Practical Guide to Cleanroom Qualification (OQ/PQ) according to ISO 14644-3

Beyond Particle Classification

Having classified a cleanroom as "ISO 7" or "ISO 8" (according to ISO 14644-1) is only the first step: the classification . But how do we know that the HVAC (Heating, Ventilation, and Air Conditioning) system that creates that cleanliness class is robust, efficient, and properly designed?

This is where plant qualification (specifically Operational Qualification - OQ - and Performance Qualification - PQ) comes into play. The reference standard for "how" to perform these tests is ISO 14644-3: Test methods .

This standard is the go-to manual for every Validation Manager and HVAC technician. Demonstrating that you have performed (and passed) these tests is critical to GMP compliance, as Annex 1 explicitly requires many of these checks (such as filter integrity and flow visualization) ¹⁵¹⁵¹⁵ .

Skipping these tests or performing them superficially means building on a shaky foundation: you may have a compliant "at rest" classification, but a plant unable to handle the contamination generated during production.

Let's see the step-by-step guide to the 5 most critical HVAC qualification tests defined by ISO 14644-3.

Phase 1 (Critical Test): HEPA/ULPA Filter Integrity Test

Why it's done: This is the most important test. We must be 100% sure that the HEPA (High-Efficiency Particulate Air) or ULPA filters, which are the heart of the cleanroom, are installed correctly and have no leaks (pinholes, holes) or bypasses in the mounting frame ^16. A damaged filter ruins the entire system.

How to perform it (ISO 14644-3 method):

1. Aerosol Generation: Upstream of the filter (in the duct or AHU), a challenge aerosol is introduced. Typically, thermal oil (e.g., PAO or DEHS) is nebulized to create uniform particles (often 0.3 µm).
2. Concentration Measurement: The concentration of this aerosol upstream is measured (it must be high enough).
3. Downstream Scanning: Using a photometer (an instrument that specifically detects aerosols), the entire downstream filter surface and, above all, the entire perimeter of the installation frame are slowly scanned.
4. Acceptance Criteria (GMP): For HEPA (H13/H14) filters, leakage detected at any point must not exceed 0.01% of the upstream concentration ¹⁷ .

Please note:

Don't confuse the overall filter efficiency (e.g., 99.97% @ 0.3µm) with the in-situ integrity test. The in-situ (scanning) test looks for localized leaks (the weak spot), not the average efficiency.

Common Non-Conformity: The inspector finds the filter integrity certificate performed at the factory by the manufacturer, but the on-site test (after transport and installation, which are the moments of greatest risk of damage) is missing.

Phase 2: Differential Pressure Measurement

Why it's done: To ensure that air always flows from cleaner areas (e.g. Grade B) to less clean areas (e.g. Grade C), and never the other way around. This creates a "pressure cascade" that acts as an aerodynamic barrier ¹⁸ .

How to perform it (ISO 14644-3 method):

1. Instrumentation: Calibrated (calibrated) differential pressure gauges are used.
2. Measurement: The stable pressure difference between adjacent rooms is measured, with the doors closed.
3. Acceptance Criteria (GMP Annex 1): Annex 1 suggests a differential of at least 10-15 Pascal (Pa) between the different grades ^19191919 .

Please note:

Differentials must be continuously monitored during production (not just during qualification) and must be equipped with alarms (visual or audible) that will activate if the pressure drops below the 20202020 limit. The qualification (OQ) must include testing of these alarms.

Phase 3 (Visual Test): Flow Visualization (Smoke Test)

Why it's done: Numbers (particles, pressure) don't tell the whole story. We need to see how the air moves. The smoke test is essential, especially in Grade A (unidirectional flow), to ensure that the airflow protects the product and washes away contamination.

How to do it (ISO 14644-3 / Annex 1 method):

1. Equipment: A "clean" smoke or fog generator is used (e.g. nebulized deionized water, glycol), which leaves no contaminating residues.
2. "At Rest" execution: It is verified that the flow is laminar (unidirectional), that it covers the entire critical area and that there are no turbulences or dead zones (e.g. stagnation behind the machine) ²¹ .
3. In Operation (Simulated) Testing: This is the key test required by Annex 1 ²² . Operator activity is simulated (e.g. arm movements, opening RABS doors) and it is verified that:
• Air does not flow from "dirty" areas (e.g. operator) to the critical point (e.g. open vial).
• Any turbulence generated by the operator is quickly removed from the airflow.
4. Documentation: It is mandatory to record the tests with video ²³ .

