Keeping clean rooms compliant

Clean rooms are critical areas in bio/pharma facilities, and it is essential that users are responsible for their care and upkeep, and familiarize themselves with the relevant regulations.

Clean rooms make aseptic processing possible. FDA prefers parenterals to be terminally sterilized, but when that is not possible without affecting the product, the agency allows aseptic processing in lieu of terminal sterilization. When a medical product is ingested, any live organisms in the drug may die before reaching the bloodstream because of the body's defence mechanisms, such as saliva and stomach acid.

Parenterals, however, bypass some of these mechanisms and must, therefore, be manufactured under cleaner conditions. Airborne particles need to be controlled to reduce the possibility of contaminant particles entering the medicinal product and ultimately, the patient. Reducing the number of airborne particles in a clean room decreases the possibility of larger particles carrying bacteria and viruses.

Defining the clean room

ISO 14644–1 (which defines clean rooms) and ISO 14644–2 (which specifies requirements to prove compliance with ISO 14644–1) have been in force for many years.^1,2 European countries have incorporated these standards, whilst in the US, ISO 14644–1 and ISO 14644–2 replaced Federal Standard 209.³

Section 2.1.1 of ISO 14644–1 defines a clean room as "a room in which the concentration of airborne particles is controlled, and which is constructed and used in a manner to minimize the introduction, generation and retention of particles inside the room, and in which other relevant parameters, for example, temperature, humidity, and pressure, are controlled as necessary". Table 1 shows the relationship between ISO 14644–1 definitions of a clean room, and FDA and European Commission (EC) requirements for facilities that use aseptic processing. Before ISO 14644–1 was adopted, clean rooms were defined in the US in simple terms such as a class 100 clean room.

Section 3.3 of ISO 14644–1 states that the clean room designation should be based on three parameters:

• classification number expressed as an ISO class

• occupancy state

• particle sizes and their related concentration.

For example, the proper definition of what was once a class 100 clean room is now ISO class 5; operational state; particle size 0.5 μm; 3520 particles/m³ .

Although ISO 14644–1 defines clean rooms for various particle sizes ranging from 0.1–5.0 μm, in bio/pharma facilities, we are mostly concerned with particles greater than 0.5 μm. Bacteria are not usually found in the air as unicellular organisms, they are normally carried on materials such as skin particles, with an average size in the air of 10–15 μm. Small inanimate particles have a low deposition rate and little chance of entering containers from the air in sufficient numbers to be a problem.⁴

Standard ISO 14644–1 classifies clean rooms only in terms of "particles per cubic meter" without differentiating between viable and non-viable particle count. US and EC GMPs is where the acceptable number of viable particles per cubic meter may be found.

Guidelines

FDA's 1987 Guideline on Sterile Drug Products Produced by Aseptic Processing was 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 requirements for sterile medicinal products.

This guide was replaced in September 2004, with the new guideline containing some notable additions:⁶

• Sterile drugs should be manufactured using aseptic processing only when terminal sterilization is not feasible.

• The classification rates clean rooms in particles/m³ 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 10–15 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 A–D). Annex 1 also addresses two particle sizes (0.5 and 5.0 μm) 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 15–20 minutes". A room may have two ISO class numbers depending on the room's occupancy state.

Figure 1 The main components of an air handing unit (AHU).

For example, a grade B room is equivalent to ISO class 7 (352000 particles, size 0.5 μm/m³ ) in its operational state and ISO class 5 (3520 particles, size 0.5 μm/m³ ) in its 'at-rest' state.

Annex 1 was updated in 2003, which contains some additions worth noting:⁷

• 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 μm 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 0.36–0.54 m/s at the working position in open clean room applications.

1. References to the old US Federal Standard 209 were removed.

1. 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.

Table 2 Air classification for various bio/pharma aseptic manufacturing steps and rooms.

Cleaning the supply air

This reduction is the first step in cleaning the supply air. However, outside air is required to satisfy personnel ventilation, room exhaust-air requirements and for positive air pressurization. A cubic foot of untreated outside air in an average industrial area could contain as many as 1200000 particles of size 0.5 μm and larger.

The maximum allowable particle counts in clean rooms varies from 100–100000 particles/ft³ 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 10–15 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 μm size while a person walking carefully with good clean clothes generates about 1000 particles.⁹ These particles may include skin flakes, hair, cosmetics, clothing particles, perspiration and respiratory emissions. A number of measures can help minimize particle generation

1. Avoid jewellery and make-up.
2. Only healthy personnel should work in a clean room to avoid spreading viruses and bacteria.
3. Finishes on the ceiling, walls and floors should prevent shedding and readily withstand wiping, and wear and tear.
4. All windows and door frames should be flush with the inside of the clean room.
5. Doors must have perimeter seals and floor sweeps to reduce air leakage.
6. 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 25–40 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

1. Temperature. One of the most important parameters to control. Typical clean room temperatures vary from 18–21þ C dry bulb for clean room classes 100 through 100000 (ISO classes 5–8).
2. 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.
3. 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 14644–1 (1999). www.iso.org

2. 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. www.fda.gov

6. 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) 24–29 (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-14644–1. International Standard ISO 14644–2. Geneva: 2002.

The original version of this aticle can be found in M. del Valle, BioPharm International, 1 (3), 50–60 (2004).

Manuel A. del Valle is director of HVAC design at Fluor Corp., USA.