Biological Dynamics Looks to Commercialize cfDNA Sample Prep through CLIA Lab, Partners
January 02, 2014
"Biological Dynamics, a five-year-old startup in San Diego, Calif., recently received a US patent
for a device that uses a dielectrophoretic microelectrode microarray to
quickly purify circulating cell-free nucleic acids directly from
unprocessed fluid samples.
The microelectrode chip-based method, developed by CEO and co-founder Raj Krishnan, is a fast and inexpensive tool for extracting vital particles from liquid biopsies, and the company is currently interested in partnering with other developers who might be keen on using the approach for cell-free DNA assays and other applications."
"In addition, Biological Dynamics is developing two oncology prognostics based on the technology: an IgVH mutation test for chronic lymphocytic leukemia (CLL) and a Bcr-Abl test for chronic myelogenous leukemia. The company will perform both tests out of its recently certified CLIA labs, which they also expect to receive CAP accreditation soon. Cell-free nucleic acids are proving to be useful measures of disease burden and treatment efficacy. In an interview with PCR Insider, Krishnan pointed to a recent NEJM study on metastatic breast cancer led by scientists at Cancer Research UK Cambridge Institute. In that two-year study, researchers created assays for cancer cells in each of 30 patients, and found that freely circulating tumor DNA in the blood correlated with tumor burden over the course of treatment. Patients were switched between treatments until one proved successful, but retrospective analysis of patient blood samples showed that tumor DNA in the blood dropped within days of initiating a useful treatment, and could thus indicate therapeutic efficacy sooner than standard measures.
Building on such studies, Krishnan said his company plans to develop tests of cancer therapy efficacy. Although Biological Dyanmics is currently further along with its CLL and CML assays (both tests are anticipated to be available by April of this year), they expect additional hematological malignancy tests will come online shortly thereafter, with solid tumor tests following sometime afterwards.
The company's approach uses a microfluidics microarray chip which Krishnan began working on while in graduate school at the University of California, San Diego. As an engineering student, Krishnan's advisor Michael Heller, former CTO of now-defunct array maker Nanogen, gave him some microchips manufactured by the company to experiment with.
'Initially the idea was that you couldn't use AC electrokinetics in high-conductivity solution, anything with a lot of salt, like blood," he said. But, "late one night I was messing around with these chips and making modifications, and I tried it, and it actually wound up working.'
Other companies in the sample-prep space use a direct-current method for dielectrophoretic filtering of nucleic acids, explained Krishnan. Boreal Genomics, for example, is developing a technique called SCODA, which it is applying to non-invasive cancer mutation monitoring, as reported in Clinical Sequencing News. Gradient elution isotachophoresis — which is being used, for example, to purify DNA from soil-contaminated buccal swabs by scientists from the National Institute of Standards and Technology — also uses direct current, according to Krishnan.
Biological Dynamic's AC chip, however, uses AC current with electrodes creating a non-uniform electric field. It is essentially a microfluidics device, in which sample runs through the chip and over the microelectrode array. The charge of cellular debris attracts or repels it relative to the field of the electrodes. A quick wash removes intact cells from the chip, and a pass of elution buffer obtains the cell-free component.
Krishnan said the process takes ten minutes from start to finish, and the isolated debris can have many uses. Their device can capture circulating cell-free debris in specific size ranges, said Krishnan, who added "luckily for us, in biology, with very rare exceptions, anything in that range is usually not supposed to be in your blood. We're talking about aggregated proteins that would identify lupus, Alzheimer's, [or] Parkinson's, cell-free circulating nucleic acids like DNA and RNA which can identify cancer, other types of particles which may be able to identify heart attacks, microRNA that are inside of exosomes, messenger RNA, or other particles," he said.
Krishnan and his collaborators have published a number of papers on the device, including two in the past 20 months in Electrophoresis. Work led by collaborator Heller and researchers at Moores Cancer Center, and published recently in Clinical Chemistry, showed the AC dielectrophoretic device's utility in processing samples from patients with CLL.
According to this study, at the end of ten minutes of AC dielectrophoresis using Biological Dynamics' device, the cfDNA was in the elution buffer while whole cells, with their genomic DNA, were in the wash buffer. In comparison, it took two separate protocols, each with ten or more steps, and almost three hours to isolate those two components by standard techniques. Isolating PBMCs required density centrifugation using a Ficoll gradient, which the authors maintained was labor intensive and costly. Importantly, the dielectrophoretic technique used 25 microliters of patient blood, while the standard protocols required one milliliter of plasma and up to 20 milliliters of whole blood, respectively. The authors also claimed the many centrifugation steps required in these protocols could themselves shear cells and generate cell-free particles and debris that may not have actually been present in the starting material.
