Mass-spec-based protein biomarker assays may still be struggling to break through to the clinic, but in the meantime they've become valuable translational research tools for pharma companies working to move drugs through the development pipeline.
At the Association for Mass Spectrometry's Applications to the Clinical Lab meeting last week in San Diego, Pfizer associate research fellow Hendrik Neubert highlighted the potential usefulness of these assays and offered some examples of his group's mass spec biomarker work.
The drug industry, Neubert noted, "has been suffering badly from drug failures." One approach to combating this problem, he said, has been to increase focus on translational research in hopes of improving drug survival rates at both the "proof-of-mechanism stage and in Phase II clinical trials," which typically investigate a compound's efficacy and dosing requirements.
Key to such efforts is developing a solid understanding of the target pathways and molecules, as well as good pharmacokinetic and pharmacodynamic models, Neubert said, and his team, he noted, has found mass-spec-based protein biomarker approaches particularly useful for such research.
"Usually it starts early on in discovery where a project team tries to elucidate a pathway that would eventually be targeted by the drug," he said. "Once that pathway has been identified, then the question is, 'What molecule in that pathway do you want to target?' Once the drug lead compounds are developed, those are fed into a preclinical PK/PD model, and once a compound moves into a clinical space, the [clinician] has an interest in monitoring closely the dose response relationship, [and] the exposure response relationship.
"Biomarker analytical input is really required at these different levels," he said. "And there are all different kinds of tools, but we've seen in our [work] that quantitative immunoaffinity LC-MS/MS approaches really have an impact."
As an example, Neubert cited work his group did using immunoaffinity MS to measure serum levels of the protein nerve growth factor – the target of Pfizer's tanezumab, a biotherapeutic currently in Phase II and Phase III clinical trials for a variety of pain indications.
The researchers built an assay to measure total NGF levels in the presence of the drug, applying it to a 50-person Phase II patient-versus-placebo clinical trial examining the effect of a single dose of tanezumab over a three-month period, as well as a second study in normal healthy volunteers that generated more than 1,600 data points on subjects' NGF levels with particularly dense sampling done during the early days of treatment to better elucidate the kinetics of the drug's "on" phase.
"The impact that this data is having in a clinical setting is tremendous," Neubert said, "because it enables the team to really link the pharmacokinetics of the drug to the occupancy of the target to the clinical outcomes. So you really have that triangular relationship with this kind of data.
"Furthermore, it really contributes to the clinical decision-making because you have the team selecting doses for future clinical trials in this indication or other indications," he added. "This sort of target-assay data is really valuable."
Because the researchers selected as a surrogate peptide one that is broadly conserved across species, they've also been able to take the assay back into the preclinical space, Neubert noted, applying it to work with monkeys, rats, and other model organisms.
"It's taking the investment that we did in the later stages and [putting] it back into early discovery," he said.
The assay starts with patient serum, from which the researchers isolate NGF using a polyclonal antibody, and then capture and digest it using a bead-based assay run on an automated, 96-well plate liquid handling format. The sample is then run through high-capacity antipeptide columns linked to a nanoflow-LC system, and the captured peptide is quantitated as a stand-in for NGF via tandem MS.
The assay runtime is about 10 minutes, and the group has thus far analyzed "well in excess of 10,000" samples on the system, Neubert noted, saying that the group feels "like we have a very good, rugged analytical system in hand."
He also described a similar immunoaffinity MS assay the group devised for measuring pepsin and pepsinogen in human saliva as part of research into acid reflux. In this case, the researchers turned to mass spec after finding that the heat-inactivation step used to kill off any residual proteolytic activity in saliva samples was also denaturing the target proteins – limiting the effectiveness of an immunoassay that they'd developed.
"We tried to develop an immunoassay for pepsin, but it just wasn't robust," Neubert said. "But in mass spec you don't care if you're denaturing your proteins because you're digesting them anyway, so it's a real advantage."
More broadly, immunoaffinity MS is valuable because it offers significantly shorter method development times than immunoassays like ELISAs, he said. The fact that the technique typically uses just a single capture antibody allows for "accelerated assay development."
This echoes remarks made by Daniel Spellman, Merck senior research biochemist for proteomics, when describing his group's label-free, differential mass spec workflow for biomarker discovery last September at Cambridge Healthtech Institute's Accelerating Development & Advancing Personalized Therapy conference. He noted that because of the relative simplicity and quickness of the mass spec workflow, Merck researchers have been able to "match [biomarker development] timelines to pharmaceutical research and the development of drugs," allowing them to "actually impact decisions" (PM 10/08/2010).
Neubert suggested that he hopes to streamline his group's immunoaffinity MS workflow even more by possibly replacing the rabbit polyclonal antibodies they currently use with alternative capture agents like aptamers.
"When we start engaging in a project, the first thing I do it look at the [protein] sequence and get antipeptide antibodies going because it takes some lead time, and you may not be successful," he said. "Having a synthetic alternative [to antibodies] that you could screen against a library like aptamers… would speed up method development tremendously."
"We've actually played with [using aptamers] a little bit," he added. "We weren't successful, but I'd like to explore it more."
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