Agilent Technologies' acquisition of Biocius Life Sciences, announced this week, has given the high-end tools vendor a high-throughput separations platform that could bolster its efforts to push its proteomics instruments into the clinic.
Biocius' RapidFire technology has traditionally been used in the form of ADME assays by pharmaceutical firms performing drug-screening work. However, said Gustavo Salem, vice president and general manager of Agilent's Biological Systems division, "one of the key opportunities" the company sees for the platform is applying it "to the clinical proteomics space."
In particular, he said, the system could be useful "as a tool for measuring intact protein modifications" and as part of mass spec-based proteomics workflows like SISCAPA, where it could allow researchers to significantly raise such assays' throughput.
Low throughput has been one of the main issues slowing mass spec-based proteomics' move from research use into the clinical lab. Protein biomarker verification and validation work requires running thousands of samples, but thus far mass-spec techniques have largely been too slow and cumbersome for this to be practical.
As Plasma Proteome Institute CEO Leigh Anderson noted in January during the Association for Mass Spectrometry's third annual Applications to the Clinical Lab meeting, "if you can't run a few thousand samples, you can't know if a biomarker is clinically relevant. This is a huge limitation that we need to overcome."
Anderson presented at the MSACL meeting work his lab had done in collaboration with Agilent to automate the SISCAPA process – of which he is an inventor. Using an Agilent Bravo liquid-handling system for sample prep attached to an Agilent 1200 series LC system and an Agilent 6490 triple quadrupole mass spec, his team built a system that could process one sample every ten minutes, a rate that he said he hoped to improve to five minutes per sample (PM 2/11/11).
The RapidFire platform, by contrast, can process samples for mass-spec analysis at a rate of one every seven seconds, offering "a potential increase in throughput that is extremely attractive," Anderson told ProteoMonitor.
While his group hasn't yet done "significant testing" of the RapidFire platform's utility in the SISCAPA workflow, he plans to "as soon as possible," he said, adding that he and Agilent "have agreed on how we're going to do [the testing of the device]. We just haven't executed it yet."
The main obstacle to using the technology for SISCAPA work is getting samples clean enough prior to running them through the RapidFire system, Anderson said. Because the platform doesn't offer the same level of separation as conventional HPLC, the samples used need to be relatively pure.
And because SISCAPA uses an immunocapture step to isolate target peptides prior to chromatographic separation, it's a better fit for the RapidFire system than other proteomic methods. However, Anderson noted, work still needs to be done to optimize the process.
"We're pretty convinced that the antibodies [used for the immunocapture] are only binding the peptides that we want, but when we run the process using magnetic beads, it's also clear that there are some sticky peptides that also bind to the magnetic beads," he said. "So that's some background that's associated with that sort of support."
"We're making rapid progress in cleaning that up, so we plan to get to the point where we're really only purifying the peptides that we want with nothing stuck to the support," he added.
Inserting the RapidFire system in the SISCAPA workflow is "an idea with some basic proof-of-concept done to date, but we've still got work to do to generate enough information to make it a truly commercial solution," Salem told ProteoMonitor.
"Obviously given our collaboration with [Anderson] in automating the SISCAPA process, you can now envision the opportunity to tie [immunocapture and the RapidFire] together prior to mass spec to create a very high-throughput workflow that will address the throughput needs you just can't do through traditional HPLC today," he said.
According to Salem, Agilent established its relationship with Anderson several years ago through work it was doing on applying its chip LC-MS systems to the protein quantitation portion of the SISCAPA method.
More recently, as the two parties began considering the method's commercial viability, the notion of automating it arose. The Biocius purchase provides the company with a platform that could be key to these automation plans.
The RapidFire system uses a series of proprietary switching valves and specially optimized chromatography cartridges that allow it to process samples at very high speed. The platform, Salem noted, isn't intended as a broad replacement for HPLC, but is useful "for applications where you don't need that [high] level of separation, which represents a lot of the work done with a mass spec."
Currently most popular as a drug-screening technology, the platform is used by 13 of the 14 top biopharmaceutical companies, Agilent said. Collaborators prior to this week's announcement, Agilent and Biocius in May 2010 jointly introduced the RapidFire 360 system for the high-throughput screening of in vitro ADME assays.
This experience as part of high-throughput pharma workflows could be useful in the mass spec-based proteomics arena, as well, Anderson suggested, noting that preparing enough samples to keep up with a machine injecting every seven seconds "is not a trivial thing."
"Based on the fact that Biocius has an existing business with this system injecting at that … throughput, the automation exists to feed a system like that," he said. As such, Anderson added, he doesn't anticipate a problem in terms of generating enough samples to do so.
In addition to the automated system Anderson presented at MSACL, researchers led by the Translational Genomics Research Institute's Matthew Rosenow have developed an automated system for SISCAPA sample prep capable of processing roughly 1,000 samples every 24 hours.
The system, which the TGen scientists began testing at the beginning of the year, uses a Tecan robotic workstation and a Thermo Scientific KingFisher Flex liquid-handling instrument to take plasma samples from cryovials and run them through the SISCAPA prep process, leaving them in a 96-well format that could then be introduced to the RapidFire system.
According to Anderson, if the platform can, in fact, be integrated into such an automated workflow it would "open up [proteomics] to much larger scale experimentation, which is the whole barrier currently inhibiting the biomarker field – particularly biomarker verification and validation."
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