Cleanroom classification is the process of “assessing the level of cleanliness against a regulatory specification” by measuring airborne particles. ISO 14644-1:2015 defines ISO Classes by maximum allowable particle counts at various sizes, and the latest draft of EU GMP Annex 1 aligns closely with these definitions. In pharmaceutical sterile manufacturing (e.g. aseptic filling), Annex 1 simply maps Grades A/B to ISO Class 5, Grade C to ISO 7, and Grade D to ISO 8. (Grade A/B are the ISO 5 (5.0) environments around filling needles; Grades C/D are background clean areas.) All classifications require measuring in both “at rest” (equipment on, no personnel) and “in operation” (with operators running the process) states.
ISO 14644-1’s classification tables give explicit numeric limits. For example, ISO Class 5 allows up to 3,520 particles ≥0.5 µm per m³, ISO 7 allows 352,000, and ISO 8 allows 3,520,000 (all at ≥0.5 µm). EU GMP Annex 1’s Table 1 uses the same numbers. It specifies that Grade A (ISO 5) must have ≤3,520 particles ≥0.5 µm per m³ in both at-rest and in-operation conditions. Grade B (also ISO 5) has the same 3,520 limit at rest and allows up to 352,000 in operation. Grade C (ISO 7) permits ≤352,000 (at rest) and ≤3,520,000 (in operation). Grade D (ISO 8) permits ≤3,520,000 at rest, with the in-operation limit set by risk assessment (not a fixed number).
Key point: If measured particle counts stay at or below the table limits for each grade/class and state, the cleanroom “complies” with the classification specification. For example, measuring 2,500 particles ≥0.5 µm per m³ in a Grade A area (ISO 5) is compliant (limit 3,520). Exceeding the limit means non‐compliance.
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ISO 14644-1 Cleanroom Classes
ISO 14644-1:2015 defines nine cleanroom classes based on particle concentration. Classes 1–4 are extremely stringent, but in pharmaceutical aseptic operations the most relevant are Class 5, 7, and 8. The standard’s table (shown below) gives the maximum particles per cubic meter for each size threshold. For example:
- ≤3,520 particles/m³ ≥0.5 µm.
- ≤352,000 particles/m³ ≥0.5 µm.
- ≤3,520,000 particles/m³ ≥0.5 µm.
Generally, only the larger size cuts (e.g. ≥0.5 µm, ≥5.0 µm) are used for compliance in pharma. ISO 14644-1 includes smaller cuts (≥0.1, ≥0.2 µm, etc.), but Annex 1 focuses on 0.5 and 5 µm for most grades.
Why particles matter: Even microscopic particles can carry microbes or disturb sterile processes. In pharmaceuticals, uncontrolled particles “can affect drug purity or sterility,” so tight particle limits are essential.
EU GMP Annex 1 Grades & Alignment
The draft EU GMP Annex 1 (rev 12) defers to ISO 14644-1 definitions. It explicitly states that Grades A and B must meet ISO Class 5 requirements, Grade C must meet ISO Class 7, and Grade D meet ISO Class 8. In practice:
- Sterile filling zones (e.g. laminar flow at needles). Particle count limit is 3,520 (≥0.5 µm) per m³ at-rest and in-operation.
- Buffer areas. Limit is 352,000 (≥0.5 µm) at-rest and 3,520,000 in-operation.
- Background areas. Limit is 3,520,000 at-rest; in-operation limits are set by the site’s risk assessment (no fixed value).
Annex 1 adds one key change on particle sizes: for Grade A/B (ISO 5), only particles ≥0.5 µm need to be counted. (This relaxes the old practice of also counting 5 µm particles in ISO 5, since such macroparticles are rare in the ultra-clean zone.) Annex 1 does recommend also monitoring ≥1.0 µm as an extra check. For Grade C and D areas, the particle-size requirements remain unchanged: counts are taken at ≥0.5 µm and ≥5.0 µm.
