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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.
Amy Ritter is Scientific Editor, BioPharm International and Pharmaceutical Technology.
Amy Ritter joined the editorial staff of BioPharm International and Pharmaceutical Technology in 2011. She received her BA in Biology from Boston University, and holds a PhD in Neurobiology from SUNY at Stony Brook. Before joining Advanstars Pharm Sciences group, she worked in the preclinical pharmacology group at Merck Research Labs, and is the author of numerous peer-reviewed scientific publications.
Articles by Amy Ritter
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