Phase 1
Design and Specification
1Define intended use and risk classification
Establish the target ISO class or EU GMP grade based on the criticality of operations performed. Aseptic filling and open product handling require Grade A/ISO 5. Risk assessment determines classification for supporting areas.
ISO 14644-1:2015 · EU GMP Annex 1 2022 · ICH Q9(R1) 2023
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2Design HVAC and airflow system
HEPA or ULPA filtration selected to achieve the target air change rate and particle removal efficiency. Unidirectional airflow required for Grade A/ISO 5 zones.
EU GMP Annex 1 2022 Section 4 · ISO 14644-4
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3Define pressure cascade and room layout
Positive pressure cascade from highest to lowest classification maintains directional airflow. Minimum differential of 10 to 15 Pascals between adjacent grades, confirmed continuously.
EU GMP Annex 1 2022 Section 4.19
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4Determine sampling locations and point count
Minimum number of sampling locations calculated per room area. Locations selected to represent worst-case zones, including areas farthest from air supply.
ISO 14644-1:2015 Annex B
Phase 2
Testing and Certification
5Conduct airborne particle count testing
Discrete particle counting performed at each location for 0.5 micron and 5.0 micron particle sizes, across as-built, at-rest, and operational states as applicable.
ISO 14644-1:2015 Annex A · ISO 21501-4
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6Perform supporting qualification tests
Airflow velocity and uniformity, HEPA filter installation leak test, room pressure differential, airflow visualization for unidirectional zones, and recovery test where required.
ISO 14644-3 · EU GMP Annex 1 2022 Section 4
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7Calculate classification using statistical method
Results evaluated using the 95 percent upper confidence limit method or sequential sampling per ISO 14644-1 Annex A. All locations must meet the target class.
ISO 14644-1:2015 Annex A.3
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Does the room achieve the target ISO class or GMP grade at every location?
YES → Certify and release
NO → Investigate and remediate
Phase 3
Documentation and Release
8Compile classification certification report
Report includes room identification, target class, test state, individual location results, instrument calibration status, test date, and technician signature.
21 CFR 211.42 · EU GMP Annex 15
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9QA review and certificate approval
Quality unit reviews the report against acceptance criteria and approves the certificate before release. Any out-of-specification result is closed via investigation first.
21 CFR 211.22 · EU GMP Annex 1 2022
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10Post classification and release room
Achieved ISO class and GMP grade posted at room entry. Room added to the qualified room list with certification date and next requalification due date tracked.
EU GMP Annex 1 2022 Section 4
Phase 4
Requalification and Maintenance
11Scheduled requalification
Frequency set per ISO 14644-2:2015: every 6 months for ISO 5 or cleaner, every 12 months for ISO 6 through 8. Full particle count testing repeated at each interval.
ISO 14644-2:2015 Table 1
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12Trigger-based requalification
Triggered outside the scheduled interval by HVAC modification, HEPA filter replacement, layout changes, extended shutdown, or investigation findings implicating room performance.
EU GMP Annex 15 · Change control procedure
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13Investigate classification failures
A failing location triggers investigation into HVAC performance, filter integrity, personnel practices, and room integrity. Root cause addressed and verified through retest.
21 CFR 211.192 · EU GMP Annex 1 2022
Requalification Frequency (ISO 14644-2:2015)
ISO 5 or Cleaner
Requalification interval: every 6 months. Applies to Grade A/ISO 5 unidirectional airflow zones used for aseptic processing.
Table 1, ISO 14644-2:2015
ISO 6 through ISO 8
Requalification interval: every 12 months. Applies to Grade B, C, and D supporting cleanroom areas.
Table 1, ISO 14644-2:2015
Trigger-Based Requalification
Required outside the scheduled interval following HVAC modification, HEPA filter change, facility shutdown, or investigation findings.
EU GMP Annex 15
Classification Failure Response Steps
Step 1 - Confirm result validity
Rule out counter calibration error, sampling technique error, or probe placement error before concluding a genuine classification failure.
Step 2 - Notify QA and evaluate room status
Assess whether the room can continue in use pending investigation based on the magnitude and location of the failure.
Step 3 - Investigate HVAC and filter performance
Review filter integrity results, airflow velocity, pressure differential trends, and recent maintenance activity.
Step 4 - Remediate root cause
Repair or replace the deficient component, whether filter, ductwork, door seal, or airflow pattern issue.
Step 5 - Retest and requalify
Repeat full particle count testing at the affected locations. The room does not return to qualified status until all locations meet the target class.
Sampling and Statistical Requirements
Minimum sampling locations
Calculated from room area per ISO 14644-1 Annex B. Larger or higher-risk rooms require additional points beyond the area-based minimum.
Sample volume
Minimum sample volume per location determined by the class limit and the smallest considered particle size, ensuring statistically meaningful counts.
95 percent UCL calculation
Where fewer than 10 samples are taken per location, the 95 percent upper confidence limit method determines compliance rather than a simple average.
Sequential sampling
ISO 14644-1 Annex A permits a sequential sampling procedure as an alternative statistical approach for classification decisions.