Xj

-Steam

Fig. 7.21. An alternative simple sample port.

the sample port and the exterior is a condensed flow of steam between valves C and D, valves A and B being closed. Valve D is connected to a steam trap to avoid condensate accumulation. To sterilize valve B prior to sampling, valve C is partially closed, valve D completely closed and valve B partially opened to allow a slow stream of steam and condensate out of the sampling port. Valve A is opened briefly to cool the pipe and the broth is discarded. Valve C is then closed and a sample is collected. Valve A is then closed and the piping is resterilized. In between collecting samples valves C and D are left partially open.

A more modern sterilizable system, as illustrated in Fig. 7.22, has been described by Werner (1992). In the closed position steam enters through the entry point 6 and passes through the sampling hole 11 and into the sampling hole 2. When sampling, the handle is pushed so that the hole in the sampling tube is in the fermenter and the vessel contents may be sampled. When the sampler is closed the unit can be resterilized.

Marshall et al. (1990) recognized that sampling requires good mechanical design and good operator practice to ensure sterility. Complex valve sequencing can lead to operator error. The automatic MX-3 rotary valve (Fig. 7.23) was developed by Marshall Biotechnology Ltd and is now marketed by New Brunswick Scientific Ltd. In this device, broth is continuously recycled to and from the fermenter and up to 12 samples can be taken automatically in sample bottles according to a pre-programmed sequence. The storage of the bottles in an integral refridgeration block re

Aseptic Sampling Werner 1992

Position during sampling

Fig. 7.22. Sterilizable sampling system tor Category 1 (Werner, 1992). Key: (1) handle for opening and closing; (2) piping for sample or condensate, respectively; (3) 'O'-ring; (4) housing; (5) spring; (6) steam inlet; (7) union nut; (8) welding socket; (9) product; (10) wall of the bioreactor; (11) port of sampling tube.

Position during sampling

Fig. 7.22. Sterilizable sampling system tor Category 1 (Werner, 1992). Key: (1) handle for opening and closing; (2) piping for sample or condensate, respectively; (3) 'O'-ring; (4) housing; (5) spring; (6) steam inlet; (7) union nut; (8) welding socket; (9) product; (10) wall of the bioreactor; (11) port of sampling tube.

duces spoilage until they can be removed for analysis. This sampler can be used for GILSP.

In all the sampling devices described above, steam is used merely to maintain aseptic conditions. If the process organism has to be contained then the sample vessel must be designed accordingly. At level 1 or B2, removal of broth should be carried out in such a way that release of micro-organisms is minimized. Such a system (Fig. 7.24) is described by Janssen et al. (1990). The sampling bottle assembly is attached to the fermenter by a double-end shut-off quickconnect B and air is vented from the vessel during sampling by a suitable membrane filter. The pipe work is steamed for 15 minutes using 1.3 bar steam with the valve E on the sample bottle closed. With valves C and D closed and A and B open a sample can be taken, then the pipe is resterilized before removing the sample bottle.

At level 2 or B3, sampling should be done in a closed system (Figs 7.25 and 7.26), and all piping coming into contact with a sample must be sterilizable with steam. The sampling vessel should be closed during transport and samples should be examined under containment

Fig. 7.23. Schematic representation of the MX-3 BioSampler rotary valve system.

conditions corresponding to those specified for the process (Werner, 1992).

Feed ports

Additions of nutrients and acid/alkali to small fermenters are normally made via silicone tubes which are autoclaved separately and pumped by a peristaltic pump after aseptic connection. In large fermenter units, the nutrient reservoirs and associated piping are usually an integrated part which can be sterilized with the vessel. However, there may also be ports which are used intermittently (Fig. 7.27). These can be sterilized in situ with steam after connection has been completed and before any additions are made.

Sensor probes

Double 'O' ring seals have been used for many years to provide an aseptic seal for glass electrodes in stainless steel housings in fermenters using GILSP. This system is also suitable for levels 1 and B2, provided that release of micro-organisms is minimized and there are adequate disinfection procedures for dealing with leakages (Werner, 1992).

Steam in

Fermenter

Steam in

Fermenter

Sample System Refinery Bottle
Sample bottle

Fig. 7.24. Sample system for level 1 (Jannsen et al, 1990).

Valve

Sterilization

Sampling

Sterilization

Removal

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