The validation of the cleanroom shall demonstrate that the production area (in which your products and components shall be exposed to the environment during its manufacturing process) is suitable and appropriate under acceptable conditions and parameters of the design in compliance with the international organization ISO under standard ISO14644.

The cleanroom validation protocol may include the following (14) challenges and testing activities:

1. Room Temperature
2. % Relative Humidity
3. Viable particulate – surface
4. Viable particulate air
5. Non-Viable particulate
6. Pressure Differential
7. HEPA Filter Integrity
8. HEPA Filters Velocities
9. Room Air Changes
10. Room Air Balance CFMs
11. Smoke Test – Airflow pattern
12. Particle Decontamination Test
13. Recovery Test – AHU shut down
14. Sound and Lighting Test

Additional criteria to be considered which affects room cleanliness should include:

• building finishes and structure
• air filtration
• location of air terminals and directional airflow
• material flow
• personnel flow
• gowning procedures
• equipment movement
• the process is carried out (open or closed system)
• outside air conditions
• occupancy
• type of product
• cleaning standard operating procedures (SOPs).

When is required an HVAC System and Cleanroom validation?

The HVAC and cleanroom validation is performed every time an Environmental Control Area (ECA) is required and used for GMP manufacturing purposes.

In case that an ECA is used for the cGMP manufacturing process, the HVAC and cleanroom validation is a mandatory requirement of each environmental controlled area as established by the Federal Register, section 501(a)(2)(B) of the Act (21 U.S.C. 351(a)(2)(B)).

It will guarantee that all necessary environmental conditions shall be available for the intended use.

The cleanroom validation must be performed after completed the facility and HVAC qualification of all equipment installation and machinery connections, supplies, air conditioning, water system, compresses air system, electric power capacity, city water, etc. with their respective commissioning, IQ’s, OQ’s and PQ’s

Typically, the HVAC validation refers and consider the commissioning or qualification of all utilities and room related to each product manufacturing operation.

What testing is required in the Cleanroom Validation?

The most common testing performed during a cleanroom validation are:

Airflow or smoke pattern

For the evaluation of this parameter, a smoke generation device is used to add a visible fume in front of the HEPA Filters or in the area in which the product shall be exposed.  The distribution of smoke is observed, documented, and recorded. It should be uniform following a laminar flow pattern in the exit direction to return ducts without any major turbulence.

Airflow velocity and changes per hour

For this test, the area of HVAC is divided into hypothetical grids and the air velocity is measured at each grid and then the average air velocity (V) is calculated. The area of the HEPA filter inlet (A) is calculated in feet and the total air volume (T) is then calculated by multiplying the average velocity of the air and the area of the inlet (T = A × V). After this, the volume of the room is calculated and the air changes per hour are obtained by dividing the total air change by the volume of the room.

Filter leak test

For the leak test of the HEPA filter, a velometer is placed at the front of the AHU system and the air velocity is checked. The air velocity should be within the higher limit of the HEPA filter.

Particle count – nonviable particle test

A particle counter is used to conduct the test. Particle count is taken at static conditions before the operation as well as operational working conditions. The particle count should be within the range as per the standards of particle classification, for example, ISO Class 7, etc.

Viable particle monitoring

Viable monitoring is performed on daily basis by employing the swab test and using nutrient agar medium for the incubation of microorganisms. The different media plates are exposed in every manufacturing section.  The microorganism count should be within the range otherwise, an investigation must be initiated to evaluate the root cause, effective corrective and preventive actions

Filter integrity test

The HEPA filter integrity is tested by injecting particles of a predetermined size (0.2 um or greater) using an aerosol generator into the HEPA filters to determine if they are retaining the aerosol particles. The 100% upward flow of the aerosol must be captured into the HEPA filter. A receptor probe that detects the aerosol is used to determine if they are passing thru the HEPA filter or not.  Each HEPA filter must be tested and monitored periodically (e.g. annually or every two years). It is important to know if they are broken.  Therefore, the amount of the aerosol detected passing thru it is monitored and documented as part of the qualification. No residues or traces of aerosol must be detected after the HEPA filter to pass the acceptance criteria of the filter integrity test.

Pressure difference

It is calculated by making use of the manometer attached to the walls of the adjacent area. The pressure difference is generally kept positive from the cleanest area to the less clean area in the range from 1 and 20 mmHg pressure.

Recovery test

The recovery of temperature and humidity conditions is checked after losing operational power conditions or doors opening. For example, the humidity and temperature are checked at the off position of the HVAC system. Then, the HVAC system is turn -on to verify how much time it takes to recover the expected conditions, the time required to stabilize the temperature and humidity is noted.  Also, this test can be done, opening the doors during some predetermined amount of time, then document the amount of time it takes to reach the expected environmental conditions.

