Recombinant proteins, processing

C. Kaiser, T. Potozki, A. Ellert and R. Luttmann, Applications of PAT - process analytical technology in recombinant protein processes with Escherichia coli, Eng. Life Sci., 8, 132-138 (2008). 

Protein Purity. During the production of recombinant proteins, process monitoring is required to assure purity levels required in every step. The recovery and purification of product from fermentation broth typically involve various procedures, such as filtration, centrifugation, and chromatography. After each purification and final step, constituent levels must be determined to ensure that the desired levels of purity have been achieved. In addition to its control function, this purity information is also frequently used to further optimize purification processes. CZE and SDS-CGE can be mostly used for the purity checks of protein products. 

Charoenrat, T., Ketudat-Cairns, M., Stendahl-Andersen, H., Jahic, M., and Enfors, S.O. (2005) Oxygen-limited fed-batch process an alternative control for Pichia pastoris recombinant protein processes. Bioprocess. Biosyst. Eng., 27, 399-406. 

Whereas recombinant proteins produced as inclusion bodies in bacterial fermentations may be amenable to reversed-phase chromatography (42), the use of reversed-phase process chromatography does not appear to be widespread for higher molecular weight proteins. 

Fig. 5. A suspension perfusion culture process having ceU recycle. System employed at Amgen using CHO cells for production of a recombinant protein.

The previous ELP fusions all are examples of protein purification in which the ELP is covalently connected to the protein of choice. This approach is suitable for the purification of recombinant proteins that are expressed to high levels, but at very low concentrations of ELP the recovery becomes limited. Therefore this approach is not applicable for proteins expressed at micrograms per liter of bacterial culture, such as toxic proteins and complex multidomain proteins. An adjusted variant of ITC was designed to solve this problem. This variant makes use of coaggregation of free ELPs with ELP fusion proteins. In this coaggregation process, an excess of free ELP is added to a cell lysate to induce the phase transition at low concentrations of... 

Londo, T., Lynch, R, Kehoe, T., Meys, M., and Gordon, N., Accelerated recombinant protein purification process development. Automated, robotics-based intergration of chromatographic purification and analysis, /. Chromatogr. A, 798, 73, 1998. 

Shokri, A., Sanden, A.M. and Larsson, G. (2003) Cell and process design for targeting of recombinant protein into the culture medium of Pichia pastoris. Applied Microbiology and Biotechnology, 60 (6), 654-664. 

When recombinant proteins are produced in a heterologous system, there may potentially be differences between the final product and the natural molecule. Hence, for each new protein produced in alfalfa, a thorough analysis of the processing, folding, assembly and post-translational modification is conducted to ensure the conformity of the purified molecules. This section describes the analysis of alfalfa-derived... 

Studies have shown that plants can make biologically active recombinant proteins through both transgenic and transient expression approaches. Although the plant post-translational machinery is similar to that of mammalian cells, there are some notable differences, e.g. differences in glycosylation, particularly the absence of sia-lation, which may impact the activity of certain proteins. The absence of mammalian enzymes may prevent complex maturation processes that are critical for the biological activity of proteins such as insulin. Fortunately these shortcomings affect the activity of only a limited number of proteins. 

When sufficiently high levels of expression and protein accumulation are achieved, efficient downstream processing protocols must be developed to insure product quality and the economic feasibility of production. As the demand for safe, recombinant pharmaceutical proteins continues to expand, the market potential of plant-produced recombinant proteins is considerable. Molecular farming can produce recombinant proteins at a lower cost than traditional expression systems based on microbial or animal cell culture, and without the risk of contamination with human pathogens. 

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