1. Introduction

1.1 Appendix 1 of Good Manufacturing Practices (GMP)

Since the implementation of the new Annex 1 of the GMP guidelines, the decontamination process as a whole (also referred to as “biocleaning” in the article) has become more clearly defined. Whether it involves linking the process to the CCS (Contamination Control Strategy) or the way the process must be structured, this revision has led to significant changes in how cleanrooms are cleaned and decontaminated in the pharmaceutical industry.

Before explaining the impact of these changes, there is one important concept that needs to be clarified. This article will focus solely on surfaces that do not come into direct contact with the product, i.e., surfaces covered by Annex 1 of the GMP guidelines. Surfaces that come into direct contact with the product are covered, among other things, by Annex 15 and may require a different cleaning and disinfection protocol.

It is also important to note that the implementation of a bio-cleaning program is specific to each environment and must be integrated into each entity’s cleanroom operating procedures. The article below outlines the main aspects of bio-cleaning in cleanrooms. (Figure 1)

Definitions 

According to the definitions taken from the GMP Guide – May 2024; Annex 1 (European Model of Good Manufacturing Practice – GMP for the Manufacture of Sterile Medicinal Products).

Decontamination. The overall process of removing or reducing any contaminants (chemicals, waste, residues, or microorganisms) from an area, object, or person. The decontamination method used (e.g., cleaning, disinfection, sterilization) must be selected and validated to achieve a level of cleanliness appropriate for the intended use of the decontaminated item (see also Bio-decontamination). 

Cleaning

A process used to remove contamination, such as product residues or disinfectant residues.

Disinfection

A process by which the number of microorganisms is reduced through the irreversible action of a product on their structure or metabolism, to a level deemed appropriate for a specific use or purpose.

Biological decontamination.

A process that eliminates viable microorganisms using a chemical sporicide.

1.2 Regulatory Framework

In the pharmaceutical industry, Good Manufacturing Practices (GMP) impose strict cleaning requirements. Specifically, they require:

The development of Standard Operating Procedures (SOPs) for each type of cleaning,

The validation of bio-cleaning methods (absence of residues, demonstrated efficacy),

Traceability of operations (who, when, how),

Regular audits to verify compliance.

Biocleaning is also a key element in the qualification of facilities and equipment (QI/QO/QP) and in batch release processes in pharmaceutical manufacturing.

In the remainder of this article, we will therefore discuss the steps involved in cleaning and disinfection.

2. Cleaning steps 

2.1 Cleaning in a cleanroom

Cleaning cycles are central to contamination control, with a very simple goal: to reduce contamination in the room to a level that allows for safe production.

The objectives of cleanroom cleaning

The cleaning phase has multiple objectives, which can be summarized as follows:

• Reduce contamination and particulate load to an acceptable level in accordance with the target ISO class.
• Remove product or solvent residues between production runs.
• Prevent cross-contamination between different batches or products.

Cleaning is therefore a fundamental step in contamination control, as it removes any contaminants present; it precedes the disinfection step and determines its effectiveness. 

In fact, a disinfectant applied to an uncleaned surface may not be effective because its action is partially inhibited by interfering substances.

These so-called non-viable contaminants are described below.

Particulate contaminants

Here we will discuss dust and fine dust. These particles generally originate from the environment, the process, and the operator. For these limits, please refer to ISO 14644-1 and the limits listed in Annexe 1.

Organic and inorganic contaminants 

Depending on the production process, various contaminants may be found on surfaces. 

Depending on the manufacturing process and the product, certain types of contaminants may have a greater or lesser impact. A risk analysis and an understanding of the process-product relationship can help determine which types of contamination need to be addressed in the bio-cleaning process.

Disinfection residues may fall into this category. Their accumulation must be controlled.

Options available for cleaning a cleanroom

Once the contaminants in the cleanroom have been identified, we can turn our attention to implementing the actual cleaning procedures. These various factors can be simply summarized using Sinner’s circle, which identifies four key areas for establishing an effective “cleaning” process (in the broadest sense).

2.2 The various solutions available depending on the use case

The selection of cleaning products for cleanrooms is a critical factor in ensuring process efficiency while preventing the introduction of new contaminants. These products must meet strict criteria regarding purity, compatibility with materials, low particulate emissions, and non-reactivity with the substances handled in the controlled environment.

The various active ingredients

There are three main categories of detergents, which can be classified based on their pH: acidic products with descaling properties, alkaline products with strong degreasing power, and pH-neutral products designed for gentle surface cleaning.

The use of pharmaceutical-grade water (filtered, deionized, or for injection) can also be included in these categories. Depending on the degree of soiling, water can be used effectively as a simple cleaner (for dusting, for example) or as a rinsing agent, which is effective against chemical detergent residues.

