Initial Considerations and Primary Recovery

With respect to the use of recombinant DNA technology and cell expression systems, a significant difference between the E. coli and mammalian cell expression systems used both academically and industrially is that whereas E. coli proteins are expressed intracellularly, industrially relevant protein targets expressed in mammalian cells are typically processed through the secretory pathway and secreted into the surrounding culture media. Therefore, the recombinant material from such in vitro mammalian expression systems may be recovered directly from the culture medium whereas in the case of expression in E.coli it is necessary to break open the cells (lysis) in order to release the intracellular recombinant protein.
1. Centrifugation and Filtration
Centrifugation and filtration are often the first step employed in any downstream bioprocessing workflow. Centrifugation is used either to remove or to collect cells and debris (collect cells where intracellular material needs to be released; remove cell debris where the biomolecule of interest is in the culture medium) before further processing is undertaken. Due to the limitations of size, batch centrifugation on a large scale is not feasible and in this case continuous-flow centrifugation can be used. Both centrifugation and filtration can be used to clarify cell culture supernatants and cell extracts; however, on a large scale traditional filtering is not possible due to the membrane size that would be required and fouling of the membrane. Cross-flow filtration has been employed to reduce both fouling of the membrane and improve the flow rate, and this approach has been successfully used to remove soluble host proteins during the recovery of inclusion bodies in E. coli cell lysates.
2 .Cell Lysis
In the case of biologics that are expressed intracellularly (e.g. in bacterial and plant cell expression systems), it is necessary to release the material so that it can be recovered and purified. The three main approaches for achieving this are enzymatic, chemical and physical lysis. Both chemical and enzymatic approaches can be used to help selectively release target products, but this requires the optimisation of each procedure for individual target molecules. Chemical approaches generally rely on the use of detergents or high pH/alkaline solutions to disrupt and lyse the cell, both of which can adversely affect the molecule of interest if these approaches are not optimised for individual targets. Enzymatic lysis can also be used to give specificity to both the process of lysis and release of the target product and therefore offers a more controlled method of cell lysis. The recent advances in the development of enzyme systems for the rupturing of bacterial and yeast cells have been well described by Salazar and Asenjo.8 Physical approaches to cell lysis at the laboratory scale include grinding (e.g. in a mortar), high pressure (e.g. in a French press) and osmotic shock. In the case of heat-stable products, heat lysis methods may also be applicable, a readily scaleable approach. Largescale physical disruption is, however, most commonly performed by either high-speed agitator bead mills or high-pressure industrial homogenisers. The equipment used derives from the paint and dairy industries, respectively.
3. Recovery of Material from Inclusion Bodies
The high-level expression of recombinant protein using E. coli expression systems often results in the formation of inclusion bodies, insoluble aggregates of material that form in vivo.Inclusion bodies usually result due to the inability of the E. coli host to fold and process correctly the polypeptide chain(s) of the recombinant protein and/or via intermolecular interactions between partially folded polypeptide chains.
Although the protein material found within inclusion bodies has no biological activity and is therefore usually of little value as is, inclusion bodies can be easily isolated from E. coli cells at high purities, thereby acting as a useful purification means. Further, it is often possible to recover the biological activity of the protein of interest once the inclusion bodies have been purified, making this a potential means of expressing and purifying target recombinant proteins. However, recovery of protein from inclusion bodies can be awkward, recoveries can be low and the process can be expensive. Further, inclusion bodies are not usually absolutely pure and there may be significant loss of protein during the recovery of material from inclusion bodies. Hence careful attention must be paid to the design of the strategies employed for the recovery of material from inclusion bodies to ensure good recovery and purification of the protein of interest.
There are four steps typically involved in the purification and biological recovery of proteins within inclusion bodies. These are the isolation of the inclusion bodies from the host E. coli cells, resolubilisation of the protein material within the inclusion bodies, refolding of the resolubilised material into a bioactive form and further purification or polishing of the protein of interest. The most important steps are the solubilisation and refolding. Inclusion bodies and therefore the proteins contained within, are usually resolubilised using high concentrations (6–8M) of chaotropic agents such as urea. The resolubilised proteins are then refolded by slowly removing the resolubilising agent while transferring the protein to an appropriate buffer system and concentration in order to reach and retain the correct biological three-dimensional structure and activity. Unfortunately, for those proteins with disulfide bonds, recovery of correctly folded and bioactive protein can often be extremely low.