Cell separation filtration

Filtration Filtration (qv) is appHed in blood cell separation to remove leukocytes from ted blood cell (RBC) and platelet concentrates. Centtifugational blood cell separators do not reduce white blood cells (WBC) in red cell and platelet products sufficiently to avoid clinical complications such as GvHD and alloimmunization. A post-apheresis filtration step is needed to further reduce the WBC load. Modem filters are capable of a 3-log reduction in white cell contamination of the blood product, eg, apheresis single-donor platelet units having a typical white cell contamination of 5 x 10 white cells in 4 x 10 platelets can be reduced to a 5 x 10 white cell contamination, a sufficiently low number to avoid severe transfusion reactions. 

Regardless of the location of the protein and its state, cell separation needs to be inemensive, simple, and reliable, as large amounts of fermentation-broth dilute in the desired product may be handled. The objectives are to obtain a well-clarified supernatant and solids of maximum dryness, avoiding contamination by using a contained operation. Centrifugation or crossflow filtration is t ically used for cell separation, and both unit operations can be run in a continuous-flow mode [Datar and Rosen, in Stephanopoulos (ed.), op. cit., pp. 369-503]. In recent years, e3q>anded-bea adsorption has become an alternative. It combines broth clarification and adsorption separation in a single step. 

Cells of C. reinhardtii were exposed to cell-free filtrates from A. flos-aquae, pure microcystin-LR or anatoxin-a, or combinations of the toxins. Both the position of the cells and the chlorophyll-a concentration of the cultures were observed for 12 days. Exposure to crude extracts as well as to combinations of the toxins significantly decreased chlorophyll levels in the cultures. Furthermore, these compounds were all capable of paralyzing the algae and thus promoted the settlement of C. reinhardtii cells. One intriguing aspect of this dynamic interaction is the separate finding that C. reinhardtii may actually induce toxin synthesis in A. flos-aquae (Kearns and Hunter 2000), essentially signaling its own demise. 

When planning an industrial-scale bioprocess, the main requirement is to scale up each of the process steps. As the principles of the unit operations used in these downstream processes have been outlined in previous chapters, at this point we discuss only examples of practical applications and scaling-up methods of two unit operations that are frequently used in downstream processes (i) cell separation by filtration and microfiltration and (ii) chromatography for fine purification of the target products. 

When bioreactors coupled to cell retention devices are used, it is also necessary to evaluate the scale-up of the cell separation equipment. In the case of the spin-filter (see Chapter 11), parameters such as filter rotation velocity and the ratio of filtration area to bioreactor working volume are particularly relevant (Deo et at, 1996). 

Filtration is a unit operation commonly employed nowadays in biotechnological processes. In this unit operation, a filter medium acts as a physical barrier to particles larger than its pores. Traditional filtration devices such as filter presses and rotary vacuum drum filters have so far found no application for the separation of animal cells. Nevertheless, membrane filters are commonly employed, as well as some alternative filter designs such as spin-filters. In the next sections, the most common types of filters used for animal cell separation will be discussed. 

Vogel JH, Kroner KH (1999), Controlled shear filtration a novel technique for animal cell separation, Biotechnol. Bioeng. 63 663-674. 

Vogel JH, Anspach FB, Kroner KH, Piret JMM, Haynes CA (2002), Controlled shear affinity filtration (CSAF) a new technology for integration of cell separation and protein isolation from mammalian cell cultures, Biotechnol. Bioeng. 78 806-814. 

Off-line determination of biomass concentration by classical gravimetric methods requires cell separation, washing steps and drying to constant weight. The separation of cells can be made either by centrifugation or by filtration. 

Most enzymes of industrial importance like amylase, protease, cellulase, etc. are extracellular. By the addition of surface-active agents, enzyme excretion through cell membranes and consequently the yield of these enzymes can be increased 38). Extracellular enzymes are separated from microbial cells by filtration and, if necessary, in addition by enrichment. Intracellular enzymes are released by disruption of the cells... 

Industrial products are mostly of the extracellular variety. They can be recovered directly from the fermentation broth. The primary recovery involves removing the cells from the broth, aptly called cell separation. Three different techniques are commonly used to achieve this goal filtration, microfiltration, and centrifugation. 

Fungal fermentations, such as those of Trichoderma or Aspergillus sp., lend themselves particularly well to cell separation by filtration through a rotary drum vacuum filter because of the ease with which the fungal mat can be shaved off by the drum s knife, renewing the filter cake surface to maintain high filtration flux. 

Microfiltration. Microfiltration, the use of tangential flow anisotropic membranes to permeate the product of choice while retaining solids, can be an attractive cell separation technique because it does not require the use of flocculants or filter aids. It is, in principle, a more technically sophisticated version of classic dead-end filtration processes. Microfiltration yields can be low due to progressive fouling of membranes. Advanced engineering has overcome many of the early... 

Figure 11. A Comparison of Crossflow Membrane Filtration, Precoat Rotary Vacuum Filtration, and Centrifugation, a, Capital Costs for Cell Separation Steps b, Total Manufacturing Costs for Cell Separation Steps (S per Kg of enzyme).

Practical Aspects of Tangential Flow Filtration in Cell Separations... 

The theory of t angential flow filtration as it applies to cell separations is discussed. Major emphasis, however, is placed on presenting the relationship of experimental results to theoretical performance. Topics highlighted are flux decay with time, effects of operating pressures and flow, membrane fouling, prefiltration requirements and filter geometries. ... 

Figure 3. Simplified cell separation schematic of tangential flow filtration system.

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