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Despite MRM-MS's Promise as a Clinical Platform, Technical Hurdles and High Costs Remain Problematic

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By Adam Bonislawski

With a growing number of multi-analyte diagnostics in development, multiple-reaction monitoring mass spectrometry has emerged as a potentially valuable technology for large clinical reference laboratories like Quest Diagnostics and Laboratory Corporation of America.

Immunoassays are currently the platform of choice for many clinical tests, but, as Nigel Clarke, director of mass spectrometry at Quest, told attendees at last year's American Association for Clinical Chemistry's annual conference, mass spectrometry is an attractive technique for its sensitivity, specificity and, perhaps most important, its multiplexing capabilities.

"With direct MRM you can potentially measure hundreds of proteins all at once in a single [mass-spectrometry] run," George Mason University professor Emmanuel Petricoin told ProteoMonitor. "That has tremendous advantages in terms of direct comparison of analytes as well as cost benefits. It's intoxicating to think about."

In terms of running multi-biomarker diagnostics, the case for MRM-MS is "overwhelming," said Leigh Anderson, CEO of the Plasma Proteome Institute. "If you're going to test for 10 proteins, there's typically a pretty small incremental cost for each of those additional analytes in an MRM assay. That's potentially a major issue going forward as we start measuring panels of proteins."

For all the technology's promise, however, a number of hurdles must be surmounted for it to reach its potential as a clinical platform. Among the primary problems is variability – in particular the variability associated with the sample-prep steps that typically precede triple-quadrupole analysis.

"Triple quadrupoles can have fantastic [coefficients of variation]," Petricoin said, "but then what's the CV of the trypsin digestion, what's the CV of the immunocapture [in the case of SISCAPA-based assays], what's the CV of the entire process? The 600-pound gorilla in the room is that the CVs of that process are actually very high."

In a paper published in this month's edition of Clinical Chemistry, researchers at the University of Washington's Department of Laboratory Medicine described an MRM-MS assay for the simultaneous quantification of the biomarkers apolipoprotein A-I and apolipoprotein B in human plasma. Using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample, they were able to achieve intraassay CVs of around 6 percent and interassay CVs of around 12 percent.

"Is that good enough to offer as an assay?" said Andy Hoofnagle, the department's director of clinical mass spectrometry and an author on the paper. "We have some [non-MRM] assays that have CVs of 7 [percent] to 15 percent over many days, so it's not way out there."

Typically, though, Petricoin said, "The LabCorps and Quests of the world are used to assays that have [CVs] in the 1-, 2-, 3-, 4-percent range. Having assays that are 20-percent variable isn't something that they're happy about. So we have to strive to get that down."

Reducing variability, however, may require increasing costs. One way to lower the CV of an MRM-MS assay is to use enough trypsin to completely digest the sample. Using that much trypsin, though, can drive per-assay costs beyond what a reference lab would like to pay.

"To drive down the CVs you have to add so much trypsin that the cost of [the assay] is $1,000 a sample, and then you can't use it in a clinic," Petricoin said. "It's like whack-a-mole."

According to Anderson, one possible way around this problem is using lower grades of trypsin.

"In proteomics we've all just submitted to marketing that says, 'Oh, we should use Promega Gold.' It's the gold standard; it's the purest; and it does cut up proteins the way you want it to," he said. "But it also turns out that there are grades of trypsin that are 1,000 times cheaper, and it appears that these vastly cheaper grades work pretty much the same for bulk applications like digesting a plasma sample."

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While acknowledging that "a significant amount" of testing and verification would be necessary to prove this, Anderson said he's "convinced it will turn out that we really can use vastly cheaper grades of trypsin, and in the relatively near term this is not going to be the dominant cost of these assays."

Hoofnagle agreed, noting that for the Clinical Chemistry study his lab used a lower-grade Sigma trypsin, "and it worked just fine."

More of an issue than trypsin prices, he suggested, are the labor costs involved and the length of time required for running the assays.

"There's a lot of mass-spec cost because we're doing nanoflow [LC], so you don't get many samples run in a day," Hoofnagle said. While his lab has multiplexed its HPLC system to allow it to run two columns at once, "we can't do that on nanoflow right now," he said. "So we're really underutilizing the mass spectrometer."

Run time "is one component of the cost structure that's turned out to be quite important," Anderson said. "You spend half a million dollars on a [mass spectrometer], and if you can run a sample every five minutes you can pay for the hardware, but if it take half an hour per sample, you just can't run enough to pay for it."

A typical immunoassay from the University of Washington costs between $10 and $15, with less frequently performed immunoassays running in the $40 to $50 range. Prices at larger laboratories like Quest and LabCorp could be somewhat cheaper due to those companies' larger volumes, Hoofnagle said.

By way of comparison, SISCAPA MRM assays — which involve an immunoenrichment step necessary for the mass spec-based quantification of lower-abundance proteins — run in the $70 to $90 range, a price that Hoofnagle said hasn't budged in the last several years.

One key to driving down costs is automation, he said, noting that "the assay isn't going to cost less than $70 unless we have an automated platform doing everything for us. Until we have true hands-free automation, it's going to be pretty expensive in terms of labor."

Even this, though, might not lower prices significantly.

"Ideally we could get [the assays] on liquid-handling stations, but those aren't free and they come with service contracts," Hoofnagle said. "I'd have to figure out how many assays we could run on the platform per year and how much the service contract is, and I don't know that it would end up being cheaper. Hopefully it would be, but I'm not sure."

Currently, clinical labs use MRM-MS primarily in tests for small molecules like vitamin D. Hoofnagle's lab is also working on an MRM assay for detecting the protein thyroglobulin — a marker for various thyroid cancers — that he said he hopes to have clinically available within the year.

MRM-MS's real potential, though, is as a platform for multi-analyte assays. While bringing down the price for a single MRM-MS assay is a challenge, lowering per-analyte prices for multiplex assays should be relatively simple.

"As you multiplex the number of proteins that you're measuring in your assay, the price per analyte drops pretty quickly," Hoofnagle said. "While people have tried to do that for immunoassays, it's pretty difficult, whereas multiplexing MRMs is pretty easy and even multiplexing SISCAPA [assays] should be pretty straightforward."

"Obviously we'd like [MRM-MS] to be competitive just measuring one analyte, because that's the most common case right now," Anderson said. "But if you're measuring more than one, it's definitely going to be cheaper than an immunoassay."

Given the regulatory questions surrounding multivariate tests, it's uncertain how long it will take for multi-protein diagnostics to become common clinical products (PGx Reporter 06/23/2010). It's clear, though, said Petricoin, that this is the direction research is moving.

"We're going to have to figure out from a regulatory side how these things will be treated, but the writing is on the wall," he said. "The era of single-marker tests is gone, and we're now into multiple-marker tests. [Multi-marker tests] are going to happen one way or another. The science is just pointing to better positive predictive value, increased accuracy for these tests."

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