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DewDiligence

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Tuesday, 12/13/2005 9:08:20 PM

Tuesday, December 13, 2005 9:08:20 PM

Post# of 252389
Keeping Up with Protein Demand

[From the 12/05 issue of Drug Discovery & Development Magazine. There’s nothing new here for regular readers of this board, but this is a reasonable overview of some of the companies involved in novel forms of protein production.]

http://www.dddmag.com/ShowPR.aspx?PUBCODE=016&ACCT=1600000100&ISSUE=0512&RELTYPE=CEL&...

>>
By Lakshmi Kamath

The number of therapeutic proteins is increasing rapidly. Advanced cell-based manufacturing technologies help when it comes to producing recombinant proteins.

Protein-based therapeutics is the newest class of chemical compounds being developed by the drug industry, and it is estimated that about 900 to 1,200 clinical candidate proteins and/or peptides are currently being investigated. In addition, 140 therapeutic proteins have been approved and 500 are in clinical trials, and it is expected that at least 50 biotherapeutics will come on to the market over the next few years.

Many of these biotherapeutics are produced using technologically advanced cell biosystems. These include microbial and mammalian systems, as well as transgenic plants and animals. These cell-based protein manufacturing technologies offer certain advantages, but they present a number of challenges as well. The use of microbes for human applications is decades old, but the advent of genetic engineering provided the means to produce recombinant proteins in bacteria and yeast, making them productive bioreactors.

The biopharma operations of Cambrex Corp., East Rutherford, N.J., offers process expertise that includes microbial and yeast cell culture. "The use of microbial cells for protein production is a well-established technology. Some advantages of using microbial cells are: they can be easily scaled up, cost of production is low, yield is high, and they are ideal for the production of low complexity proteins," says Peter Van Hoorn, PhD, the company's executive vice president.

Escherichia coli (bacterial) and Saccharomyces cerevisiae (yeast) make up about 40% of the therapeutic protein production market. "We typically use E. coli, S. cerevesiae, and P. pastoris for production of our proteins," says Van Hoorn. "Each of these systems is amenable to production of different types of proteins." For example, insulin, growth hormones, and growth factors can be easily produced in E. coli.

With microbial and yeast systems, production and recovery processes are robust. However, recovery can be challenging in E. coli as the target protein tends to be intracellular and requires breaking open the cells, unlike yeast, where proteins are typically excreted into the growth media. "At Cambrex, we express our proteins in the outer membrane or periplasm of E. coli, enabling us to use a gentle lysis technique to recover larger amounts of the target protein."

A major limitation of bacterial and yeast systems is their inability to confer human glycosylation patterns on their proteins. Glycosylation is a process where complex sugar structures called glycans are added to the surface of many therapeutic proteins. It plays a key role in determining protein folding and stability, half-life, and the biological activity of a therapeutic protein in vivo.

GlycoFi Inc., Lebanon, N.H., offers a yeast manufacturing platform that addresses the glycosylation problem via a protein-expression technology that permits precise control over it, says Tillman Gerngross, PhD, the company's chief scientific officer. "We have engineered a library of yeast strains that can produce one specific, or a combination of, pre-engineered sugar patterns on a protein, so it will achieve the desired therapeutic effect. These cell lines can easily be adapted to commercial-scale production."

Mammalian cell systems

Mammalian cells are the most widely used expression systems for production of complex human glycoproteins. About 60% of therapeutic protein products, especially antibodies, are produced in mammalian cells. The Chinese hamster ovary (CHO) cell line dominates this category as host cell of choice. Other cell lines such as baby hamster kidney (BHK), human embryo kidney (HEK-293), and human retinal cell lines have also been approved for recombinant protein production.

Van Hoorn says mammalian cells offer several advantages, including proper protein folding, assembly, and post-translational modification which results in protein that is superior in terms of quality and efficacy. "We use a variety of mammalian cells including CHO, PER.C6, and the GA cell line. There is a trend now to use cell lines that are of human origin rather than mouse or hamster derived, as human cell lines possess the cell mechanism to allow production of target protein with human glycosylation patterns."

