Efficient Small-Scale Production of Proteins - - BioPharm International

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Efficient Small-Scale Production of Proteins


BioPharm International


The genetics and physiology of gram-negative E. coli are very well understood. Its enormous proliferation potential makes it an attractive system for protein production. One major disadvantage of E. coli is that protein folding is often not comparable to the protein in its genuine human form. Many efforts are being made to improve this issue, for instance, by tagging secretion signals on the protein to avoid its accumulation in inclusion bodies and to simplify purification. Nevertheless, if mammalian glycosylation is required to make a protein active, E. coli cannot be used.

Yeast

The most commonly used yeasts are S. cervisiae and P. pastoris. They are a link between prokaryotic and higher eukaryotic expression systems. They offer a combination of low-generation periods and low requirements in terms of culture conditions with a eukaryotic folding and secretion apparatus. Yeast does glycosylate proteins, which may be sufficient for some products. On the other hand, non-mammalian glycosylation is potentially immunogenic, and if that is a concern for a particular protein, then yeast is perhaps a poor choice.

Insect cells

Protein production in insect cells has become more and more relevant for research and development applications. Insect cells are easy to transfect with baculovirus vectors to yield high amounts of protein — with post-translational modifications which are quite close to, but not as complex as, in human systems. Most relevant cell lines are Sf-9 and Sf-21 (from Spodoptera frugiperda) and HighFive (from Trichoplusia ni).

Mammalian Cells

Despite comparably high production costs, 60–70% of all recombinant protein pharmaceuticals are produced in mammalian cells.3 The major reason is that post-translational modifications of the recombinant proteins largely correspond to the genuine human patterns, and that correspondence is indispensable when it comes to mediating immunological effector functions (e.g., recombinant antibodies). Protein production rates in mammalian cells are relatively low, however, so many efforts are being made to improve those rates.

Protein Production in Mammalian Cells

The development of a large-scale manufacturing process for recombinant proteins in mammalian cells usually follows a well established procedure: gene transfer, selection of transfected cells, production testing and preservation, establishing cultivation, and protein purification. The high yields obtained in today's processes are the result of 20 years of research that have led to a better understanding of gene expression, metabolism, growth, and apoptosis delay in mammalian cells. Overall, efforts have led to improvements in vectors, gene transfer, host-cell engineering, medium development, screening methods, and process engineering and protein purification. However, although these improvements have given to a leading role to mammalian cells in the large-scale production of therapeutic proteins, little improvement has been made with respect to small-scale protein production in mammalian cells.

Cell culture in suspension

Most cell lines used for large-scale protein production grow in suspension, and reach higher cell densities than adherent cell types. Furthermore, it is much easier to scale up the protein production process for cells grown in suspension. The most frequently used cell lines for protein production are chinese hamster ovary (CHO), suspension CHO, baby hamster kidney (BHK), mouse myeloma (NS0), human embryonic kidney (suspension 293), or human retinal cells (PER.C6).

Commercial media of high quality are available for cell culture. Fetal bovine serum, added at a concentration of 1 to 20%, is still widely used for regular propagation of mammalian cells. When it comes to therapeutic protein production, however, cell culture processes are executed in serum-free media. Modern media compositions support excellent cell culture performance in the absence of serum-provided peptides, growth factors, and an undetermined collection of proteins, lipids, carbohydrates, and small molecules. Major reasons for excluding serum are its undefined character and the risk of transmitting adventitious agents (e.g. bovine viruses and prions).

Gene transfer


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