Common Non-Conformity: The company only performs the smoke test "at rest" (with the system empty), which is flawless. The inspector requests the "in operation" video and discovers that the operator's movement creates turbulence that pushes air from the aisle directly onto the filling point.

Phase 4: Measuring Air Speed and Air Changes/Hour

Why it's done: To ensure that the air volume handled by the HVAC is sufficient.

• Unidirectional Flow (Grade A): Air speed is measured to ensure the washing "piston" effect.
• Turbulent Flow (Grade B, C, D): Air changes per hour are calculated to ensure dilution of contamination.

How to perform it (ISO 14644-3 method):

1. Grade A: Using a calibrated anemometer, the speeds are measured at several points (approximately 15-30 cm below the filter).
• GMP criterion: The average velocity is typically in the range 0.36 – 0.54 m/s ²⁴ .
2. Grades B/C/D: The total air flow rate is measured (with a flow meter or by measuring the velocities in the ducts) and divided by the volume of the room.
• GMP Criteria: There are no fixed numbers, but as a reference (from ISPE/ISO 14644-4 guidelines), a Grade B requires >40-60 changes/hour, a Grade C >20 changes/hour ^25252525 .

Phase 5: Recovery Time Test

Why it's done: This test demonstrates the system's robustness . It measures how quickly the cleanroom can return to its "at rest" state after being contaminated (simulating an "in operation" event, such as a door opening or intense activity).

How to perform it (ISO 14644-3 method):

1. Contamination: The room is brought "out of specification" (or to a level 100 times higher than the "at rest" limit) generating particles ²⁶ .
2. Measurement: The particle counter is started and the time (in minutes) required for the particle concentration to return steadily below the "at rest" limit is measured.
3. Acceptance Criteria: Annex 1 (old version) suggested a recovery time of 15-20 minutes ²⁷ . Although the new revision is less explicit, this remains an excellent industry benchmark for demonstrating HVAC efficiency.

Watch out for: A long recovery time (>20 minutes) is a serious symptom. It indicates that the system has insufficient air exchange or poorly designed flows (dead zones) and is unable to handle the contamination generated during normal operation.

📦 Box: Practical Checklist for OQ/PQ Qualification (ISO 14644-3)

Before signing the HVAC qualification protocol, check:

• [ ] HEPA Filter Integrity: Performed on site ? Result <0.01% on all filters and frames?
• [ ] Differential Pressures: Is the cascade correct (e.g. B > C > D)? Are the values stable and >10 Pa?
• [ ] Pressure Alarm Test: Do alarms go off if the differential drops below the setpoint?
• [ ] Air Velocity (Grade A): Is the flow unidirectional and in the range 0.36-0.54 m/s?
• [ ] Parts/Hour (Grades B/C): Is the number of parts sufficient and documented (e.g. >20/h)?
• [ ] Smoke Test (Video): Is there an "at rest" and "in operation" video?
• [ ] Smoke Test (Result): Does the video demonstrate the absence of critical turbulence or reverse flows?
• [ ] Recovery Test: Performed? Is the recovery time acceptable (e.g. < 20 minutes)?
• [ ] Instruments: Are all the instrumentation used for testing (anemometer, photometer, particle counter, pressure gauge) calibrated (calibrated)?

Conclusion

Cleanroom qualification doesn't stop with the particle counting of ISO 14644-1. It's the dynamic and physical tests of ISO 14644-3 (filter integrity, pressures, smoke tests, recovery) that provide true evidence that the HVAC system is robustly designed, correctly installed (OQ), and capable of maintaining the required cleanliness during operations (PQ).

Performing these tests rigorously and documenting them (especially with smoke test videos) is the only way to be ready for a GMP inspection and ensure product protection.

To learn more about testing methodologies, interpret results, and build audit-proof HVAC qualification protocols compliant with ISO 14644 and Annex 1, discover the complete guide " Operational Guide to the ISO 14644 Series " on GuideGxP.com.