By Ben Butkus
iCubate, a spinout of the HudsonAlpha Institute of Biotechnology, has developed a benchtop, fully integrated cartridge-based molecular diagnostic platform capable of running highly multiplexed assays using the company's amplicon-rescued multiplex, or ARM, PCR method.
The company is hoping to differentiate itself from other molecular diagnostic shops primarily through the ARM-PCR technique, which iCubate has shown can amplify up to a quarter of a million unique targets in one reaction — although a typical multiplexed diagnostic assay would interrogate just a few dozen – with a high degree of sensitivity and specificity.
In addition, the company has adopted a strategy of opening its platform to other assay developers, whom iCubate hopes will develop additional content for the system under a revenue-sharing program, Jian Han, president and CSO of iCubate, told PCR Insider.
"We felt the need to open the platform up so more users could develop assays, so that's what we did earlier this year," Han said. "We call this iCubate 2.0, like Web 2.0, where users generate content, and we build an online store, and have a profit-sharing [program] where the developer gets 70 percent [of assay sales]."
The roots of iCubate and the ARM-PCR technology extend to another multiplex PCR method that Han invented in the 2000s called target-enriched multiplex, or TEM-PCR, which uses nested gene-specific primers tagged with a universal set of so-called SuperPrimers to improve the multiplexing of PCR by enabling consistent amplification conditions for different primer sets and eliminating background amplification from high primer concentrations.
In the early part of that decade, a company that Han founded called Genaco developed a multiplexed assay based on TEM-PCR for several respiratory pathogens to address the SARS outbreak.
In 2006, Qiagen acquired Genaco, primarily for the TEM-PCR method, and further developed and commercialized the original multiplex respiratory panel as the ResPlex panel. In addition, Diatherix Laboratories, a Genaco successor that is also associated with HudsonAlpha, has developed and commercialized several infectious disease panels based on TEM-PCR.
Han, meanwhile, worked for Qiagen for about a year before moving to HudsonAlpha, where he still serves as an investigator. While at HudsonAlpha, Han developed ARM-PCR, which he calls a faster, more sensitive version of TEM-PCR. The Huntsville, Ala.-based institute applied for patents on the technology and licensed them to Han's latest company, iCubate, for commercialization. HudsonAlpha was awarded its first US Patent covering the ARM-PCR technique in August (PCR Insider, 8/16/11).
Compared to TEM-PCR, ARM-PCR increases assay sensitivity and multiplexing capability by increasing nested primer concentrations and omitting the universal SuperPrimer from the initial PCR reaction with the nested primers, according to the company.
However, because it also employs an amplicon rescue step, it requires two rounds of PCR, which, if set up in the open, would increase the chances of amplicon contamination and false positives. Thus, for use in molecular diagnostic applications, the method is best used with a closed, sample-to-answer platform, which iCubate and local engineers have also recently developed out of necessity.
iCubate showcased its platform at the Association for Molecular Pathology meeting and exhibition last month in Grapevine, Texas. Called the iCubate System, it comprises an iC-Reader, an iC-Processor, and a computer with pre-installed iC-Report software.
At the core of the iCubate System is the closed cassette, which contains pre-loaded assay reagents and can collect liquid waste. Up to four cassettes can be loaded into the iC-Processor, which automatically processes the cassettes by performing nucleotide extraction, PCR amplification, hybridization, and washing. Because ARM-PCR is an endpoint PCR method, the platform uses hybridization array-based detection of amplification products.
But iCubate sees its platform only as a means to the end of driving adoption of the ARM-PCR technique as widely as possible.
"The unique thing about our technology is not really the iCubate platform, it is the multiplexed PCR," Han said. "Like people in the software industry say, 'If you're really serious about developing software, then develop your own hardware;' and that's what we did. We developed a platform to adopt the multiplex PCR."
The company has many current and potential competitors when considering the plethora of companies developing automated, sample-to-answer molecular diagnostic platforms. However, the two companies whose technologies most closely align with iCubate's are Idaho Technology and Seegene, both of which offer platforms based on highly multiplexed amplification technologies, and both of which are commercializing infectious disease diagnostics based on those methods.
Han said that iCubate's value proposition is that it fulfills all six terms that he believes describe the ideal molecular diagnostics platform: "extraction, amplification, detection, multiplexing, automation, and a closed system."
"Many companies develop a platform doing one or more, but not all six," he said. "I think Idaho Technology has beautifully done a fully integrated, multiplex, sample-to-answer [platform] … but their amplification technology is not as robust as ours. But we also have our issues. So I think that there is always room to improve and have a second- or third-generation instrument."
iCubate claims that the upper limit of ARM-PCR multiplexing is in the range of 250,000 targets or so. In fact, Han and colleagues at HudsonAlpha have conducted immune repertoire sequencing projects demonstrating this capability; and, as reported in May by PCR Insider sister publication Clinical Sequencing News, are attempting to sequence the immune repertoires of 10,000 individuals representing 100 diseases. Han has also founded a company called iRepertoire to further commercialize the technology for sequencing sample prep.
However, for the purposes of molecular diagnostics, most assay panels comprise just a few dozen targets, although iCubate is open to the possibility of early adopters of its technology developing more complex panels with greater numbers of targets — and as such began the iCubate 2.0 program.
"We now have more than 200 people … in China, Korea, and the US … registered to be our developers," Han said. "We are really rolling this out, and are trying to get more people interested in multiplex PCR to develop their own applications — sometimes they are applications we haven't even thought about. Food safety; bioterrorism — there are a lot of other applications out there, not necessarily medical."
iCubate is also developing its own assay panels, primarily in an attempt to offer up "some example multiplex panels," Han said. "Most of those are assays that we were familiar with from the Genaco days … so they were easy to develop and validate. We are going to roll out a limited number, because we don't want to compete with our developers — no more than 20 panels, and then we'll stop."
The company does plan to pursue regulatory approval for some of the most basic panels, and in fact last year submitted a pre-investigational device exemption to the US Food and Drug Administration for a multiplex sepsis panel. "We got a good response, and adjusted our clinical trial approval strategy, so we are going to submit [an application] next year," Han said.
The company netted an approximately $3 million equity investment from HudsonAlpha when it was founded; and since then has raised another $7.5 million from non-institutional angel investors in two separate rounds, Han said.
iCubate also plans to reap some revenue from development partners, to whom it expects to sell iCubate systems and consumable cartridges for assay development. Han said that iCubate recently won a bid from the Chinese Center for Disease Control and Prevention, which has acquired an iCubate system and consumable cassettes to develop various assay panels of interest. "So we'll have revenue even before an FDA approval," he said.
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