Lc

LC Level control

TC Temperature control

LC Pressure sensor

LC Flow sensor

Air vent

Safety relief D TC 4a valve

Kill tank

Water u

Aseptic drain pump

Sump

Fig. 5.13. A vessel for the batch sterilization of liquid waste from a contained fermentation (Jansson et al, 1990).

(ii) Diffusion

Extremely small particles suspended in a fluid are subject to Brownian motion which is random movement due to collisions with fluid molecules. Thus, such small particles tend to deviate from the fluid flow pattern and may become impacted upon the filter fibres. Diffusion is more significant in the filtration of gases than in the filtration of liquids.

(iii) Electrostatic attraction

Charged particles may be attracted by opposite charges on the surface of the filtration medium.

(iv) Interception

The fibres comprising a filter are interwoven to define openings of various sizes. Particles which are larger than the filter pores are removed by direct interception. However, a significant number of particles which are smaller than the filter pores are also retained by interception. This may occur by several mechanisms — more than one particle may arrive at a pore simultaneously, an irregularly shaped particle may bridge a pore, once a particle has been trapped by a mechanism other than interception the pore may be partially occluded enabling the entrapment of smaller particles. Interception is equally important a mechanism in the filtration of gases and liquids.

Filters have been classified into two types — those in which the pores in the filter are smaller than the particles which are to be removed and those in which the pores are larger than the particles which are to be removed. The former type may be regarded as an absolute filter, so that filters of this type (provided they are not physically damaged) are claimed to be 100% efficient in removing micro-organisms. Filters of the latter type are frequently referred to as depth filters and are composed of felts, woven yarns, asbestos pads and loosely packed fibreglass. The terms absolute and depth can be misleading as they imply that absolute filtration only occurs at the surface of the filter, whereas absolute filters also have depth and filtration occurs within the filter as well as at the surface. Terms which bear more relationship to the construction of filters are 'non-fixed pore filters' (corresponding with depth filters) and 'fixed pore filters' (corresponding with absolute filters).

Non-fixed pore filters rely on the removal of particles by inertial impaction, diffusion and electrostatic attraction rather than interception. The packing material contains innumerable tortuous routes through the filter but removal is a statistical phenomenon and, thus, sterility of the product is predicted in terms of the probability of failure (similar to the situation for steam sterilization). Thus, in theory, the removal of microorganisms by a fibrous filter cannot be absolute as there is always the possibility of an organism passing through the filter, regardless of the filter's depth. Also, because the fibres are not cemented into position an increase in the pressure to which the filter is subjected may result in movement of the material, producing larger channels through the filter. Increased pressure may also result in the displacement of previously trapped particles.

Fixed pore filters are constructed so that the filtration medium will not be distorted during operation so that the flow patterns through the filter will not change due to disruption of the material. Pore size is controlled during manufacture so that an absolute rating can be quoted for the filter — i.e. the removal of particles above a certain size can be guaranteed. Thus, interception is the major mechanism by which particles are removed. Because fixed pore filters have depth they are also capable of removing particles which are smaller than the pores by the mechanisms of inertial impaction, diffusion and attraction and these mechanisms do play significant roles in the filtration of gases. Fixed pore filters are superior for most purposes in that they have absolute ratings, are less susceptible to changes in pressure and are less likely to release trapped particles. The major disadvantage associated with absolute filters was the resistance to flow they present and, hence, the large pressure drop across the filters which represents a major operating cost. However, modern absolute filter cartridges which have been developed by many filtration companies contain pleated membranes with very large surface areas, thereby minimizing the pressure drop across the filter.

It is important to realize that filters should be steam sterilized before and after operation (see Chapter 7). Thus, the materials must be stable at high temperatures and the steam must be free of particulate matter because the filter modules are particularly vulnerable to damage at high temperatures. Thus, the steam itself is filtered through stainless steel mesh filters rated at 1 /um.

Filter sterilization of fermentation media

Media for animal-cell culture cannot be sterilized by steam because they contain heat-labile proteins. Thus, filtration is the method of choice and as may be appreciated from the previous discussion, fixed pore or absolute filtration is the better system to use. An ideal filtration system for the sterilization of animal cell culture media must fulfil the following criteria:

(i) The filtered medium must be free of fungal, bacterial and mycoplasma contamination.

(ii) There should be minimal adsorption of protein to the filter surface.

(iii) The filtered medium should be free of viruses.

(iv) The filtered medium should be free of endotoxins.

