Inside the Diagnostics Industry: Technology Advancements Bringing Resurgence of Interest in Commercialization of Paper-Based POC Diagnostics

Paper-based diagnostics are not that new. Dipstick assays and lateral flow tests, like home pregnancy tests, date back to the 1960s and 1970s. Yet there was never a broad proliferation of the technology to encompass a multitude of clinical applications. While there has always been hope that paper-based products’ ease of use, rapid time to results, and low cost would make them prime targets to increase the accessibility of medical care, particularly in resource-limited settings, the reality is that diagnostics manufacturers have not pursued the strategy. “The principles today are the same, but what’s changed is that we’ve refined more ways to detect more things,” says Andrew Warren, a Massachusetts Institute of Technology (MIT) graduate student and lead author of a recent paper on the development of a paper-based diagnostic for cancer. “Today we have expanded our thoughts as to what else it is useful for and moved beyond a binary yes/no. With nanotechnology we have a good sense of how to do this in a robust manner.” There has been a resurgence of interest in paper-based diagnostics resulting from advances in the technology led by renowned chemist George Whitesides’ group at Harvard University. Patterning paper can be fabricated in 2-D or 3-D to transport the fluidics and patterned channels and wells now allow for filtering and multistep reactions. Recently, paper-based microfluidics has emerged as a point-of-care platform that is capable of bringing sophisticated multiplex assays to resource-limited settings without the need for any large equipment. Use Cases One of the leaders in the paper-based diagnostics space is Cambridge, Mass.-based Diagnostics For All (DFA), a 6-year old spinoff based on technology from Whitesides’ lab. DFA was established as a nonprofit and is funded through philanthropic grants. It holds the exclusive worldwide license for Whitesides’ patterned paper technology. Describing the technology as “elegantly simple,” DFA says that patterned paper-based technology is not only inexpensive but also requires minimal training to use, practically no sample preparation, and no electricity or additional equipment to process a sample. In addition to the liver function assay (see box on page 6) that DFA says will be the first approved paper-based microfluidics test, they are working on other paper-based diagnostics. Developed through a grant from the Bill & Melinda Gates Foundation, the second product in the DFA pipeline is a low-cost, rapid diagnostic test for immune markers to determine successful vaccination against tetanus and measles. In a U.S. Defense Department, Defense Threat Reduction Agency-funded effort in conjunction with Harvard University, DFA is developing nucleic acid amplification- and immunoassay-based paper-microfluidic devices for Brucella abortus. Commercialization Whitesides has been quoted as saying that ‘‘if the science of something is still interesting, the ‘something’ is probably not ready to be a product.” The technology behind paper-based diagnostics, particularly microfluidics, has come a long way in just the last few years. Currently,  though, researchers are in the midst of finding the best use cases for the technology, including which clinical conditions are most in need of these increasingly sophisticated assays and in which populations. But historically, commercialization and adoption of these tests has been plagued, despite good intentions, by a lack of a business case supporting their use. “There has been a lot of promise in the field, but not a lot of delivery,” writes Ali Kemal Yetisen from the University of Cambridge (United Kingdom) in a critical review published February 2013 in Lab on a Chip. “Diagnostics for resource-limited settings does not always receive attention from large companies while limited research activity in smaller biotechnology companies and academia struggle to make an impact in creating real world products, scaling up and achieving market penetration. . . . This aspect parallels the disinterest of industrial partners in these applications due to the limited market and low profit margins.” These low margins are also accompanied by low manufacturing and development costs. Yetisen and colleagues say in the review that low-cost lateral flow tests can be manufactured for 10 cents to $3 per test, and development costs for lateral-flow immunoassays range from $30,000 to $100,000 per test if the target analyte and necessary antibodies are available. Paper-patterning costs are estimated to be less than 1 cent to 3 cents, including the price of the paper, although a large number of tests are needed to achieve economies of scale for production. “It will be very challenging, yet not impossible, for a biotechnology or a diagnostic company, research group or investors to capture value from paper-based microfluidics,” writes Yetisen. “Different strategies need to be considered based on the geographical placement (developed or the developing world) of the product and also on the therapeutic area (established or new). If the market is mature for a particular therapeutic area, then expecting a shift from current diagnostic methods to paper-based microfluidics is unrealistic.” But Yetisen and colleagues do offer optimism that recent advances in capillary-based microfluidics might improve the translation of hope into products. While the DFA’s liver function assay was specifically designed to be used in underserved areas, there has been quite a bit of interest in the developed world. “We are seeing a dual-market use case,” says Patrick Beattie, director of operations at DFA. “Pharmaceutical companies are interested in the assay as a companion diagnostic and for trials, although no official partnerships have yet been established.” Beattie says that regulatory submission will depend on the ultimate market selected and could range from U.S. Food and Drug Administration approval to CE-mark, World Health Organization prequalification, or local regulatory approval. The interest and cooperation from what Beattie describes as “traditional” diagnostics manufacturers is welcome, given that some in the laboratory and diagnostics industry see sophisticated, highly sensitive, point-of-care technology as a threat. “What we are developing is disruptive and will be viewed by some as negative, but we have had great discussions with traditional diagnostics manufacturers. They see us an opportunity,” Beattie tells DTET. “Being a nonprofit, for us it is important to focus on technology development and not manufacturing and distributions, which could get in the way of our own success. It is not simple to build a diagnostic company. We want to take advantage of the market-based focus of a potential partner. ” Looking Ahead While all involved in paper-based diagnostics are on a mission to make the tests commercially available to underserved markets as rapidly as possible, they believe they are able to push the technology to new levels of complexity. “We are taking clinical chemistry tests that have been performed on a machine and cartridge-based system and moving them to solid, disposable platforms,” says Beattie. “The approaches we are using is to look at what internists do in the developed world and see what isn’t available in the developing world. An obvious next step is renal function testing and normal blood chemistry.” Side Box: The Process of Making Paper-Based Microfluidic Below outlines the steps in DFA’s making of its liver function paper-based microfluidic assay.

