Primary containment: Operation and equipment Work with viable micro-organisms should take place in closed systems (CS), which minimize (m) or prevent (p) the release of cultivated micro-organisms Treatment of exhaust air or gas from CS Sampling from CS Addition of materials to CS, transfer of cultivated cells Removal of material, products and effluents from CS Penetration of CS by agitator shaft and measuring devices Foam-out control
Secondary containment: Facilities
Protective clothing appropriate to the risk category Changing/washing facility Disinfection facility Emergency shower facility Airlock and compulsory shower facilities Effluents decontaminated Controlled negative pressure HEPA filters in air ducts Tank for spilled fluids Area hermetically sealable
* Unless required for product quality, — , not required; + , required.
m, minimize release. The level of contamination of air, working surface and personnel shall not exceed the level found during microbiological work applying Good Microbiological Techniques.
p, prevent release. No detectable contamination during work should be found in the air, working surfaces and personnel.
Federation for Biotechnology were aware of this problem with non-recombinant micro-organisms and produced a consensus list (Frommer et al., 1989).
Most micro-organisms used in industrial processes are in the lowest hazard group which only require
GILSP, although some organisms used in bacterial and viral vaccine production and other processes are categorized in higher groups. There is an obvious incentive for industry to use an organism which poses a low risk as this minimizes regulatory restrictions and reduces the need for expensive equipment and associated containment facilities (Schofield, 1992). The costs of some containment design features are discussed in Chapter 12.
In this chapter, where appropriate, a distinction will be made between equipment which is used to maintain aseptic conditions for GILSP and those modifications which are needed to meet physical containment requirements in order to grow specific cultures which have been classified in higher hazard groups. Details on design for containment have been given by East et al. (1984), Flickinger and Sansone (1984), Walker et al. (1987), Turner (1989), Hambleton et al. (1991), Leaver and Hambleton (1992), Vranch (1992) and Werner (1992).
Overall containment categorization
In this chapter the emphasis will be on containment levels which might be obtained from particular designs of fermenters and associated equipment. However this is only one aspect of assessment. To meet the standards of the specific level of containment it will also be necessary to consider the procedures to be used, staff training, the facilities in the laboratory and factory, downstream processing, effluent treatment, work practice, maintenance, etc. It will be necessary to ensure that all these aspects are of a sufficiently high standard to meet the levels of containment deemed necessary for a particular process by a government regulatory body. If these are met, then the process can be operated.
In fermentations with strict aseptic requirements it is important to select materials that can withstand repeated steam sterilization cycles. On a small scale (1 to 30 dm3) it is possible to use glass and/or stainless steel. Glass is useful because it gives smooth surfaces, is non-toxic, corrosion proof and it is usually easy to examine the interior of the vessel. Two basic types of fermenter are used:
1. A glass vessel with a round or flat bottom and a top flanged carrying plate (Fig. 7.4). The large glass containers originally used were borosilicate battery jars (Brown and Peterson, 1950). All ves-
Fig. 7.4. Glass fermenter with a top-flanged carrying plate (Incel-tecli L.H. Reading, England).
sels of this type have to be sterilized by autoclav-ing. Cowan and Thomas (1988) state that the largest practical diameter for glass fermenters is 60 cm.
2. A glass cylinder with stainless-steel top and bottom plates (Fig. 7.5). These fermenters may be sterilized in situ, but 30 cm diameter is the upper size limit to safely withstand working pressures (Solomons, 1969). Vessels with two stainless steel plates cost approximately 50% more than those with just a top plate.
At pilot and large scale (Figs 7.6 and 7.7), when all fermenters are sterilized in situ, any materials used will have to be assessed on their ability to withstand pressure sterilization and corrosion and on their potential toxicity and cost. Walker and Holdsworth (1958), Solomons (1969) and Cowan and Thomas (1988) have discussed the suitability of various materials used in the construction of fermenters. Pilot-scale and industrial-
Fig. 7.5. Three glass fermenters with top and bottom plates (New Brunswick Scientific, Hatfield, England).
scale vessels are normally constructed of stainless steel or at least have a stainless-steel cladding to limit corrosion. The American Iron and Steel Institute (AISI) states that steels containing less than 4% chromium are classified as steel alloys and those containing more than 4% are classified as stainless steels. Mild steel coated with glass or phenolic epoxy materials has occasionally been used (Gordon et al., 1947; Fortune et al., 1950; Buelow and Johnson, 1952; Irving, 1968). Wood, plastic and concrete have been used when contamination was not a problem in a process (Steel and Miller, 1970).
Walker and Holdsworth (1958) stated that the extent of vessel corrosion varied considerably and did not appear to be entirely predictable. Athough stainless steel is often quoted as the only satisfactory material, it has been reported that mild-steel vessels were very satisfactory after 12-years use for penicillin fermentations (Walker and Holdsworth, 1958) and mild steel clad with stainless steel has been used for at least 25 years for acetone-butanol production (Spivey, 1978). Pitting to a depth of 7 mm was found in a mild-steel fermenter after 7-years use for streptomycin production (Walker and Holdsworth, 1958).
The corrosion resistance of stainless steel is thought to depend on the existence of a thin hydrous oxide film on the surface of the metal. The composition of this film varies with different steel alloys and different manufacturing process treatments such as rolling, pickling or heat treatment. The film is stabilized by chromium and is considered to be continuous, non-porous, insoluble and self healing. If damaged, the film will repair itself when exposed to air or an oxidizing agent (Cubberly et al., 1980).
The minimum amount of chromium needed to resist corrosion will depend on the corroding agent in a particular environment, such as acids, alkalis, gases, soil, salt or fresh water. Increasing the chromium content enhances resistance to corrosion, but only grades of steel containing at least 10 to 13% chromium develop an effective film. The inclusion of nickel in high percent chromium steels enhances their resistance and improves their engineering properties. The presence of molybdenum improves the resistance of stainless steels to solutions of halogen salts and pitting by chloride ions in brine or sea water. Corrosion resistance can also be improved by tungsten, silicone and other elements (Cubberly et al., 1980; Duurkoop, 1992).
AISI grade 316 steels which contain 18% chromium, 10% nickel and 2-2.5% molybdenum are now com
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