NEW YORK (GenomeWeb) – Evotec said this week that it has achieved a milestone in its protein biomarker work with Roche, as the drugmaker selected for phase I clinical trial use a response prediction marker developed through the two firms' collaboration.
Significantly, Roche plans to use mass spectrometry for measuring that marker and related maker signatures in the clinical setting, Dirk Ullman, Evotec's executive VP, lead discovery, told ProteoMonitor.
Evotec's Proteome Profiling platform, which the company acquired through its 2011 purchase of proteomics firm Kinaxo, is mass spectrometry based. However, Ullman noted, it was uncertain whether Roche would choose to continue using mass spec for clinical measurement of markers identified through the collaboration, or if it would instead transfer them to an immunoassay platform, as is common practice in protein biomarker research.
While mass spec offers potential advantages in terms of selectivity and assay development, the technology has historically presented challenges in terms of sensitivity, reproducibility, and throughput. Given these challenges, immunoassays have been, and largely remain, the preferred technology for protein quantitation in clinical settings.
Mass spec has made gains, in recent years, however, with researchers continuing to push forward the technology's capabilities and firms including Quest Diagnostics, Mayo Medical Laboratories, and Integrated Diagnostics offering mass spec-based protein tests. Roche's decision to move the Evotec markers forward on mass spec is another indication of the technology's growing clinical momentum.
According to Ullman, the drugmaker settled on multiple-reaction monitoring mass spec after comparing "over a dozen assay methods" for measuring the markers.
"The decision was in the end solely made by Roche, because they are the owner of the results and the owner of the clinical projects," he said, noting that the drugmaker "was quite strong in MRM technology."
Development of the MRM assays has been done jointly by the two companies, Ullman said. "Of course, in the ultimate clinical setting these type of measurements need to be done under certified conditions, and so it [remains] to be decided where these types of final sample analysis will be done. But from the discovery setting and the validation setting towards clinical approval, we have done this jointly."
Interestingly, Evotec has developed MRM assays for the markers not using a conventional triple quadrupole, but, rather, a high resolution instrument – Thermo Fisher Scientific's Q Exactive. While triple quads have traditionally been the preferred instrument for MRM assays, a number of researchers have in recent years begun trying targeted protein quantitation on high-res machines, the Q Exactive in particular.
Because high-res machines are able to collect data on a wide range of ions, they have the potential for easier assay development and better specificity. In triple quad-based MRM assays, the first quadrupole isolates a target precursor ion, which is then fragmented in the second quadrupole, after which a set of preselected product ions are detected in the third quadrupole.
By contrast, high-resolution approaches typically use the upfront quadrupole of a Q-TOF or Q Exactive machine to isolate a target precursor ion, but then monitor not just a few but all of the resulting product ions. Because of this, researchers don't have to determine upfront what the best transitions to monitor will be, significantly reducing assay development time.
The larger number of product ions monitored using high-res machines should also improve the specificity of the analysis, since more transitions will be available to confirm a peptide ID. This might also reduce the effects of co-isolating background peptides.
Triple quads, on the other hand, generally offer higher sensitivity for targeted quantitation than hi-res machines. Ullman noted, however, that few studies had, in fact, done head-to-head comparisons of the sensitivity offered by the two approaches.
"We believe that the sensitivity we can achieve on the Q Exactive is very nice," he said, noting that the instrument was particularly good for measuring the multi-protein signatures the companies were working on.
"It's the high resolution [of the Q Exactive], not the sensitivity – which is fair enough – for [measurement of] the protein signatures where we think the Q Exactive is the best at the moment," he said. Evotec also uses the Q Exactive for its discovery work.
Evotec's Roche collaboration began in 2011, with the company originally planning to identify markers using the PhosphoScout phosphoproteomics platform that it acquired in its purchase of Kinaxo.
It ultimately, however, moved away from looking specifically at protein phosphorylation and toward protein expression more generally, Ullman said.
Protein phosphorylation is a key area of research within cancer proteomics given the role of phosphorylation in cell signaling and the prominence of kinase inhibitors within drug development pipelines. Phosphoproteomics has been limited by technical challenges, however, particularly in clinical settings. Most notably, the lability of the modification makes it susceptible to alteration if samples are not preserved immediately after biopsy – a requirement that can be difficult to meet within conventional surgical workflows.
Such technical challenges were partially behind the decision to move away from looking for phosphoproteomic markers in the Roche collaboration, Ullman said, but, he added, the most important factor was that the phosphoproteomics signatures they generated didn't prove sufficiently predictive.
"We have done studies and successful biomarker identification studies in oncology that are based on phosphoproteomics, and there is no reason why this shouldn't be possible," he said. "It is very dependent on the complexity of the signatures, the peptides you can generate, and the sensitivity you can achieve within the proteomic digest. But it is possible."
Evotec's Roche collaboration was initially a three-year deal slated to end this year, but Ullman said the two companies are currently "working on an extended collaboration plan," that would tackle, among other projects, further validation of mass spec assays to the response prediction markers the firms have identified.