Next-Gen Expression Systems - Pharmaceutical Technology

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Next-Gen Expression Systems
More sophisticated biological expression systems expand the functionality of the traditional systems for protein synthesis.


Pharmaceutical Technology
Volume 35, Issue 6, pp. 36-39

Most of the biopharmaceuticals that have reached the market in the United States and the European Union are produced in just a few expression systems. A recent report in the September 2010 issue of Nature Biotechnology indicated that 32 of the 58 biopharmaceuticals approved between 2006–2010 were produced in mammalian expression systems (mostly Chinese hamster ovary [CHO] cells), and 17 were produced in Escherichia coli (E. coli). The remainder were produced in yeast (4 in Saccharomyces cerevisiae, 1 in Pichia pastoris), transgenic animals, insect cells, plants, or by direct synthesis (1).


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Choosing the best expression system depends on the complexity of the product, the batch size, and cost constraints, but each system comes with tradeoffs. E. coli was the first system used for commercial production, with the marketing of human recombinant insulin in 1982 (2). The bacterium is easy to culture, proliferates rapidly, and can be grown in inexpensive, defined media. In the nearly 30 years since, manufacturers have continually improved the system, optimizing culture conditions, scaling production volumes, and boosting protein yields. E. coli expression systems are limited, however, in the types of proteins they can produce, and are best used for simple proteins with few posttranslational modifications. E. coli does not produce glycosylated proteins, for example, nor can it correctly produce large proteins with complicated folding patterns, or multiple disulfide bonds. E. coli does not secrete proteins, requiring the manufacturer to take extra purification steps, including cell lysis, centrifugation, multiple filtrations, and denaturing and subsequent refolding of the protein. Gram-negative bacteria such as E. coli also contain endotoxins, which must be removed.

Mammalian cells have become the system of choice for antibody production, which requires most of the posttranslational modifications that can't be achieved in E. coli. Mammalian cells—CHO cells being the most widely used— are best equipped to secrete glycosylated, multi-subunit proteins that will fold and assemble correctly. They are amenable to genetic manipulation, and lines exist that have been optimized for ease of transfection, increased protein expression, and high-density culture. The major drawback to mammalian cells is that compared with microorganisms, they are more difficult to culture, require expensive culture media and a low-shear environment, and they often produce lower yields. There are also biosafety concerns around the transmission of viruses that can be pathogenic in humans.

Some more recent entrants on the market strive to address some of these drawbacks. Most notably, PerC.6, a transformed human cell line jointly developed by Crucell and DSM for use as a manufacturing-scale expression system, increased expression yields so dramatically, that it shifted manufacturers' concerns from producing adequate protein yields to having adequate capacity to efficiently purify the increased titers. PerC.6 can be grown in suspension cultures at relatively high densities in serum-free medium, and according to company literature, can achieve yields of up to 8 g/L in standard fed-batch cultures, and 20 g/L using DSM's proprietary culture system, which are levels comparable to those of microbial expression systems.


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