Several filter manufacturers now supply absolute filtration systems for the sterilization of animal cell culture medium. Such systems consist of membrane cartridges which are fitted into stainless steel, steam steril-izable modules. The membranes for media filtration are constructed from steam sterilizable hydrophilic material and are treated to produce a filtrate of particular quality. For example, if minimal protein adsorption is a major criterion then a specially coated filter membrane is used. It would be very difficult to construct a single filtration membrane which would fulfil all four criteria cited above. Thus, a series of filters are used to achieve the desired result. The example shown in Fig. 5.14 is provided by Pall Process Filtration Ltd and illustrates a system to produce sterile, mycoplasma free serum and consists of four filters arranged in sequence. The first filter is a positively charged polypropylene pre-filter with an absolute rating of 5 /xm for the removal of coarse precipitates, clot-like material and other gross contaminants; the second filter is also positively charged polypropylene but with an absolute rating of 0.5 /xm for bulk microbial removal, deformable gels, lipid-based materials and endotoxin reduction; the third filter is a single layered, nylon/polyester positively charged filter with a 0.1-yu.m absolute rating for further microbial and endotoxin removal and optimum protection of the final filter; the fourth filter is similar to the third and has the same rating, but is double layered and removes mycoplasmas, gives absolute sterility and final endotoxin control. Thus, the combination of four filters gives a sequential removal of decreasingly small particles and prolongs the life of the final filter. If it is necessary to remove viral contamination then a final 0.04-^.m nylon/polyester filter would be added.

Similar systems may be used in downstream processing of animal cell products where the rating and properties of the filters would be optimized for the particular process. Figure 5.15 illustrates a system for the removal of cells and cell debris from an animal-cell fermentation broth. The pre-filter is a polypropylene 1.0-/Am rated filter to remove the bulk of the cells and debris and the second filter is an hydroxyl modified nylon/polyester 0.2-/um rated filter giving absolute cell removal with minimal protein adsorption.

Compressed air top pressure k

Vent filter

Bulk pooling tank

Bulk pooling tank

To call culture vessel or filling line

Fermenter

Filter 1

Filter 2

To furthor downstream processing and purification

Fermenter

Fig. 5.14. Filtration system for the provision of sterile, mycoplasma free serum.

Filter 1. 5/im absolute rated prefilter for removal of coarse precipitates.

Filter 2. 0.5/im absolute rated prefilter for bulk bioburden removal.

Filter 3. 0.1/xm absolute rated single layer prefilter for futher bioburden and endotoxin removal.

Filter 4, 0.1/^.m absolute rated double layer final filter for absolute sterility, mycoplasma removal and further endotoxin control. (Pall Process Filtration Ltd., Portsmouth, U.K.)

Filter sterilization of air

Fig. 5.15. Filtration system for the removal of cells and cell debris from an animal cell culture fermentation.

Filter 1. 1.0/tm absolute rated prefilter for bulk cell and cell debris removal.

Filter 2. 0.2/xm absolute rated single layer 'Bio-Inert' filter for final bioburden removal.

(Pall Process Filtration Ltd., Portsmouth, U.K.)

tion plant for an 85 m3 fermenter. Smith (1981) cited the use of absolute filtration in the sterilization of air for the ICI biomass continuous fermenter.

Aerobic fermentations require the continuous addition of considerable quantities of sterile air (see Chapter 9). Although it is possible to sterilize air by heat treatment, the most commonly used sterilization process is filtration. Fixed pore filters (which have an absolute rating) are very widely used in the fermentation industry and several manufacturers produce filtration systems for air sterilization. These systems, like those for the sterilization of liquids, consist of pleated membrane cartridges designed to be accommodated in stainless steel modules. A selection of such cartridges and holders is shown in Fig. 5.16 and a sectioned filter unit is shown in Fig. 5.17. The most common construction material used for for the pleated membranes for air sterilization is PTFE, which is hydrophobic and is therefore resistant to wetting. Also, PTFE filters may be steam sterilized and are resistant to ammonia which may be injected into the air stream, prior to the filter, for pH control. As was seen for the filter sterilization of liquids it is essential that a prefilter is incorporated up-stream of the absolute filter. The prefilter traps large particles such as dust, oil and carbon (from the compressor) and pipescale and rust (from the pipework). The use of a coalescing prefilter also ensures the removal of water from the air; entrained water is coalesced in the filter (air flow being from the inside of the filter to the outside) and is discharged via an automatic drain. Figure 5.18 illustrates the layout of such a filtration unit showing the steam sterilization system and Fig. 5.19 is a photograph of the air steriliza-

