By Ben Butkus
Part two of a two-part series. Part one, which covered new technologies under development by Life Technologies and RainDance Technologies, is available here.
For the past several years, researchers looking to use digital PCR — an extremely sensitive and precise version of quantitative real-time PCR — had one vendor to choose from, South San Francisco-based Fluidigm. But the company's monopoly on the market is likely short-lived as several other companies are either developing digital PCR tools or are actively surveying the landscape.
In addition to established PCR players like Life Technologies, which plans to launch an early version of a digital PCR platform by the end of the year, a number of startups are looking to make a name for themselves in this still-emerging market.
However, in addition to competition from larger, entrenched vendors, these firms will need to negotiate a complex intellectual property landscape that one player described as "thorny."
While dPCR is a relatively new focus area for both Life Technologies and RainDance Technologies, it will be the sole focus of Pleasanton, Calif.-based startup QuantaLife.
Founded in 2008 to commercialize technology from Lawrence Livermore National Laboratory in nearby Livermore, Calif., QuantaLife has been operating in stealth mode over the last two years as it develops a dPCR platform that uses emulsion-generated microdroplets to partition individual PCR reaction volumes.
QuantaLife's vice president of marketing, Michael Lucero, has been involved in the development of both real-time PCR and digital PCR, with prior stints at Applied Biosystems and Fluidigm. Lucero was also CEO of Stokes Bio prior to Life Tech's acquisition of the Irish startup last year.
In an interview with PCR Insider, Lucero echoed the comments of Gordon Janaway, product manager for real-time PCR reagents at Life Tech, that dPCR partitioning must be done cheaply in order for it to become a viable platform technology.
"All the things people say are great about dPCR are dependent on how many partitions you can divide it into," Lucero said. "So the question is, 'How do you do that so that it’s cost-effective and easy?'"
QuantaLife's technology, he said, involves making an emulsion that creates "tens of thousands of monosized droplets … each with a volume of about one nanoliter. Ten thousand of those droplets equal 10 microliters, which is the typical sample size for a customer."
QuantaLife's method aims to make dPCR easy to use and compatible with many current PCR protocols. To make droplets, a sample in aqueous form is placed into a droplet generator, which is "an inexpensive plastic consumable," Lucero said. "The sample goes in, oil goes in, you put it in the machine, and our droplet generator creates an emulsion for you. It does it in batch, so you could do multiple samples at the same time."
Then, he said, a user places the emulsion into a single PCR-compatible tube; or, each emulsion could be placed in a well of a traditional multi-well plate for even higher throughput.
"Then you put it on whatever thermal cycler a customer has; carry out PCR; and all the droplets are read as negatives or positives," Lucero said. "Some of the droplets will have target in them, and they'll amplify and produce a strong signal. The ones that don't have target will just have the background fluorescence."
He added that QuantaLife's readout instrument can be described as being similar to a flow cytometer, what he called a "droplet cytometer that …very quickly reads the droplets, so you can read 10,000 droplets in less than a minute."
Lucero said that this scale of partitioning is "very different than what's been available" with platforms like Fluidigm's, and is necessary to get meaningful quantitative results.
"If you can take a single sample of standard size; divide that into 10,000 different reactions; and then count every single reaction for the target of interest — that's the kind of thing that's going to turn things around," Lucero said. "It's going to make things much more quantitative. It's also going to limit the background and increase the relative concentration of something rare in a sample."
The upshot is that QuantaLife plans to offer price points that are "similar to what real-time PCR is today," Lucero said. "I think it's just as important an innovation for PCR as real-time PCR was. And it's actually an improvement on real-time PCR in the same way that real-time PCR was an improvement over old PCR."
Application-wise, QuantaLife will take aim at many of the same markets that Life Tech is eyeing. These include copy number variation detection and copy number alteration, as well as detecting point mutations in cancer to aid in uncovering new treatment targets or developing companion diagnostics.
"It's looking for the needle in a haystack — we call it rare-event detection," Lucero said. "That's where dPCR has made some headlines already, because you can detect something that's overwhelmed by a background that's very similar. If you partition the sample into all those reactions you can detect the needle."
Meanwhile, although Life Tech's Janaway said that gene expression using dPCR is in "the distant future" because of the huge number of replicates it would necessitate, Lucero said that QuantaLife's platform will enable such studies in the much nearer term.
"Plain old gene expression — we can do it better than anybody else," Lucero said. "There are always a couple ways to show you're good at something, and one way is single-cell gene expression. People have been making a big deal out of that, but using real-time PCR. Digital PCR is really the way to go."
