Working in a cleanroom depends on precision, discipline, and consistent compliance. Every action, from handling materials to removing waste, has a direct impact on product quality and ultimately on regulatory confidence. During a recent annual cleanroom behaviour training session, a client asked me to address a series of practical questions on how to apply these principles in day-to-day operations. I thought it would be useful to share ten of these, drawing on both regulatory expectations and the lessons learned from real-world cleanroom practice.
- When should sterile wipes be used as barriers, and when are they unnecessary?
Sterile wipes are often used as a barrier between gloved hands and non-sterile surfaces such as carts, chairs, or door handles. The intent is to minimise the risk of transferring contamination from those surfaces into critical areas. However, the benefit is quickly lost if the same wipe is reused for extended periods, as it can itself become a source of contamination. A good practice is to replace wipes at defined intervals (for example, every 30 minutes, or sooner if they become visibly soiled or after multiple contacts).
That said, it is worth questioning whether wipes are always necessary. If surfaces have already been disinfected with peroxide or sterile IPA immediately before use, the additional barrier may provide little extra protection. In these cases, the focus should shift to frequent glove sanitisation, especially after contact with Grade C (ISO 7) surfaces and before transferring materials into Grade A (ISO 5). This approach ensures control without introducing unnecessary handling steps that might create more risk than benefit.
- What do I need to know about disinfecting before Grade A (ISO 5)?
Note: This question was asked in relation to use of manufacturing steps performed within Biological Safety Cabinets.
The objective is always to protect the Grade A environment, and that means non-sterile items must undergo a validated disinfection step at the point of transfer. Gloves should also be sanitised at appropriate points, so they do not become a contamination vector. For routine transfers from Grade C (ISO 7) into Grade A (ISO 5), regulators expect items and gloves to be disinfected, using a validated contact time, immediately before entry. Each transfer should be treated as a fresh risk and managed accordingly.
That said, not everything needs to be wiped on every occasion. Pre-sterilised components with double or triple sterile wraps are controlled through packaging design and aseptic unwrapping, not through additional IPA application inside Grade A. Similarly, items sterilised by autoclave, depyrogenation, or VHP-decontaminated pass-throughs rely on those validated processes as the kill step. Over-sanitisation can be counterproductive, adding unnecessary handling risk. The real control lies in applying the right disinfectant, at the right point, with validated contact time and aseptic technique.
- Why does operator movement matter in a cleanroom?
Airflow is the unseen safety net of every cleanroom. Unidirectional flow ensures that filtered air sweeps across the workspace, carrying particles away from critical sites and protecting exposed product. When this flow is disrupted, even briefly, contamination risk increases. Something as simple as a sudden arm movement can generate turbulence that pulls particles back into the clean zone, undermining the very purpose of Grade A protection. For this reason, operator movements must remain slow, controlled, and deliberate to preserve airflow integrity.
Regulators expect this to be more than theory. Smoke studies are required to demonstrate that airflow patterns remain intact under real operating conditions, and inspectors routinely review them to assess facility design and operator discipline. These studies make the invisible visible, showing how equipment layout and operator behaviour influence laminar flow. The evidence they provide forms the basis for practical rules: minimise unnecessary talking, move with intention, and avoid leaning over open product. In cleanroom operations, airflow is the first line of defence, and protecting it is central to sterility assurance.
- How should particle excursions be handled?
Even short-term spikes in particle counts must be treated seriously. Regulators expect every excursion to be investigated and justified, with clear evidence that potential product impact has been assessed and documented in a scientifically sound way. Dismissing an alert-level excursion without explanation undermines both the contamination control strategy and regulatory confidence in the site’s state of control.
While a single fluctuation may be linked to a specific event such as an operator intervention, a material transfer, or an HVAC adjustment, repeated excursions point to a loss of process control that requires deeper investigation. Trending data over time is essential to distinguish isolated anomalies from systemic issues. The key is to demonstrate that particle monitoring is not a box-ticking exercise, but a meaningful safeguard that ensures the cleanroom consistently protects product quality.
