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Affinity-Based Platforms Challenging Mass Spec for Protein Biomarker Discovery

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NEW YORK – While mass spectrometry has traditionally dominated proteomics research, newer affinity-reagent based platforms are making inroads in the space with promises of high-throughput, highly multiplexed analyses.

This is particularly true for protein biomarker discovery research led by investigators outside of proteomics who may not share the field's historic commitment to mass spec approaches and are therefore more likely to opt to run their samples on platforms from companies like Somalogic or Olink, suggested Robert Moritz, director of proteomics at the Institute for Systems Biology.

"What I see is that a lot of MDs and researchers in control of large repositories are now making [grant] proposals that bypass traditional proteomics groups as they have systems now from either Somalogic or Olink that will produce data and present results to them in a rapid fashion and at a depth that makes it difficult for [mass spec] to compete on numbers," Moritz said.

A number of proteomics researchers have questioned the specificity of Somalogic's assays in particular, but several users of the platform said they have seen good reproducibility of results, and some have questioned the consistency of data quality in mass spec proteomics experiments.

Affinity reagent-based approaches have long played a substantial role in protein biomarker discovery work. Perhaps most prominently, Myriad RBM has offered multiplexed immunoassays for almost two decades and has long been a popular option for researchers doing protein biomarker discovery. The company's panels measure only several hundred proteins, however, making it a fairly targeted approach to biomarker research.

By contrast, Boulder, Colorado-based Somalogic currently measures 5,000 proteins on its SomaScan platform. Olink measures more than 1,000 proteins using its proximity extension assay technology (PEA) and plans to expand that to around 3,000 proteins by the end of the year. This combination of broad panels and high throughput make them compelling alternatives to mass spec-based approaches, which are typically lower throughput and which some researchers outside the field still view with a measure of skepticism.

These factors have led even some proteomics researchers with long histories in mass spec to predict that firms like Somalogic and Olink will capture a significant portion of the protein biomarker discovery market.

"In general, I think that Somalogic and especially Olink will largely replace [mass spec]-based methods for discovery in plasma, CSF, and other biofluids due to [their] speed and very small sample amounts required for analysis," said Steven Carr, senior director of proteomics at the Broad Institute.

He noted that while these platforms are somewhat expensive on a per-sample basis, "you don't need a [mass spec] system and people who are expert in how to run it."

Karsten Suhre is a professor of physiology and biophysics at Weill Cornell Medicine and director of the Bioinformatics Core at Weill Cornell's Qatar campus. He has made extensive use of the SomaScan platform as part of his work combining genomic, metabolomic, and proteomic data from large population studies to study genetic and environmental factors of various diseases.

He said that he also uses mass spectrometry and that the Qatar proteomics core has three recent generation Thermo Fisher Scientific Orbitrap instruments but noted that mass spec was not well suited to the large-scale studies his work focused on.

"In my line of research where hundreds and thousands of blood samples need to be analyzed, mass spec is presently not an option as measurement times are too long," he said. "Also, mass spec of blood samples is still hampered by the large dynamic range that need to be covered."

He compared the relationship between affinity-based approaches like SomaScan and mass spec to that of genotyping arrays versus whole-genome sequencing.

"The latter provides much more detail but is not tractable on a population scale," he said.

Paola Sebastiani, a professor of biostatistics at Boston University, cited similar points in explaining why she felt SomaScan was better suited than mass spec to her work looking at the genetics and proteomics of aging.

"The issue with mass spec, from what I understand, is that you can go in depth and increase your sensitivity if you put in hours and hours of instrument time," she said. "I think there is a scalability problem with the technology."

Mass spec's throughput and sensitivity limitations are well documented. And while the technology has made strides in recent years, it is still far from being able to analyze tens to hundreds of thousands of plasma samples at a depth of around 5,000 proteins as Somalogic has done with its SomaScan platform. For instance, top labs running the most advanced mass spec plasma proteomic workflows are currently able to quantify between 500 and 1,000 proteins using assays that take around 15 minutes per sample.

