NEW YORK – With its first US Food and Drug Administration clearance of an instrument and assay, Akonni Biosystems now plans to build up a menu of diagnostic panels for complex diseases that require detection of tens to hundreds of biomarkers.
The Frederick, Maryland-based firm intends to capitalize on the low cost of its technology, higher multiplexing capabilities compared to most competitors, and the potential to test for nucleic acids, proteins, or perform enzymatic reactions using its proprietary array-based technology.
The firm's TruDiagnosis platform was cleared by the US Food and Drug Administration earlier this month along with a pharmacogenetic test for three alleles to help guide blood thinner treatment.
Akonni has been developing multiplexed infectious disease assays — including ones for tuberculosis and influenza — for more than 15 years, but the company ultimately chose to take a PGx test through FDA as its first assay for strategic reasons, said Akonni CEO and Foundaer Charles Daitch.
"We had to take some baby steps, and the first step was to get our hardware and software through," Daitch said.
The amplification and detection module of the system, called TruArray, is microarray-based but with some novel twists, he said.
Most microarrays essentially consist of ligands or oligos printed onto a two-dimensional surface, which can limit the diffusion. It is also harder to extrapolate assays from the tube-based research and development stages into 2D arrays. Akonni's technology, however, uses printed hydrogel drops containing pre-synthesized oligos.
"It creates more of a 3D binding reaction, which is 99 percent water," Daitch said. The hydrogel drop is about 100 microns in diameter, he said, with a volume of approximately 0.2 nanoliters.
This topography, and a pore network structurally inherent in the hydrogel material, allows the probe anchored within to behave more like it would in its native aqueous environment, and the reaction's kinetics are more efficient, as if it were happening in a "little nano test tube," Daitch said.
The rest of the microarray is fairly standard, but the detection step in the TruArray consumable also brings in novel, proprietary technologies.
The microarray is printed in a flow cell chamber in a microfluidic cartridge, with an area of about 1 centimeter squared, although it can be made smaller, Daitch said. This engenders a 20- to 40-microliter reaction volume, containing hundreds of hydrogel nano test tubes.
"We tried to keep it as simple as possible," Daitch said, so there are no valves or electrowetting or any other "fancy plumbing" that can increase the cost of a microfluidic consumable.
Akonni instead uses a materials-based approach to allow the fluid to wick into the chamber, making it technically valve-less, from an engineering standpoint, Daitch said, without even wax or silicone oil valves.
Furthermore, unlike other cartridge-based systems, the PCR step for a molecular assay in the system is done in the same chamber as the microarray. An isothermal hybridization step allows detection, also within the chamber.
"It translates to a simpler workflow … and we're using very simple microfluidic designs," Daitch said.
Overall, this makes the device very inexpensive to manufacture. "That's really where we wanted to make a difference: mid-multiplexing for complex diseases, and doing it in a cost effective way without sacrificing the quality of the diagnostic result," he said.
The FDA clearance did not include the firm's sample preparation technologies, called TruTip extraction and the TruTip automated workstation, but rather it mimicked a predicate clearance and relied on Qiagen sample prep kits.
Still, Daitch said he believes Akonni's ability to multiplex assays with between 50 and 250 targets puts the firm in a different category than many other multiplex diagnostic systems on the market.
"We're not low-plex, and we're certainly not a high-density microarray," he said. The firm is now targeting complex disease conditions that require hundreds of biomarkers, he added.
Importantly, the arrays are also biomarker agnostic. Akonni can design them to detect nucleic acids, peptides or antibody proteins, or it can even immobilize enzymes for biochemical reactions — although each type of assay would have to be performed in a separate microfluidic cartridge — which adds a level of potential assay diversity not seen in most systems.
A number of studies have recently been published describing and evaluating Akonni's system.
In Lab on a Chip, the Akonni team described a "lab-on-a-film" version of the firm's technology that detected multidrug-resistant tuberculosis from sputum extract, using the automated TruTip workstation for extraction, with high sensitivity.
This version of the TB test had 203 gel elements and included probes for 37 mutations, deletions, or insertions across five genes, including an internal control, according to the study. Printing the gel elements on flexible film cost 500 times less than microarray glass, and the assay had a sensitivity of 32 colony-forming units per milliliter in a small set of samples.
And, in collaboration with researchers at Harvard Medical School, Akonni published a proof-of-concept study in PLoS One showing that the TruTip technology could be used to extract TB pathogen from sputum samples.
Furthermore, a team made up of members from PATH, the Bill and Melinda Gates Foundation, and the US Centers for Disease Control and Prevention recently evaluated eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings by sending test developers blinded panels containing TB, influenza A, and Salmonella typhimurium samples.
