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C&EN Global Enterp 2018.96:36-40. Downloaded from pubs.acs.org by UNIV OF NORTH DAKOTA on 11/22/18. For personal use only.
Making biologics on demand A team at the University of Maryland, Baltimore County, has developed this Bio-MOD system, easily packed in a suitcase for transport, to synthesize biopharmaceuticals on-site.
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Systems make flipping the switch on biopharmaceutical production faster and easier, potentially enabling personalized medicine CELIA HENRY ARNAUD, C&EN WASHINGTON
C R E D I T: JA M ES K EGLE Y ( P H OTO) ; S H UT T E RSTO C K ( I CO N S )
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s a U.S. Army doctor stationed in Afghanistan in 2003, Geoffrey Ling had a hard time accessing medicines his patients needed. “The combat support hospital was nothing more than a tent city,” he says. “When you’re in a situation like that, you have limited supplies.”
In particular, biopharmaceuticals, like insulin, were hard to obtain and store. “I wanted to have a machine that could make any drug at any time in any quantity for providers in situations like I was in, austere situations where they have to take care of patients,” Ling says. “Instead of carrying around boxes and boxes of drugs that they may never use, they would just carry some basic reagents and make what they needed.” When he joined the U.S. Defense Advanced Research Projects Agency (DARPA) as a program manager, he found himself in a position to do something about it. In 2010, DARPA leadership let him start a battlefield medicine program. He challenged researchers to come up with ways of manufacturing drugs, both small molecules and biopharmaceuticals, in under 24 hours. Earlier this year, two DARPA-funded research teams—one from Massachusetts Institute of Technology and one from the University of Maryland, Baltimore County—answered the second half of Ling’s call, reporting modular systems capable of manufacturing protein therapeutics on demand. The systems and others like them are expected to have far-reaching effects: In addition to allowing biopharmaceuticals, also known as biologics, to be produced in remote settings for military physicians, the same type of technology might make it easier and cheaper to supply drugs and vaccines around the globe. And in developed countries that already have access to biologics, the underlying technologies could pave the way for truly personalized medicines.
Answering the call One of DARPA’s goals in synthesizing biologics on demand is to eliminate the need for refrigerating drugs. “Insulin was at the top of my list because insulin requires refrigeration,” Ling says. Cold storage needs energy, and that’s a big problem when you are dealing with extreme environments that lack infrastructure, like the military faces, he adds. But making insulin and other biologics on the spot and then giving them to
patients immediately would eliminate the need for a refrigerator. DARPA also wants to reduce the need to stockpile biologics as countermeasures in the event of chemical, biological, radiological, or nuclear attacks. Such drugs rarely need to be used, and they need to be replaced as they expire. “There’s a time clock on those molecules, and they may become less and less active” during storage, says Brad Ringeisen, deputy director of DARPA’s biological technologies office and program manager for battlefield medicine. “This concept of making something where you need it, when you need it was really attractive to us because you might be able to greatly reduce the need to stockpile.” Smaller stockpiles could be kept for immediate use, and more drugs could be made on the spot in response to epidemics or attacks. Biologics are normally synthesized in batches in large-scale reactors that are thousands of liters in volume by cells genetically engineered to produce desired proteins. In the case of Escherichia coli bacteria, the cells must be lysed to recover the proteins. In the case of yeast or Chinese hamster ovary (CHO) cells, the cells secrete the proteins, which simplifies purification. Switching one of these systems from making one molecule to another can take months. Such largescale reactors are most efficient when used to produce proteins for a large patient population. Companies aim to optimize their supply chain over an approximately twoyear time frame, but getting such forecasts right can be challenging. “Traditionally, the manufacture of broader classes of biopharmaceuticals—from enzymes to hormones to cytokines—requires one-off, bespoke processes and unique facilities designed for each molecule,” says J. Christopher Love, a chemical engineer in the Koch Institute for Integrative Cancer Research at MIT. He leads one of the DARPA-funded teams that developed a system to meet Ling’s challenge. Love and his group wanted to design an on-demand biologics manufacturing system that could
In brief Accessing biopharmaceuticals can be challenging in remote areas. Researchers funded by the U.S. Defense Advanced Research Projects Agency are trying to change that. They are coming up with ways to quickly ramp up production of biologics when they are needed. One project focuses on producing proteins in a benchtop system. The other is packing its system into a suitcase to take it on the road. Read on to learn more about these projects and others that are making it faster and easier to manufacture biologics.
