Keeping clean rooms compliant

There are many regulations covering clean room compliance. Here, the author looks at some of the main issues
regarding this topic.
Nov 01, 2006
By Manuel A.del Valle
Pharmaceutical Technology Europe
Volume 18, Issue 11

Guidelines
FDA's 1987 Guideline on Sterile Drug Products Produced by Aseptic Processingwas the original
document that defined clean rooms.5 It defined only two types of clean rooms critical (class
100) areas where sterilized items are handled and controlled (class 100000) areas where
unsterilized items are handled.
Table 1 Environmental
This guide was replaced in September 2004, with the new guideline containing some notable
requirements for sterile
additions:6
medicinal products.
Sterile drugs should be manufactured using aseptic processing only when terminal sterilization is not feasible.

The classification rates clean rooms in particles/m 3 and states their ISO class number. It also adds viable particle counts using microbiological
settling plates in addition to the original air sampling method.

"Controlled" classification has been replaced by "supporting clean areas" for areas handling unsterilized items. Under this definition, class 1000 and
class 10000 as well as the previously mentioned class 100000 areas have been added.

An air flow rate of 20 air changes per hour (AC/h) is still expected for class 100000 (ISO 8) clean rooms, but the rate is not specified for class 1000
or class 100000 clean rooms.

Poly-alpha-olefin (PAO) was used as an alternate aerosol to dioctylphthalate (DOP) for filter integrity testing of high-efficiency absorbency (HEPA)
filters.

Filter scanning for HEPA filter leak testing should be conducted at a position about 2 in. (50 mm) from the face of the filter.

For HEPA filters in the critical (sterile) area, the air velocity is measured 6 in. (150 mm) from the filter face or at a defined distance close to the
work surface.

The particle count during filling/closing operations is still taken no more than 305 mm away from the worksite, within the air flow.

Written standard operating procedures (SOPs) for microbiological environmental monitoring should address elements such as alert and action
levels.

Blow-fill-seal applications should be performed in class 100000 (ISO 8) rooms or cleaner, and that the critical zone should meet class 100 (ISO 5)
particulate and microbiological standards.

Procedures for aseptic connections, which expose a product or product contact surfaces, should be performed under unidirectional airflow in a class
100 (ISO 5) environment, in a class 10000 (ISO 7) or better room.

States that drains are considered inappropriate for classified areas of the aseptic processing facility, other than Class 100000 (ISO 8) areas.

Pressure differential between rooms of different classes should be 1015 Pa, and 12.5 Pa between a classified and nonclassified room.
 Annex 1

Annex 1 of the EC good manufacturing practice (GMP) guide defines clean rooms in terms of grades
(namely grades AD). Annex 1 also addresses two particle sizes (0.5 and 5.0 m) and two occupancy
states (operational and 'at-rest') and says: "The particulate conditions for the 'at rest' state should be
achieved in the unmanned state after a short clean-up period of 1520 minutes". A room may have two
Figure 1 The
ISO class numbers depending on the room's occupancy state.
main
For example, a grade B room is equivalent to ISO class 7 (352000 particles, size 0.5 m/m3 ) in its
components of
operational state and ISO class 5 (3520 particles, size 0.5 m/m3 ) in its 'at-rest' state.
an air handing
Annex 1 was updated in 2003, which contains some additions worth noting: 7
unit (AHU).
The 'in operation' and 'at rest' states should be defined for each clean room or suite of clean rooms.

It now allows for one 5 m particle/m3 or larger for grade A zones, both 'at-rest' and in operation, as well as for a
grade B zone 'at-rest'.

The air velocity for a grade A area from 0.45 m/s 20% was replaced by a range of 0.360.54 m/s at the working
position in open clean room applications.

References to the old US Federal Standard 209 were removed.

Terminal (ceiling) HEPAs should be provided for Grades A, B and C. This is very important because before this EC
Guideline update, HEPAs for Grade C could have been located inside the AHU, but not any more. This now makes Grade C
rooms more expensive to build.

Classifying the facility areas

Classifying an entire facility class A (ISO 5) would ensure product purity, but doing so is unnecessary and too expensive.
Table 2, which shows recent trends in the biotech industry, relate clean room classifications to various manufacturing
operations, based on an operational state (when the product is actually being manufactured).
Some of these classifications are fixed (e.g., for ISO class 5, grade A is required where sterilized items
are exposed to room air), while others may vary depending on the contamination risk assessment by
manufacturers. For example, downstream purification is sometimes performed in grade B clean
rooms while at other times grade C suffices. The potential of product contamination is reduced by
minimizing the number of particles in the clean room.
Cleaning the supply air
Table 2 Air
This reduction is the first step in cleaning the supply air. However, outside air is required to satisfy
classification for
personnel ventilation, room exhaust-air requirements and for positive air pressurization. A cubic foot
various bio/pharma
of untreated outside air in an average industrial area could contain as many as 1200000 particles of
aseptic
size 0.5 m and larger.
manufacturing
steps and rooms.
 The maximum allowable particle counts in clean rooms varies from 100100000 particles/ft 3 though clean rooms have a
number of systems (including air handling units [AHUs] and HEPA filters) to ensure the air supplied will be cleaner than
required.8
Another potential external source of contamination is the AHU and the ductwork distributing the conditioned air (Figure 1).
Typical AHU construction includes aluminium interior panels with welded joints at the floor; cooling coils with stainless steel
coil frames (that prevent rust); easy-to-clean direct-drive plenum fans (to eliminate the possibility of particles from fan belts
entering the air stream); an access section between each AHU component (filter, fans, coils); and a
covered floor drain at each access section.
Terminal HEPA filters at the room's ceiling pick up any dust particles introduced into the duct via
ductwork access doors or generated by normal oxidation of galvanized sheet metal.
A further external source of contamination is adjacent rooms with doors, pass-throughs or other
openings connecting to the clean room. Reducing contamination requires specifying sealed, flush light
fixtures; sealing all walls, ceiling and floor penetrations, and pressurizing the rooms to prevent air
leakage. A pressure differential of 1015 Pa between a clean room and a connecting room of lower
cleanliness classification satisfies both US and European GMPs.
Minimizing particle generation
The biggest source of airborne particles in a clean room is personnel who generate both viable and
nonviable particles. A walking person with normal clothing generates about 13500 particles of 0.5 m
size while a person walking carefully with good clean clothes generates about 1000 particles. 9These particles may include
skin flakes, hair, cosmetics, clothing particles, perspiration and respiratory emissions. A number of measures can help
minimize particle generation

