Duckweed flourishes as cleanroom cash crop



PITTSBORO, N.C.—What's green, grainy and grows in a cleanroom?

It's not some morphing microbiological contaminant, or even mold, for that matter. It's duckweed—a.k.a. pond scum—and it is the bread and butter of Biolex Inc., a biotechnology company that uses the green aquatic plant, Lemna, to develop proteins that are used to manufacture drugs that treat such serious illnesses as hepatitis.

Duckweed, or Lemna, is the very heart of Biolex Inc.'s patented technology, the Lemna System, which was developed at North Carolina State University. Genetically altered, proteins recovered from Lemna are the basis for a host of interferon-based drugs that can treat such illnesses as hepatits.
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Once home to a hosiery plant that harkens back to North Carolina's textile days, the unassuming building located on the outskirts of Research Triangle Park houses a young and expanding biopharmaceutical company that has tweaked contamination-control technology to create environments better than the habitats where duckweed grows.

Duckweed, the smallest of flowering plants, grows while floating in still or slow-moving fresh waters. But at Biolex, it flourishes in jars and trays that are racked in brightly illuminated, 15 by 15-foot growth rooms that operate at sub-ISO Class 9 cleanroom levels.

"The plant likes a calm environment," says David G. Spencer, Biolex's chief operating officer and vice president of research and development. "Inside the growth rooms, the air streams out and up. There are six air changes per hour. You want enough air changes, but you don't want to cause any evaporation media. We also have temperature and humidity controls."

The growth rooms are kept at 78 degrees F. Biolex also geared the rooms to be easily upgraded to comply with the Food and Drug Administration's (FDA) current good manufacturing practices (cGMPs).

"For GMPs, we can change the surfaces for enhanced cleanability, and the HVAC already comes powered to drive through HEPA filtration," says Spencer. "So, essentially we are making our own convirons."

And these "convirons"—controlled habitats—are perhaps the key factor in an alternative to open-field environments, says John Irick, senior vice president for corporate and business development.

Open-field environments, like those used to grow bio-engineered corn, have a host of proposed regulatory requirements, including cGMP-approved planters and harvesters, according to Biolex officials. Aside from being subject to regular inspections by the Department of Agriculture (USDA), the field itself is isolated to reduce chances of cross-pollinating other crops, and requires a 50-foot fallow perimeter. Once the field is harvested, no food crop or plant can be planted in the next season.

In addition, open-field crops leave important "growth-influencing environmental factors uncontrolled." These variables include light intensity and variation, temperature, water and nutrient availability and air quality. Other variables include herbicides, pesticides, animals, insects and plant pathogens.

"These are things that we do not have to contend with at all," Irick says, of the Biolex Lemna Expression System, marketed as the LEX System. "This plant is totally contained and it will not propagate viral or pathogenic prions."

Lynn Dickey, vice president of research, says the process of genetically altering duckweed to yield proteins is relatively easy. The plant itself is clonal, so it grows without seeds or pollen. It doubles its biomass in 36 hours, producing a host of proteins that it secretes into a simple media of water and organic salts.

"It simply floats on top of the media," Dickey says. "It can tolerate all kinds of buffers and pHs. We can customize the media for the protein if we had to, and that's a nice added feature."

For transformation, Biolex dips duckweed into a specific agrobacterium, which creates a callus tissue, Lemna, that has turned white and died since it is not resistant to antibiotics in the media. Blossoms from the callus tissue reveal the transformed plant.

"Within five weeks, we start to get our first transgenic plant," Dickey says. Transgenic tissues are then batch-cultured in aseptically sealed vessels under artificial lights. The vessels and media, however, are safe from pathogens.

"There are no known plant viruses that can infect duckweed," Spencer says. "Even if there were any, it wouldn't have any relevance to humans. If an animal or human virus got into a culture with Lemna, there would be nothing for it to replicate off of, so it would die under its normal conditions of instability."

Biolex founder Anne-Marie Stomp developed the roots of the LEX System at North Carolina State University. In March 2000, Biolex was granted an exclusive, worldwide license from the university to use the patented technology.

At the time of this report, Biolex continued to receive venture capital and was in the middle of engineering a 14,000-square-foot GMP operation, which will include additional growth rooms and recovery, purification and inoculum areas.

Growth rooms will remain at the sub-ISO Class 9 levels, while chromatography areas will be rated at ISO Class 7 and final fill will be completed in ISO Class 6 or Class 5 hoods.

Once complete, vessels filled with inoculum secreted by duckweed go straight to a growth room. The transgenic duckweed then goes to a recovery area, where it is bio-massively separated from the media. The media then passes through an ISO Class 7 purification suite.

"For growth areas, we do not anticipate having to go under [ISO Class 8]," Spencer adds. "We will reserve those [higher] cleanliness levels for our recovery and purification areas."