By Ben Butkus
Eppendorf Array Technologies, a subsidiary of lab equipment provider Eppendorf, has restructured its microarray business to focus on developing a real-time, multiplex PCR array for molecular diagnostics applications such as infectious disease testing and companion diagnostics, according to a company executive.
Eppendorf has already completed a pre-clinical validation study using a late-stage prototype of the platform to identify microbes that cause ventilator-associated pneumonia, and has initiated additional pre-clinical studies with hospitals in Germany and Belgium, the executive said.
In addition, the company is seeking an industrial partner with regulatory expertise to help commercialize the platform by sometime in 2012, Wilhelm Plüster, managing director of Eppendorf Array Technologies, told PCR Insider this week.
EAT, a provider of microarray technology such as the DualChip product line since 1999, made the decision recently to stop selling microarrays by the end of this year to focus on RAP's development. "We restructured and see much more potential for this technology than ordinary low-density arrays," Plüster said. "With this, we position ourselves more in the PCR field than in the microarray field."
The platform, called real-time array PCR, or RAP, combines the multiplexing capabilities and detection specificity of microarrays with the speed, sensitivity, dynamic range, and potential for quantitative results characteristic of real-time qPCR, according to Eppendorf.
According to Plüster, the RAP platform was "100 percent developed in house" by combining EAT's existing microarray expertise in Namur, Belgium, with real-time PCR capabilities housed in Eppendorf's Hamburg, Germany, headquarters.
In addition, the company was able to tap into existing expertise in the area of laboratory plastics to develop a "simple, plastic consumable cartridge" on which the PCR reactions are arrayed.
The end result is a platform comprising the plastic consumable, a combined thermal cycler/microarray detection instrument, and data-acquisition and -analysis software. The platform also uses a proprietary genetically engineered DNA polymerase optimized for the high salt buffer conditions that are needed to hybridize amplicons on the microarray surface during annealing.
"The microarray in this case is only used as a detection modality," Plüster said. "It is multiplexed PCR, but instead of … being limited by the number of fluorophores, we can detect a lot of amplicons and different mutations within amplicons on the microarray, in real time."
The proprietary detection technology makes it possible to discriminate surface-bound fluorescence from unbound label in solution, which "makes buffer changes, washing, or pipetting steps obsolete," Eppendorf said.
"It's using fluorescently labeled primers … but you get the spatial discrimination on the microarrays," Plüster said. "Instead of real-time PCR, where you need to have that discrimination in the [fluorophore] wavelengths, here we can work with a one-color system, and are almost unlimited with the number of probes we can put on the microarrays."
The first-generation platform does not yet have automated sample-prep capabilities, so users must manually prepare master mix and DNA samples and add them to the RAP cartridge. Following that, "all of the other steps act directly like real-time PCR," Plüster said. "Instead of having a tube, you have our cartridge; and you apply your master mix, place it in our instrument, and press start. All of the liquid handling and movement and microarray analysis is fully automated."
In its pre-clinical validation study using a multiplex panel for ventilator-associated pneumonia, Eppendorf was able to simultaneously detect anywhere from 30 to 50 DNA targets in one assay. However, the company said that a single, multiplexed assay currently has the potential to detect as many as 100 targets.
In addition, the company has pre-clinical studies underway with the Robert-Bosch Hospital in Stuttgart, Germany; as well as with partners of the BioWin Healthcare Cluster of Wallonia in Belgium.
Data from these studies will likely not be available until early next year, Plüster said, but early results indicate that RAP can identify 95 percent of expected targets in pure bacterial isolates, spiked-in bronchial lavage, and infected clinical samples in less than six hours, as opposed to the typical 48- to 72-hour window typical of classic microbiology methods.
"For diseases like pneumonia and sepsis, that's too late," Plüster said. "You need to have an answer within the first six hours so you know what antibiotic you need to apply."
As such, Eppendorf sees the platform as being highly marketable for multiplex PCR testing of gene panels, such as a plethora of microbial and antibiotic-resistance genes or a collection of biomarkers to predict a patient's response to cancer treatments. "The biggest market where we see the best fit is in molecular diagnostics, because we will provide ease of use and a [fast] time to result," Plüster said.
Eppendorf joins a host of other companies developing highly multiplexed PCR microarrays, although not all of its potential competitors are immediately tackling molecular diagnostics. Examples include Wafergen, Akonni Biosystems, and Aquila Diagnostic Systems.
Many large life science tool vendors, such as Life Technologies, Fluidigm, and Qiagen already offer multiplexed PCR systems, but these generally use multiwell plates or microfluidic compartments as opposed to microarrays.
Plüster said that Eppendorf has applied for 14 patents on its technology so far, and has been granted eight of them, including one this week, US Patent No. 7,829,313 (see IP Watch, this issue).
And although RAP has potential as a research platform, Eppendorf is narrowing its focus for the time being on bringing it to market as an in vitro diagnostic device, primarily because the market for PCR-based molecular diagnostics is "progressing quite rapidly," Plüster said.
Since Eppendorf has little to no clinical development expertise, it is seeking a partner with clinical experience to help bring RAP to market. "Eppendorf's main focus is on research applications," Plüster said. "To bring this to a certified IVD product, it takes time. This is why we are looking for a partner for commercialization."
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