- What’s the best way to manage waste in ISO 5 areas?
Grade A (ISO 5) areas should remain clear and always uncluttered to protect unidirectional airflow and minimise contamination risk. Waste is not just untidy — it disrupts airflow, sheds particles, and can obstruct first air, all of which undermine product protection. For that reason, waste should never be allowed to accumulate on work surfaces or near exposed materials.
The best practice is a “clean as you go” approach, with removal at natural process breaks or when receptacles reach about two-thirds capacity. This balance ensures trash does not build up, while also avoiding unnecessary process interruptions. The principle is simple: maintaining an uncluttered workspace is not cosmetic, it is an essential part of sterility assurance.
- What are the required temperature and humidity levels in a cleanroom?
Cleanrooms are commonly operated at 18–22°C and 30–60% relative humidity, but these values are industry norms rather than regulatory requirements. The actual ranges should be defined through risk assessment, considering product stability, process needs, and facility capability.
Regulators expect the chosen parameters to be scientifically justified and controlled as part of the contamination control strategy. High humidity above 70% can increase risks such as microbial growth and condensation, while excessively low humidity may introduce issues like static build-up or operator discomfort. The key is to demonstrate that environmental conditions are set to protect product quality first, while also enabling operators to perform reliably.
- Is cleaning validation always required?
Cleaning validation is a regulatory expectation, and deficiencies in this area are among the most common findings in FDA 483s and warning letters. The purpose is not simply to “tick a box” but to prove that the cleaning process consistently prevents cross-contamination and effectively removes residues of both product and cleaning agents.
What matters is scientific evidence that the chosen method is robust across different conditions, equipment, and product types. Validation demonstrates that equipment and facilities can return to a controlled, hygienic state that supports sterility assurance and batch release. Without this evidence, manufacturers cannot be confident that the product residues from one batch will not compromise the next. In short, cleaning validation underpins both compliance and product safety, making it one of the most critical elements of any contamination control strategy.
- What do I need to know about glove sanitisation and use?
Gloves are one of the main potential contamination vectors in cleanrooms because they’re constantly in contact with surfaces and materials, which is why they should be sanitised frequently, and especially after touching carts, surfaces and materials.
While it’s important to sanitise regularly, it’s not advised to sanitise right before glove testing, as this can give inaccurate results and won’t represent the real working conditions, which is important to monitor effectively.
And of course, gloves should always be changed if torn, dirty, damaged or have been outside of the cleanroom.
- Can marker pens be used safely in Grade A (ISO 5)?
Note: This question was asked in relation to use of manufacturing steps performed within Biological Safety Cabinets.
Markers can be used in Grade A (ISO 5), but they must be controlled like any other item. Ideally, they should be pre-sterilised; if that is not possible, they must be low-lint, non-porous, and thoroughly disinfected with a validated agent before entry. Once inside, they should be handled aseptically and never used over open product or critical surfaces.
- When is re-cleaning required after a cleanroom has been disinfected?
A cleanroom does not lose its status the moment someone re-enters. Once a full disinfection cycle has been completed and the required contact time has elapsed, the room remains in a validated clean state for a defined period, known as the hold time. This duration is established during Environmental Monitoring Performance Qualification (EMPQ), which demonstrates how long the environment can reliably remain under control after cleaning. Brief entry within this period, such as retrieving extra materials, is acceptable provided there is no open product, proper gowning is maintained, and no critical surfaces are touched.
Re-cleaning becomes necessary if the validated hold time has been exceeded, if the cleaning process was not carried out correctly, or if activities occur that compromise the clean state. Examples include gowning breaches, contamination risks, or open product handling in the room. The emphasis should always be on what the EMPQ has validated the room to tolerate, rather than assuming any entry automatically requires a new cleaning cycle.
Final Word
Working in a cleanroom is about more than compliance — it is about developing consistent habits that safeguard the product, protect the operator, and ultimately ensure patient safety. Focusing on these essentials not only keeps you inspection-ready but also builds confidence that your daily practices support sterility assurance and regulatory expectations.