Making bacterial extracts suitable for chromatography

Despite the exotic appearance and odor of bacterial extracts, it should be remembered that they are predominantly water with only a few percent w/v solids. The twin objectives of preparation prior to chromatography are, therefore, clarification and concentration. It is not the purpose of this chapter to describe cell disruption techniques, but it is worth noting that the method used, whether physical, mechanical, or chemical, can affect the properties of the extract and subsequent clarification, for instance, in relation to viscosity on membrane filtration. The concentration stage can incorporate a primary purification step, for example, in the form of a protein precipitation/resuspension or a batch binding of protein, normally to an ionexchange matrix. The more recent development of fast-flow chromatographic matrices does, however, allow larger process volumes to be applied directly to columns, for example, 200-L bed vol columns can operate at loading flow rates of up to 1000 L/h. The general techniques used for clarification and concentration are summarized in Table 1, and practical examples are described as they relate to pilot and large-scale processing rather than laboratory techniques. Table 1 General Techniques Used for Clarification and Concentration Physical Chemical Clarification Centrifugation Two phase Filtration (P)H Polymers Concentration Filtration Precipitation Batch binding.