Avoid jewellery and make-up.

Only healthy personnel should work in a clean room to avoid spreading viruses and bacteria.

Finishes on the ceiling, walls and floors should prevent shedding and readily withstand wiping, and wear and tear.

All windows and door frames should be flush with the inside of the clean room.

Doors must have perimeter seals and floor sweeps to reduce air leakage.

Equipment should be placed so that it is easy to maintain and clean.

Removing particles
 The air distribution portion of the air conditioning system helps remove particles from a clean room as they are generated.
Air is the supplied from the ceiling in a nonaspirating manner to reduce room air turbulence.
This is typically accomplished by using ceiling terminal HEPA filters. Air is removed from the room with low wall returns
strategically located behind equipment or operations that generate particles. Dead spots (no airflow areas) in the room
should be avoided.
Airflow rate also helps to remove generated particles. For example, the number of AC/h in a clean room where powder
operations are performed should be greater than the flow rate in a room that handles liquids in closed vessels and piping.
For ISO 5 (class 100 or grade A) areas in operation, an air flow rate of 0.45 m/s 20% satisfies both US and EC GMP aseptic
guides. For ISO 8 (class 100000 or grade C) areas in operation, an air flow rate of 20 AC/hour is satisfactory. There are no air
flow rates mentioned either in the US or in the EC GMP aseptic guidelines for other clean room classes such as ISO 6 (class
1000), ISO 7 (class 10000 or grade B), nor ISO 9 (pharmaceutical grade or grade D). For years, this author has been
successfully using air flow rates of 120 AC/hour and 60 AC/hour for clean rooms of type ISO 6 and 7, respectively.

Some pharmaceutical companies have used 2540 AC/h for ISO 7 clean rooms when dealing with liquids in closed
containers and piping. For ISO 9 clean rooms, this author recommends a minimum of 15 AC/h and HEPA filters inside the
AHU to satisfy EC grade D areas in the 'at-rest' condition.
Wiping down with chemical agents at specific times (such as at the end of a day or a batch) is also necessary for clean
rooms. Fumigation with agents such as formaldehyde and vapourized hydrogen peroxide can also be used as a final
cleaning method for areas largely contaminated with viable organisms.
Other environmental parameters
Some other factors that should be considered include

Temperature. One of the most important parameters to control. Typical clean room temperatures vary from 1821 C
dry bulb for clean room classes 100 through 100000 (ISO classes 58).

Relative humidity (RH). Biopharm products can normally withstand a wide range of humidity. Keeping the humidity
over 30% in the winter helps reduce static electricity; keeping the RH below 60% in the summer helps reduce the growth of
live organisms and rust on equipment.

Pressure. Clean spaces should be positive in relation to adjacent, less clean spaces. Exceptions to this would be clean
rooms handling live organisms requiring biocontainment and rooms where solvents, flammables or potent compounds are
handled.
These rooms should be negative to adjacent rooms whether the adjacent rooms are cleaner or not. Typically, this is
accomplished by using airlocks of the pressure-bubble type where static pressure in the airlock is higher than all the rooms
it serves. Conditioned air to these pressure-bubble airlocks is supplied from a clean, non-contaminated source.
Summary
Maintaining clean rooms is a complex task. You must not only know the relevant country building codes and
pharma/biopharma industry GMPs, but also be able to meet them. It is a constant battle to keep clean rooms truly clean.
References
1. ISO 146441 (1999). http://www.iso.org/
2. http://www.emea.eu.int/
3. Federal Standard 209E Airborne Particulate Cleanliness Classes in Cleanrooms and Clean Zones, General Services
Administration, 11 September (1992).
4. G.J. Farquharson and W. Whyte, "The design of clean rooms for the pharmaceutical industry," in W. Whyte, Ed., Clean
room design (John Wiley and Sons Ltd, Chichester, UK, 1992).
5. http://www.fda.gov/
6. http://www.fda.gov/Cder/guidance/5882fnl.htm
7. http://ec.europa.eu/
8. ASHRAE International. Method of testing general ventilation air-cleaning devices for removal efficiency by particle size.
Standard 52.2-1999. Atlanta GA:1999.
9. ISPE, Baseline pharmaceutical engineering guide, Vol. 3: Sterile manufacturing facilities Chart A3-1: Number of particles
generated per second. Tampa, FL: First ed.; 1999 Jan.
10. J.L. Vesper, BioProcess International, 1 (2) 2429 (2003).
11. International Organization for Standardization, Clean rooms and associated controlled environments Part 2:
Specifications for testing and monitoring to prove continued compliance with ISO-146441. International Standard ISO
146442. Geneva: 2002.
The original version of this aticle can be found in M. del Valle, BioPharm International, 1 (3), 5060 (2004).
Manuel A. del Valle is director of HVAC design at Fluor Corp., USA.