Mold Risk in Cleanrooms: A Holistic Control Approach

By adopting best practices and cross-functional collaboration, manufacturers can strengthen contamination control strategies and safeguard both product quality and patient safety.

Background: The Criticality of Contamination Control

Mold is a resilient and ubiquitous contaminant that poses a significant risk to pharmaceutical cleanrooms if not properly controlled. In this article, practical prevention and control strategies are discussed to support manufacturers in developing robust, compliant mold control programs that protect product quality and patient safety. In the rigorous world of pharmaceutical manufacturing, maintaining cleanroom environments is paramount to ensure product quality and patient safety.

The three pillars of contamination within the pharmaceutical industry include particulate (physical), chemical (including endotoxin and mycotoxin), and microbiological contamination—a concept well established in GMP guidance and industry standards. EudraLex Volume 4 Annex 11 and Pharmaceutical Inspection Convention/Pharmaceutical Inspection Co-Operation Scheme (PIC/S) Annex 12 consolidate this imperative by requiring a documented, facility-wide contamination control strategy that addresses them explicitly.

PIC/S Annex 1 also elevates the importance of molds (fungi) and spore-forming microorganisms control as indicative of overall contamination control with statements that include the following:

• “9.11; iv. — Particular attention should be given to organisms recovered that may indicate a loss of control, deterioration in cleanliness or organisms that may be difficult to control such as spore-forming microorganisms and molds.”
• “9.31 — following the isolation of organisms that may indicate a loss of control, deterioration in cleanliness or that may be diffi-cult to control such as spore-forming microorganisms and molds.”

The consumption and administration of contaminated drug products present varying levels of risk to patients and manufacturers. Mold-contaminated products have been recalled globally due to risk of serious infections, strong, unpleasant odors, etc., causing temporary facility shutdowns and product shortages. More importantly, mold-contaminated products have caused death and illness. In 2012, the New England Compounding Center disaster killed over 60 people and made nearly 800 ill due to fungal contamination of an injectable steroid.3

Such occurrences of contaminated drug products highlight the importance of contamination control processes and are the reason this remains an area of focus for regulators. Pharmaceutical manufacturers are responsible for ensuring the quality of their products. This includes protecting the drug product from contamination through holistic, science-based control measures. To systematically address contamination risks, manufacturers can leverage risk management tools such as Risk-Based Manufacturing of Pharmaceutical Products (Risk-MaPP), which provides a structured approach to identify potential hazards, assess their likelihood and impact, and define appropriate control measures and monitoring activities in line with ICH Q94 principles.

The ubiquity of mold in day-to-day general environments means that anyone or anything that enters classified areas can serve as a vector for contamination. Once mold enters the pharmaceutical cleanroom, eradication and control can be challenging due to its resilience to heat and chemicals. Once spread to new environments, mold can remain dormant for extended periods of time. This article explores the sources and risks of mold contamination, its impact on cleanroom environments, and effective holistic strategies for prevention and control.

Understanding Mold Growth and Proliferation

Mold is a type of fungus that is ubiquitous in our environment and can be found in air, plants, soil, and water. In nature, mold is considered one of nature’s greatest recyclers as it breaks down complex organic materials, such as decaying matter, into simpler forms through the release of enzymes.

Mold typically grows on the surface of materials, but beneath the surface it develops a complex root -like network called mycelium, which consists of fine filamentous structures known as hypha or hyphae. Unique among common microbiological contaminants, mold often becomes visible to the naked eye due to the formation of long branching structures called conidiophores, which gives mold its fluffy or fuzzy haired appearance. At the end of the conidiophores are spores (i.e., conidiospores), which allow mold to spread and proliferate through asexual reproduction (see Figure 1).

These spores act like microscopic seeds that disperse easily through air currents, in an equivalent way to how dandelion seeds are carried by the wind. Mold spores can be transferred by air movement caused by differential pressures or by personnel carrying them on clothing or equipment such as tools or trolleys. Once settled on a surface, spores can germinate and grow if the right conditions are present. Optimal mold growth depends on four key requirements: a source of nutrients, sufficient water/humidity, suitable temperature, and oxygen. Understanding these four parameters is critical for controlling the presence of mold in pharmaceutical cleanrooms (or any other location) (see Figure 2).

