Special Reports
March / April 2017

Understanding Cleanliness Classifications for Life Science Facilities
Gordon J. Farquharson
Norman A. Goldschmidt
Understanding Cleanliness Classifications for Life Science Facilities

In recent years we have observed misunderstanding and confusion over correlation between the 2004 US Food and Drug Administration (FDA) environmental cleanliness requirements for sterile product manufacture 1 and those of the European Medicines Agency EudraLex Volume 4, Annex 1.2

This misunderstanding, which began before the demise of Federal Standard 209, has been exacerbated by the introduction of the ISO cleanliness classification system standards in 1999 (14644-1:1999) and most recently in the 2015 revisions (14644-1:2015). The situation has since become more critical because the Annex 1 requirements in are replicated virtually verbatim in the Pharmaceutical Inspection Co-operation Scheme (PIC/S) and World Health Organization (WHO) good manufacturing practice requirements, which are used by regulators around the world. Such broad adoption of virtually identical regulatory guidance based on the European Union (EU) system has also aggravated the situation.

The strong similarities between the systems are likely to have led to the common misapplication of designations and incorrect correlation between classification systems. In addition, subtle differences in the requirements can cause even greater confusion:

  • Both EU and PIC/S require “in-operation” and “at-rest” classifications.
  • Both EU and PIC/S specify a “cleanup” or “recovery” time and qualification thereof.
  • The FDA standard classifies and monitors airborne particles at a single size threshold of ≥ 0.5 μm; EU and PIC/S use two size thresholds: ≥ 0.5 and 5.0 μm.
  • EU and PIC/S airborne ≥ 5.0 μm particle concentration limits for grade A cleanliness do not align with ISO 5 class limits.
  • Designation and qualification of a class below ISO 8 in operation (Grade C) as ISO 8 at rest (Grade D) vs. ISO 9 in operation or “controlled, not classified” (CNC).

Before beginning a discussion of differences within modern classification systems, it is perhaps advisable to review space classification before harmonization efforts led to the current systems.

ISPE has attempted to bridge these somewhat confusing differences with a single composite cleanliness grading system intended to satisfy international regulatory bodies and make good scientific sense.

FS 209

The classification of space by airborne particulate concentration began with Federal Standard 209 in 1963 and is the source for the classifications still used by the US Pharmacopeia and FDA (Table A): Other countries established standards that further complicated the terms and nomenclature affecting the design and cleanliness classification of global cleanroom facilities (Table B).

As you can see, our current issue of aligning two standards is small by comparison to the historical alignment of diverse global standards. What’s more, the current underlying harmonized standard for space classification serves to align regulations to a great degree. To fully understand the similarities and differences between regulatory requirements, we must understand the harmonized space classification system on which they are based.

Table A: FS 209: Space classification by particle concentration per cubic foot
Particle size, μm
Class 0.1 0.2 0.3 0.5 5.0
1 35 7.5 3 1 -
10 350 75 30 10 -
100 - 750 300 100 -
1,000 - - - 1,000 7
10,000 - - - 10,000 70
100,000 - - - 100,000 700

ISO 14644-1:1999 (superseded): This standard defined classes of cleanliness by airborne particle count concentration following a decimal system. The classes are illustrated in Table C for a series of size ranges. The relationship between ISO class number, particle number concentration, and reference particle size is defined in the standard by the formula Cn = 10N × (0.1 μm/D)2.08, where Cn is the particle count, N is the ISO class, and D is the particle mean diameter in μm.

ISO 14644-1:2015: This standard replaces ISO 14644-1:1999, but serves the same purpose as the prior standard (Table D), with some notable changes:

  • There is no class limit particle count specified for ≥ 5 μm particles at ISO 5 due to the uncertainty of counting these large particles at low concentration.
  • Monitoring ≥ 5 μm particles at low concentration must be done in concert with another particle size; the “macro particle” descriptor M should be added to communicate the uncertainty of the reading.
  • The decimal classification system must be adjusted to 0.5 class increments (e.g. ISO 4, ISO 4.5, ISO 5)

Although this common standard now harmonizes space classifications in regulations across the world, differences in interpretation can result in some ambiguity.