Sterilization of fermenter exhaust air

In many traditional fermentations the exhaust gas from the fermenter was vented without sterilization or vented through relatively inefficient depth filters. With the advent of the use of recombinant organisms and a greater awareness of safety and emission levels of allergic compounds the containment of exhaust air is more common (and in the case of recombinant organisms, compulsory). Fixed pore membrane modules are also used for this application but the system must be able to cope with the sterilization of water saturated air, at a relatively high temperature and carrying a large contamination level. Also, foam may overflow from the fermenter into the air exhaust line. Thus, some form of pretreatment of the exhaust gas is necessary before it enters the absolute filter. This pretreatment may be a hydrophobic prefilter or a mechanical separator to remove water, aerosol particles and foam. The pretreated air is then fed to a 0.2-(¡.m hydrophobic filter. Again, it is important to appreciate that the filtration system must be steam sterilizable. Figures 5.18 and 5.19 illustrate the prefilter and mechanical separator systems respectively.

The theory of depth filter;,

Although most fermentation companies rely upon the pleated membrane, fixed pore (absolute rated) filter

• 5.16. A selection of absolute membrane filter cartridges and stainless steel housings (Fall Process Filtration, Portsmouth, U.K.).

systems it is necessary to consider the theory of depth filtration. Aiba et al. (1973) have given detailed quantitative analysis of these mechanisms but this account will be limited to a description of the overall efficiency of operation of fibrous filters. Several workers (Ranz and Wong, 1952; Chen, 1955) have put forward equations relating the collection efficiency of a filter bed to various characteristics of the filter and its components. However, a simpler description cited by Richards (1967) may be used to illustrate the basic principles of filter design.

If it is assumed that if a particle touches a fibre it remains attached to it, and that there is a uniform concentration of particles at any given depth in the filter, then each layer of a unit thickness of the filter should reduce the population entering it by the same proportion; which may be expressed mathematically as:

Steam

Steam

Fig. 5.18. Dual hydrophobic filter system for the sterilization of off-gas from a fermenter (Pall Process Filtration Ltd., Portsmouth, U.K.).

where N is the concentration of particles in the air at a depth, x, in the filter and K is a constant. On integrating equation (5.13) over the length of the filter it becomes:

where N() is the number of particles entering the filter and N is the number of particles leaving the filter.

On taking natural logarithms, equation (5.14) becomes:

Equation (5.15) is termed the log penetration relationship. The efficiency of the filter is given by the ratio of the number of particles removed to the original number present, thus:

where E is the efficiency of the filter. But:

(N{)~ N)/N0 = i - (N/Nq). (5.17) Substituting N / Nq = e~Kx Thus:

The log penetration relationship [equation (5.15)] has been used by Humphrey and Gaden (1955) in filter design, by using the concept X>0, the depth of filter required to remove 90% of the total number of particles entering the filter; thus:

If N0 were 10 and x were X90, then N would be 1:

Steam

Turbosep™

Heater

Heater

PTFE sterile filter

Condenser

Steam

Turbosep™

Condenser

Fig. 5.19. A mechanical separator and hydrophobic filter system for the sterilization of off-gas from a fermenter. Left. Cut-away diagram. Right. Equipment arrangement, showing steam supply. V1-V6, valves; O, steam, traps (Domnick Hunter Ltd., Birtley, Co. Durham, U.K.).

2.303( — 1) = -KXgo, therefore, X90 = 2.303/K. (5.19)

Consideration of equation (5.15) indicates that a plot of the natural logarithm of N / N0 against x, filter length, will yield a straight line of slope K. To obtain the information for a plot of this type, data must be gleaned for the removal of organisms from an air stream by filters of increasing length which would involve assessment of microbial levels in the air entering and leaving the filter. Humphrey and Gaden (1955) and Richards (1967) described equipment with which such assessments could be recorded.

The value of K is affected by the nature of the filter material and by the linear velocity of the air passing through the filter. Figure 5.20 is a typical plot of K and Xt)tl against linear air velocity from which it may be seen that K increases to an optimum with increasing air velocity, after which any further increase in air velocity results in a decrease in K. Table 5.3 (Riviere, 1977) summarizes the effects of linear air velocity on the removal of a range of micro-organisms with a variety of filter materials.

Fig. 5.20. The effect of increasing linear air velocity on K and X90 of a filtration system (Richards, 1967).

The increase in K with increasing air velocity is probably due to increased impaction, illustrating the important contribution this mechanism makes to the removal of organisms. The decrease in K values at high air velocities is probably due to disruption of the filter, allowing channels to develop and fibres to vibrate, resulting in the release of previously captured organisms.

Table 5.3. X90 values for the removal of a range of micro-organisms by a variety of filtration materials (Humphrey, 1960; Rivierre, 1977)

Filter material Diameter Micro-organism Air speed X9()

Filter material Diameter Micro-organism Air speed X9()

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