Interpreting Classification Tables
The ISO and Annex tables list maximum particle counts. To interpret them: locate your cleanroom’s class/grade, then compare measured counts at each particle size to the table entry. For example:
The Annex 1 Table 1 (shown above) covers only 0.5 and 5 µm sizes. Notably, for Grade A/B the 5 µm column is marked “not specified” – reflecting the new focus on 0.5 µm particles. (However, Annex 1 permits assessing 5 µm counts in Grade A/B if justified by risk/historical data.) Table footnotes also note that Grade D in-operation limits must be risk‐based.
In short, if your particle count <= the tabulated limit for that state and size, the cleanroom classification is met. All comparisons should use the same sample volume and methodology specified in ISO 14644‑1 (typically counting at least 1 cubic meter of air per sample).
Classification Procedure & Risk Assessment
Sampling and States: Classification is performed by sampling air in all specified locations with calibrated counters. ISO 14644-1 sets the minimum number and placement of sample points. Annex 1 (4.28) instructs that for aseptic filling areas (Grades A/B) you should add sampling points at critical processing locations (e.g. point-of-fill, stopper bowl, etc.) based on a documented risk assessment. In other words, use ISO 14644-1 as the baseline, then use a Classification Risk Assessment to ensure you include all critical zones in the ISO 5 area. (This is separate from the ongoing Monitoring RA used for routine environmental monitoring.)
Classification must cover both “at rest” (equipment running, no operators) and “in operation” (with simulated or actual aseptic process). Annex 1 explicitly says initial classification should occur during simulated operations or an aseptic process simulation. This yields the in-operation data. (For comparison, “at rest” is defined as full utilities and equipment on, but no personnel or activity.)
Measurement: Particle counters typically sample a fixed air volume (e.g. 1 m³) and count particles in size bins. Record the total count ≥0.5 µm (and ≥5.0 µm in Grade C/D). Annex 1 requires counting all particles ≥0.5 µm and ≥5.0 µm for classification (both states); note that the Grade A/B rows simply waive the ≥5.0 µm requirement.
Compliance Checklist & Key Points
- Grade vs. ISO class: Verify the required class. Grades A/B → ISO Class 5; C → ISO 7; D → ISO 8
- Sampling plan: Use ISO 14644-1:2015 guidelines for sample locations and volumes. Include extra points in Grade A/B around the fill needle, determined by risk assessment.
- States: Always test both at-rest and in-operation. Initial classification should ideally be during simulated production.
- Particle sizes: In Grade A/B count ≥0.5 µm (Annex 1 no longer mandates 5 µm). In Grades C/D count both ≥0.5 µm and ≥5.0 µm. Optionally, include ≥1.0 µm for additional monitoring in ISO 5 zones.
- Compare to limits: Check measured counts against the Annex 1 Table 1 or ISO 14644-1 tables. For example, ISO 5/Grade A max is 3,520 ≥0.5 µm; ISO 7/Grade C is 352,000 (at-rest).
- Risk assessment: Document a Cleanroom Classification RA to ensure all critical zones are tested. For Grade D in-operation, set limits via risk evaluation.
- Documentation: Maintain classification reports (particle counts, locations, sampling volumes) and RA justification. These are key for audits.
By following these guidelines, pharmaceutical manufacturers can interpret the ISO/Annex classification tables and demonstrate compliance. In sterile filling lines, for instance, meeting Grade A/B (ISO 5) criteria ensures the fill zone remains within strict particle limits, thereby protecting product sterility. Compliance hinges on rigorous measurement and comparison to the published limits – if counts stay below the tables, values, the cleanroom classification is satisfied.