As published earlier this year, also in PLoS One, the authors "noted significant differences in performance among these technologies and identified those most promising for potential further development."
They found Akonni's assays to be the most accurate, but also noted that they required a significantly longer turnaround time compared to other technologies due to a three-hour DNA microarray hybridization step after amplification.
The same team had also evaluated the Akonni sample preparation technology compared to five others, and in a separate PLoS One publication demonstrated that technologies from Akonni and MolBio produced the best results.
The long road to FDA
Akonni has been developing its diagnostic system since 2002, and has been describing the gel drop technology inside its TruDiagnosis system for at least a decade.
The recently-published studies are in addition to a body of earlier literature produced by the firm and others detailing assays for influenza detection and typing, methicillin-resistant Staphylococcus aureus (MRSA), extensively drug-resistant (XDR) tuberculosis, as well as extraction of circulating fetal DNA using the TruTip technology.
The firm previously won a $3.2 million Phase II Small Business Innovation Research grant from the National Institute of Allergy and Infectious Diseases in 2009 to work on multiplex respiratory testing in collaboration with New York's Wadsworth Center, among others, and with Wadsworth it was also funded to develop a meningitis and encephalitis panel.
A partnership with Harvard University to develop multiplex tuberculosis identification and resistance testing assays in a closed consumable format garnered $29 million in NIH funding five years ago.
Daitch said that Akonni has been dedicated to global health since its founding, and that having simpler workflows, low cost, and being able to look at complex diseases could make a difference in low-resource settings.
Tuberculosis, for example, is deadliest in countries with the lowest resources, he said, and efficiently detecting drug resistance is an obstacle to effective treatment.
That said, "If you are going to launch in the tuberculosis world, there are several things you have to be sensitive to," he noted, including performing extensive studies to make sure the diagnostic markers in the test are representative of the resistant TB strains in a given geography.
Other molecular diagnostics that detect genetic mutations conferring drug resistance have faced some challenges in predicting prevalence of different markers when designing their assays. For example, a few years back the Cepheid GeneXpert MTB/RIF test started missing a mutation in Swaziland that was occurring in 30 percent of TB patients.
That problem comes down to the bio-discovery stage of assay development, Daitch said, but is also impacted by the limit of the number of targets in a technology. A qPCR platform-based assay is limited in how it can be modified to add or change targets, and to increase the multiplexing, a system would need to increase the number of optical detection channels or split the sample, but both of these have drawbacks.
Microarray-based tests, on the other hand, can more easily add targets, Daitch said, and Akonni also benefits because the 3D hydrogel makes it more straightforward to translate lab results to the microfluidic.
Now, Akonni is trying to finalize the set of markers that will be used on its array for XDR TB, which includes resistance to rifampin, isoniazid, fluoroquinolone, streptomycin, pyrazinamide, and aminoglycosides, Daitch said.
The TB test remains in the pipeline, but the firm "didn't want to go out first with that one" because it is a much more intense clinical study, Daitch said. "You have thousands of samples, sites all over the world, and we just don't have the financing to execute a clinical study like that," he said.
The clinical study for the warfarin test, on the other hand, was straightforward, and allowed Akonni to get the instrument, software, and consumables through FDA as a first step.
Daitch said TB may also not be the very next assay in its pipeline, but it is in the top five, and, he added, it is supported by two current NIH grants.
The other tests in the pipeline include multiplexed assays for hospital-acquired infections with antimicrobial resistance that are focused on different symptoms or syndromes. Upper respiratory and lower respiratory panels detecting resistance in both viral and bacterial targets are in the pipeline, as are panels for enteric pathogens, bloodstream infections, and meningitis and encephalitis. The firm is also considering developing tests for rare diseases that require multiplex testing.
"We want to detect complex diseases. We don't want to compete with real-time PCR, which is low-plex, and we don't want to compete with sequencing … We really have a place in antibiotic resistance," he said. At a breakpoint above 100 markers, Akonni thinks it will make a difference in the clinical diagnostics world.
In terms of specific costs, Akonni has only just received clearance and Daitch expects that having additional menu will drive future uptake. He estimated that its unique design features will help the firm compete on quality with other mid-multiplex tests, but be somewhere around an order of magnitude less expensive.
"We can now start to compete with some of the entrenched multiplexed platforms," he said, adding, "I don't want to pretend we are going to come up and eat their lunch or anything like that" but, he said, he thinks that there is so much new data and new biomarkers every day so that it is unlikely there is going to be any one platform that can solve everyone's problems.
"There are plenty of disease conditions out there for us to find our niche and make a difference," Daitch said.