Manuel A.del Valle

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 

 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. 

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.

  European countries have incorporated these standards, whilst in the US, ISO 14644–1 and ISO 14644–2 replaced Federal Standard 209.

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 

 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/m

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.

  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. 

Guideline on Sterile Drug Products Produced by Aseptic Processing

 was the original document that defined clean rooms.

  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:

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

 The classification rates clean rooms in particles/m

  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 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/m

 ) in its operational state and ISO class 5 (3520 particles, size 0.5 μm/m

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

The 'in operation' and 'at rest' states should be defined for each clean room or suite of clean rooms. 

  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. 

 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 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.			

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/ft

 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.

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. 

Articles

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.