Quick Table:
| Grade | ISO class | Particle sizes to monitor (for classification) | At-rest limit (particles / m³, ≥0.5 µm) | In-operation limit (particles / m³, ≥0.5 µm) | ≥5.0 µm requirement | Typical pharma locations / examples | Sampling / notes |
| A | ISO 5 | ≥0.5 µm (≥1.0 µm optional) | ≤ 3,520 | ≤ 3,520 | Not specified in Annex 1 (≥5 µm counts are typically negligible; may be recorded if justified) | Laminar flow at fill needle, immediate fill zone (aseptic filling) | Include all critical processing points (point-of-fill, stopper bowl) — follow Classification RA. |
| B | ISO 5 | ≥0.5 µm (≥1.0 µm optional) | ≤ 3,520 | ≤ 352,000 | At-rest: none specified; In-operation: ≤ 2,930 (where measured/required) | Surrounding zone to Grade A (operator work positions during aseptic processing) | At-rest = equipment on, no personnel; in-op = normal staffing/operations. Sample critical points. |
| C | ISO 7 | ≥0.5 µm and ≥5.0 µm | ≤ 352,000 (≥0.5 µm) / ≤ 2,930 (≥5.0 µm) | ≤ 3,520,000 (≥0.5 µm) / ≤ 29,300 (≥5.0 µm) | Both sizes required | Clean/working areas for less-critical stages (fill-line support areas) | Use ISO 14644-1 sampling plan (number/placement of points); count both size channels. |
| D | ISO 8 | ≥0.5 µm and ≥5.0 µm | ≤ 3,520,000 (≥0.5 µm) / ≤ 29,300 (≥5.0 µm) | In-operation limits are risk-based (set by site RA) | Both sizes required for classification | Background/minor support areas surrounding the sterile zone | For in-operation performance, define limits via risk assessment; include support-area sample points. |
Also Read: Numeric Comparison Table
ISO 14644-1:2015 vs EU GMP Annex 1 | Particle Limits (Particles per m³)
Note: ISO uses Classes 1–9, Annex 1 uses Grades A–D.
| Annex 1 Grade | Equivalent ISO Class | State | ≥0.5 µm (particles/m³) | ≥5.0 µm (particles/m³) |
| A | ISO 5 | At-rest | 3,520 | Not defined* |
| A | ISO 5 | In-operation | 3,520 | Not defined* |
| B | ISO 5 | At-rest | 3,520 | Not defined* |
| B | ISO 5 | In-operation | 352,000 | 2,930 |
| C | ISO 7 | At-rest | 352,000 | 2,930 |
| C | ISO 7 | In-operation | 3,520,000 | 29,300 |
| D | ISO 8 | At-rest | 3,520,000 | 29,300 |
| D | ISO 8 | In-operation | Set by facility RA | Set by facility RA |
ISO 14644-1:2015 — Particle Limits (Numeric Only)
Particles/m³
| ISO Class | ≥0.1 µm | ≥0.2 µm | ≥0.3 µm | ≥0.5 µm | ≥1.0 µm | ≥5.0 µm |
| ISO 1 | 10 | 2 | — | — | — | — |
| ISO 2 | 100 | 24 | 10 | 4 | — | — |
| ISO 3 | 1,000 | 237 | 102 | 35 | 8 | — |
| ISO 4 | 10,000 | 2,370 | 1,020 | 352 | 83 | — |
| ISO 5 | 100,000 | 23,700 | 10,200 | 3,520 | 832 | 29 |
| ISO 6 | 1,000,000 | 237,000 | 102,000 | 35,200 | 8,320 | 293 |
| ISO 7 | — | — | — | 352,000 | 83,200 | 2,930 |
| ISO 8 | — | — | — | 3,520,000 | 832,000 | 29,300 |
| ISO 9 | — | — | — | 35,200,000 | 8,320,000 | 293,000 |
Direct Side-by-Side (Numeric Only)
| ISO Class (≥0.5 µm) | Particles/m³ | Annex 1 Grade | At-rest (≥0.5 µm) | In-operation (≥0.5 µm) |
| ISO 5 | 3,520 | Grade A | 3,520 | 3,520 |
| ISO 5 | 3,520 | Grade B | 3,520 | 352,000 |
| ISO 7 | 352,000 | Grade C | 352,000 | 3,520,000 |
| ISO 8 | 3,520,000 | Grade D | 3,520,000 | Risk-based |
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