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narrow internal diameter. This generates high shear forces. As the cells emerge from the outlet point, they also experience an instantaneous drop in pressure, to normal atmospheric pressure. The combination of high shear forces and drop in pressure serves to rupture most microbial cells with relative ease. Additional disruption methods include agitation in the presence of abrasives, such as glass beads.
After cellular disruption, the cell debris is generally removed by centrifugation, leaving behind a dilute solution of crude (unpurified) protein product. If the producer cell secretes the product, the initial stages of downstream processing are less complex. After fermentation, the cells are removed by centrifugation or filtration, leaving behind the product-containing fermentation media.
The next phase of downstream processing usually entails concentration of the crude protein product. This yields smaller product volumes, which are more convenient to work with and can subsequently be processed with greater speed. Concentration may be achieved by inducing
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Figure 3.19. (a-d) Photographic representation of various industrial-scale centrifuges. Photos courtesy of
Alfa Laval Separation AB, Sweden
product precipitation using, for example, salts such as ammonium sulphate or solvents such as ethanol. However, ultrafiltration is the more usual method employed (Figure 3.20).
Ultrafiltration membranes are usually manufactured from tough plastic-based polymers, such as polyvinyl chloride or polycarbonate. A range of membranes are available which display different cut-off points (Figure 3.20). Membranes displaying cut-off points of 3, 10, 30, 50 and 100 kDa are most commonly used. Thus, if the protein of interest displays a molecular mass of 70kDa, it may be concentrated effectively by using an ultrafilter membrane displaying a molecular mass cut-off point of 50 kDa. Ultrafiltration is a popular method of concentration because:
• high product recovery rates may be attained (typically of the order of 99%);
• processing times are rapid;
• process-scale ultrafiltration equipment is readily available, and running costs are relatively
After concentration, high-resolution chromatographic purification is usually undertaken. A variety of different chromatographic techniques are available, which separate proteins from each other on the basis of differences in various physiochemical characteristics (Table 3.18.) Detailed description of the theory and practice underlining these systems go far beyond the scope of this text, and are freely available in the scientific literature.
In general, a combination of two to four different chromatographic techniques are employed in a typical downstream processing procedure. Gel filtration and ion exchange chromatography are amongst the most common. Affinity chromatography is employed wherever possible, as its high biospecificity facilitates the achievement of a very high degree of purification. Examples include the use of immunoaffinity chromatography to purify blood factor VIII and lysine affinity chromatography to purify tPA. A selection of affinity systems are presented in Table 3.19. In addition to separation of contaminant proteins from the protein of interest, most such high-resolution chromatographic steps will also facilitate the removal of non-proteinaceous contaminants, as discussed later.
As with most aspects of downstream processing, the operation of chromatographic systems is highly automated and is usually computer-controlled. While medium-sized process-scale chromatographic columns (e.g. 5-15 litres) are manufactured from toughened glass or plastic, larger processing columns are available that are manufactured from stainless steel. Process-scale chromatographic separation is generally undertaken under low pressure, but production-scale high-pressure systems (e.g. process-scale high pressure liquid chromatography (HPLC)) are sometimes used, as long as the protein product is not adversely affected by the high pressure experienced. A HPLC-based ‘polishing step’ is sometimes employed, e.g. during the production of highly purified insulin preparations.
Final product formulation
High-resolution chromatography normally yields a protein that is 98-99% pure. The next phase of downstream processing entails formulation into final product format. This generally involves:
• addition of various excipients (substances other than the active ingredient(s) which, for example, stabilize the final product or enhance the characteristics of the final product in some other way);
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Figure 3.20. Ultrafiltration separates molecules on the basis of size and shape. (a) a diagramatic representation of a typical laboratory-scale ultrafiltration system. The sample (e.g. crude protein solution) is placed in the ultrafiltration chamber, where it sits directly above the ultrafilter membrane. The membrane in turn sits on a macroporous support which provides it with mechanical strength. Pressure is then applied (usually in the form of an inert gas), as shown. Molecules larger than the pore diameter (e.g. large proteins) are retained on the upstream side of the ultrafilter membrane. However, smaller molecules — particularly water molecules—are easily forced through the pores, thus effectively concentrating the protein solution [see also (b)]. Membranes can be manufactured that display different pore sizes, i.e. have different molecular mass cut-off points. (c) An industrial-scale ultrafiltration system. Photo courtesy of Elga Ltd, UK