Us FDA Application Of ISO Classifications

The 2004 FDA Guidance for Industry cited at the beginning of this article provides some clarification of the FDA use of ISO grades. At all grade levels the FDA assumes a particle size of ≥ 0.5 μm and that classification and monitoring occur with the room in operation. The guidance also assigns unique definitions to some classifications:

ISO 5: A space that has been classified to meet ISO 14644-1 requirements (3,520 particles/cubic meter) for airborne 0.5 μm particulate in the in-operation state. These spaces are constructed with a “flushing” or “sweeping” generally unidirectional airflow that protects critical areas, with a suggested velocity of 0.45 meters per second ±20%, or as justified and qualified via airflow visualization.

ISO 9: A space that has been classified to meet ISO 14644-1:1999 requirements (35,200,000 particles per cubic meter) for airborne 0.5 μm particulate in the in-operation state. This classification does not actually appear in FDA guidance but is found in some FDA-regulated facilities.

Terms and Definitions

Ambient environment: Environmental conditions where no HVAC systems are present.

Uncontrolled (UC): Areas where HVAC systems may be present, but no claim is made or qualified for the specific control of particulate, temperature, or humidity. These areas are sometimes referred to as “general” or “comfort-controlled” areas within pharmaceutical facilities such as office and technical space. May also be designated “not controlled (NC).”

Classified space: Areas in which HVAC systems are designed to reduce airborne contaminants below a specified level as defined in ISO 14644-1 (tested per ISO14644-2,3) and both temperature and RH are controlled more tightly than in the ambient environment. These areas must be performance verified/qualified. They may be tested to meet ISO requirements for airborne 0.5μm particulate and viable organisms in the “in-operation” state to meet US FDA requirements, or they may be tested to meet ISO requirements for airborne 0.5 and 5.0 μm particulate as well as viable organisms in both the “in-operation” state as well as the “at-rest” states to meet EMA and PIC/S requirements. Where EMA and PIC/S requirements are to be satisfied, the transition between the two states should take place in 15–20 minutes. This can be verified via the “recovery test” as specified in ISO 14644-3.

Recovery: A test defined in ISO 14644-3 that challenges room environmental performance by measuring the time required for contamination to reduce by 1 to 2 log after particle generation in the space ceases. ZLG Aide Memoire 07121104 Grade E: A classified space that satisfies the airborne viable microorganisms requirement of < 250 CFUs per cubic meter.

ZLG Aide Memoire 07121104 Grade E: A classified space that satisfies the airborne viable microorganisms requirement of < 250 CFUs per cubic meter.

ZLG Aide Memoire 07121104 Grade F: A classified space that satisfies the airborne viable microorganisms requirement of < 500 CFUs per cubic meter.

Table B: Historical comparison of classification systems
Particles per cubic meter ≥ 0.5 μm US FS-209E, 1992 US FS-209E equivalent per cubic foot EU EudraLex Vol.4 Annex 1, 1997 France AFNOR, 1989 Germany VDI 2083, 1990 Britain BS 5295, 1989 Japan JISB 9920, 1989 ISO 14644-1 and CEN 243
1                
3.5         0   2 2
10 M1              
35.3 M1.5 1     1   3 3
100 M2              
353 M2.5 10     2   4 4
1,000 M3              
3,530 M3.5 100 B: at rest
A: at all
times		 4,000 3 E or F 5 5
10,000 M4              
35,300 M4.5 1,000     4 G or H 6 6
100,000 M5              
353,000 M5.5 10,000     5 J 7 7
1,000,000 M6              
3,530,000 M6.5 100,000     6 K 8 8
10,000,000 M7              
Table C: ISO 14644-1:1999 air quality classes
Particles per cubic meter (cubic foot), by size
ISO Class 0.1 μm 0.2 μm 0.3 μm 0.5 μm 1 μm 5 μm
1 10 2 0 0 0 0
2 100 24 10 4 (0.1) 0  
3 1,000 237 102 35 (1) 8  
4 10,000 2,370 1,020 352 (10) 83  
5 100,000 23,700 10,200 3,520 (100) 832 29
6 1,000,000 237,000 102,000 35,200 (1,000) 8,320 293
7       352,000 (10,000) 83,200 2,930
8       3,520,000 (100,000) 832,000 29,300
9       35,200,000 (1,000,000) 8,320,000 293,000

