Aseptic Areas

ASEPTIC AREAS    

A)                Additional Requirements

                      i)  Clothing

The operative is a potential source of microorganisms and it is imperative that steps are taken to prevent this contamination. The operative must wear sterile protective headwear totally enclosing hair and beard, spectacles, powder-free rubber or plastic gloves (often two pairs are worn), a non-fibre-shedding facemask (to prevent the release of droplets) and footwear. A suitable garment is a one or two-piece trouser suit. Fresh sterile clothing should be provided each time a person enters an aseptic area.

           ii)  Entry to aseptic areas

Entry to an aseptic suite is usually through a ‘black–grey– white’ changing procedure (Figure 23.4), where white represents the highest level of cleanliness. Movement from ‘black’ to ‘white’ is via two changing rooms, the ‘grey’ area also serving as an entry to the cleanroom (Figure 23.4). There are several types of entry system in use. More details may be found in Whyte (2010).

           iii)              Equipment and operation

Any articles entering the aseptic area should ideally be sterilized, but may be disinfected. In order to achieve this, articles should be transferred via a double-ended sterilizer or hatch (i.e. with a door at each end). If they are not to be discharged directly to the aseptic area, they should be (1) double-wrapped before sterilization; (2) transferred immediately after sterilizing into a clean environment until required; (3) transferred from this clean environment via a double-doored hatch (where the outer wrapping is removed) to the aseptic area (where the inner wrapper is removed at the workbench). Hatches and sterilizers must be designed so that only one door may be opened at any one time. Solutions manufactured in the cleanroom may be brought into the aseptic area through a sterile 0.22 μm membrane filter.

Workbenches, including laminar flow units, and equipment, should be disinfected immediately before and after each work session. Equipment must be of the simplest design possible for the operation being performed. Aseptic manipulations must be carried out in the grade A air of a laminar flow cabinet or isolator. Speed, accuracy and economy of movement are essential features of good aseptic technique. It is therefore essential that workers are well trained and motivated and familiar with the task in hand. Observation and microbiological monitoring of the operator and of the environment are very important. Under no circumstances must living microorganisms, including those used for vaccine preparation  and for biological monitoring be introduced into the aseptic area.

B)                Environmental Monitoring

Monitoring of the environment is essential during manufacturing. It ensures that environmental requirements are being met and also helps spot trends.

Air is monitored for particles and microorganisms. Microorganisms are usually sought using settle plates or active samplers, such as the slit-toagar sampler. Settle plates rely on organisms falling from the atmosphere and settling onto an exposed agar plate. After a specified time (usually 4 hours) the plate will be covered and incubated. A slit-to-agar sampler draws in a specified volume of air, forcing organisms onto the surface of an agar plate. This latter method is able to give a viable count per volume, but organisms may be damaged and hence rendered non-viable by the capture process. Limits of viable counts for different grades of air are shown in Table 23.4. One of the limitations of traditional microbial detection is the time taken to culture bacteria and fungi. There is a great deal of interest in developing rapid (Denyer,  2007)  or  instantaneous  (Jiang,  2009) methods of microbial detection.

The nature of contamination can be informative. For example, the presence of Staphylococcus spp. suggests human-borne contamination. The adequacy of changing facilities and gowning would then be checked. In contrast, Bacillus spores would suggest environmental contamination and the entry of equipment into the cleanroom would be scrutinized.

Glove prints are taken by applying four fingers and a thumb to an agar plate. This ensures that disinfection of gloves is adequate. Surfaces may be monitored by swapping or by using contact plates. The latter have the advantage of providing a quantitative measure of surface contamination, but there is a risk of leaving agar deposits on the surface (Butson & Hawitt, 2008).

C)          Eliminating Human Intervention

The greatest source of contamination in the cleanroom comes from the operating staff (Whyte & Hejab, 2007). Movement of staff can increase particle shedding and disrupting laminar airflow. It is not surprising, therefore, that modern practices seek to minimize or even eliminate humans from the aseptic production area. This can be achieved by the use of automation, of isolators and of restricted access barriers (RABs).

All aseptic packaging should be carried out in a grade A environment with a grade B background (Table 23.2). Advances in technology now permit the production of self-contained workstations, or isolators, which incorporate many of the design principles of cleanrooms and laminar flow cabinets.

               i) Isolators

The isolator both protects the product from contamination by the operator and the operator from any hazardous materials. Direct interaction between the operator and the product is minimized by providing a grade A laminar flow of air with a positive pressure, the internal space being accessed by means of a glove/ sleeve system (Figure 23.5). A grade D background  is considered adequate for such operations. A fuller account of isolators is given by Midcalf et al. (2004).

     ii)       Restricted access barrier systems

Restricted access barrier systems (RABS) provide a level of control intermediate between an isolator and a cleanroom (Agalloco & Akers, 2006). They allow for easier intervention than an isolator but require a grade B background.

    iii)     Blow–fill–seal technology

Blow–fill–seal units are purpose-built pieces of equipment which carry out these three steps in a continuous process within a controlled environment. Containers are formed from thermoplastic granules and blown to form containers which are then filled and heat-sealed. These units are fitted with a grade A air shower and operated in a grade C environment for aseptic manufacture and a grade D background for products which are to be terminally sterilized.