Lucero's vision of dPCR-driven gene expression studies involves isolating single cells in wells, preparing them for PCR, and "counting messages." QuantaLife's technology, he said, will allow users to partition the messages from different alleles for something Lucero calls "allele-specific gene expression. It really illustrates the kind of things you can do with dPCR that are more precise, and provide more information."
Finally, Lucero said that dPCR's development will be very closely tied to that of next-generation sequencing.
"If you're making discoveries with next-gen sequencing, you are in need of a technology for validation and follow-up work that matches the capabilities of sequencing," he said. "It's sort of what happened with microarrays for gene expression and TaqMan real-time PCR. Microarrays were used for global discovery; and then TaqMan came along and replicated a lot of the work in large numbers of samples, one gene at a time. The discoveries that were made with microarrays were validated with TaqMan."
As such, Lucero said that QuantaLife is already "working with people at some of the large genome projects, and [is] already doing some of the validation work," though he declined to elaborate due to the early-stage nature of the collaborations.
As for the company's dPCR platform, Lucero said QuantaLife plans to field beta units by the end of this year. "They'll actually be sold into user sites. And then we'll have our instrument out for commercial release in the early part of 2011."
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BEAM Me Up
Another company seeking to exploit digital PCR's "needle-in-a-haystack" capabilities is Hamburg, Germany-based Inostics, which is using technology licensed from Johns Hopkins University to create assays for cancer mutation detection and discovery and companion diagnostic development.
Specifically, Inostics, whose name stands for "individualized diagnostics," is using technology known as "BEAMing" — short for beads, emulsion, amplification, and magnetics —developed in the laboratory of Johns Hopkins researcher Bert Vogelstein.
In BEAMing technology, single-molecule PCR is performed on magnetic beads in water-in-oil emulsions, with each magnetic bead coated with thousands of copies of DNA molecules with an identical sequence.
According to Inostics' website, the technology can detect and enumerate mutant and wild-type DNA when present at ratios greater than one in 10,000; and its sensitivity and selectivity is "100-fold higher than conventional technologies."
Representatives from Inostics could not be reached for comment. However, in an interview with PCR Insider, Vogelstein provided some details about the technology's development and its capabilities.
"It started out with diagnostic applications," Vogelstein said. "In the early 1990s, our group started to look for mutations in clinical samples. The challenge we faced then, which dPCR in part overcomes, is that the number of mutant molecules in a clinical specimen is often very low compared to the number of normal or wild-type molecules present."
Vogelstein and colleagues first developed a method to look at single molecules in individual wells of a multi-well PCR plate. "We used 384-well plates, and … you could fill about 25 percent of the wells. If you filled more than that … then in some of the wells you would get more than one molecule per well."
This allowed his team to examine around 100 molecules simultaneously, and "even that was an advance because it did allow us to look at samples which only had one or a few mutant molecules," Vogelstein said.
However, such a signal-to-noise ratio was still not appropriate for clinical applications, "because it’s really not enough to be able to see if there is a statistically significant difference."
They later developed the BEAMing technology as a way to look at millions or even hundreds of millions of molecules at once. "Basically we convert single molecules to single beads, but now each bead has 100,000 identical copies of the starting molecule, and you can look at these beads after hybridizing to a wild-type or mutant probe," Vogelstein said. "You can look at them in a [fluorescence-activated cell sorter], and the whole thing becomes quite feasible."
Vogelstein said that the basic BEAMing technology also forms a core part of two commercially available sequencing instruments – the Roche 454 Genome Sequencer FLX and the Life Tech Applied Biosystems SOLID platform. "The reason for that is they need to sequence individual clusters, or molecules, and this is one of the few ways to get those clusters in high enough numbers to do what they need," Vogelstein said.
QuantaLife's Lucero said that the BEAMing technology works well, but called it "ridiculously hard. You have to make an emulsion, break an emulsion, and then read all these beads in a flow cytometer."
Vogelstein said he has heard similar criticism from others, and that Inostics has made further refinements to the core technology, but that he still doesn't understand why the technique is viewed as being difficult to implement. "The [SOLID sequencer] uses this exact technology just the way we describe, and they have hundreds of machines out there. I don’t know why people have a hard time. It’s really almost quite as simple as mixing oil and water."
Regardless, the user friendliness of BEAMing may not be an important point, since Inostics knows how to use the technology and is offering services such as mutation detection, mutation discovery, and companion diagnostic development, rather than developing a commercial dPCR system for sale to end users.