In addition to this collaboration on CLL, Krishnan said, Biological Dynamics scientists are working with other researchers at the Moores Cancer Center to obtain sample sets enabling them to correlate cfDNA with other diseases. They are pursuing the hypothesis that concentration of high molecular weight debris in and of itself may be correlated with therapeutic outcome in cancer treatment. Krishnan and his collaborators are also currently preparing a manuscript on a study using their device in relation to heart attack, as well as working on a grant to pursue its use in diagnosing traumatic brain injury.
As the utility of measuring cfDNA, miRNA, and other biomarkers from liquid biopsy becomes more apparent, there does appear to be a market for improved sample prep. A recent PLOS One paper showed a head-to-head comparison of four commercially available kits that can pull cell-free particles from samples. The two best performers — Qiagen's QIAamp DNA Blood Mini Kit and Circulating Nucleic Acid Kit — reliably filtered out cfDNA, but required several hours and a number of processing steps to do so. None of the kits in the study proved very good at filtering out miRNA, and varying the processing altered results. These kits also required a relatively large volume of starting sample. Dielectrophoretic devices, like Krishnan's, promise to equivalently separate out molecules of import in minutes, directly from unprocessed blood.
The company has had sufficient funding thus far. With David Charlot, CTO and co-founder of the company, Krishnan won $42,000 in an entrepreneurial challenge at UCSD. He said that "eventually all of that money went back to UCSD to license the technology and the patent and to start the initial company, but it was a huge bonus." After that, they began fundraising, and raised an undisclosed amount of money in Series A and Series B rounds. Then, they asked themselves, "Can we actually start a CLIA lab?," Krishnan said. "Our investors were awesome; they gave us enough money so we could start a CLIA lab, and [we will] actually start offering this test to people once we have the validation done." Biological Dynamics obtained CLIA certification for its lab in August of 2013.
However, the microfluidics microarray chip is a useful tool for all sorts of applications, Krishnan emphasized. While Biological Dynamics is currently focusing on lab-based prognostic tests for oncology, it is also seeking partners interested in using the device to measure circulating cell-free biomarkers, among other uses.
'We're always interested in collaborations of different natures,' he said. 'A patent only lasts 20 years to begin with, so you can't develop everything yourself. In fact we welcome and we want relationships with other key contributors or opinion leaders or companies that are interested in different areas. When we see a willing partner, we're more than happy to either give them chips or license them the technology, or whatever level of collaboration that they want to have with us.'"
http://www.genomeweb.com/pcrsample-prep/biological-dynamics-looks-commercialize-cfdna-sample-prep-through-clia-lab-partn
The microelectrode chip-based method, developed by CEO and co-founder Raj Krishnan, is a fast and inexpensive tool for extracting vital particles from liquid biopsies, and the company is currently interested in partnering with other developers who might be keen on using the approach for cell-free DNA assays and other applications."
"In addition, Biological Dynamics is developing two oncology prognostics based on the technology: an IgVH mutation test for chronic lymphocytic leukemia (CLL) and a Bcr-Abl test for chronic myelogenous leukemia. The company will perform both tests out of its recently certified CLIA labs, which they also expect to receive CAP accreditation soon. Cell-free nucleic acids are proving to be useful measures of disease burden and treatment efficacy. In an interview with PCR Insider, Krishnan pointed to a recent NEJM study on metastatic breast cancer led by scientists at Cancer Research UK Cambridge Institute. In that two-year study, researchers created assays for cancer cells in each of 30 patients, and found that freely circulating tumor DNA in the blood correlated with tumor burden over the course of treatment. Patients were switched between treatments until one proved successful, but retrospective analysis of patient blood samples showed that tumor DNA in the blood dropped within days of initiating a useful treatment, and could thus indicate therapeutic efficacy sooner than standard measures.
Building on such studies, Krishnan said his company plans to develop tests of cancer therapy efficacy. Although Biological Dyanmics is currently further along with its CLL and CML assays (both tests are anticipated to be available by April of this year), they expect additional hematological malignancy tests will come online shortly thereafter, with solid tumor tests following sometime afterwards.
The company's approach uses a microfluidics microarray chip which Krishnan began working on while in graduate school at the University of California, San Diego. As an engineering student, Krishnan's advisor Michael Heller, former CTO of now-defunct array maker Nanogen, gave him some microchips manufactured by the company to experiment with.
'Initially the idea was that you couldn't use AC electrokinetics in high-conductivity solution, anything with a lot of salt, like blood," he said. But, "late one night I was messing around with these chips and making modifications, and I tried it, and it actually wound up working.'
