Efficient Small-Scale Production of Proteins

Feb 09, 2006

Over the last three decades, numerous protein expression systems have been developed with various quality requirements on large and small scales. Huge steps have been made in large-scale protein production in mammalian systems while the small-scale mammalian systems are expensive and inflexible. Thus, small-scale production is done in simpler expression systems, sometimes sacrificing the quality of the proteins. However, relief is on the way.

New developments in mammalian cell transfection, however, allow the highly efficient transfection of suspension cells relevant for protein production. This not only facilitates a simpler approach for the generation of stable production cell clones, it also turns protein production in transiently transfected cells into a true alternative for small-scale protein production. This allows scientists to use proteins resembling the original human protein for all kinds of applications, potentially leading to better results in pharmaceutical research and development.

Once a new gene is discovered, the corresponding protein, the actual trigger of cellular functions, needs to be characterized, e.g., by crystallography or NMR techniques. Subsequently, the protein's role within the cellular metabolism must be unravelled. Finally, the protein may be investigated as a target for the development of small molecule drugs or it may become a drug candidate by itself.

The demand for recombinant proteins is increasing. More and more proteins are required for research applications and numerous new protein-based drugs are in pre-clinical or clinical testing, constituting an increasing need for new clinical-grade expression systems and large-scale production capabilities.

Table 1. A Short Overview of Applications Associated with Protein Production.
While some analytic applications require only sub-milligram amounts of research-grade material, clinical applications often require clinical-grade material on a multi-kilogram scale (Table 1). Protein research requires the production of recombinant proteins in quite different amounts and qualities.

Mammalian Cells are the Best

Although transgenic plants and animals may one day become commercially competitive platforms for the large-scale manufacturing of proteins, almost all biologics on the market today are manufactured in either a mammalian or microbial expression system.1,2 For clinical applications, cultivated mammalian cells are the dominant system for producing recombinant proteins. Proteins produced in mammalian systems are properly folded, assembled, and post-translationally modified. In contrast, research-grade material is often produced in much simpler expression systems such as E. coli. Although for many applications it would be beneficial to use proteins that are closer to the original human pattern, simpler expression systems are often chosen because they are much cheaper and more flexible for production of small amounts of proteins.

This article is a brief introduction to different expression systems, focussing on mammalian cells and the latest developments in the transfection of mammalian cells. These new transfection technologies can speed up the generation of stably transfected mammalian cell clones significantly by direct transfection of suspension cells and can turn transient protein production from mammalian cells into a true alternative to small-scale protein production in simpler expression systems. For the first time, high-quality proteins can be expressed quickly and flexibly in mammalian cells and are thus available for standard R&D applications.

Alternative expression systems

Figure 1. A Brief Overview of the Relative Advantages of Various Expression Systems.
Let us review the four dominant sources of proteins. As a rule of thumb lower level organisms are the fastest and cheapest systems for protein expression. E. coli doubles about every 20 minutes and yeast every hour, whereas mammalian cells will take a day or more. However, E. coli often yields incorrectly folded and modified protein. In contrast, mammalian cells are expensive and produce comparably lower yield, but a high quality protein product (Figure 1).


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