Resultados de aplicación de post test

The results of sampling in and around the fermenter area are summarised in Table 6.2.

TABLE 6.2 Summary of results of sampling in fermentation area

Sample ID Comments Cells collected

(in cyclone)

FI Background, before inoculation n/d*

F2 2 h after inoc. n/d*

F3 5 h after inoc. n/d*

F4 During sampling of ‘live’ fermenter 4.7 + 1.1 X 1 0^

F5 After fermenter contents steam n/d*

sterilised

F6 During sampling of sterilised fermenter 2.7 ±0.3 X 105

F7 1.5 h after sampling from sterilised n/d*

fermenter

F8 3.5 h after sampling from sterilised n/d*

fermenter

*n/d (none detected, i.e. <2 x 10^ cells in the cyclone). Where cells detected, data shown ± SEM (n = 2). All samples were taken at a position 2 m from the fermenter, with the cyclone inlet situated 1 . 1 m above ground level.

The results indicate that during fermentation to a high cell density, there is no evidence of any release of process organisms to the air surrounding the fermenter. This is an expected result considering that the fermenters used are designed to operate to a high degree of containment and incorporate double mechanical seals with steam traces. However, there is evidence of a release of plasmid material during the use of the contained sampling array on two occasions. The amount of material captured equates to the equivalent of less than 0.5 pL of live cell broth in both instances. It was observed during both sampling events that after decoupling of the sampling array (Figure 6.5) a small amount of liquid leaks out. This liquid is likely to comprise mainly condensed steam, but there is a possibility that there is also a minuscule amount of material derived from the broth. Temperature mapping studies carried out as part of the commissioning program for the plant suggest that any cells that remained in the pipe work o f the sampling array would be steam sterilised by the time o f decoupling.

Fermenter Steam

Sample bottle (pre-sterillsed)

FIGURE 6.5 Schematic representation of the contained sampling array using at GWMRC Bioprocessing Pilot Plant. The pre-sterilised sample bottle and piping are connected to the sampling line via the couplings A and B. The line is steamed through before and after taking the sample. It is thought that there is a possibility that after steaming after collection of the sample a small quantity of the sampled broth might remain in the pipework, particularly in the vicinity of the valve to the sampling bottle. Glaxo Wellcome have temperature mapped the pipework during steam sterilisation right up to the 'weir' of the diaphragm valve to validate that any residual cells would be heat deactivated.

On the second occurrence of a release during sampling, the contents of the fermenter itself had been sterilised. Analysis of a sample of the sterilised broth indicates that per mL there is 1000 fold less pQR701 detected than there was in an unsterilised broth sample. The PCR method is therefore less sensitive to cells that are steam sterilised

Chapter 6. Validation o f containment in a bioprocess pilot plant

within the bulk of the fermenter. This may be due to enzymatic ‘nicking’ o f DNA occurring as the temperature slowly rises within the media. It is thought possible that cells that are killed by a sudden temperature rise, for example within the sampling line, are less susceptible to nicking as cellular proteins are rapidly denatured. In this case, the PCR might be equally sensitive to these killed cells as it is to viable cells.

The use of a microfiltration step before PCR to distinguish plasmid that is contained within cells from plasmid that is extracellular showed that with both samples being discussed here, target plasmid was not detected in the filtrate. It was hoped that the ratio of intra- to extracellular pQRTOl plasmid concentration measured in the samples might indicate the condition o f the cells which are released into the air and detected. For instance, steam sterilisation might have caused the intra/extracellular ratio to rise as lysis occurs in parallel to cell death. Since the pQRTOl concentration in each sample was found to be entirely intracellular (or at least non-0.45 pm filterable) then it can be concluded that this method is unable to distinguish steam killed cells from those that are viable. This is a failing of the currently available methodology as it is envisaged that there will be occasions when killed cells are present in the environment which could give rise to a false positive result for the presence of live cells. Notably, however, the concentration of killed cells in the air surrounding the sampling port is reduced to background levels within 1.5 h after the original sample (Table 6.2, samples F6, F7, F8).

Since it is recognised that sampling fi’om fermenters is an operation that might give rise to breach of containment, the design of sampling ports has received some consideration (Cameron et al, 1987; Hambleton et al, 1991; Leaver and Hambleton, 1992). Cameron et al (1987) have in fact monitored the containment effectiveness of a sampling port with a B. subtilis spore suspension in a fermenter using a cyclone as the air sampling device. In this study, bacteria were detected (by culture) at times coincidental with the operation of the sample valve. It should be noted that the port was not steam sterilised and it was thought that some of the seals were subject to wear, which may have accounted for the release of culturable cells. However, Hambleton and co-workers have suggested that where high containment is required, the use o f secondary containment features may be necessary (Hambleton et a l,\9 9 \). A contained sampling device that is of interest with regard to this study is supplied by Bioengineering AG (based on European patent 0172838) and is described as an aerosol-fi’ee high containment sampling mechanism. In this device, a needle that pierces the sample container and retracts after sampling is enclosed and steamed before the next sample container is attached. It would seem that the propensity for aerosol generation (even o f steam-killed cells) is reduced relative to that for the sampling array used at GWMRC (Figure 6.5). The use of sampling devices

such as these in a fermentation plant might solve the problem o f false positives that would arise by QPCR detection of aerosol release.

In summary, it seems most likely that non-viable cells were detected during fermenter sampling. However, at present, it is not possible to distinguish between viable and non- viable cells. This is a refinement that will need to be addressed if sampling is likely to occur in areas where viable and non-viable cells may be released and the former present a hazard whilst the latter is, to a certain degree, acceptable. There is some discussion of possible future work in this regard in Section 7.1.1.3.