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Pressure BioSciences Receives $850K to Develop Sample Prep for Mitochondria

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While technology development in proteomics has overwhelmingly focused on mass spectrometry and to a lesser degree separation sciences, comparatively little work has been done on sample preparation.
 
But last month, Massachusetts biotech Pressure BioSciences announced it had been awarded an $850,000 Phase 2 Small Business Innovation Research grant to further develop its pressure cycling technology, according to CEO and founder Richard Schumacher.
 
The two-year award, which follows a $149,000 Phase 1 SBIR grant received in September 2006, is designed to enable the PCT technology to develop automated and reproducible methods of extracting protein biomarkers, subcellular molecular complexes, and organelles such as mitochondria, from cells and tissues.
 
This ability, in turn, could improve new-drug development and hasten personalized medicine.
 
By developing the technology, South Easton-based Pressure Biosciences is trying to tackle a sometimes overlooked and less-glamorous part of the proteomic research process.  
 
In the years since mass specs changed the way researchers study proteins, much, if not most, technology development in proteomics has gone into designing instruments with greater analytical capabilities. But according to Schumacher, the fervor has left floundering without upgrades a technology — sample preparation — at the front end of the discovery process.
 
“My God, there’s been billions of dollars put into the analysis part,” Schumacher said. “We have $500,000 mass specs, we have $100,000 and $50,000 pieces of equipment. We have all kinds of beautiful analytical equipment and software that have come to the market in the last 20 years to help us analyze samples.
 
“But if you look at the first part of getting an analysis … what’s happened in the sample preparation market in the last 20 years?” he asked. “Hardly anything.”
 
As a result, proteins yielded from mass spec studies can differ wildly from lab to lab and within the same lab. Also, researchers will sometimes find what they’re looking for, but at other times the resulting proteins are being denatured and destroyed, Schumacher said.
 
Pressure BioSciences’ solution is its PCT sample-prep technology, which depends on the use and manipulation of ultra-high pressure to induce lysis for the extraction of cell components. Biological samples are placed on a lysis disk within single-use processing tubes called PULSE, which stands for Pressure Used to Lyse Samples for Extraction, manufactured by Pressure BioSciences.
 
After adding appropriate buffers and solvents, the tubes are placed in the company’s Barocycler, a self-contained, high-pressure instrument.
 

“… if you look at the first part of getting an analysis … what’s happened in the sample preparation market in the last 20 years? Hardly anything.”

As pressure in the Barocycler increases, the sample is moved from the sample chamber of the PULSE tube through the lysis disk into its fluid-retention chamber. Pressure is quickly released and the sample, which is now partially homogenized, is pulled back through the lysis disk. This process breaks up cellular structures releasing nucleic acids, small molecules, and proteins.
 
Like heat and chemicals, pressure can be easily manipulated on a sample, but it acts homogeneously on the sample instantly. With heat- and chemical-based methods, it takes longer to achieve homogeneity.
 
The result is that the Pressure BioSciences technology “can, more than any other sample-preparation method on the market … exquisitely control the preparation of the sample,” Schumacher said.
 
With its Phase 1 SBIR grant, the company was able to demonstrate that the technology could be used to isolate mitochondria from cells and tissues “by selectively breaking the plasma membrane and leaving mitochondria intact,” said Alexander Lazarev, vice president of R&D for Pressure BioSciences.
 
Partially based on that Phase 1 work, the company last month filed a US patent application covering the technology and method.
 
For the Phase 2 grant, the company will collaborate primarily with Bruce Kristal at the department of neurosurgery at Brigham and Women’s Hospital. In the first stage of the grant, researchers will try to show that mitochondria are physiologically active after tissue samples have undergone preparation by PCT methods, Lazarev said.
 
In the second stage of the project’s work, Lazarev said he and his team will validate the extraction methods for particular tissues “of high interest for mitochondrial research.” He said these include brain, liver, skeletal muscles, cardiac muscles, and adipose tissues.
 