Finally, there are very specific product categories with limited applications that are highly targeted to local issues, such as enzymatic products.

The appropriate choice of product therefore depends largely on the type of contamination to be removed and must be specified in the decontamination protocols issued by the CCS. To ensure maximum effectiveness, the use of detergent is generally combined with mechanical cleaning, such as a wipe or cleanroom mop.

Wipes and technical textiles 

Textiles used in cleanrooms must be designed to minimize the release of particles and fibers. They are typically made from low-lint synthetic materials and packaged in a cleanroom.

There are several types of cleanroom textiles:

• Knitted polyester: highly durable, low-pilling, suitable for high-wear areas.
• Microfiber: high absorbency, effective at trapping fine particles.
• Nonwoven (cellulose-polyester, polypropylene): used in less critical areas or for pre-cleaning operations.
• Pre-impregnated: a fabric that has already been saturated with detergent or rinse solution, ensuring even application and minimizing dosing errors.

2.3 Conclusion

As we have described earlier, the cleaning process is essential for controlling contamination in cleanrooms.

Cleaning in cleanrooms must be carefully planned and evaluated based on the specific requirements of each environment, and must take into account several factors, including: 

• Chemical compatibility: Products must be compatible with the materials present (resins, stainless steel, polymers, etc.) to prevent corrosion or degradation.
• Storage and traceability: Products must be stored under controlled conditions, with full traceability (batch number, opening date, expiration date).
• Operator training: Improper use (overdosing, inadequate rinsing, mixing of products) can lead to residues or undesirable reactions.

In addition to using a cleaning product, it is also very important to choose the right fabric to achieve the best possible results.

Finally, it is also important to emphasize the importance of cleaning prior to the disinfection step. Indeed, as described in Appendix 1 of the GMP guidelines, it is essential to begin the disinfection step on a surface that has been cleaned beforehand; otherwise, the disinfection step may be ineffective.

3. Disinfection in a cleanroom

Disinfection is the process by which the number of microorganisms (viable contamination) on a surface is reduced through the irreversible action of a chemical agent on their structure or metabolism, to a level appropriate for a specific use. 

In sterile manufacturing, disinfection is part of the CCP and always follows a preliminary cleaning step to remove dirt and organic residues. The disinfection protocol must be documented, validated on-site, and monitored. The product alone is not enough; it is the combination of the product, the method, the surface, and the routine that ensures control.

Key disinfection practices involve prioritizing the reduction of the microbial load on critical surfaces and equipment. It is important to combine broad-spectrum action against bacteria, yeasts, and fungi with periodic sporicidal treatments. These protocols must be documented and monitored, with the rotation of disinfectant classes adjusted to limit microbial resistance.

3.1 Regulations and Standards: Framework and Practical Implications

Disinfectants (also known as biocidal products) are strictly regulated to ensure human safety and environmental protection, and to reduce the risks associated with improper use (toxicity, emissions, byproducts). Three main European legislative frameworks apply and must be verified for any biocidal product:

• REACH Regulation (EC) No. 1907/2006 (amended by (EU) No. 453/2010): Registration, Evaluation, Authorization, and Restriction of Chemicals. It affects the availability of active ingredients and imposes safety requirements on manufacturing and marketing.
• CLP Regulation (EC) No. 1272/2008: Classification, labeling, and packaging of substances and mixtures. It mandates safety data sheets (SDS), pictograms, hazard statements, and packaging/labeling requirements.
• Biocidal Products Regulation (BPR) (EU) No. 528/2012: It regulates the authorization of biocidal products marketed in the EU. It requires the evaluation of active substances and the authorization of formulations based on their intended use.

The BPR categorizes uses into 22 product types (PTs) that define a product’s scope of authorization. Among these, the most relevant for cleanrooms include PT2 (Disinfectants and algaecides not intended for direct application to humans or animals) and PT4 (Surfaces in contact with food and feed). 

Claims regarding the efficacy of biocidal products must be supported by standardized tests, typically conducted in accordance with EN 14885. The process is generally divided into three phases:

Phase 1: Basic suspension assays to demonstrate intrinsic activity.

Phase 2: Step 1 (realistic suspension) and Step 2 (tests on carriers). This is where most of the practical evidence is generated. Phase 2 tests include suspension methods with organic load (simulating clean/dirty conditions) and surface tests that replicate actual application (e.g., use of wipes, DSVA tests). Phase 2 tests evaluate not only log reduction but also the effect of application mechanisms and the risk of contaminant transfer.

Phase 3: In-use testing or testing under real-world conditions, which confirms performance in the operational environment (materials, wiping technique, effective contact time).