1 cubic meter = 35.2 cubic feet When referring to US FDA guidance only the 0.5 μm particle size (highlighted) is measured

© ISO. This material is reproduced from ISO 14644-1:1999 with permission of the American National Standards Institute (ANSI) on behalf of the International Organization for Standardization. All rights reserved. ISO 14644-1:1999 is an archived ISO document. It cannot be referred to as an approved ISO standard.
Table D: ISO 14644-1:2015 classification of air cleanliness by particle concentration

Maximum allowable concentrations

Particles per cubic meter for particles equal to and greater than the sizes shown [a]
ISO Class 0.1 μm 0.2 μm 0.3 μm 0.5 μm 1 μm 5 μm
1 10[b] [d] [d] [d] [d] [e]
2 100 24[b] 10[b] [d] [d] [e]
3 1,000 237 102 35 [b] [d] [e]
4 10,000 2,370 1,020 352  83[b] [e]
5 100,000 23,700 10,200 3,520 (100) 832 [d–f]
6 1,000,000 237,000 102,000 35,200 (1,000) 8,320 293
7 [c] [c] [c] 352,000 (10,000) 83,200 2,930
8 [c] [c] [c] 3,520,000 (100,000) 832,000 29,300
9 [c] [c] [c] 35,200,000 (1,000,000) 8,320,000 293,000
  1. All concentrations in the table are cumulative, e.g., for ISO Class 5, the 10,200 particles shown at 0.3 μm include all particles equal to and greater than this size.
  2. These concentrations will lead to large air sample volumes for classification. Sequential sampling procedure may be applied; see Annex D.
  3. Concentration limits are not applicable in this region of the table due to very high particle concentration.
  4. Sampling and statistical limitations for particle in low concentrations make classification inappropriate.
  5. Sample collection limitations for both particles in low concentrations sizes greater than 1 μm make classification at this particle size inappropriate, due to potential particle losses in the sampling system.
  6. In order to specify this particle size in association with ISO Class 5, the macroparticle descriptor M may be adapted and used in conjunction with at least one other particle size (see C.7).
  7. This class is only applicable for the in-operation state.

© ISO. This material is reproduced from ISO 14644-1:2015 with permission of the American National Standards Institute (ANSI) on behalf of the International Organization for Standardization. All rights reserved.

EU, PIC/S, and WHO

EudraLex Volume 4, Annex 1 requirements for sterile products stipulate in-operation and at-rest airborne particle count limits at both ≥ 0.5 and 5.0 μm particle sizes. It further directs that spaces recover from the in-operation to the at-rest state after a 15–20-minute cleanup period. Some other unique definitions are:

Grade A: A classified space that satisfies European Medicines Agency (EMA) and PIC/S requirements to meet:

  • ISO 5 measured via airborne ≥ 0.5 μm particulate
  • ISO 4.8* measured via airborne ≥5.0 μm particulate in the in-operation and at-rest states
  • Airborne viable microorganisms < 1 colony forming unit (CFU) per cubic meter

These spaces are normally unidirectional flow with a suggested air velocity of 0.36–0.54 meters per second. These spaces surround product only and must be absent of people.

Grade D: A classified space that satisfies EMA and PIC/S requirements to meet ISO 8 measured via:

  • Airborne 0.5 and 5.0 μm particulate in the at-rest state only
  • Airborne viable microorganisms < 200 CFUs per cubic meter.

Aside from these differences in particle size and operational state, there is a good deal of alignment between the classification systems for the US, EU, PIC/S, and WHO. We summarize these similarities and differences in Table E.

