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Mayo Clinic Developing MALDI-MS Assay for Monitoring Plasma Cell Disorders


NEW YORK (GenomeWeb) – The Mayo Clinic is developing a MALDI mass spec assay for monitoring levels of monoclonal proteins (M-proteins) in patients with plasma cell disorders like multiple myeloma.

The test, called Mass-Fix, is designed to replace the gel electrophoresis and immunofixation electrophoresis assays that are currently used for measuring patients' M-protein levels and will provide higher sensitivity and throughput as well as lower costs, David Murray, a clinical pathologist at Mayo's Rochester, Minnesota campus and one of the developers of the assay, told GenomeWeb. He said Mayo aims to have the assay in routine use around the middle of next year.

M-proteins are established markers for plasma cell disorders and a key analyte for diagnosis and management of these diseases. Murray noted that Mayo has used gel electrophoresis-based assays for measuring the proteins since 1964, with relatively little changes in the technology over time.

The assay is fairly time consuming and laborious, he said. "It takes a couple of hours to run one. You have to stain it. You have to scan it. Gels have never really been fully automated. So it seemed like [the assay] was ripe for a technology change."

Mass spec, given its growing role in protein biomarker work and protein quantitation, was an obvious choice. Murray and his colleagues first tried to measure M-protein levels using standard shotgun proteomic techniques, but found that these were not sensitive enough to reliably detect the protein.

They then tried looking at the intact protein, but, he noted, glycosylation on its heavy chain caused problems for their analysis.

"Then we came up with the idea of just using the light chain," he said. They developed an assay using an electrospray QTOF instrument that, Murray said, "worked really well." But while the assay was faster than the existing gel electrophoresis methods, it was still slower than they hoped for, largely due to the 15 minute liquid chromatography gradient required upfront of the mass spec analysis.

"So then we decided to look at whether we could make it work on a MALDI TOF," Murray said. "And we did some initial work that showed that we could."

The MALDI assay didn't provide the resolution and sensitivity of the QTOF assay, but because it didn't require chromatography, it was significantly faster, with cycle times of around one minute per sample. It was also more sensitive than the gel electrophoresis-based methods.

Another benefit of the MALDI approach, as opposed to gel-based methods, is the ability to identify interferences from monoclonal therapeutics, which, Murray noted, are increasingly used to treat patients with plasma cell disorders.

"New interferences from monoclonal therapeutics are coming up all over the place in our field," he said. "If someone is on a monoclonal therapeutic, with gel electrophoresis, you are never really sure, but if you have the exact mass [from mass spec measurements], you can say, 'Well, this could be rituximab, this could be daratumumab."

The assay uses nanobodies, the antibody fragments developed by biotech firm Ablynx, to enrich M-proteins prior to analysis. This, Murray said, is key to the assay because enriching with conventional antibodies would lead to inferences that would complicate the analysis.

"Nanobodies have a different mass than normal immunoglobulins from sheep or other species," he said. "And we found that if you are going to do this reduction step [to separate the M-protein light and heavy chains], if you have sheep [antibodies] in there, it will also reduce [those proteins] and cause interferences in the assay, whereas a single-domain nanobody won't do that."

The Mayo researchers described the assay in a paper published last month in Clinical Chemistry. Murray said that it is ready for clinical use but that he and his colleagues are continuing to develop it for use in a high volume laboratory like theirs.

"We are building our own software to display the spectra the way we want them displayed and then generate databases to put the patient data in," he said. "Right now, everything is done by paper files. This is our chance to get everything electronic."

The researchers are using a Bruker Microflex LT instrument for the assay. This is the same instrument used in Bruker's MALDI Biotyper clinical microbiology system, and Murray said he and his colleagues did some initial development of their assay on the MALDI Biotyper system housed in Mayo's clinical microbiology department.

They have since purchased their own Microflex LT, but Murray suggested that other facilities interested in running the M-protein assay could use MALDI Biotyper systems previously purchased by their clinical microbiology colleagues.

"Those who are not high volume like us, they could use the same instrument to do this [assay] and their biotyping, as long as there is no interference between the two, which I can't imagine why there would be," he said. "I think that is something that could easily be done."

In past interviews with GenomeWeb, some researchers have suggested that the widespread adoption of MALDI systems like the Biotyper and BioMérieux's Vitek MS instrument for clinical microbiology could help MALDI mass spec spread into more conventional clinical protein quantitation.

For instance, SISCAPA Assay Technologies CEO Leigh Anderson suggested that such instruments represented "a very interesting case of mass spectrometry getting into clinical use almost under the radar ... and an interesting potential avenue to introduce mass spec protein assays."

One advantage, Murray said, is that the Biotyper and Vitek MS systems have received 501(k) clearance from the US Food and Drug Administration. While Mayo plans to offer the MASS-FIX test as a laboratory-developed test initially, it hopes to ultimately take it through FDA approval, and, he said, "the fact that Bruker and BioMérieux went through FDA clearance is really going to help us get through the FDA at some point."

Murray said he and his colleagues are also continuing to develop a QTOF version of the test using a Sciex 5600 TripleTOF. He said that test would be used for patients whose treatment had  driven their M-protein levels below the levels detectable by MALDI. Currently, he said, this is a small population of patients, but it is growing as the field advances.

Given the smaller patient population involved, throughput would not be as big of an issue for such an assay, he added. "Once you get to that point, you are not talking about hundreds of samples, so the turnaround time is not as important."

In fact, Murray said, the bulk of samples comes not from patients with a plasma cell disorder, but with a monoclonal gammopathy of unknown significance, or MGUS. These are patients where M-proteins have been detected but who have not progressed to a plasma cell disorder. He said that around 3 percent of the population over 50 falls into this category, and that these patients must have their M-protein levels checked annually.

Mayo has filed patents on the MASS-FIX assay and plans to market the test to non-Mayo labs in addition to running it in house. Murray said he anticipates that reimbursement will be a struggle initially, but that widening use of the test will help will this effort.

"It's complicated right now," he said. "We have gone after CPT codes, but it is hard to get one when you are the first one in. There are generic MALDI codes out there, but they don't reimburse as well as gel electrophoresis, so that is sort of a problem. It is something we are trying to work on, but we need multiple people to start adopting the technology before we can get a CPT code."

He noted that the cost savings provided by the assay, compared to the gel-based methods, gives Mayo some room to work out these issues, though he did not say how much he expected the test to save. He also said that, as one of the world's leading centers for myeloma research and treatment, Mayo is better suited than most to drive adoption.

That said, while Mayo's clinicians are largely onboard with the new test, doctors are not immediately comfortable with mass spec assays, Murray said.

"I always say that if I had to do it over again, I would never show a hematologist multiply charged spectra," he joked. "There is a lot of education that has to happen, and people have to get used to the technology."

In fact, Murray said, the name of the test, Mass-Fix, was deliberately chosen to recall the conventional immunofixation technique used for M-protein measurements.

"I didn't like the name Mass-Fix at all, but our physicians told me that if you don't put the work 'fix' in there, no one is going to know what you are talking about," he said.