• Paper is printed on a sheet with wax defining areas for 55 tests. Assembled tests include a sandwich of two such sheets.
• The sheets are baked for 30 seconds at 130 degrees Celsius to allow the wax to melt completely through the paper’s 0.2 millimeter thickness.
• The test wells are wax-free circles 2 millimeters across.
• Specified amounts of the reactant chemicals are deposited on each sheet. The first sheet’s reagents react with enzymes, while the second sheet receives dyes that change color if exposed to products released by the first reactions.
• The two sheets are fused together with adhesives in a press.
• A protective laminate is affixed to the top of the package.
• Completed tests are cut into individual squares.

(Adapted from MIT Technology Review) Side Box: Paper-Based Microfluidic Liver Assay Routine monitoring of liver function for patients on anti-retrovirals (ATV) and therapy for tuberculosis is the standard of care. However, this monitoring for liver toxicity is “severely limited” in the developing world, because of expense and lack of access to modern laboratory instrumentation. While access to ATVs has expanded in underserved parts of the world, there has not been similar improvement in access to monitoring tests. Diagnostics For All (Cambridge, Mass.) is validating and looking for a partner to commercialize a liver function assay, in what will likely be the first approved paper-based microfluidic  diagnostic, the company says. The assay monitors for drug-induced liver injury through serial measurements of serum transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]). The 3-D multilayered design allows a single 30 ml to 35 ml sample of whole blood to be split into five separate  “streams” of plasma, which are tested with five independently optimized assays in parallel. The test requires no preanalytical sample prep, no additional agents, and no reader and returns results in 15 minutes, at ambient temperature. The researchers say that when the test is mass produced, the cost could be less than 10 cents per test. According to a study published in Science Translational Medicine (September 2012), an earlier version of the liver assay was used to test 223 blood samples obtained by venipuncture and 10 finger-stick samples from healthy volunteers. The test allowed visual measurements of AST and ALT, in both whole blood and serum with more than 90 percent accuracy. “Our test performed well compared to automated methods, even in specimens that were obtained from critically ill patients with multiple derangements in other analytes and that were up to 5 hours old at the time of testing,” write the authors, led by Nira R. Pollock, M.D., Ph.D., associate medical director, Infectious Diseases Diagnostic Laboratory at Beth Israel Deaconess Medical Center in Boston. “The experiments presented here have firmly established proof of concept and clinical relevance and will allow us to move into clinical field studies.” Side Box: Paper-Based Tests for Cancer, Blood Clots Among the unmet global health needs are noninvasive methods to diagnose noncommunicable diseases, which are becoming an increasing burden, in low-resource areas. MIT researchers have developed exogenous agents that can serve as synthetic biomarkers, measurable in urine and quantifiable by a companion paper test, for noncommunicable diseases including cancer and blood clots. According to a mouse-model study published Feb. 24 in the Proceedings of the National Academy of Sciences, the test relies on injected nanoparticles (synthetic biomarkers) that interact with tumor proteins called proteases or thrombin in clots. At the diseased tissue, local up-regulated proteases (matrix metalloproteinases or thrombin, respectively) cleave their surface coat of peptides, releasing hundreds of reporters that are easily detectable on a paper strip from a sample of the patient’s urine. From injection to results is about one hour in mice, and the researchers expect similar time to results in humans.  The researchers are also working on a nanoparticle formulation that could be implanted under the skin for longer-term monitoring. While testing in humans is necessary, senior author Sangeeta Bhatia, M.D., Ph.D., said in a statement that the technology would likely first be applied to high-risk populations, such as those with a personal or family history with cancer, although eventually, she would like to see it used for early detection in developing nations. She said that applications in the United States and other developed countries could be “transformative,” enabling image-free cancer detection in a home or pharmacy clinic.