The table’s colored areas show areas of alignment between regulations, while the yellow areas show the differences between the US and other classification systems. Although US, EU, PIC/S, and WHO systems have four identified classes, they do not map directly, as each has one class the others do not.

ISPE has attempted to bridge these somewhat confusing differences with a single composite cleanliness grading system intended to satisfy international regulatory bodies and make good scientific sense.

To meet these objectives, the ISPE HVAC Community of Practice suggested that the ISPE grading system, originally proposed in the second edition of Sterile Products Manufacturing Baseline™ Guide, be modified as shown in the 2013 Baseline Guide Volume 6: Biopharmaceutical Facilities (Table F and Table G).

* The use of ISO 4.8 in lieu of ISO 5 for 5.0 μm particles in Grade A appears to be the result of aligning this limit on the lowest number possible, working ISO 14644 sample size calculations backward using a 1 cubic meter sample size.

This unified system can help eliminate common misunderstandings about classification alignment and clarify the intent of environmental control within facilities.

Revised ISPE Grades

The ISPE Sterile Guide team has suggested a further refinement, replacing the ISPE grades with a US/EU designation as follows:

Grade 8 (ISO 8/Grade C): A classified space that satisfies FDA requirements for:

  • ISO 8 measured via airborne 0.5 μm particulate in the in-operation state
  • EMA and PIC/S requirements to meet ISO 8 measured via airborne 0.5 and 5.0 μm particulate in the in-operation state
  • ISO 7 measured via airborne 0.5 and 5.0 μm particulate in the at-rest state, with a 15–20-minute transition between states
  • Airborne viable microorganisms < 100 CFUs per cubic meter

Grade 7 (ISO 7/Grade B): A classified space that satisfies FDA requirements for:

  • ISO 7 measured via airborne 0.5 μm particulate in the in-operation state
  • EMA and PIC/S requirements to meet ISO 7 measured via airborne 0.5 and 5.0 μm particulate in the in-operation state
  • ISO 5 measured via airborne 0.5 and 5.0 μm particulate in the at-rest state, with a 15–20-minute transition between states
  • Airborne viable microorganisms < 10 CFUs per cubic meter

Grade 5 (ISO 5/Grade A): A classified space that satisfies FDA requirements for:

  • ISO 5 measured via airborne 0.5 μm particulate in the in-operation state
  • EMA and PIC/S requirements to meet ISO 5 measured via airborne 0.5 μm particulate
  • ISO 4.8 measured via airborne 5.0 μm particulate in the in-operation and at-rest states
  • Airborne viable microorganisms < 1 CFU per cubic meter. These spaces are normally unidirectional flow with an air velocity of 0.20–0.45 meters per second.

Controlled not classified with local monitoring (CNC+/Grade D): Areas where HVAC systems are designed to reduce airborne contaminants below the level of the ambient environment and in which both temperature and relative humidity (RH) are controlled more tightly than in the ambient environment. Claims for environmental control in these areas are related to both system design and system performance; installation qualification and operational qualification are common.

These areas are typically qualified to meet ISO 8 requirements at rest only, and to control temperature and humidity within a specified band. They are monitored for viable particulate during operation to provide background information for investigations and to assure adequate layers of closure. These areas are generally aligned with PIC/S designation Grade D and airborne viable microorganisms < 200 CFUs per cubic meter.

Controlled not classified (CNC): Areas where HVAC systems are specifically designed to reduce airborne contaminants below the level of the ambient environment and both temperature and RH are controlled more tightly than in the ambient environment. Claims for environmental control in these areas are related to the design of the system; installation qualification is common. No claim is made or qualified for the specific control of particulate. Typical systems will have heating, cooling, and filtration meeting minimum efficiency reporting values of 13 or better. These areas are sometimes referred to as “pharmaceutical” or “clean” areas within pharmaceutical facilities.

Temperature controlled: Areas where HVAC systems are specifically designed to control both temperature and (where applicable) RH more tightly than in the ambient environment. Temperature and RH are usually qualified in these areas and temperature mapping is expected. This is designation is typically found in warehouse spaces, cold rooms, and logistics. Some companies apply the CNC designation for these areas.

