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In the past workers have mainly concentrated on single unit operations rather than considering processes as a whole and the effect that unit operations have on others further downstream. Interactions include: nucleic acids and cell debris released during cell dismption can affect fractional precipitation and chromatography stages; increased cell rupture to maximise intracellular product release can result in debris too fine to sediment which then passes through subsequent steps. Such interactions make it essential to have a process view of downstream processing. Interactions between different unit operations in general and especially those involved in protein purification are discussed below.

1.5.1 Fermentation and product recovery

The essential increases in titre achieved as a result o f strain improvement and optimisation o f culture conditions have led to the situation where fermentation rather than product recovery considerations have dominated process development. Hence downstream recovery processes often prove adequate but less tiian ideal. Rosen (1983) pointed out that 45 % of equipment costs can be associated with product recovery compared with 14 % for fermentation. Also the ratio o f recoveiy to fermentation costs is 2.0 for an enzyme. Hence it is important that sufficient effort is devoted to downstream as well as fermentation aspects. An essential part of tins is that o f process interactions. Important parameters o f fermentation that affect protein recovery have been reviewed by Fish and Lilly (1984).

1. Harvest time. Enzyme titre in a batch culture may be at a maximum for no more than an hour, for example gramicidin S synthetases in Bacillus brevis (Kula et al, 1982). Reduction in titre may be due to depletion o f carbon or nitrogen which can result in increased protein

turnover. In defined media, cell lysis is more likely. This can lead to enzyme inactivation and consequent product loss.

2. Growth medium. The type of growth medium can affect the cell wall strength which has a significant effect on cell disruption for the recovery o f an intracellular product. The rate of release of p-galactosidase fi’om E.coli by passage through a high pressure homogeniser was faster when the culture was grown on glycerol with mineral salts instead o f a complex medium (Gray et al, 1972). This is thought to be due to the availability o f components in complex media for building strong cell walls which are not present in defined media.

3. Age of culture. The rate of protein release during disruption was highest for bacteria harvested early in the exponential phase and decreased as further growth occurred until it was several times less for cells recovered in the stationary phase (Wang et al, 1979). Similarly Candida utilis grown at a high specific growth rate in cyclic batch culture was easier to disrupt in a high pressure flow device than when grown at a lower specific growth rate in continuous culture (Engler and Robinson, 1981).

1.5.2 Homogenisation and centrifugation

The physical characteristics o f the feed material to the centrifuge depend on the operating parameters o f the homogeniser. In particular, the total cell debris particle size distribution and viscosity of the suspension have a major effect on centrifuge performance. Homogenisation at high pressures and for repeated passes maximises product release but also leads to the production o f sub-micron sized cell debris (Keshavarz et al, 1990) and the release o f nucleic acids and proteoglycans (Mosqueira et al, 1981). These cause an increase in suspension viscosity making debris recovery more difficult. The interaction between the homogenisation step and the cell debris clarification step is shown in Figure 1-4 (Clarkson et al. 1993b).

100

c?

20

1.0 1.5 2.0 2.5 3.0 3.5 4.0

Particle diameter (|i m)

The theoretical separation limit of a centrifiige can be calculated fi*om the centrifuge critical particle diameter, dc, which is a function o f the design and operating conditions of the machine. In theory, cell debris particles with a diameter greater than or equal to dc will be recovered whereas particles smaller than dc will not. Figure 1-4 shows the equivalent particle diameters above which lie 90 % (dgo) and 50 % (dso) of the total particle population. Figure 1-4 shows how the centrifuge throughput must be reduced in order that the d^o line and the dc lines intercept. At a centrifuge throughput o f 100 Vh the dc line intercepts with the dgo line at a protein release o f 25 %. Hence for the efficient removal o f cell debris the homogeniser must be operated so that most the product is not released. If the centrifuge throughput is halved then the homogeniser can be operated to give 48 % protein release.

1.5.3 Protein precipitation and centrifugation

Precipitation is sometimes perceived as an unreliable process. This is because performance is affected by a number o f factors one o f which is the variable composition o f the feed stream resulting fi'om variations in the fermentation. The main contaminating substances that interfere with precipitation are fermentation broth, nucleic acids and cell debris (Richardson et al, 1989).

When considering the precipitation process it is essential to remember the subsequent centrifugation step for precipitate removal. This is an analogous situation to the homogenisation/centrifugation interaction. For efficient clarification o f the process stream after precipitation it is important to maximise the precipitate particle size, strength, and density difference in relation to the process liquid. Different precipitation reactor designs also impact on centrifuge clarification efficiency; discussed earlier (Section 1.4.3).