• Nutrients: Mold requires an appropriate carbon source as nutrients.
• Water/humidity: Damp or humid conditions promote mold growth.
• Temperature: Many mold species are mesophilic, thriving at temperatures between 18°C–25°C. However, certain types of molds can grow at lower and higher temperatures, too. For example, Cladosporium species grow best within this range but can also grow at lower temperatures, albeit at a slower rate.6 Temperature within cleanrooms is usually controlled between 20°C–25°C.
• Oxygen: Most mold species are aerobes and require oxygen for growth. Reducing oxygen levels in cleanrooms is not advised due to the obvious impact on personnel safety.

Potential Routes of Ingress

Mold can enter cleanroom environments through various pathways, including but not limited to incoming materials, equipment, personnel, and facility infrastructure.

• Materials and equipment: Material transfer processes—such as wooden pallets, cardboard, trolley wheels, or maintenance tools—can harbor mold if not properly controlled or cleaned.
• Personnel: Operators can carry mold spores on their clothing, skin, or personal items if the gowning strategy is not well designed and/or not executed correctly.
• Facility design: Poor facility integrity—such as gaps in doors or windows, and air or water leaks in the building envelope—can allow external contaminants to enter classified spaces.
• Poor internal facility configuration and material choices can allow pooling of water or create uncleanable recesses and enclosures that can allow mold to gain a foothold within the facility.
• Heating, ventilation, and air conditioning (HVAC) design: Insufficient filtration, improper pressurization, poor choice of HVAC materials, and insufficient ventilation can allow external contaminants to enter classified spaces and can provide a location for mold to proliferate, increasing the risk of contamination.

Figure 1: The structure of mold.5

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Figure 2: Requirements for mold growth.

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Control Through Monitoring

Environmental monitoring (EM) programs should be designed to provide assurance that microbial contamination is controlled, and to detect excursions from environmental limits or microorganisms that may indicate a loss of control. EM programs should be based on comprehensive risk assessments to establish appropriate sampling locations, frequencies, monitoring methods, and incubation conditions. Nonselective media such as Tryptone soya agar, soybean casein digest medium, or selective media like Sabouraud dextrose agar can be used to recover and detect mold. Analysis of EM and microbial identification data should be performed periodically to identify potential trends. For example, an analysis of microbial identification data can help identify shifts in microbial flora, which may be indicative of a loss of control.7

Never locate cleanrooms directly against an exterior wall. Always provide a ventilated and inspectable gap, such as a service corridor, between exterior walls and cleanroom areas to serve as an extra barrier against moisture infiltration.

Investigating Mold Contamination

Effective investigation of mold contamination requires a structured, science-based approach that goes beyond routine microbiological testing. Although there is often a tendency to assign ownership of microbial out-of-specification events solely to the quality control microbiology department, robust investigations should always involve a multidisciplinary team. This team typically includes representatives from manufacturing, engineering, microbiology, and quality assurance to ensure that all potential sources and contributing factors are assessed.

A comprehensive investigation should start with a thorough review of EM data and relevant trends. Trend analysis helps identify recurring deviations, persistent hot spots, or systemic weaknesses that may contribute to mold ingress or proliferation. Additionally targeted EM can be deployed to pinpoint potential sources. Visual tools, such as measles maps5—facility diagrams overlaid with contamination recovery points—help illustrate spatial trends and focus the investigation on specific areas, equipment, or processes.

Root cause analysis tools such as fishbone (Ishikawa) diagrams, the 5 Whys method, and failure mode and effects criticality analysis (FMECA) can help teams systematically identify the most probable root causes and contributing factors. Corrective and preventive actions (CAPAs) must be evidence-based and proportionate to the identified risks. CAPAs should never be implemented prematurely; cleaning or disinfection alone is not sufficient unless the source of the contamination is confirmed and eliminated. Once the root cause has been addressed, it is essential to verify the effectiveness of the actions taken. Effectiveness checks—such as follow-up EM, additional trend reviews, and visual inspections—help confirm that the contamination has been fully resolved. Where the root cause cannot be determined immediately, intensified cleaning, disinfection, and enhanced monitoring can help locate persistent sources.

Implementing Prevention and Control Strategies

To minimize the risk of mold contamination, manufacturers should adopt a comprehensive, multi-layered prevention and control program.

