How to Use an Ultrasonic Fogger to Conduct a Smoke Study in a Pharmaceutical Barrier Isolator?
Pharmaceutical companies needing a smoke study in a large Barrier Isolator, ISO Suite or cleanroom could use Ultrasonic AFM40 Cleanroom Fogger, which is easy to start up using WFI (water for injection), sterile water or high-purity DI water. And the AFM40 has two 80mm fog outlets to get more fog out These liquids are pure enough to produce a pure fog for airflow visualization. The fog droplets are about 8-10 microns in diameter and exit the fogger at about 65-70F temperature. It poses no chemical risks, does not affect oxygen levels, and can be precisely controlled in terms of fog control in direction, duration and how the fog is disseminated in the area using a stream fog, or Fog Rakes to spread the fog density out in a wide fog pattern. The AFM40 is particularly well suited to smaller to medium-sized isolators, to studies being conducted during or adjacent to aseptic processing qualification activities, and to situations where the study protocol needs to be executed quickly with minimal setup time. If much higher levels of fog volume and fog purity is required, consider the Apollo 100 LN2 Cleanroom Fogger with dual 80mm fog outlets; or consider the Apollo 150 LN2 Cleanroom Fogger with three 80mm fog outlets for maximum fog in a smoke study. Both of these LN2 cleanroom foggers produce a range of ultrapure fog from low volume for small smoke studies to high volume for large airflow visualization in smoke studies. Here is a cleanroom fogger comparison of each of our cleanroom foggers.
Understanding the Regulatory and Technical Context
Before walking through the procedure, it helps to be clear about what regulators and standards actually expect. GMP Annex 1 (the 2022 revision) places significant emphasis on the Contamination Control Strategy (CCS) and specifically calls for airflow visualization studies as part of the qualification of aseptic processing environments. ISO 14644-3 provides technical guidance on airflow visualization testing methods, and ISO 14644-1 establishes the classification framework within which barrier isolators and Restricted Access Barrier Systems (RABS) operate. USP <797> and USP <800> reference airflow requirements for compounding environments, and while they don’t prescribe a specific fog method, the underlying principle of demonstrating unidirectional airflow and first air protection is consistent across all of these frameworks.
What none of these documents do is mandate ultrasonic fogging specifically. What they require is that you demonstrate airflow behavior under representative conditions. How you generate the tracer aerosol is largely a methodological choice, and the choice of ultrasonic fogging for an isolator is usually a practical one: it’s controllable, relatively low-risk, and easy to stop quickly if something goes wrong during the study.
Pre-Test Preparation
Good smoke studies don’t happen by accident. The preparation phase is where most of the protocol weaknesses show up if they’re going to show up at all.
Water quality verification. This is non-negotiable. Ultrasonic foggers aerosolize whatever is in the water, and if that water contains minerals, endotoxins, or microbial contamination, those contaminants become airborne particulates and residue on product-contact surfaces. Use water for injection (WFI) or, at minimum, high-purity deionized water with a confirmed conductivity and TOC specification that aligns with your contamination control strategy. Document the water lot, source, and any in-process testing. Some sites run a residue check by fogging onto clean glass and examining under UV light before the formal study.
Equipment setup and inspection. Inspect the fogger unit itself before use. If there’s any visible residue in the reservoir from a previous use, clean and rinse it with WFI before filling. The transducer should be clean and undamaged. Confirm the output rate is appropriate for the volume of the isolator. A unit that produces fog faster than the enclosure can carry it away will over-fog quickly and obscure the visualization rather than support it.
System qualification checks. Confirm the isolator is operating within its qualified parameters before the study begins. That means verifying that the HEPA filtered supply is functioning, that the pressure cascade is within specification, and that glove ports are in their normal operational configuration. The air change rate should be at normal operational settings, not elevated or reduced. You want the smoke study to reflect the conditions under which product is actually manufactured.
Documentation and video setup. You need video. Still images are useful for reports but are rarely sufficient on their own to demonstrate dynamic airflow behavior. Position cameras to capture the critical process zone from multiple angles if possible. Lighting is often the limiting factor inside isolators because the enclosure walls can create glare. Test your lighting setup before the study begins. A high-contrast background behind the area of interest, if you can arrange it, makes fog patterns significantly more visible on video. Document the camera positions and lighting setup in your study protocol so the observation conditions are reproducible.
Personnel in gloves. If the study is intended to represent aseptic manipulations, then someone should be at the glove ports during at least some of the fog introduction phases. Arm insertion through glove ports is one of the most significant sources of turbulence in an isolator environment, and a study conducted without any glove port interaction doesn’t fully characterize the airflow behavior under representative conditions.
