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Novilytic Using $1.5M SBIR Grant to Develop Immunoenrichment Tech for Clinical Mass Spec

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NEW YORK (GenomeWeb) – Sample prep firm Novilytic plans to use funds from a recently awarded National Institutes of Health grant to develop affinity reagents aimed at improving the performance and lowering the cost of clinical mass spec assays.

The company said last week that it won an NIH Phase II Small Business Innovation Research grant worth up to $1.5 million. Fred Regnier, formerly a professor of chemistry at Purdue University and Novilytic's co-founder and CEO, said that the firm will use the funds to refine a nanoparticle-based immunoenrichment approach to simplifying sample separations upfront of mass spec analysis.

The award is Novilytic's fourth SBIR grant, following a $99,537 grant in 2011, a $970,380 grant in 2014, and a $147,170 grant in 2016.

A primary goal of the work will be to integrate antibody-based enrichment into the company's Noviplex cards sample collection platform, essentially an enhanced version of dried blood spots. Registered this year with the US Food and Drug Administration as Class I Medical Devices, the cards consist of a series of membranes that take blood from either a fingerstick or venipuncture and remove red blood cells via size filtration, producing a precise amount of plasma and accounting for differences in sample hematocrit values. According to the company, the cards are able to collect 2.5 µL aliquots of plasma from 25 µL of whole blood in three minutes with variation in the volume collection of less than 5 percent.

The system is one of a number of microsampling approaches under development, as interest in such sample formats is on the rise among researchers and clinicians. Competitors in the space include Phenomenex spin-off Neoteryx, whose Mitra Microsample Device similarly collects blood microsamples rapidly and without volumetric hematocrit bias.

Like conventional dried blood spots, these microsample formats can be stored and shipped without refrigeration, meaning they can be sent through standard mail, significantly reducing shipping and storage costs. This, along with the fingerstick format, could enable more flexible patient sampling, allowing people to, for instance, take a sample in their own home and mail it directly to a lab without having to go to a doctor's office or clinic for a blood draw.

Key to such applications is the emergence of mass spec as a technology for clinical work. The small volume of microsamples make them poorly suited for running panels of immunoassays, but mass specs can, in theory, run reasonably large multiplexes of proteins or other analytes in microsamples.

Particularly for protein tests, however, mass spec throughput remains a limited factor for clinical work, Regnier said, noting that while the instrument itself is extremely fast, the upfront sample prep process remains a challenge.

"Mass spectrometry identifies and analyzes things in milliseconds, but, ironically, it takes hours to prepare a sample for analysis," he said. "Sample preparation is a huge logjam in proteomics, and you have to overcome that before mass spectrometry will get involved in clinical diagnostics."

Mass spectrometers are commonly used for clinical assays, but they make up only a small slice of a market still dominated by immunoassays. This is due in significant part to their lower throughput and, consequently, higher costs, suggested Tim Woenker, Novilytic's COO.

While an immunoassay system typically runs hundreds of thousands of assays a year, a mass spec can run around 10,000 to 20,000 assays per year, at most, Woenker said. "That's a huge productivity and cost advantage for immunoassays."

A large proportion of mass spec sample prep steps can be done beforehand in batch processes, increasing throughput, but even so, liquid chromatography separation prior to mass spec analysis remains a major limitation.

"LC separation for proteomics can take an hour," Woenker said. "That's a ridiculous throughput. That's just way too slow."

To tackle this problem, Novilytic is using antibody-based enrichment of target analytes prior to LC to reduce the sample complexity and thus the amount of separation time required.

It's a common technique in the field, with a number of clinical researchers and companies including SISCAPA Assay Technologies and Thermo Fisher Scientific using antibody-based enrichment upfront of LC to improve the throughput and performance of mass spec assays.

Novilytic's approach uses antibody enrichment followed by treatment of the sample with what the company has termed its Antigen Sequestering Transport Particles (ASTPs), which are nanoparticles that bind the protein-antibody complex. Once these particles have bound the target protein-antibody complexes, the samples can be run on size exclusion chromatography (SEC). As the name suggests, SEC separates particles based on their size, with larger particles moving more quickly through the column and eluting first. Because of the large size of the ASTP-antibody-protein complexes compared to other analytes in a typical blood sample, these molecules will be among the first to come off the column, eluting within around 60 seconds, Regnier said.

That would allow for chromatographic separation times of around a minute, as opposed to the 15 to 30 minutes or more currently required for many clinical proteomic assays.

Novilytic will not develop or offer the antibodies used in these analyses, but just the ASTPs for binding the antibody-protein complexes. Regnier said it plans to both integrate the ASTPs into its Noviplex cards and to develop them to be added to samples in solution for use with conventional venipuncture samples.

"Our approach is to allow people to use any antibody they want, and after they've gotten that immune complex, then we take over and get it cleaned up for them and get it into a mass spectrometer with minimal effort on their part and minimal understanding of immunology and analytical chemistry," he said.

While the approach is theoretically applicable to digested proteins, Regnier said the company is developing the ASTP approach for capturing intact proteins. It also is applicable to small molecule targets like Vitamin D, he said.

The company intends the ASTP approach to be used for analysis of moderately to highly abundant molecules, Woenker said, adding that this is where he and Regnier believe most clinically used markers will fall.

"We're not going for the lowest things in the grass here," he said. "We believe most of the clinically relevant biomarkers that get validated will be higher abundance, more in the middle-range nanogram-per-milliliter level, both for protein-based and small molecule biomarkers. The reason for that is that to get really down deep, you have to go to nano-LC, and that's just not going to work in the clinical lab. You have to have higher throughput and good sensitivity, but not heroic sensitivity, I guess I would put it, to get diagnostically useful information."

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