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.
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.
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).
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).