Fog Introduction Technique
This is the part where things go wrong most often in practice, usually because the engineer introduces the fog too fast or in the wrong location.
Position the fogger so that it introduces fog at or near the HEPA supply air level, not at the work surface. The intent is to allow the fog to be entrained into the existing airflow pattern rather than injecting it against that pattern. If you introduce fog at a point of high velocity downward flow and aim the fogger upward, you’ll create turbulence that isn’t representative of normal operations and your video will show disturbed airflow that the isolator isn’t actually producing.
For a standard horizontal or vertical unidirectional flow isolator, start with the fog introduced upstream of the critical process zone, at the perimeter of the HEPA supply face. Let the fog develop for a few seconds before making any interpretations. The first several seconds after fog introduction often show a transient that isn’t representative of steady-state behavior.
Introduce the fog in short bursts when possible rather than continuous output, particularly at first. This lets you see how the airflow carries each pulse and gives you time to observe without the enclosure becoming uniformly saturated with fog.
Once the baseline flow pattern is established from an empty setup, introduce representative equipment and containers. Observe how the fog behaves around obstructions. Dead zones often appear as areas where fog accumulates or stagnates while the surrounding flow continues to move. Reflux patterns typically appear as fog moving counter to the primary flow direction, often near enclosure walls or behind taller equipment.
Observing Airflow Characteristics
First air protection. The most critical observation in any aseptic barrier isolator smoke study is whether first air, meaning unobstructed air directly from the HEPA filtered supply before it contacts any surface, actually reaches open product and container closures. Fog introduced upstream of an open vial or syringe filling position should visibly sweep over and around that position without interruption from reflux or eddy currents. If the fog consistently bypasses the critical surface or if backflow from downstream positions occasionally interrupts the flow pattern, that’s a finding that needs to be addressed before the isolator is used for aseptic processing.
Unidirectional airflow verification. ISO 14644-3 describes unidirectional airflow as parallel flow at a consistent velocity across the working zone. In practice, perfect parallelism doesn’t exist, but the expectation is that flow direction remains consistent and that no large-scale recirculation or stagnation zones exist within the critical process zone. The smoke should move in a reasonably coherent direction. Where it doesn’t, the deviation should be documented and evaluated in the context of the process being performed there.
Pressure cascade integrity. While the smoke study doesn’t directly test pressure differentials, it can provide indirect evidence of pressure cascade behavior at interfaces such as the transfer hatch. Watch how fog behaves when the transfer hatch door is opened during a simulated transfer operation. If fog moves from the lower-classification zone toward the isolator interior rather than the reverse, that’s relevant to your contamination control strategy and should be documented.
Glove port interaction. Arm insertion causes a temporary pressure and flow disturbance in most isolator configurations. Observe whether this disturbance resolves within a reasonable time after the arms are stationary. Some level of transient disruption is expected and acceptable; persistent turbulence that doesn’t resolve while arms are in place at the glove ports is more concerning.
Limitations of Ultrasonic Foggers in Isolator Applications
Ultrasonic fogging works well in this application within specific conditions, and it’s worth being clear about where those conditions break down.
Droplet evaporation. Ultrasonic foggers produce relatively fine droplets, but they are still liquid aerosols and they will evaporate. In a warm, dry environment or one with high air velocity, the fog may dissipate before you’ve had time to make useful observations. This is a real limitation in some isolator configurations, particularly those running at higher air change rates or elevated temperatures. The fog becomes lighter as droplets evaporate, which can cause it to rise and stratify rather than following the actual air movement.
Residue risk. If the water quality is poor, the mineral content and other dissolved solids in the water remain on surfaces after the water evaporates. Even with WFI, if you over-fog an area repeatedly, you may deposit enough residue to be detectable on product-contact surfaces. This is why studying with the actual process setup present, rather than a clean empty isolator, increases the stakes around water purity. Some validation teams choose to conduct the smoke study after completing media fill simulation runs or on a separate, dedicated study isolator to avoid any possibility of residue contamination of the production unit.
Over-fogging. Too much fog obscures rather than reveals. When the enclosure becomes uniformly saturated, you lose the contrast needed to distinguish flow patterns. This is especially problematic in smaller isolators. Less fog, introduced more strategically, almost always produces better visual data than filling the enclosure with as much fog as the unit can generate.