Facility Construction and Design

All surfaces should be smooth, sealed, and designed to withstand repeated cleaning and disinfection. Joints must be properly sealed to prevent water pooling, which can promote mold growth. Damaged or cracked surfaces should be promptly repaired to prevent development of contamination due to accumulation of moisture and dirt.

Never locate cleanrooms directly against an exterior wall. Always provide a ventilated and inspectable gap, such as a service corridor, between exterior walls and cleanroom areas to serve as an extra barrier against moisture infiltration. Avoid using wood inside walls, above ceilings, or within any cleanroom envelope. Likewise, do not use paper-faced gypsum board in cleanrooms; instead, specify moisture resistant, antimicrobial (preferably glass-faced) gypsum board only. This significantly reduces the availability of nutrients that can support mold growth.

All mechanical equipment installed above cleanrooms should be fully sealed with watertight finishes or equipped with appropriate containment pans. Ensure that pans are installed beneath floor drains and drain lines to catch leaks or condensation. Do not rely on general construction air plenums or positive pressure return-air plenums in cleanroom areas. Use only sealed sheet metal ductwork to maintain proper air control.

Airflow and Ventilation

Interstitial spaces above cleanrooms should always be ventilated to avoid stagnant areas that can accumulate moisture. Clean rooms should always be positively pressurized to adjacent uncontrolled spaces. Pressure differentials should generally prevent backflow from less-clean to more-clean spaces within cleanroom environments. Terminal high-efficiency particulate air (HEPA) filters at the cleanroom boundary can prevent contamination from entering the cleanroom via HVAC, even in low-grade cleanrooms.

Because mold spores can spread through passive or active means, they may become airborne and settle onto surfaces or products, especially in areas with poor ventilation. HVAC is an important tool to dilute airborne mold spores in cleanrooms and to transport them out, safely capturing them on dry filters. Airflow visualization studies can help verify airflow direction and detect any dead spots where mold could grow. Properly sealed walkable ceilings provide an added defense against leaks and facilitate safer access for inspection and maintenance.

HVAC Systems

Mold spores can disperse through air currents. Well-designed air handling systems, appropriate pressure differentials, and air visualization studies can help ensure consistent airflow and minimize stagnant zones where mold might grow. Controlling room relative humidity below 60% also helps limit mold proliferation while avoiding levels so low that static build-up becomes a hazard.8

Air handling unit (AHU) outdoor air sections must be adequately sized and designed to prevent wind-driven rain or snow from entering the system. Snow control is particularly challenging and typically requires very low-velocity louvers to minimize risk. Snow melting and drain systems in plenums can prevent water leakage. Whenever possible, secondary AHUs should be designed for sensible heat control only; however, they may still produce condensate during start-up phases. This should be anticipated during commissioning to prevent excess moisture from migrating into cleanrooms.

HVAC maintenance is essential to ensure that the system operates as intended and to limit contamination risks. Pre-filters should be inspected regularly and replaced as needed, ensuring there is minimal bypass of unfiltered air. Bag or pocket filters are often preferred over pleated filters because they tend to dry more quickly if exposed to moisture, reducing the potential for mold growth inside the system. Air filters should be installed in gasketed frames to maximize mold spore removal. High-efficiency or HEPA final filters in AHUs can provide the greatest assurance of exclusion of airborne mold spores from the HVAC system. HEPA filters should be protected from water and excessive relative humidity above 80%.

For coils that are wet for extended periods or for coil banks larger than six rows, consider installing more robust intermediate filters—such as MERV 13 or F8 filters (ASHRAE 62.1)—to help capture additional particulates before they reach critical cooling components. Cooling coils should be designed for low system air velocities to avoid moisture carryover into downstream ductwork. The use of UV-C light or other coil-cleaning technologies can also help keep coils and drain pans clean and dry when they are routinely exposed to moisture.

Coil condensate and AHU floor drain traps must be properly sized and configured for actual system pressures to ensure effective drainage. A preventive maintenance program must be in place to inspect and confirm that these traps do not become clogged or cause water backup, which could otherwise increase humidity and support mold proliferation. Proper maintenance of HVAC filters, coils, ductwork, and drain pans can play a critical role in reducing the spread of airborne mold spores throughout the cleanroom environment.4

...although 70% alcohol is effective against vegetative bacteria and yeasts, it is not sufficient alone for mold, as it may not fully eliminate the root structure.