Mammalian cells systems do pose challenges, Van Hoorn says, including high investment and manufacturing costs, the need for complicated facilities, and the potential for transmission of viral or pyrogenic remnants carried over in the final product.

Transgenic plants

Plant-made pharmaceuticals (PMPs) is an emerging field, and recent improvements in plant-expression platforms have made this technology more attractive for commercial applications. Ventria Bioscience in Sacramento, Calif., has a platform called ExpressTec which uses self-pollinating crops such as rice and barley for protein production. Scott Deeter, PhD, Ventria's chief executive officer, says the main constraint of a plant-based production system is in achieving high expression levels. They were able to identify a method that instructs the plant to replace its own protein with the target protein and produce it in large quantities.

Ventria Bioscience has successfully produced human lysozyme using ExpressTec technology. "We can produce 1% of lysozyme biomass in our plants and 50% of this

click to enlarge
Cell-Based Protein Expression Systems at a Glance
is in a soluble format. This type of production level is about 25 times greater than other plant-production systems." Deeter says that the technology's key feature is that it offers the ease of large-scale protein production, so they are able to produce 10 kg of lysozyme per acre.

It's also cheaper. A best-case cost scenario with mammalian cell culture is $200 per gram of protein compared with less than $10 per gram with ExpressTec. Deeter says that not all proteins are suited to their technology and that the need for mammalian cell culture will continue to exist.

"The acceptance curve for plant-based protein manufacturing is increasing. We anticipate that in the next three to five years we can achieve very large recombinant protein production," Deeter says.

Biolex Inc., Pittsboro, N.C., offers another plant-based system. The LEX System uses a genetically engineered aquatic plant, Lemna, which grows very quickly and doubles its biomass, thus allowing the company to scale up to a multi-ton format rapidly, says Lynne Dickey, PhD, the company's vice president of research. "It is a clonal plant and the original transformant can be converted to a production line within six months. Compared to other plant-based systems such as rice, the LEX System offers better genetic stability. The plant is grown in defined media containing water and inorganic salts, resulting in complete absence of animal-derived media contaminants in end-product."

Obviously, the use of plant cells to produce valuable protein is increasing. However, PMP proponents still have to deal with the public perception of genetically engineered plants, and while low production costs can be an advantage, several factors such as low levels of recombinant protein expression, strict adherence to regulatory compliance requirements, and waste disposal issues could contribute to higher costs.

Transgenic animals

Transgenic animals are also being explored as an alternative to the traditional bacterial fermentation or mammalian cell-culture methods. The technology is ideal for production of difficult-to-express complex proteins that need to be produced in large kilogram quantities, says Tom Newberry, vice president of corporate communications for Genzyme Transgenics Corp. (GTC), Framingham, Mass. GTC prefers goats for their transgenic technology because they offer a good balance in terms of speed of development and yield per liter of milk. They have successfully produced more than 85 proteins.

"Some examples of human blood proteins we have produced are anti-thrombin, alpha1 A trypsin, and alpha1 anti-trypsin." GTC also offers Fusion Platform Technology, which enables the production of fusion antibodies.

"Typical investment costs for a standard cell culture bioreactor facility can range between $300 million and $500 million, whereas producing a transgenic animal can be as low as $50 million. The final protein product is uniform, linearly scalable, and can better match supply capacity to product demand. It takes about 18 months to produce the founder animal and up to three years for a commercial-scale product. This is very competitive to building a traditional cell-culture manufacturing facility." But, like any emerging technology, use of animals has its hurdles. Producing a transgenic animal can end up being cost-prohibitive as success rates in producing these animals are very low. In addition, regulatory rules are stringent and the safety of proteins produced from animal sources is still a concern.
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