Temperature and humidity uniformity test

The uniformity of temperature and humidity are monitored by employing a calibrated datalogger, chart recorder, thermocouples, thermometer, RTD, manometer, etc. The two parameters are monitored in a continuous manner, documented in the format and stabilization is ensured within the specified limit.

Fresh air determination

The fresh air intake is observed at the inlet on the fresh air damper. The total air change is calculated. The intake of fresh air is divided by the total air change in the room and multiplied by 100 to obtain the percent fresh air intake on each cycle by the HVAC system in all the individual rooms.

How determine or calculate the number of samples required to classify a cleanroom as per ISO 14644: 2015?

The number of samples to be taken on each cleanroom depends on the area.

The ISO 14644 standard can be used to determine the sampling plan and the number of sampling points to use during the HVAC system validation.  The ISO standards are not free or public domain since it has copyrights.  Refer to the following table as an example.

For a complete table and more information about the iso 14644-1:2015, refer to httpss://www.iso.org/standard/53394.html

 How to prepare a cleanroom validation protocol?

Some people perform the validation strategy following this approach:

The cleanroom validation PQ protocol may include, but, is not limited to the following sections and information, (as applicable)

1. Purpose
2. Scope
3. General Definitions
4. Safety
5. Reference Documents
6. Responsibility
7. Exceptional Conditions and Discrepancies
8. Strategy: Environmental Monitoring Instructions

• Room Temperature And % Relative Humidity Monitoring
• Differential Pressure Monitoring
• Non-Viable Particulate Monitoring
• Viable Particulate Monitoring
• Air Flow Monitoring and Balancing
• Room Airflow Pattern Test – Smoke Test
• HEPA Filter Air Velocity Test
• HEPA Filter Integrity Test
• Particle Decontamination Test
• Recovery Test – Hvac System Shut-Down and Restore Test
• Room Air Changes Test
• Sound Pressure and Lighting Test

9. Product Disposition
10. Room/Area Certification Parameters Not Meeting Requirements
11. Documentation
12. Approvals

What is the ISO 14644?

The ISO 14644 is a group of several international standards dedicated to bringing the guidelines related to the ECA Environmental Controlled Area management.

It establishes the classification of air cleanliness in terms of the concentration of airborne particles in cleanrooms and clean zones.

ISO 14644 Standards were first formed from the US Federal Standard 209E Airborne Particulate Cleanliness Classes in Cleanrooms and Clean Zones.

The ISO 14644 is composed of the following documents:

• ISO 14644-1: Classification of air cleanliness
• ISO/DIS 14644-1.2(2014): Classification of air cleanliness by particle concentration
• ISO 14644-2: Specifications for testing and monitoring to prove continued compliance with ISO 14644-1
• ISO/DIS 14644-2.2(2014): Monitoring to provide evidence of cleanroom performance related to air cleanliness by particle concentration
• ISO 14644-3: Test Methods
• ISO 14644-4: Design, Construction, and Start-up
• ISO 14644-5: Operations
• ISO 14644-6: Vocabulary
• ISO 14644-7: Separative devices (clean air hoods, gloveboxes, isolators, and mini environments
• ISO 14644-8: Classification of airborne molecular contamination
• ISO 14644-9: Classification of surface particle cleanliness
• ISO 14644-10: Classification of Surface Cleanliness by Chemical Concentration
• ISO 14644-12: Classification of Air Cleanliness by Nanoscale Particle Concentration

For more information, refer to httpss://www.iso.org/standard/53394.html

For a preview, refer to httpss://www.iso.org/obp/ui/#iso:std:iso:14644:-1:ed-2:v1:en

What is the US FED 209D?

The FED-STD-209 E Airborne Particulate Cleanliness Classes in Cleanrooms and Clean zones was a federal standard concerning the classification of air cleanliness, intended for use in environments like clean rooms. The standard-based its classifications on the measurement of airborne particles.

The time before the ISO 14644 was implemented, this legacy document established the standard classes, and provides for alternative classes, of air cleanliness for cleanrooms and clean zones based on specified concentrations of airborne particles. It prescribes methods for verifying air cleanliness and requires that a plan be established for monitoring air cleanliness. It also provides a method for determining and describing concentrations (U descriptors) of ultrafine particles.

The standard was canceled on November 29, 2001, by the United States General Services Administration (GSA). The document was superseded by standards written for the International Organization for Standardization (ISO).