Oxygen displacement is not a concern. Unlike liquid nitrogen-based fog systems or CO2-based generation methods, ultrasonic water foggers do not displace oxygen. This is a meaningful advantage in a contained space like an isolator where personnel are working through glove ports. There is no need for oxygen monitoring or personnel evacuation protocols with ultrasonic water-based fogging.
Troubleshooting Common Problems
Fog dissipates too quickly. This usually indicates that the air velocity is high, the relative humidity inside the isolator is very low, or both. Check environmental conditions before starting. If the isolator is operating in a particularly dry or warm environment, you may need to pre-condition the enclosure by allowing some fog to saturate the internal atmosphere slightly before conducting formal observations. Alternatively, consider a glycol-based aerosol if the protocol allows it, since glycol droplets are more stable than pure water at common pharmaceutical cleanroom conditions.
Visibility is too low. Before adjusting anything, check your lighting. Poor contrast between the fog and the background is more often a lighting problem than a fog density problem. Increasing fog output when visibility is the issue often just makes things worse. Try adjusting the angle or intensity of your light source first.
Fog is sinking. Water fog sinks when the droplets are too large or when the enclosure air is significantly warmer than the fog output temperature. Larger droplets settle faster than smaller ones. If your unit is producing visible mist that drops immediately rather than following horizontal or vertical air currents, the output may be too coarse for useful flow visualization in that environment. Switching to a unit that produces smaller droplet sizes, or slightly warming the water reservoir, can help.
Excess turbulence from positioning. If the fogger itself is generating turbulence visible in the video, reposition it so that the output nozzle or diffuser is oriented parallel to, rather than against, the primary airflow direction. The fogger should be a passive source of tracer aerosol that the existing airflow carries, not a competing airflow source.
Post-Test Purge and Cleanup
After the smoke study is complete, allow the isolator’s ventilation system to purge the residual fog completely before accessing the interior. Document the purge time. Wipe down all internal surfaces and equipment with a validated disinfectant or WFI-dampened wipe as appropriate for the isolator’s surface materials and your contamination control procedures. Inspect the HEPA supply filters visually if accessible. In most cases, a properly conducted study with WFI will leave no detectable residue, but a wipe-down is a reasonable precaution and good practice for maintaining the isolator’s environmental baseline.
Remove the fogger reservoir contents and rinse the reservoir with WFI. Do not leave water sitting in the reservoir between uses, particularly in an aseptic environment, as standing water in fogging equipment is a known microbiological contamination risk.
When Ultrasonic Fogging is the Right Choice, and When It Isn’t
For most pharmaceutical barrier isolator smoke studies, an ultrasonic fogger using WFI or high-purity DI water is a practical, well-controlled, and appropriate method. It produces neutral-density aerosol under typical cleanroom conditions, poses no chemical risk, does not affect oxygen levels, and can be precisely controlled in terms of output volume and duration. It’s particularly well suited to smaller to medium-sized isolators, to studies being conducted during or adjacent to aseptic processing qualification activities, and to situations where the study protocol needs to be executed quickly with minimal setup complexity.
Where ultrasonic fogging becomes less ideal: in very large barrier isolators, RABS and ISO Suites with high air volumes and fast air change rate; the ultrasonic fog may not be visible long enough to be useful in environments with a water-based fog. If you need a much more visible fog providing ultrapure fog at much higher fog volumes, combined with fog volume control and fog velocity control, then consider our two best LN2 Foggers for your ISO smoke study or USP 797 controlled smoke study. The Apollo 100 LN2 Cleanroom Fogger produces up to 16 cubic meters fog per minute total output through two 80mm fog outlets, which equates to 8 cubic meters of fog through each of the two fog outlets. The travel distance of LN2 fog is greater than ultrasonic fog since the ultrapure droplets are 2-3 microns in diameter at a much higher fog density. The Apollo 150 LN2 Cleanroom Fogger provides with three 80mm fog outlets at 24 cubic meters ultrapure fog per minute total output, which equates to 8 cubic meters of ultrapure fog through each of the three 80mm fog outlets.
The bottom line is that the fog method combined with the right cleanroom fogger is the right method to achieve a certified airflow smoke study. What matters is whether the smoke study produces clear, certified, visual evidence of airflow uniformity; clearly describing airflow turbulence and airflow dead zones, which can now be corrected in order to certify the smoke study. This supports a GMP Annex 1 airflow visualization requirement, USP 797 requirement and ISO smoke study requirement. Ultrasonic fogging using pharmaceutical-grade water, properly positioned and carefully documented on video, achieves this goal for smaller smoke studies; while the LN2 Cleanroom foggers achieve the certification goals in larger area, smoke studies.
