What are cleanroom grades A, B, C and D, and how do they compare with ISO 14644 classes?

1What the four grades are.

EU GMP Annex 1 sets out “four grades of cleanroom/zone” for the manufacture of sterile products. The current text came into operation on 25 August 2023, apart from point 8.123 on lyophilisers, which followed on 25 August 2024.¹ EudraLex lists it as “fully applicable since 25 August 2024”.²

• Grade A is “the critical zone for high-risk operations”, such as the filling zone, stopper bowls, open primary packaging and aseptic connections. It is normally provided by localised airflow protection, such as unidirectional airflow workstations within a RABS or isolator, and unidirectional airflow should be qualified across the whole grade A area.
• Grade B is the background cleanroom for grade A during aseptic preparation and filling, where grade A is not an isolator. Its air pressure differences are monitored continuously.
• Grades C and D are for less critical stages of aseptic manufacture, as backgrounds for isolators, and for preparing and filling terminally sterilised products.¹

Annex 1 gives examples. Aseptic assembly of filling equipment, aseptic filling and loading a lyophiliser are grade A work. Preparing solutions to be filtered is grade C. Cleaning equipment and assembling cleaned components before sterilisation are grade D.¹ The background for an open isolator should generally be at least grade C, for a closed isolator at least grade D, and for a RABS at least grade B.¹ WHO’s 2022 GMP for sterile products, revised with the EU and PIC/S as a harmonised text, uses the same grades and classification limits.³

2How a grade is classified.

Classification is part of cleanroom qualification. It measures total particles of 0.5 µm and larger and of 5 µm and larger, both “at rest” and in operation, against Table 1 of Annex 1.¹ At rest means utilities and HVAC running and the main equipment installed but not operating, with no one in the room. In operation means the room working in its defined mode “with the maximum number of personnel present performing or simulating routine operational work.”¹

For grade D in operation, the manufacturer sets limits from a risk assessment and routine data. Classification including 5 µm particles may be considered for grades A and B where the contamination control strategy (CCS) or historical trends indicate it.¹ The PIC/S, EMA and WHO interpretation of the revised annex, in force from 28 October 2026, confirms that ≥5 µm limits are “not a default requirement” at classification of grade A or of grade B at rest, but that monitoring ≥5 µm particles against defined limits is required in routine operation.⁴

The at-rest limits should be reached after a “clean up” period at the end of operations, with a guidance value of less than 20 minutes.¹ Qualification also sets microbial limits for each grade: no growth in grade A, and for air samples 10, 100 and 200 CFU/m³ in grades B, C and D.¹ Grade A and B areas are requalified at least every 6 months and grade C and D areas at least every 12 months.¹

3How the grades compare with ISO 14644-1 classes.

ISO 14644-1:2015 classifies air cleanliness by the concentration of airborne particles with threshold sizes from 0.1 µm to 5 µm. ISO says it “cannot be used to characterize the physical, chemical, radiological, viable or other nature of airborne particles.”⁵ Annex 1 doesn’t assign ISO classes to its grades. It points to the ISO 14644 series for qualifying cleanrooms and to ISO 14644-1 for the minimum number and position of sampling locations.¹

The ≥0.5 µm limits in Annex 1 are the same concentrations FDA’s 2004 aseptic processing guidance gives for ISO 5 (3,520 per m³), ISO 7 (352,000) and ISO 8 (3,520,000).¹⁶

FDA’s guidance says an ISO 5 particle concentration “is equal to Class 100 and approximately equals EU Grade A.”⁶ The match is only for ≥0.5 µm particles. Annex 1 adds its own ≥5 µm limits, microbial limits and continuous particle monitoring of grade A throughout critical processing (9.16 and 9.17), and FDA’s table adds microbial action levels, so a room’s ISO class alone doesn’t establish its grade.¹⁶

4How FDA classifies aseptic areas.

In the United States, 21 CFR 211.42(c)(10) requires aseptic processing areas to have, as appropriate, HEPA-filtered air under positive pressure, a system for monitoring environmental conditions, and systems for cleaning and disinfecting the room and equipment.⁷ FDA’s 2004 guidance, which contains nonbinding recommendations, sets out how it expects those areas to be classified.⁶

• FDA puts “most emphasis on data generated under dynamic conditions”, with personnel present and operations under way.
• The critical area, where sterilised product, containers and closures are exposed, should be ISO 5, measured at locations normally not more than one foot from the work site during filling and closing.
• The area immediately next to the aseptic line should meet at least ISO 7 under dynamic conditions. ISO 8 is appropriate for less critical activities, such as equipment cleaning.
• An isolator’s interior should meet ISO 5, and an ISO 8 background “is commonly used”.⁶

FDA has also said that a sterile manufacturer should not rely solely on ISO 14644-1 and 14644-2 to qualify its facility. It should use them with FDA’s regulations and guidance, and “appropriate measures augmenting ISO’s recommendations (e.g., with microbiological data)” would likely be expected.⁸

5How it runs in Seal.

In Seal, environmental monitoring keeps each sample with its location, collection window, sampled volume, media and incubation, so the collection time, not the read date, identifies which work was running. Changes to the sampling plan, frequencies and review rules become new approved versions, and earlier collections keep the plan they were taken under.

Cleanroom cleaning and disinfection models each room as zones and surfaces, with its area grade, and runs contact time as a clock from completed wetting of each zone. Production start can be configured to block while a required surface is still inside its contact interval, and return to use is a signed decision on the assembled evidence.