Clarification techniques

Proteins can be produced by a number of different routes such as fermentation, tissue culture, and by extraction from plasma or plants. Whatever route is chosen, the raw protein-bearing stream is likely to be a complex mixture containing both dissolved species and particulate material. The target protein will be present at very low concentration and with a host of contaminants such as cells or cell debris, DNA, proteins and polysaccharides, and a large quantity of water. Such a mixture is very difficult to treat using the highly selective processes that are required to obtain the target product at high purity since the presence of particulate material impairs their function. The first challenge of protein purification is therefore to convert the complex fermentation broth which is a mixture of dissolved and suspended solids into a form that is amenable to further purification. Although there is much interest in direct recovery of protein from such materials, the most frequent first step currently is to clarify the raw protein source to remove suspended matter. It is then possible to use a range of highly selective techniques to purify the target protein. There are a number of clarification techniques that can be adopted and the choice of which to use depends on both the source of raw feed and the scale of operation. There are two main classes of process; sedimentation and filtration. Sedimentation can be carried out under normal gravity conditions or, as is almost always the case for biological streams, using a centrifuge. Filtration can be performed using either conventional filter media or using membrane filters for removal of finer particles. The aim of this chapter is to describe these methods, and their underlying principles, the advantages of each are discussed, and examples of equipment are presented. Practical advice is presented on how and when to use each technique. Sedimentation processes operate primarily on the basis of density differences between the various components of a mixture. They are most commonly applied to suspensions of solid in liquid, but also to disengage immiscible liquids. If there is no density difference between particulates and the suspending medium, sedimentation cannot occur.