Equipment and Materials

Material transfer processes should be supported by risk assessments that identify contamination hazards and define critical control points. Cleaning and disinfection steps should be validated and documented. Materials such as cardboard and wooden pallets should be managed carefully, as they can serve as a food source for mold. They should not be allowed beyond the initial warehouse. Facility flows should be designed to reduce the risk of transport of contamination by application of multiple cleaning/disinfection and gowning steps with separation between steps to minimize the risk of contaminant transfer.

Cleaning and Disinfection

A validated cleaning and disinfection program is vital for controlling surface bioburden. When developing the program, consider elements such as a review of the activities and processes within the facility, personnel and material flow, and trends from EM and microbial identification data. The program should include appropriate product rotation to maintain broad-spectrum efficacy against bacteria, bacterial spores, yeasts, and molds.

Disinfectants must have proven fungicidal activity. For example, although 70% alcohol is effective against vegetative bacteria and yeasts, it is not sufficient alone for mold, as it may not fully eliminate the root structure. Cleaning and disinfection activities should take place in an appropriate location that will serve to reduce the risk of recontamination from the environment or contact with less-clean materials and individuals.

Personnel Practices

Personnel flows and gowning processes must be designed to minimize contamination risks. Separate gowning areas for entry and exit, proper gown selection based on cleanroom classification, and clear hygiene protocols all help prevent personnel from introducing mold. Comprehensive GMP training reinforces awareness of how personal practices impact contamination control. Gowning steps should each take place in an appropriate location that will serve to reduce the risk of recontamination from the environment or contact with less-clean individuals.

Practical Case Examples

The following examples illustrate how assessing and adjusting current practices can significantly reduce the risk of mold contamination.

Cold Room Cleaning

A pharmaceutical company observed recurring mold growth at the base of cold room racking uprights. Their routine cleaning process used water and detergent, which inadvertently introduced excess moisture, creating favorable conditions for mold. Following a review, the cleaning procedure was revised to a dry wipe approach with routine inspections for signs of contamination. By eliminating unnecessary moisture, the risk of mold proliferation was significantly reduced.

Facility HVAC System Redesign

At another site, a pharmaceutical plant experienced extensive mold growth within a GMP corridor due to water ingress—HVAC units were housed above the corridor within a mechanical space. An undersized exterior air louver failed to deflect wind-driven rain, which entered the ductwork and infiltrated the corridor wall, leading to hidden mold growth. Regulatory authorities identified the issue, resulting in costly remediation and extended downtime. The long-term solution involved relocating the air intake to the roof and installing a properly sized louver with correct sealing and slope, preventing future water ingress and safeguarding the cleanroom environment.

Facility Envelope Separation

At yet another site, a pharmaceutical plant experienced mold recoveries in low-grade current GMP spaces due to deficiencies in the original building envelope. Disassembly of the entire building envelope was impractical, necessitating the establishment of an interior boundary within the building to protect the cleanrooms. Grade D and controlled not classified corridor materials of construction were improved with a mix of cladding and cleanroom panels forming a nearly airtight boundary to protect the cleanrooms from the ambient environment.

Conclusion

Mold contamination remains a critical challenge in pharmaceutical cleanrooms, demanding a proactive, science-based, and cross-functional approach. Although its sources are diverse and its resilience is significant, the risk can be effectively managed through robust facility design, stringent environmental controls, and disciplined cleaning and sanitization programs validated for efficacy against fungi. Equally important is fostering a culture of shared responsibility from engineering, production, quality, and microbiology. This includes ensuring that investigations are thorough and that CAPAs are rooted in sound risk assessment and implemented with measurable follow-up.

By combining best practices in facility construction, HVAC design, material and personnel flow, and training, manufacturers can strengthen their contamination control strategies and maintain compliance with ever-evolving regulatory expectations. These practices will also help ensure cleanroom environments remain mold-free. Ultimately, protecting cleanrooms from mold means more than safeguarding product quality and patient safety. It is about sustaining trust and upholding the highest standards of pharmaceutical manufacturing.

Acknowledgments

The authors thank the following individuals for their contributions to this article.

Nick Haycocks, RoNiK LLC, Independent Consultant

Norm Goldschmidt, Genesis AEC, President

Mark Muench, Abbvie, Assoc. Director Global Utilities & Facilities

John Tomanovich, Camfil, Life Sciences Segment Manager

Jim Quinn, CRB Group, Senior Mechanical Engineer