Control, Coordination Help Scale Up New Enzymes

By Agnes Shanley, Editor in Chief

After years of focusing on research and development, Diversa Corp. (San Diego, Calif.) is scaling up production of its first commercial enzymes, using recombinant techniques to express them in different strains of yeast. Bacterial expression systems will also be used in the future, according to the company. Fermic S.A. de C.V., a contract manufacturer in Iztapalapa, near Mexico City, is dedicating a portion of its manufacturing capacity to Diversa, which, in turn, is co-investing in capital equipment at the site.

A new downstream processing facility has been built at Fermic's site, dedicated to Diversa's new enzymes. At this point, one enzyme is already in commercial production, and another two have been scaled up and are ready to move into production pending FDA approval. Within the next five years, both partners expect to be making over $100 million worth of enzymes per year at the site.

Theirs is no standard toll arrangement, says Patrick Simms, Diversa's senior vice president for commercial process development and operations. In fact, the groundwork for the scale-up project was laid about two and a half years ago, says Susan Oliver, Diversa's associate director of manufacturing and engineering, who currently divides her time between San Diego and Iztapalapa. All told, about 25 people in California and Mexico, including operators, are now dedicated to the enzyme manufacturing project, she says, including four Fermic staffers, who work full-time on the Diversa project and report directly to her.

Diversa had worked with a number of companies in the U.S. that offer contract fermentation services, but their focus tends to be on large volume, low-cost projects, Simms says. Typically, after three to five years, such companies want to do something else with their facilities. "There's a low ebb of available capacity out there," he says. The new agreement with Fermic, a certified cGMP facility that specializes in fermentation, will lock in production capacity for the long term. Initially, hundreds of thousands of liters in fermentation capacity, and an equivalent amount of separations capacity, will be involved, but the total will be increased as needed, by adding modular equipment.

Currently, Phytase, an enzyme that Diversa codeveloped with Danish drugmaker Danisco, is being produced in full-scale 90,000-L quantities at Fermic's facilities. The enzyme helps degrade phytic acid in animal feeds so that animals can absorb phosphate more readily. Diversa has applied for FDA approval of Quantum, another phytase enzyme, and plans to ramp up production upon approval; other enzymes are also ready to move into full-scale production, once they receive a green light from FDA and other global regulatory agencies.

Finicky Organisms

Scaling up these bioprocesses has presented a number of challenges because the host organisms tend to grow, thrive and reproduce in very specific environments. "Processes coming from the lab are finicky," says Samun Dahod, director of process development at Diversa. "In the lab, you might be using nutrients that cost $5 per kilo, or you might use deionized water, neither of which is cost-effective or practical in manufacturing."

Any changes, from switching raw material suppliers to using water with a different mineral composition, can reduce protein expression and limit production, Simms says. "Downstream, in recovery, the scale of the plant equipment is so different from that in the laboratory or pilot plant," he says. "The processing time for each step is quite different--for example, many days in the plant versus one to two days of recovery in the pilot plant. Chemical engineers working on scale-up have to work through issues such as microbial contamination and stability."

To improve results, it is critical to make the fermentation process as robust as possible. The proper "scale down" approach is essential, Dahod says, and all processes at Diversa's fermentation pilot plant duplicated Fermic plant conditions closely, using equipment and materials as similar as possible to those that would be used at the manufacturing site. Being able to use a second, larger pilot plant onsite at Fermic's facility helped with the scale-up process, says Fermic engineer Nazario Neira.

Computer control has also been essential to developing, and improving, a robust fermentation process. However, optimizing control systems for bioprocesses can be tricky. "You have to consider the living organism's growth cycle and metabolic characteristics," says Diversa process development scientist Song Liu. In addition, he says, the control system must be responsive enough to sense changes in a timely manner and take corrective actions. In some cases, he says, sampling times as short as 3 seconds are required.

The computer control system must also be able to distinguish between changes due to external mechanical factors such as aeration, agitation or pressure, and biological changes occurring due to the metabolism of the organisms involved. "Typically, we have to use very simple variables, such as dissolved oxygen, oxygen uptake rate, CO2 evolution rate, and pH changes, to control something that's very complex," says Dahod. "It's essentially coordination and keeping up with the organism," he adds. "You have to figure out, as the process moves along, how to keep mechanical parameters in synch with what's happening, biologically, within the cell."

Diversa and Fermic use a two-tiered system for control. The first tier establishes setpoints for process variables such as temperature or back pressure on the fermenter, and then uses basic feedback loops to control these variables. For example, a back-pressure sensor sends readings to the computer, and, if any of them exceed or fall short of setpoint values, the computer sends a signal to a control valve on the fermenter's exhaust line, increasing or decreasing the pressure.