality of wet contact time highlights the importance of documenting it adequately during a cleaning and disinfection session to meet both quality and compliance requirement.
• Other Factors: In addition to the above obvious factors there are other less obvious factors which are equally important and have impact of the efficacy of the chemical agents. Such factors are:

1. frequency of disinfection,
2. rotation of disinfection (for more information refer to Hollands, 2021),
3. technique of cleaning and disinfection (for more information refer to Sandle, 2016)
4. state of repair and storage of accessories used for the purpose of cleaning and disinfection,
5. disinfectant residue removal,
6. hold times of disinfectants,
7. transfer process of cleaning and disinfection materials in and out of the cleanroom,
8. training of cleaning crew

G. Does your SOP have the following?

Since we now understand what factors could affect the efficacy of disinfection it is important to incorporate the elements within the cleaning and disinfection SOP with sufficient details so that the information is not open to interpretation and the steps could be performed in a consistent and standardize manner each time every time. Sometimes it might be prudent to have a bilingual SOP to make it more effective and easier to follow.

• Preparation: The types of chemical agents to be used and their concentration.
• Regimen: The frequency and rotation of disinfectants.
• Regimen: The frequency of cleaning and disinfection.
• Materials: A list of suitable cleaning materials and their expiration.
• Application Methods: Step by step cleaning methods.
• Wet Contact Times: Length of time surfaces must stay wet to be effective.
• Residual Removal: Steps to ensure the chemical agent is adequately removed.
• Transfer: Process for the transfer of chemical agents and related materials such as mops, buckets, etc. in and out of the clean room(s).
• Expiration: Hold times for chemical agents.

H. What are Some of the Best Practices of Cleaning and Disinfection?

I. What is the Importance of Training and Periodic Walkthrough?

Cleaning and disinfection consists of:

• several variables
• manual in nature
• performed during odd hours
• often work performed by contractors
• language barrier
• potential of high turnover

Therefore, it is needless to say the importance of training (not only read and understand of the SOP but to have On-the-Job Training) and oversight for a critical task like cleaning and disinfection. It is important to note that for any manual activity it is key to ensure that it is performed in a standardized and consistent manner to ensure its effectiveness.

EudraLex Volume 4 Guidelines on Good Manufacturing Practice Specific to Advanced Therapy Medicinal Products states within section 3.2 Training

• Cleaning and maintenance personnel should receive training relevant to the tasks performed, in particular on measures to avoid risks to the product, to the environment, and health risks.
• Training can be provided in-house. The effectiveness of training should be periodically assessed. Records of training should be kept.

In addition to the training periodic walkthrough of the facility to observe cleaning and disinfection activities is equally value added from an oversight standpoint to implement a robust and effective cleaning and disinfection program. A checklist such as below could be used to record and learn about the strength and weakness of both the SOP and the training program.

• Gowning performed appropriately for area being cleaned
• Wipe down of Cleaning Cart into clean space performed appropriately, as applicable
• Cleaning agents and supplied used within expiration
• Order of room cleaning is aligned with the procedure
• Sequence of cleaning is aligned with the procedure
• Proper technique used when cleaning
• Following proper cleanroom behaviors
• Mops/buckets changed out appropriately between rooms or cleanroom grades, as per procedure Required contact times for disinfectants met as defined in the procedure
• Copy of effective SOP or Job Aid present during cleaning
• Logbook entries made for cleanings, as required per procedure

In conclusion the answering the final question why do the details matter? The details matter because

• The number of microorganisms matters
• The diversity of the microorganisms matters
• The sources and location of the microorganism matter
• The mode of action and the application of the disinfectant matters
• The manual nature of the cleaning and disinfection matters
• All the other factors matter!

References

1. Azab, W.El. (2020) A refresher on disinfectant wet contact time, Cleanroom Technology. https://www.cleanroomtechnology.com/news/article_page/A_refresher_on_ disinfectant_wet_contact_time/167596 accessed April 04.
2. Berman, J.J. (2012) “Class Bacilli Plus Class Clostridia” Chapter 12. In Berman, J. Taxonomic Guide to Infectious Diseases. Academic Press, pp.65-71.
3. Center for Disease Control and Prevention (CDC) (2008) Introduction, Methods, Definitions of Terms. Guideline for Disinfection and Sterilization in Healthcare Facilities. https://www. cdc.gov/infectioncontrol/guidelines/disinfection/introduction.html accessed April 04.
4. EudraLex Volume 4 (2013) Good Manufacturing Practice (GMP) Guidelines. Part 1 Basic Requirements for Medicinal Products
5. EudraLex Volume 4 (2017) Guidelines on Good Manufacturing Practice Specific to Advanced Therapy Medicinal Products, Section 3.2 Training
6. EudraLex Volume 4 (2020) Good Manufacturing Practice (GMP) Guidelines, Revised Annex 1 Manufacture of Sterile Medicinal Products, Disinfection. https://www.gmp-compliance.org/ files/guidemgr/2020_annex1ps_sterile_medicinal_products_en.pdf accessed April 04.
7. Hollands, W. (2021) Disinfectant rotation and the frequency of use of a sporicidal agent. American Pharmaceutical Review. https://www.americanpharmaceuticalreview.com/ Featured-Articles/581815-Disinfectant-Rotation-and-the-Frequency-of-Use-of-a-Sporicidal-Agent/ accessed April 04.
8. Saha, R. (2019) Disinfectant Efficacy: How can we make it effective? American Pharmaceutical Review. https://www.americanpharmaceuticalreview.com/Featured-Articles/364046-Disinfectant-Efficacy-How-Can-We-Make-It-Effective/ accessed April 04
9. Sandle, T. (2011) A Review of Cleanroom Microflora: Types, Trends, and Patterns, PDA Journal of Pharmaceutical Science and Technology, Vol. 65, No. 4, July–August 2011, pp392-403.
10. Sandle, T. (2013) “Application of Disinfectants and Detergents in the Pharmaceutical Sector”. In Sandle, T. The CDC Handbook: A Guide to Cleaning and Disinfecting Cleanrooms. Grosvenor House Publishing: Surrey, UK, pp. 168-197.
11. Sandle, T. (2016) Pharmaceutical Facility Sanitization: Best Practices Considered. American Pharmaceutical Review. https://www.americanpharmaceuticalreview.com/Featured-Articles/184449-Pharmaceutical-Facility-Sanitization-Best-Practices-Considered/ accessed April 04.
12. United States Code of Federal Regulation Sec. 211.56 Sanitation.
13. United States Environmental Protection Agency (EPA) (2022) Pesticide Registration Manual: Chapter 4-Additional Consideration for Antimicrobial Products. https://www. epa.gov/pesticide-registration/pesticide-registration-manual-chapter-4-additional-considerations accessed April 04.
14. United States Pharmacopoeia (USP) (2021) USP <1072> Disinfectants and Antiseptics. 

Disclaimer: This article reflects the views and opinion of the author and should not be construed to represent any company’s views or policies. My communication represents my own best judgment.

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