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A key advantage of cell-free systems is speed. “If you need something in a hurry and it’s truly patient specific, you need it made on demand in a few hours. This is the only way you can realistically do it,” Rao says. In contrast, Love’s cell-based system can do end-to-end production of hundreds to thousands of doses of protein biologics in about three days. The other advantage of cell-free systems is that refrigeration is not needed during transport. The freeze-dried cell extracts that Bio-MOD uses are like powdered milk, Rao says. Fresh milk is a perishable product that has to be refrigerated. “You need a cold chain,” he says. “Once people figured out you could make powdered milk, it revolutionized nutrition throughout the world. You could have something that was shelf stable. You just added water when you needed it, and it was ready.” The cell extracts work similarly. “You just add buffer and DNA; in four to six hours, the expression of the protein product is done,” Rao says. Rao’s group has shown that even cells from human blood can serve as a source of cell-free extracts (Sci. Rep. 2018, DOI: 10.1038/s41598-018-27846-8). He sees such extracts being particularly useful for making vaccines. In such instances, the researchers could draw blood from individuals, make the extract, use it in the BioMOD system to produce a protein such as a vaccine antigen, and inject it back into the same person. This process would be especially helpful for dealing with outbreaks and epidemics. If you could make the vaccine at the point of care, you could administer it to all the people in the vicinity right away and “nip potential outbreaks in the bud,” he says. Injecting the vaccine back into the same
Protein mix Cell-free protein manufacturing follows the general workflow shown here. Freeze-dried cell extracts and DNA blueprints (plasmids) are mixed and rehydrated to complete the synthesis. Cells Break open cells, extract protein-making machinery, and discard cell debris Plasmids
Freeze-dry Store or transport at room temperature Freeze-dry
Make DNA constructs encoding proteins
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Mix & rehydrate
Produce protein
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Love points out that his be easily switched from team’s InSCyT system has producing one molecule all the components of conto another. Doing that has ventional biomanufactur“really required thinking ing, just at a smaller scale. deeply about the biology of “We think a lot about rightthe host cell that produces scale manufacturing,” he the products,” Love says. says. “What we’ve demonThe system he and his strated has all the same team came up with, called elements for manufacturing Integrated Scalable Cythat one might generally exto-Technology, or InSCyT, pect in a typical facility.” uses Pichia pastoris yeast InSCyT consists of three cells to produce various modules. Fermentation drugs in a benchtop manutakes place in a production facturing system (Nat. Biomodule. The cell culture technol. 2018, DOI: 10.1038/ Granulocyte colonymedium from that module, nbt.4262). Pichia’s genome stimulating factor is one with the protein product, is small enough, Love says, of the proteins that’s that for about $30, you can been made on demand. It flows through tubing to a sequence the whole thing. treats radiation exposure. chromatography system in a purification module. “The accessibility of that Finally, the purified protein is filtered in a biology makes it possible to think about formulation module. tuning the host to make the molecules of In contrast with Love’s system, the othinterest with the precision that’s neceser DARPA-funded system to meet Ling’s sary for biopharmaceuticals.” With new challenge works without cells. Called sequencing and genome-editing tools, Bio-MOD, for Biologically-derived Mediresearchers can quickly adapt the yeast to cines on Demand, the system, designed by make new proteins. chemical engineer Govind Rao at UMBC Knowing the yeast’s biology well also and his team, fits in an 89-cm suitcase enables Love and his team to predict what (Nat. Biomed. Eng. 2018, DOI: 10.1038/ proteins from the host cells might cons41551-018-0259-1). The researchers are taminate their biologic product. Typically, now working on an even smaller version the biopharma industry programs CHO that fits in a briefcase. cells to pump out selected biologics. The Instead of using cells, Bio-MOD works industry uses CHO cells because they with freeze-dried extracts from CHO cells. are mammalian cells that can perform The extracts contain gene transcription the posttranslational modifications often and translation machinery from the cells needed for therapeutic proteins. But these that Bio-MOD then harnesses to synthecells, more complex than yeast, generate size protein therapeutics. Compared with about 2,000 contaminant proteins in Love’s system, Rao’s is similarly modular, addition to the desired therapeutic. With yeast, only about 200 host proteins end up including single-use protein production and purification modules that can be inin the cell culture medium with the prodserted into the suitcase. uct, making purification easier.
