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Digital PCR Startup Enumerix's Ultra-dPCR Method Massively Expands Droplet Number

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NEW YORK – Enumerix, a new digital PCR company from a team of genomics industry all-stars, has come out of stealth mode with a low-cost digital PCR method that can generate 30 million droplets per reaction, massively expanding the dynamic range over current technologies.

Called Ultra-dPCR, the firm expects to launch a commercial product based on the method by the end of next year initially focused on the research and noninvasive prenatal testing markets.

The firm is promoting its technology as next-generation sequencing-like, and, as a proof of concept of the broad dynamic range, it has developed an NIPT assay that detects a total of 222 loci in chromosomes 13, 18, and 21 in a single-tube workflow.

Enumerix was founded in 2019 by Stephen Fodor, Christina Fan, and Ari Chaney, a trio that formerly worked together at single-cell analysis firm Cellular Research.

The team has some impressive bona fides in the genomics space. Fodor previously cofounded Affymetrix in 1992, while Fan is known as a co-inventor of NIPT along with Stephen Quake. Chaney, Fodor, and Quake cofounded Cellular Research in 2011. That firm was acquired in 2015 by Becton Dickinson as part of a broader genomics strategy for single-cell research, while Affymetrix was acquired in 2016 for $1.3 billion by Thermo Fisher Scientific.

Aaron Solomon, Enumerix's general manager, said in an interview that the team "had great success at Cellular Research, and decided it was so much fun, they wanted to do it again."

The Enumerix digital PCR method does not require microfluidics, but rather uses inexpensive consumables paired with standard laboratory equipment.

The droplet generator is a plastic device with pores, Solomon said.

This piece fits into a standard 200-microliter tube strip. A sample is loaded along with Enumerix's proprietary emulsion substance, then the tube strip is loaded into an adapter that allows it to be put into any standard lab centrifuge.

"It gets spun for 20 minutes, and the centrifugal force creates a showerhead of droplets coming out that go into the PCR tubes," he said.

The firm's emulsion chemistry was designed to settle droplets to the bottom of the tube, allowing for more efficient heating and cooling in a standard thermal cycler. It is also clear, so it is straightforward to perform 3D imaging of the tube using the firm's proprietary Light Sheet scanner instrument.

The increased number of partitions can increase the dynamic range of detection compared to other methods. Solomon said the reactions have zero dead volume and can be loaded with four times as much sample as other technologies, both of which can increase sensitivity. This might be particularly important in detection of rare molecules, such as for liquid biopsy applications.

As described in Analytical Chemistry this week, the imaging instrument samples 500 frames of the emulsion droplets and compiles these to generate a 3D reconstruction of the PCR tube. Custom software then defines the partitions and counts the fluorescent droplets.

The Light Sheet is currently configured to detect four colors, and Solomon said the firm can easily use combinatorial methods to detect 12 targets in a single assay. "We can do some other tricks to get beyond that," he said.

The overall run time is two hours to process 48 samples, and the workflow also lends itself to automation.

Solomon said the total cost of the consumables per sample is less than $10. Enumerix plans to commercialize the consumables, the reader instrument, and the proprietary software to image the tubes, with the cost of the Light Sheet scanner being "comparable with other scanners that are on the market in the digital PCR space," Solomon said.

While other instruments operate over four logs of dynamic range, Solomon said the Ultra-dPCR extends that range by up to two logs. "It means we can provide additional sensitivity, and we don't have to titrate into the proper detection concentration," he said.

Janice Lai, the firm's director of engineering working on the Light Sheet instrument, was also with the team at Cellular Research, as was Eleen Shum, head of product development at Enumerix and first author on the Anal Chem study.

Shum said in an interview that some systems require users to estimate the molecules in a sample and dilute them before loading, "but in our assay, you just throw it in and it will capture all the molecules."

The 222-plex assay measures trisomies 13, 18, and 21, which are actionable and reimbursable, Solomon said.

The NIPT space is an early target market for Enumerix, but the system could also be useful in the research and translational market to measure copy number variations and transcript levels, and to count cell transfection, Solomon also said. In the clinical space, it lends itself to liquid biopsy methods and detection of minimal residual disease, as well as cell and gene therapy technologies that require counting transfected cells and looking for off-target transfection.

"We think of ourselves as partners with the CLIA labs that will be developing these tests," Solomon said.

Overall, the fast, inexpensive, straightforward workflow can also help enable broader access to genetic testing, and potentially greater equity in testing, he said.

Disruptive potential

The Ultra-dPCR system seems to fill a gap between NGS's detailed data with expensive and slow workflows and qPCR's speed and low cost but with fewer targets.

The digital PCR space has also been growing lately. Major players Bio-Rad Laboratories and Stilla Technologies are now competing with new dPCR technologies from Qiagen, Thermo Fisher Scientific, and Roche, and they are all vying for more color channels and higher multiplexing per droplet using fewer instruments.

Raindance — a firm acquired by Bio-Rad in 2017 — claimed 10 million droplets with its technology, and a few startups over the years, such as Espira and Lamprogen, have used centrifugal force in their droplet generation combined with microfluidic chips. But Enumerix would conceivably have an advantage in terms of the number of droplets generated, as most commercial systems generate tens of thousands of droplets per reaction. 

Solomon said having 30 million droplets can also eliminate "multiple occupancy" problems where more than one molecule gets amplified in a single droplet. This problem requires other technologies to deploy Poisson statistics in order to derive counts of positive droplets.

"We can do true molecular counting of millions of molecules," he said.

And, although the Light Sheet scanner only has four colors, Solomon said the low cost of the consumable could allow users to run an assay across multiple tubes. 

The digital PCR space has also been rife with lawsuits over the years, and, relatedly, single-cell patents granted to Fan and Fodor had been part of a BD lawsuit against 10x Genomics that has since been settled.

But Enumerix has developed and licensed a broad IP portfolio that includes droplet generation, the scanning technology, and some NIPT applications, Solomon said, and has worked closely with its counsel to determine that the firm is free to operate in the space.

Shum noted that the consumable is also "fundamentally different" from other technologies, which would avoid the IP issues that other firms have had over microfluidics and droplet generation methods.

Enumerix currently has some academic collaborations, but none yet publicly disclosed. An earlier version of its Anal Chem paper was originally posted to bioRxiv in August.

To date, the Palo Alto, California-based firm has been self-funded, but Enumerix plans to start raising money to fund commercialization, and to go fully commercial by the end of next year, Solomon said.

"We think we have a platform that is broadly applicable," he said, adding that the technology "strikes the right balance and addresses some of the challenges that have been holding back digital PCR up until now."

Enumerix currently expects to have instruments at sites by the summer, and has been eyeing a few conferences to exhibit at, as well as hopefully present data and posters.

And, although digital PCR startups seem to be acquired fairly quickly these days — last year Thermo Fisher acquired Combinati and Bio-Rad acquired Dropworks, for example — Solomon said that Enumerix doesn't see that as its endpoint.

"We're in it to win it," he said.

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