Vogelstein also said that his lab routinely uses BEAMing technology to perform dPCR-based experiments in his lab, particularly diagnostic applications.
One point on which Vogelstein differs from some of his dPCR contemporaries is the idea that it will be useful for everyday gene expression studies. "It could be," he said, "but it might be a little bit of overkill for those applications. I think its real power lies in its ability to detect rare events. That’s where the signal-to-noise ratio is really a key feature."
As with any cutting-edge life science invention with the potential to be a platform technology, the dPCR intellectual property landscape is already shaping up to be complicated — and individual players have begun staking their claims.
RainDance Technologies' CEO Roopom Banerjee told PCR Insider that the dPCR IP landscape is shaping up as "pretty thorny," but added that he believes RainDance has "a great position in droplets."
"We take IP pretty seriously," Banerjee said, adding that the company has 68 patents issued or pending "to do any sort of biology in microdroplets;" and another 400 in-licensed patents around general microfluidic technology.
Meantime, QuantaLife's IP estate revolves primarily around IP owned by LLNL and based on technology developed by LLNL researcher Bill Colston and others.
According to Lucero, Colston and colleagues "came up with this idea of partitioning samples with droplets, and doing droplet chemistry, and married it to PCR. They wrote a patent, and that's the founding patent of [QuantaLife]. It pre-dates all other droplet chemistry type things."
Lucero said that QuantaLife is one of two companies co-exclusively licensing pertinent technology from LLNL, although he declined to name the other party, citing legal agreements.
Colston and others at the institution have previously collaborated on microdroplet PCR with RainDance Technologies scientists, including in a 2008 Analytical Chemistry paper.
When asked about RainDance's connection to LLNL, Banerjee told PCR Insider that he was not at liberty to discuss any potential link to the institution due to confidentiality concerns.
As of the publication of this article, LLNL's Industrial Partnerships Office describes on its website a technology available for licensing entitled "Microdroplet PCR and Sample Preparation Systems and Methods," and cites two Analytical Chemistry papers co-authored by Colston, though not the aforementioned RainDance collaborative effort.
It is unclear whether this technology is still available for licensing. An LLNL spokesperson confirmed that both QuantaLife and RainDance have each licensed one patent from the institution, but declined to comment on whether the deals were exclusive or not, citing confidentiality agreements.
As for Inostics, "the IP is relatively simple," Vogelstein said. The first paper using the term "digital PCR" in the scientific literature, he said, is a 1999 effort published in the Proceedings of the National Academy of Sciences by Vogelstein and Kenneth Kinzler, simply titled "Digital PCR."
Vogelstein said that Johns Hopkins has been awarded a patent "surrounding dPCR in general," and has several other applications based on the technology.
He added that Inostics has a license to the patent, as does Exact Sciences, which has a license to use the technology specifically in stool-based diagnostics and prenatal testing.
Meantime, QuantaLife's Lucero points to a 1992 Biotechniques paper authored by researchers from Flinders Medical Institute in Australia as the first definitive description of dPCR in the literature.
It is unclear whether any of the researchers associated with that publication or the Flinders Medical Institute filed for patents surrounding their discoveries, although no such patent could be found in a search of the USPTO database.
And finally, Life Technologies may be entering the digital PCR space with the broadest IP position of all players. When Life Tech acquired Cytonix last year, it said that it did so primarily for the company's IP surrounding microfluidics-based digital PCR technology.
In addition to Janaway's statement that the Cytonix acquisition provided "foundational IP" around PCR, in a separate interview John Gerace, vice president and general manager of PCR systems for Life Tech, told PCR Insider that the IP covers the partitioning of chemical or biological reactions into separate reaction vessels, either in wells or droplets.
Indeed, the earliest published patent in the USPTO assigned to Cytonix and describing such claims, No. 6,143,496, has an application filing date of April 1997 and was awarded in November 2000.
Further, Cytonix states on its website that it co-invented digital PCR methods and technology along with researchers at the National Institutes of Health in 1995. It received an exclusive license for this IP from NIH in 2006.
"But it doesn't do us justice just calling out the Cytonix IP," Gerace said. "We have legacy IP in our portfolio on different elements of how to practice digital PCR. So there is existing IP within our portfolio, as well as IP that we recently acquired through Cytonix, Biotrove, and the Stokes Bio acquisitions.
"That sort of ties our strategic play together," Gerace added. "In order to make a real presence in the digital space, to transform that space, we have assets that we're deploying against it."