The practical implications of these standards are straightforward and must be incorporated into disinfection procedures. It is essential to select products that comply with GMP, REACH, and CLP regulations, to retain certificates and technical data sheets, and to document in-use validation (protocols, neutralization, and shelf life of prepared solutions). The management of dilutions and storage must also be incorporated into the procedures.

Annexe 1 requires additional precautions depending on the zone level. For example, in zones A/B, products must be sterile, or justification for non-sterility must be provided in accordance with the CCS. It is also necessary to justify the storage of dilutions and to regularly monitor the program to detect deviations. 

3.2 Active ingredients: characteristics and considerations

The choice of active ingredients is based on the required spectrum of activity, material compatibility, and operator safety. A pragmatic strategy involves combining broad-spectrum biocidal products with periodic sporicidal treatments, alternating between different classes of agents to reduce the risk of microbial adaptation.

Below is a summary of the most commonly used active ingredients and their properties, which will help you select the appropriate biocides in accordance with established GAPs. (Figure 4)

Practical considerations

In addition to selecting the active ingredient, several practical considerations must be taken into account. Ready-to-use formulations are particularly advantageous because they minimize preparation errors and offer a longer application window under actual usage conditions. The addition of surfactants, additives, or stabilizers can affect wetting, penetration, and the stability of the active ingredient, as well as the presence of residues after use. 

It is also crucial to verify the compatibility of materials (stainless steel, aluminum, polymers, coatings, HEPA filters) through testing and to prioritize testing on representative surfaces, while validating procedures in accordance with GCP.

It is essential to conduct on-site tests to measure effective contact time, assess neutralization and residues, verify the impact on surfaces (corrosion, tarnishing), and, if necessary, monitor using local strains. It is also important to document the shelf life of prepared solutions, storage conditions, replacement frequency, and safety instructions, including the use of personal protective equipment (PPE) and ventilation.

Work-Study Program

Appendix 1 recommends alternating between different types of disinfectants with distinct modes of action. In addition, their combined action must be effective against bacteria, yeasts, and molds.

This rotation should be tailored to the production environment, the contaminants present, and applicable constraints. For example, daily cleaning can be performed using a narrow-spectrum product, such as alcohol or AMQ depending on the surface, supplemented by a periodic rotation (weekly or biweekly) that includes an oxidizing agent or a chlorine-based product to ensure sporicidal action. The frequency should then be adapted to the local microbial load and revised based on environmental monitoring. 

3.3 Conclusion

Disinfection in cleanrooms, as required by Annex 1, is a technical and regulated process that requires a documented, validated, and monitored strategy. It includes preliminary cleaning, the rotation of disinfectant classes—including a sporicide—in-use validation on representative surfaces, and testing against local strains. The choice of disinfectant must take into account the required spectrum of activity, material compatibility, safety, and regulatory requirements (BPR/REACH/CLP).

It is essential to consider and document the concentration, contact time, temperature, pH, presence of organic matter, application method, and applied dose. In practice, it is crucial to optimize and control the parameters of the “Sinner’s circle”—chemistry, mechanics, time, and temperature—for each product-surface combination.

4. Conclusion

Controlling contamination in cleanrooms is a complex and sensitive issue. This approach is an integral part of a pharmaceutical company’s procedures and SOPs, particularly within a Contamination Control Strategy as defined in Annex 1 of the GMP guidelines.

Among the principles outlined, we have emphasized the importance of two of these steps: cleaning and disinfection.

Cleaning, a process designed to reduce physical contamination in the environment, is carried out using specialized cleaning products in combination with mechanical action performed using a cloth suitable for cleanrooms. 

Once the surface has been cleaned, the disinfection process can begin. This requires the use of a biocidal product whose active ingredient targets the microbial load present in the area.

In addition, it is important not to overlook the steps that come before cleaning and disinfection, such as tidying up and decluttering work areas. 

References

• 1. GMP Guide, Appendix 1: Manufacturing of Sterile Preparations. ANSM. https://ansm.sante.fr/uploads/2024/06/13/20240613-guide-bpf-2024-2.pdf
• 2. European Chemicals Agency (ECHA). https://echa.europa.eu/fr Applied Microbiology and Biotechnology, 63(5), 534–541.
• 3. EU Regulations: REACH, CLP, BPR  Official Journal of the European Union / ECHA. https://echa.europa.eu/fr
• 4. EN 14885 standard, CEN/AFNOR. Journal of Food Science, 72(7), pp. 233–239.
• 5. Bharti, B., et al., 2022. Recent advances in sterilization and disinfection technology: A review. Chemosphere, 308, p. 136404. Journal of Food Protection, 58(5), 499–504.