C R E D I T: O MA R BE R M UD E Z /MI T
person would reduce or even eliminate the need for screening for viruses or immunogenicity. Rao’s system has some drawbacks. Cell-free production systems are generally suited to making small amounts of proteins. And more purification is required to remove the cell debris. DARPA’s Ringeisen sees the benefits of both the MIT and UMBC systems, as well as their distinct applications. Rao’s freeze-dried product “can be stored pretty robustly on the shelf at relatively elevated temperatures and humidity,” Ringeisen says. “You can imagine that being used in a very field-forward, austere kind of environment.” Ringeisen sees Bio-MOD as appropriate for special operations missions involving small numbers of individuals who need protection from specific threats. Love’s benchtop system, on the other hand, could be used to replace biopharmaceutical stockpiles. The infrequency of chemical and other types of attacks means those stockpiles are rarely used, Ringeisen says. “You could almost think of this as additive manufacturing for pharma, where you could produce what you need when you need it.” He envisions scenarios in which an outbreak or exposure event leads to switching on production in nearby facilities. “Chris Love has shown that after about 24 hours he is able to grow up these Pichia yeast strains and start producing
“I wanted to have a machine that could make any drug at any time in any quantity.” —Geoffrey Ling, former doctor, U.S. Army, and current CEO, On Demand Pharmaceuticals hundreds of doses a day just with his laboratory-scale operation. You can imagine scaling that up to make thousands of doses a day.” Both projects demonstrated that they could produce a variety of molecules, including protein therapeutics and antigens for vaccines. Among them was granulocyte colony-stimulating factor (G-CSF), a protein that is administered in response to radiation exposure. A generic version of G-CSF has already been synthesized and been U.S. Food & Drug Administration approved, so it’s a well-understood protein therapeutic that the MIT and UMBC teams used to test their systems.
Cell-free innovations Organizations besides DARPA are interested in on-demand biomanufacturing to make it easier and cheaper to provide biologics around the globe. Most of those other organizations and researchers are
focused on designing cell-free systems because of their ability to work in a multitude of environments. The first time that freeze-dried cell extracts were used to produce proteins was by Bradley C. Bundy’s group at Brigham Young University in 2014 (BioTechniques, DOI: 10.2144/000114158). Around the same time, James Collins and coworkers at MIT also created freezedried cell extracts, which they eventually applied to on-demand, portable biomanufacturing (Cell 2016, DOI: 10.1016/j. cell.2016.09.013). Bundy’s team has focused on using extracts from E. coli, an abundant and common type of bacterium. “We’re using an E. coli-based system because we want this to be a very low-cost system so more people can have access to lifesaving protein therapeutics,” Bundy says. Bundy has recently started collaborating with Rao’s team. Collins says his cell-free system is aiming to improve global health. “It would open up portability for health care workers in
With the InSCyT benchtop system from researchers at MIT, biologics can be produced within 24 hours. NOVEMBER 12, 2018 | CEN.ACS.ORG | C&EN
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glycosyl groups, that are hard for cell-free low-resource areas,” he says. “It would systems to add. Earlier this year, a team open up portability for military personnel led by Michael C. Jewett of Northwestor space travel or hikers or athletes.” But ern University and Matthew P. DeLisa of for larger companies, he says, “outside of Cornell University reported a method for offering easy-to-use, inexpensive research getting E. coli cell-free extracts to add sugtools for making molecules on the spot, I ars to proteins in a single reaction mixture don’t think our platform would figure into (Nat. Commun. 2018, DOI: 10.1038/s41467production.” That’s because large companies typically need to produce sizable quan- 018-05110-x). Jewett is now collaborating with Rao’s Bio-MOD team. tities of biopharmaceuticals and need sufAnd there are costs associated with acficient resources to run the corresponding live-cell bioreactors. Cell-free platforms are quiring cells to make the extracts and with purifying them after they’re made. “You better suited for producing modest quantihave to start with cells to get to extracts,” ties in areas of limited resources, he says. MIT’s Love says. “And all that cell debris Perhaps validating Collins’s skepticism, has to be removed.” Breaking open, or lysonly one mainstream biopharma coming, the cells to get the gene transcription pany—Sutro Biopharma—is focusing on and translation machinery leaves behind cell-free systems. On the discovery side, unwanted membranes. the company uses cell-free production to quickly make molecules for testing. For example, it uses such systems to make