This unified system of classification, whether by stating the grade and ISO number or by referencing both the ISO class and the EU grade can help eliminate common misunderstandings about classification alignment and clarify the intent of environmental control within facilities.

In closing, the authors offer terms and definitions to clarify any remaining confusion regarding common classification and environmental control terminology.

Table E: Comparison of regulatory requirements
FDA In-operation (particles per cubic meter) Active air action EU, WHO, PIC/S In-operation (particles per cubic meter) At-rest (particles per cubic meter) Active air action
ISO USP 0.5 μm Limits Grade 0.5 μm 0.5 μm 0.5 μm 5.0 μm Limits
ISO 5 100 3,520 1 A 3,520 20 3,520 20 < 1
ISO 6 1,000 35,200 7 N/A          
ISO 7 10,000 352,000 10 B 352,000 2,900 3,520 29 10
ISO 8 100,000 3,520,000 100 C 3,520,000 29,000 352,000 2,900 100
N/A N/A N/A N/A D N/A N/A 3,520,000 29,000 200

 

Table F ISPE cleanliness grading system: Environmental control requirements in regulations

ISO Class

USP particles per cubic foot

US FDA in-operation limit, particles per cubic meter

EU and PIC/S grade

 EU and PIC/S

ZLG “aide-memoire” 07121104

Active air action limits, colony-forming units per cubic meter
In-operation limit, particles per cubic meter At rest limit, particles per cubic meter
≥ 0.5 μm ≥ 0.5 μm ≥ 0.5 μm ≥ 5.0 μm ≥ 0.5 μm ≥ 5.0 μm
ISO 5 100 3,520 A 3,520 20 3,520 20 N/D < 1
ISO 6 1,000 35,200 N/D 35,200 290 3,520 29 N/D 7
ISO 7 10,000 352,000 B 352,000 2,900 3,520 29 N/D 10
ISO 8 100,000 3,520,000 C 3,520,000 29,000 352,000 2,900 N/D 100
ISO 9 1,000,000 35,200,000 N/D N/A N/A N/A N/A N/D N/D
N/A N/A N/A D N/A N/A 3,520,000 29,000 N/D 200
N/D N/A N/A N/D N/A N/A N/A N/A E 250
N/D N/A N/A N/D N/A N/A N/A N/A F 500
CNC* N/A N/A N/D N/A N/A N/A N/A N/D N/A
U/C* N/A N/A N/A N/A N/A N/A N/A N/D N/A
* Not ISO classes; these are common designations without a standard definition

 

Table G ISPE Cleanliness Grades†

ISPE grade

 In-operation limit, particles per cubic meter At-rest limit,
particles per cubic meter

Active air action limits, colony-forming units per cubic meter
≥0.5 µm ≥ 5.0 µm ≥ 0.5 µm ≥5.0 µm
Grade 5 3,520 20 3,520 20 <1
Grade 6 35,200 290 3,520 29 7
Grade 7 352,000 2,900 3,520 29 10
Grade 8 3,520,000 29,000 352,000 2,900 100
CNC+* N/A N/A 3,520,000 29,000 200
CNC* N/A N/A N/A N/A N/A
UC* N/A N/A N/A N/A N/A
† 2013 Biopharmaceutical Facilities Baseline Guide
* Not ISO classes; these are common designations without a standard definition


Notes on Tables F and G

  • N/D = not designated
  • Values may be averages; EU and PIC/S require measurement of particles up to and including 0.5 μm and 5 μm, the US standard requires 0.5 μm, hence the table incorporates both to ensure compliance with the most stringent requirement.
  • Samples from Grade 5 areas should normally show no viable organisms.
  • Recovery from the in-operation to the at-rest state should be verified to occur within 15–20 minutes for ISPE grades 6, 7, and 8. The recovery test as defined in ISO 14644-3:2005 and IEST RP003 may be carried out to verify a one or two log reduction test. Recovery testing may also be performed for informational purposes.
  • At-rest figures are given to support recovery and “static” room classification testing. Maintenance of these levels during idle (not in use) periods is not intended
    .