June 15, 2009 (Vol. 29, No. 12)

Dnano Seeks to Replace Cell-Based Manufacturing with a Cell-Free Method

Therapeutic proteins such as insulin or monoclonal antibodies are manufactured in bacteria, yeast, or mammalian cells. Many proteins, however, cannot be expressed in these cells because they are too large or toxic to the cells that produce them. The mission at Dnano is to replace cell-based protein manufacturing with an easier and cheaper cell-free method. The company’s P-Gel (short for protein producing gel) promises to generate high yields of new proteins at lower costs. The scientists at Dnano predict that the P-gel platform will transform protein production into simple solid-state chemical reactions, similar to how PCR improved DNA synthesis.

The company’s name represents its ability to blend DNA and nanotechnology. Co-founder and CSO Dan Luo, Ph.D., views DNA not only as a genetic material, but also as a generic material. “The natural shape of DNA is a scaffold,” Dr. Luo says. He took advantage of the structure to build hydrogels made entirely from DNA and water, where linear DNA molecules are crosslinked with branched DNA to form a 3-D matrix. Then Dr. Luo’s team pioneered ways to exploit DNA’s inherent genetic functions.

They discovered that genes could be dropped inside the hydrogel without encapsulating them. “We have the luxury of linking genes to the gel matrix because both are made of DNA,” adds Dr. Luo, “and DNA ligates with DNA easily.” This feature makes the P-gel system unique.

The DNA sequences within the P-gel easily join to the ends of plasmids that code for a desired protein. Plasmids with genes for this protein are integrated throughout the P-gel. Genes locked into the P-gel are protected from damage. To increase the surface area for reaction, tiny drops of the P-gel are molded into pads about one square millimeter by 20 microns thick. Several hundred pads are placed in a solution of amino acids and protein-making machinery such as ribosomes extracted from living cells. Because genes are packed close together, enzymes taking part in the transcription process remain close by and perform quickly.
Genes concentrate in the P-gel at much higher levels than they can in solution, where DNA precipitates out, Dr. Luo says. “The gel gives higher yields and expression advantages,” says Alan Biloski, Ph.D., co-founder and CEO. According to the company, researchers at Dnano have produced 16 functional proteins in the P-gel at yields 300 times higher than current solution-based methods. The manufactured proteins include reporter proteins such as green fluorescent protein (GFP), kinases, light- and heavy-chain antibodies, hormones, highly repetitive proteins, and membrane proteins. All were successfully produced with complete fidelity of amino acid sequences and full functionality, Dr. Biloski says.

Dnano does not intend to manufacture and sell proteins like GFP, which are made cheaply in cell-based systems. The production of GFP in the P-gel was done as a proof of concept. “If you can produce proteins in bacteria, that’s the cheapest way to go,” comments Dr. Biloski.

Enzymes used in detergents, for instance, are made in bacteria for about $1 per gram, “and we can’t compete with that,” he adds. Instead, Dnano plans to capture the market for the thousands of proteins that cannot be expressed in bacteria to bring totally new proteins to the market.

DNA hydrogels were patterned in CORNELL shapes at centimeter scale (top) and stained with DNA-specific fluorescent dyes (bottom).
(Originally published in Nature Materials, 2006)

Multiple Benfits

So far, each protein gene inserted in the P-gel matrix has worked successfully, regardless of the protein’s size, type, or function. “This appears to be a universal and versatile production system,” says Dr. Biloski. “Moreover, the P-gel method makes it possible to make proteins that cannot be made in cell-based systems, including large, insoluble proteins and proteins that would kill E. coli or cells currently used to manufacture them.”

Because no living organisms are involved in the P-gel system and protein expression relies totally on enzymatic reactions, most proteins can be produced within the gel with stable production rates. Currently, 15–50 milligrams of protein per milliliter of reaction are made in the P-gel, and the company is scaling up to kilogram amounts.

The P-gel can be reused multiple times, making it suitable for automated multi-pass protein expression systems. Since the P-gel method lends itself to setting up highly consistent cGMP processes, it also may reduce regulatory hurdles for therapeutic proteins. Patients, too, may benefit, as cheaper generic drugs are manufactured in P-gels, Dr. Biloski says.

There are many benefits to the P-gel system, according to Dnano. It eliminates the need to build expensive cell-based manufacturing facilities; it reduces production costs to levels similar to that for small molecule drugs; it eliminates worries about contamination by prions and viruses, and it allows for continuous sampling during protein expression. “You can take out samples and analyze them to prove that the protein made at the beginning process is identical to the end process. You can’t do that with sealed cell-based systems,” explains Dr. Biloski.

It takes about a day to make a protein in the P-gel, compared to weeks and months for cell-based systems. After a protein is synthesized in the P-gel, separation and purification are easier as well. Cell membranes and other components of the cell-based systems such as carbohydrates and lipids do not have to be removed. Straightforward chromatography is often sufficient to purify proteins made in the P-gel.

Dnano is producing proteins for the R&D divisions of some large pharmaceutical and biotechnology companies in pilot studies. It plans to expand services to therapeutic protein manufacturing and/or provide nonexclusive licensing for the pharmaceutical and biotech industries. In the future, the company plans to produce investigational proteins as drug targets, biomarkers, or therapeutic agents. Eventually, it may retain the option to manufacture and market generic protein drugs. “Our method is simple and fast, and we have no doubts that we can transfer it to other people’s laboratories,” concludes Dr. Biloski.

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