Other companies in the sample-prep space use a direct-current method for dielectrophoretic filtering of nucleic acids, explained Krishnan. Boreal Genomics, for example, is developing a technique called SCODA, which it is applying to non-invasive cancer mutation monitoring, as reported in Clinical Sequencing News. Gradient elution isotachophoresis — which is being used, for example, to purify DNA from soil-contaminated buccal swabs by scientists from the National Institute of Standards and Technology — also uses direct current, according to Krishnan.
Biological Dynamic's AC chip, however, uses AC current with electrodes creating a non-uniform electric field. It is essentially a microfluidics device, in which sample runs through the chip and over the microelectrode array. The charge of cellular debris attracts or repels it relative to the field of the electrodes. A quick wash removes intact cells from the chip, and a pass of elution buffer obtains the cell-free component.
Krishnan said the process takes ten minutes from start to finish, and the isolated debris can have many uses. Their device can capture circulating cell-free debris in specific size ranges, said Krishnan, who added "luckily for us, in biology, with very rare exceptions, anything in that range is usually not supposed to be in your blood. We're talking about aggregated proteins that would identify lupus, Alzheimer's, [or] Parkinson's, cell-free circulating nucleic acids like DNA and RNA which can identify cancer, other types of particles which may be able to identify heart attacks, microRNA that are inside of exosomes, messenger RNA, or other particles," he said.
Krishnan and his collaborators have published a number of papers on the device, including two in the past 20 months in Electrophoresis. Work led by collaborator Heller and researchers at Moores Cancer Center, and published recently in Clinical Chemistry, showed the AC dielectrophoretic device's utility in processing samples from patients with CLL.
According to this study, at the end of ten minutes of AC dielectrophoresis using Biological Dynamics' device, the cfDNA was in the elution buffer while whole cells, with their genomic DNA, were in the wash buffer. In comparison, it took two separate protocols, each with ten or more steps, and almost three hours to isolate those two components by standard techniques. Isolating PBMCs required density centrifugation using a Ficoll gradient, which the authors maintained was labor intensive and costly. Importantly, the dielectrophoretic technique used 25 microliters of patient blood, while the standard protocols required one milliliter of plasma and up to 20 milliliters of whole blood, respectively. The authors also claimed the many centrifugation steps required in these protocols could themselves shear cells and generate cell-free particles and debris that may not have actually been present in the starting material.
In addition to this collaboration on CLL, Krishnan said, Biological Dynamics scientists are working with other researchers at the Moores Cancer Center to obtain sample sets enabling them to correlate cfDNA with other diseases. They are pursuing the hypothesis that concentration of high molecular weight debris in and of itself may be correlated with therapeutic outcome in cancer treatment. Krishnan and his collaborators are also currently preparing a manuscript on a study using their device in relation to heart attack, as well as working on a grant to pursue its use in diagnosing traumatic brain injury.
As the utility of measuring cfDNA, miRNA, and other biomarkers from liquid biopsy becomes more apparent, there does appear to be a market for improved sample prep. A recent PLOS One paper showed a head-to-head comparison of four commercially available kits that can pull cell-free particles from samples. The two best performers — Qiagen's QIAamp DNA Blood Mini Kit and Circulating Nucleic Acid Kit — reliably filtered out cfDNA, but required several hours and a number of processing steps to do so. None of the kits in the study proved very good at filtering out miRNA, and varying the processing altered results. These kits also required a relatively large volume of starting sample. Dielectrophoretic devices, like Krishnan's, promise to equivalently separate out molecules of import in minutes, directly from unprocessed blood.
The company has had sufficient funding thus far. With David Charlot, CTO and co-founder of the company, Krishnan won $42,000 in an entrepreneurial challenge at UCSD. He said that "eventually all of that money went back to UCSD to license the technology and the patent and to start the initial company, but it was a huge bonus." After that, they began fundraising, and raised an undisclosed amount of money in Series A and Series B rounds. Then, they asked themselves, "Can we actually start a CLIA lab?," Krishnan said. "Our investors were awesome; they gave us enough money so we could start a CLIA lab, and [we will] actually start offering this test to people once we have the validation done." Biological Dynamics obtained CLIA certification for its lab in August of 2013.
However, the microfluidics microarray chip is a useful tool for all sorts of applications, Krishnan emphasized. While Biological Dynamics is currently focusing on lab-based prognostic tests for oncology, it is also seeking partners interested in using the device to measure circulating cell-free biomarkers, among other uses.
'We're always interested in collaborations of different natures,' he said. 'A patent only lasts 20 years to begin with, so you can't develop everything yourself. In fact we welcome and we want relationships with other key contributors or opinion leaders or companies that are interested in different areas. When we see a willing partner, we're more than happy to either give them chips or license them the technology, or whatever level of collaboration that they want to have with us.'"
http://www.genomeweb.com/pcrsample-prep/biological-dynamics-looks-commercialize-cfdna-sample-prep-through-clia-lab-partn
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