antibody-drug conjugates. “Because it’s fast, we can evaluate many On-demand biomanufacturing has the different constructs,” says Shabbir Anik, potential to lower the cost of producing chief technical operations officer at Sutro. biologics for smaller patient cohorts, even Still, Sutro is also using cell-free manto the point of individual patients. ufacturing to take products through to In the Netherlands, that goal is well commercialization. The firm says one on its way to becoming reality. Huub advantage of a cell-free system is that it Schellekens and coworkers at Utrecht Unican use the same expression system for versity have been running a pilot program multiple new products. The company in which they make biological therapeutics makes the cell extract on demand for individitself and places it in ual patients. inventory. Within 24 Economics is one of hours of loading exthe drivers. Prices are tract into a bioreactor, increasing at the same Sutro can produce protime that the average teins. With cell-based effectiveness of new systems, it can take drugs is decreasing, days or weeks to have Schellekens says. The product. Sutro’s first current biopharmaantibody-drug conjuceutical industry “is gate manufactured by not really designed for the cell-free process is the next step in pharnow in a Phase I clinimaceutical care, which cal study. is personalized mediSutro also wants to cine,” he says. use cell-free systems Schellekens’s team to make biopharmais working on a method ceuticals that would for making therapeuotherwise be difficult tics for individual pato make in cells. For tients. “We are mainly example, such systems producing biologics in can be engineered to the hospital pharma—Huub Schellekens, professor cy,” Schellekens says. add nonnatural amino acids to proteins. Or of medical biotechnology, Utrecht “We can produce most they can make proteins University expensive drugs at 5% that are toxic to cells. of their current prices “We’ve made things like cytokines and and still make a profit.” Such a model can growth factors and peptides that degrade be thought of as the biologic equivalent of cellular systems,” Anik says. a compounding pharmacy. But cell-free systems face challenges Schellekens and his group are starting too. Many protein therapeutics are decwith treatments for lymphoma, a cancer orated with sugars, also called glycans or of the white blood cells that can become
Making it personal
“There will always be a need for a pharmaceutical industry for large patient populations and for mass production of drugs.”
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resistant to antibody therapies. Because of this resistance, doctors need another way to target a patient’s cancer cells. Once a new biomarker for the cells is found, Schellekens’s team could produce a bespoke monoclonal antibody to match it and be ready to treat the person. The idea is to make the amount needed for a year’s worth of treatment. The dosage forms made by the pharmaceutical industry often result in the need to discard unused products. “If you make it for an individual patient, we can give him or her the exact amount they need. We don’t need to throw away anything,” Schellekens says. The approach is a “no-brainer” for expensive orphan drugs, such as some enzyme replacement therapies, Schellekens says. But there will still be a place for a pharmaceutical industry. “There will always be a need for a pharmaceutical industry for large patient populations and for mass production of drugs,” Schellekens says. His team is focusing on off-patent drugs for now, but he thinks he will be able to sidestep patent issues regardless. Such treatment is known as “magistral” treatment, meaning it was made or prescribed to fit the needs of a particular case. “Magistral treatment is driven by the obligation every doctor has to treat patients,” Schellekens says. “As a medical doctor, I’m obliged to treat patients in need. That is a duty that cannot be overwritten by somebody on economic grounds.” Schellekens sees such drug production being implemented on a regional basis for general applications and in specialized treatment centers such as cancer centers. “It would be very inefficient if every hospital were to make all possible biologics.” Back in the U.S., efforts are ongoing to advance the DARPA-funded technologies. Ling, the former army doctor and DARPA program manager, is spearheading one of them. He’s now the CEO of On Demand Pharmaceuticals, a start-up that’s licensing technologies for both small-molecule drugs and biologics. “I want to see this work go to fruition,” Ling says. “The only way you’re going to get to patients is if a commercial enterprise decides to go ahead and make the thing and actually get it distributed.” Love’s team is in the process of forming its own spin-off to move its platform toward commercial development. But he’s unwilling to provide details at this time. Ringeisen anticipates that the systems DARPA is funding could be operational within 18 months. That’s an ambitious timeline, Ringeisen admits. But, he adds, “if it weren’t ambitious, it wouldn’t be DARPA.” ◾