In a statement, Kristal said that the goal of the grant is to “increase the standardization of mitochondrial isolations and to increase yield and quality of mitochondria from tissues difficult to work with. … We also will examine the ability of the [technology] to facilitate biochemical studies of mitochondrial constituents that are often otherwise limited by solubility limitations.”
 
If the company successfully validates the method, then it can be made commercially available to the public, Lazarev said. The plan is to eventually commercialize the product created from the Phase 2 study as a kit for mitochondrial isolation and eventually for other organelles.
 
Under Pressure
 
Work developing the PCT technology was initially begun when Schumacher was at Boston Biomedica, a company he founded in 1986 and where he was CEO. That company began as a diagnostics firm, but then branched into other areas, which confused some of its investors, Schumacher said.
 
Because Boston Biomedica had its irons in so many fires, investors couldn’t figure out whether the company was an instruments firm, a clinical laboratory, or an HIV therapeutic firm. The PCT technology only further blurred its identity, Schumacher said.
 
“There was pressure from the environment, from the people from the [investment community] that we were just too complicated,” Schumacher said.
 
Eventually, in 2004, Boston Biomedica was split up and its different businesses sold off. Schumacher kept the PCT platform and with it formed Pressure BioSciences. Today, its shares are traded on the Nasdaq.
 
Though mass specs have monopolized the lion’s share of proteomics and protein research technology development, Pressure BioSciences officials said that balance of power may be slowly changing. Indeed, at several conferences during the past year, speakers and thought leaders in the field have emphasized the need to improve sample collection and sample-prep methods.
 
Still, Pressure BioSciences officials said that techniques commonly used today haven’t changed much through the centuries: in essence, it consists of grinding tissue samples. And according to Schumacher, the research community still doesn’t fully believe that improvements to sample-prep techniques are necessary.
 
“We deal with a lot of people who not only are skeptical because they’ve never heard of using high pressure, but now they’re saying, ‘I don’t have a problem. I’ve been using my homogenizer, my bead beaters, my sonicators for five, 10 years, and I don’t have a problem,’” he said. In fact, those researchers may be destroying or throwing away valuable data and spectra, Schumacher said.
 
“People have never thought or heard of using pressure that is twice what you get at the deepest part of the ocean on your lab bench. And then it’s mind-boggling to think that that pressure is more gentle than any of the sample-prep methods that are used,” he said.
 
Paul Pevsner, an associate professor of pharmacology at the New York University School of Medicine, has two Barocyclers, which he’s been using to help him detect protein biomarkers associated with in vitro fertilization, cancer, and stroke.
 
The PCT technology produces higher and more consistent yields, he said, and reduces the time for tryptic digestion from overnight to about 45 minutes. It also allows him to see new proteins he hadn’t seen using other sample-prep methods.
 
In a paper submitted for publication dealing with work in in vitro fertilization, Pevsner and his colleagues describe two newly discovered proteins that are present only in competent embryos, which would eventually become babies. The proteins, which he found using the PCT technology, had never been seen before, he said, and could be biomarkers predictive of successful in vitro births, he said.
 
“It’s pretty startling stuff,” Pevsner said. “We’ve tried every kind of extraction technique known to man, and we just don’t do any of the others” any longer.
 
As of March 31, Pressure BioSciences had placed 40 Barocyclers — 22 instruments sold, rented, or leased, and 18 with collaborators. The instrument comes in two models.
 
Last year, Pressure BioSciences also launched the ProteoSolve LRS kit, a detergent-free protein-extraction method that uses novel chemistry to fractionate lipid and protein components from samples. It is also developing ProteoSolve SB, a method for extracting all four major molecule types, including DNA and RNA, from samples.
 
While the market for Pressure BioSciences’ instruments is “relatively small,” the platform will be “very important” for new drug development, Lazarev said. “In every major pharmaceutical company, there is a group that works on mitochondrial metabolism now,” he said.
 
Schumacher added that the technology has applications for other research areas, including forensics, bio-terror, and basic research.
 
“Anybody who has to study a protein, anybody who has to study and RNA or DNA molecule should look at this technology because it could enhance it,” Schumacher said.

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