Late in 2013, a deadly outbreak of disease in Meliandou, Guinea, took the life of an 18-month-old boy. Members of the boy’s immediate and extended families started dying, too. By March 2014, public health scientists identified the disease as Ebola. This outbreak in West Africa killed more than 11,000 people by early 2016. Perhaps, surprisingly, one of the treatments comes from proteins transiently expressed and produced in tobacco.

San Diego-based Mapp Biopharmaceutical developed ZMAPP, which is a cocktail of three monoclonal antibodies to treat Ebola. The company says the antibodies “work to prevent the spread of the disease within the body.” This experimental drug arose from a public-private partnership, involving Defyrus, Kentucky BioProcessing, Mapp Biopharmaceutical, the Public Health Agency of Canada, the U.S. Army Medical Research Institute of Infectious Diseases and others.

In August 2014, Kentucky BioProcessing shifted exclusively to producing the monoclonal antibodies for ZMAPP. “By the first part of October, we had produced—under GMP—the three monoclonal antibodies that make up the cocktail,” says Hugh Haydon, president of Kentucky BioProcessing. So, it only took the company about six weeks to crank up its tobacco-based protein expression system to make the necessary components.

Advances in a variety of protein-expression systems make it easier than ever for scientists to produce proteins for research and medicine.

Treatments from tobacco

When talking about producing GMP lots of each of the ZMAPP antibodies in about six weeks, Haydon says, “Those people who are knowledgeable of protein expression might even find that unbelievable, but it is representative of the pace at which this system moves.” He adds, “This speed is one of the great features of the system.”

In Kentucky BioProcessing’s method, a host tobacco plant starts life like any other. “It’s simply a plant—nothing special,” Haydon says. About three weeks after seeding, the plant gets transfected with what Haydon calls “a genetic construct of choice.” He adds, “Once that construct has been introduced, the plant recognizes a foreign gene, and it starts to manufacture the protein associated with the inserted genetic material.” In just a few days after transfection, the plant is harvested to extract the target protein. In the ZMAPP case, the three monoclonal antibodies were produced separately and pooled later to make up the treatment cocktail.

In addition to being fast and capable of simultaneously producing multiple proteins, this system can produce most kinds of molecules. “In terms of classes or types, we’ve shown an ability to produce all types of proteins—monoclonal antibodies, vaccine antigens, hormones, growth factors and so on,” Haydon explains. “Like any other system, though, there might be certain proteins that would not behave well in our system.”

In addition, this system produces proteins that are free of mammalian pathogens. “Plants are not susceptible to mammalian pathogens,” Haydon explains. “So, when you think about producing a protein with potential to be introduced in humans, there is a safety factor, because you don’t have any risk of human pathogens being included.”

Applying Escherichia coli

Lots of hosts—bacteria, insects, mammalian systems, plants and yeast—can be used to produce proteins. The bacterium E. coli is one of the most common systems.

At the University of Wisconsin-Madison School of Medicine and Public Health, Richard Burgess, James D. Watson Professor Emeritus of Oncology, and members of his lab usually express proteins with the pET/BL21(DE3) pLysS system, which was developed by F. William Studier, an emeritus staff member of the biology department at Brookhaven National Laboratory. In using this technique, says Burgess, “my lab has found that this system gives consistently excellent results.”

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Sometimes, one system works better than another. As Burgess points out, “In some cases, it helps to use the E. coli host BL21(DE3) Rosetta2 that is supplemented with rare transfer RNAs when expressing certain human or viral proteins as it increases the level of expression.” He adds, “We generally are not worried about formation of inclusion bodies, because we are very good at refolding proteins solubilized from inclusion bodies.”

Mammalian methods

At NC State University’s biomanufacturing training and education center, senior scientist Driss Elhanafi, uses the mammalian Chinese hamster ovary (CHO) cells to produce therapeutic proteins. CHO cells are the dominant platform that produces recombinant proteins with the closest quality to human cells, which enhances efficacy and minimizes immunogenicity. Tremendous advances in terms of cell-line development, growth media and culture-control engineering led to a routine production of 2–5 grams per liter of cell culture. Elhanafi uses Thermo Fisher Scientific’s DG44 system. “It is widely used by the industry as it allows the achievement of higher yield, and it is well accepted by regulatory agencies, [such as the U.S. FDA],” he says. Moreover, the DG44 system generates multi-copies of the gene of interest inside cells, and that drives the overproduction of the protein of interest.

CHO cells are the dominant platform that produces recombinant proteins with the closest quality to human cells, which enhances efficacy and minimizes immunogenicity.

On the tricky side, Elhanafi notes that it’s “a lengthy process.” As he says, “depending on the target protein, it takes several months to isolate the best producer clone.”

Mammalian cells can also produce vaccines. For example, Madin-Darby Canine Kidney (MDCK) cells make an efficient alternative for flu-vaccine production, which traditionally uses millions of eggs a year in a cumbersome process. In 2016, the FDA approved Seqirus’ Flucelvax, which is the first cell culture–based flu vaccine.

Algal advances

Tobacco is not the only plant host that can be used to produce proteins. Some scientists use algae. “They naturally accumulate proteins at high levels,” says Stephen Mayfield, director of the California Center for Algae Biotechnology at the University of California, San Diego. “They are easy to transform for recombinant protein expression, and you can grow them either photosynthetically or by fermentation.”

Taking an algal angle on protein production provides other benefits. As Mayfield points out, algae “are eukaryotic, so they can express complex proteins.” As an example, he notes, “You can produce proteins in the chloroplast so they stay within the cell for oral applications and you can secrete proteins for easy purification.” Like tobacco, algae pose no risk of incorporating a human pathogen in a protein product. “As plants,” Mayfield states, “they lack mammalian viruses, prions or other toxins.”

Whereas some systems can boast a growing history of use in protein expression, algal systems are just getting started. “The platform is still relatively new and needs additional development to be cost competitive with existing systems,” Mayfield says. “There are no protein products from algae that have gone through all of the FDA regulatory process, although some are getting close.” For example, he indicates that some proteins produced in algae are “in animal trials for use as human and animal replacements for antibiotics.”

Whereas some systems can boast a growing history of use in protein expression, algal systems are just getting started.

Given the range of protein-expression systems available, scientists can explore multiple options for specific problems. Some work better on certain problems than others, and some provide different benefits and challenges. Even a product known for causing health problems can be used to create lifesaving treatments. The way that something gets used can make all the difference.

Image: Kentucky BioProcessing. At Kentucky BioProcessing, scientists use tobacco to produce proteins, and some of them can even save lives.