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Protein Power: "Salt cloud" concept shows promise for developing new drugs and foods faster, at lower cost

NEWARK, DE.--Tucked inside an electrically charged "salt cloud," tiny proteins can avoid slipping through molecular filters, which may make it easier to purify and separate them from other proteins, engineers with Millipore Corp., Genentech and the University of Delaware reported today.

Someday, proteins for "magic bullet" type medicines, more nutritious foods and new industrial enzymes might be made at lower cost, by using a molecular filter that exploits the salt-cloud concept, says Prof. Andrew L. Zydney of UD's Department of Chemical Engineering.

The new technology, now in development, was disclosed Oct. 12 at the International BioTherapeutics '99 conference in Washington, D.C.

"In the laboratory, the results have been promising," says Robert van Reis, a distinguished engineer with Genentech's Department of Recovery Sciences in South San Francisco.

"This is the first time that anyone has been able to use a membrane-based system to carry out highly selective separations, while also maintaining a high-volume throughput," van Reis says. "Biotechnology is heading toward much larger batch sizes. This increases the desire to find economical ways to purify proteins. One way to achieve this is to concentrate, buffer exchange and purify proteins in a single step, and that's exactly what this new membrane technology may be able to do."

If it pans out, the joint research could even be useful for purifying drugs based on antibodies produced using recombinant DNA methods, says Ralf Kuriyel, research and development manager at Millipore in Bedford, Mass.

In addition to removing protein impurities, the system could also simplify the process of removing contaminants and other impurities such as DNA and viruses, Kuriyel says.

And, Zydney has described the system's potential effectiveness for separating whey proteins from milk, and for recovering recombinant proteins. Whey proteins could be used in new foods and medicines, ranging from improved infant formula and antibiotic agents to growth factors and hormones, Zydney says.

To date, the researchers have achieved overall yields of 94 percent, with purification factors approaching 1,000-fold for model protein systems, meaning that 99.9 percent of all impurities are removed by a single membrane separation step.

"That's a very, very powerful separation," Kuriyel says. "And, those results were achieved using two molecules that were relatively similar in size. This technology could ultimately open up a multitude of applications."

Membranes for mass production

Current strategies for protein purification rely heavily on chromatography, in which a moving sample is adsorbed or bound by a stationary material, usually a porous resin in the form of small, spherical beads.

In contrast, the new approach sends a sample directly through the microscopic pores of a polymeric membrane. (The research team currently uses a specially modified Biomax(tm) membrane, which Millipore plans to market by 2000, according to Kuriyel.)

Ramping up production using this strategy is a simple matter, Zydney says: "If you're using a membrane that's one-tenth of a square meter in size, you can easily add additional membranes to create an array offering, say, 100 square meters worth of separation power."

In addition, he says, if the results obtained with model systems prove successful, "We could potentially reduce the total number of steps required for purification, which should, in turn, reduce the overall cost of the process."

Overcoming obstacles

Researchers have been using membrane-based systems for concentrating differently sized proteins since at least the 1960s, Zydney says. But, attempts to separate two proteins of similar size just never worked, and "invariably offered poor yields, with minimal or no purification," Zydney explains.

Now, Millipore, Genentech and UD researchers have teamed up to overcome this problem, by exploiting the salt cloud that naturally surrounds proteins under certain conditions. "Charged proteins in water are surrounded by a diffuse cloud of oppositely charged ions," Zydney explains. "This ionic cloud increases the effective size of the proteins."

As a result, he says, the charged proteins are too large to squeeze through the molecular-sized membrane pores, which are more than 1,000 times smaller than the width of a single human hair.

Adding a charge to membranes further improves the separation process, by electrically repelling any similarly charged proteins. In this way, Millipore's Biomax(tm) membranes dramatically improve yield and purification and help prevent pore-fouling, Kuriyel says.

A key to the system is Zydney's approach for manipulating electrical interactions by adjusting the feedstream's acidity (pH) and salt concentration. By changing the solution pH, the researchers can adjust the charge on different proteins, effectively "dialing in" the most appropriate conditions for a given separation, Zydney reports.

Also crucial to the system's efficiency is a strategy, invented by van Reis and patented by Genentech, for choosing the optimal pressure for the filtration process. Previously, Zydney says, "People had tended to use the highest possible pressure, to increase the flow rate and boost their yield. Then, Genentech engineers discovered that optimal results could actually be achieved at lower pressures. It was a real breakthrough."

"We're using a combination of strategies that could open the door to many new products based on therapeutic proteins," he says. "At this point, we have successfully demonstrated the effectiveness of these techniques using a variety of model systems. The next step is to implement the technology on real feedstreams, in actual commercial processes. That work is currently under way."

Millipore and Genentech provide support for Zydney's research. Serving on the UD project team were a number of current and former graduate students, including Skand Saksena, Narahari Pujar, Manoj Menon and Douglas Burns. Genentech's team included van Reis, Gizette Sperinde, Jeff Brake, Poonam Mulherkar, Adeyma Arroyo, Leah Frautschy and Beth Goodrich. The Millipore group includes Kuriyel, Shishir Gadam, Scott Orlando, Glen Bolton and Steve Pearl.

Contacts: UD--Laura Overturf, (302) 831-1418,;
Millipore--Mike Estabrook, (781) 533-2897,;
Genentech--Stephanie Ashe, (650) 225-5464

Oct. 12, 1999