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An essential piece of on-line monitoring is rapid solids removal prior to assay (van de Merbel et al., 1996). These include whole cells and particles deliberately generated in downstream processing such as cell debris and protein precipitates. The latter would redissolve during subsequent dilution causing overestimation of the liquid phase solubility. The integrity of the soluble constituents must be preserved (Mattiasson and Haakanson, 1993). Although centrifugation is mechanically complex it is more effective with saturated protein solutions compared to microfiltration which experiences polarisation and fouling (section 1.1.2). Blinding of the filter membrane reduces the transmission leading to inaccurate process values. Scaled-down versions of both may be applied to the sample stream and are discussed below.

1.2.1.1 Centrifugation

The development of a prototype miniature centrifugal separator or 'microcentrifuge' has been achieved for the automated clarification of samples (Richardson et al., 1996a). The salient features were a specially designed 20 mm i.d. cup shaped bowl pneumatically driven at high speed having rapid acceleration and deceleration, shown in Figure 1.4, p. 57. The sample batch was introduced and after the spin period the supernatant was recovered from the stationary bowl. A high pressure water cleaning cycle with accompanying suction was then instituted (section 2.4.2).

The microcentrifuge was found to obey basic theory in terms of the angular velocity and sedimentation distance using clarified yeast homogenate / ammonium sulphate solution system. A nominal pelleting time defined as the time required to

attain a 0.01 fraction unsedimented was measured by optical density at 650 nm. It was related to the reciprocal of the square of the bowl speed at 33,000 and 60,000 rpm (equation 1.13) for a fixed volume 0.5 ml sample at 70% overall feed saturation. Similarly there was a proportionality which intercepted the origin between the pelleting time varying from 15-45 s and the logarithmic settling distance ratio ln(Rd/Ri) with sample volumes from 0.3-0.9 ml at 80% overall feed saturation and a constant speed of 30,000 rpm.

Solubility profiles were consequently determined for total protein by the dual UV wavelength method as adapted to FIA (section 1.2.2). It was observed that the microcentrifuge left a small residual turbidity in the supernatant after a 1 min spin time compared to a laboratory centrifuge run at 10,000 g for 30 min. This was imperceptible with the précipitants 25 % w/v PEG 6000 or elsewhere absolute ethanol, the sedimentation being particularly efficient. The offset was thus not believed to be an inherent facet of the machine. The spin time also appeared sufficient using ammonium sulphate solution. It was concluded that owing to the low aggregate size (as well as a density difference <100 kg/m^ between the protein precipitates and the suspending fluid) the loosely adhering outer layer would have resuspended on braking.

The microcentrifuge unit has been investigated for the on-line analysis of excreted metabolite concentration from recombinant Escherichia coli fermentations (Turner et al., 1994). The problem of abnormal supernatant turbidity existed, comprising more solids than were acceptable to HPLC, which is considered later in section 5.2.

1.2.1.2 Filtration

The evolution of sample filters for downstream processing is associated with those used in fermentation (Lorenz, 1987). However the latter devices always require aseptic operation to be maintained. An early study here of on-line measurement involved extracellular hydrolase enzymes (Kroner and Kula, 1984). The culture broth

was circulated over a steam-sterilisable ultrafiltration or microporous membrane obtaining a representative sample stream. The filter type and running conditions, such as the pore size, the relative flow rates and the stirrer speed, were chosen for a given feed. A continuous system incorporating the on-line monitoring of alkaline protease from the fermentation of Bacillus licheniformis was possible.

The dynamic filtration sampling device was optimised, resulting in a unit that interfaced a process to an analyser (Kroner and Papamichael, 1988). It consisted of two plates between which a variety of standard 90 mm diameter filter membranes were able to be inserted. Pore sizes of 0.2 or 0.45 /xm were recommended for enzymes. The volume was approximately 175 ml on the retentate side, but only

1.1 ml on the filtrate side to minimise the delay time. This was slightly more than 2 min with a salt tracer. The recycling feed flow rate was typically 10-60 1/hr whilst that of the filtrate was 0.1-1.5 ml/min, at a comparatively low pressure differential of 0.1-0.3 bar. An adjustable magnetic stirrer in the upper chamber rotating at up to 1000 rpm enabled good mixing and controlled the formation of the membrane sublayer. Other modules have seen many applications for instance during bacteria, yeast and mycelia fermentations (Gam et al., 1989).

The devices allow continuous sample preparation having no serious retention of proteins, even where complex media is present containing a high level of solids. The capacity to adjust the operational parameters extends the long-term stability. The design of the membrane area to filtrate side dead volume ensures a short delay time with a fast response to process concentration fluctuations. The most suitable filter may be selected and the units are easily accessed. Despite this the transmission has been observed to vary for different proteins (Stamm et al., 1989). In addition the retention of contaminants including the antifoam agent polypropylene glycol were altered by conditions such as temperature. The feed throughput is large though may be reduced or taken periodically for a lower frequency of analysis.