'About time for a new approach'

Leigh Anderson, CEO of SISCAPA Assay Technologies, has long been both an important figure in the development of mass spec-based proteomic technologies and a critic of the technology's throughput limitations.

"Having spent a long time in the biomarker work with mass spec and before that with 2-D gels, I'd say it's about time for a new approach to [protein biomarker] discovery," he said.

Some, however, raise questions about the specificity of Somalogic's reagents and whether they might under some circumstances be either binding to off-targets or failing to bind to their stated targets.

"Specificity is super important," said Jennifer Van Eyk, principal investigator for research and director of the Advanced Clinical Biosystems Institute in the Department of Biomedical Sciences at Los Angeles' Cedars-Sinai Medical Center. "You have to know what you're quantifying. You can be accurately measuring something but have the wrong identification, and I think that in discovery that is deadly both in the fact that you have potentially identified the wrong protein but also in that you have closed off research into that protein. What happens if that protein actually was super important but you misidentified it?"

Antibody-based platforms like those from Rules-Based Medicine, Meso Scale Discovery, and Olink use two antibodies to detect target proteins, which give them increased specificity. Somalogic's Somamer-based system uses only a single capture agent, however, which has led to concerns about the technology's specificity.

Somamers are a modified form of aptamers, nucleic acid-based reagents capable of binding proteins or other targets and which can then be readout via technologies like PCR, arrays, or next-generation sequencing. Somalogic has worked to combine two forms of specificity into the reagents by developing the molecules so that they would exhibit slow dissociation rates from their targets [on the order of an hour or more] in the case of true hits and fast dissociation [on the order of seconds] in the case of off-target binding.

This, combined with the molecules' affinity for their targets provides two-dimensions of specificity in a single molecule, which the company said means they can offer highly specific detection without requiring a secondary detection molecule as in a sandwich immunoassay.

However, a number of studies have found issues with Somamer specificity. In work done using SomaScan as part of the UK's Interval study, researchers found that 14 percent of the Somamers showed non-specific binding, either to a protein other than the target protein (7 percent) or to a protein isoform (7 percent).

In a study published in 2018 in Diagnostics, a team led by researchers at the University of Tennessee Health Science Center compared SomaScan measurements with ELISA measurements for three proteins linked to renal disease, FGF23, FGFR1, and FGFR4. They found that while the correlation between the assays was "good to near-perfect" for FGF23 and FGFR4, the assays for FGFR1 were poorly correlated.

Somalogic is working to characterize the target binding and likely off-targets of its reagents, but the company acknowledges potential challenges.

"We're pretty confident in what the platform is measuring, which is the availability of the specific epitope that [Somamer] binds to," Alan Williams, the company's chief development and operations officer, said last year. "The challenge is that the availability of that epitope can be impacted by all sorts of things. It can be impacted by the fact that there's a complex formation that either exposes or hides the epitope. There could be post-translational modifications that actually change the epitope such that the [Somamer] no longer binds."

Ultimately, the company believes the platform's advantage stems in large part from its throughput, which it expects will let researchers make measurements in large cohorts and observe reproducible signals that they can train against specific endpoints.

"In a number of cases we're using retrospectively collected samples where there are hard outcomes, and we are able to essentially identify signatures on the platform that are associated with those outcomes," Williams said.

Suhre said that he was aware of the specificity concerns but said he thought they were overstated.

"There are examples where probes bind non-specifically," he said. "But I see [SomaScan] as a discovery platform. Target specificity can be established for the few proteins that come as being of interest in a second step."

Sebastiani, who said she has no financial or other relationship with Somalogic, said that she has been impressed with the specificity and reproducibility of results she has seen from the SomaScan system.

She added that in the future SomaScan would be her first choice of platforms for protein biomarker discovery work, though she noted that in a world where time and money were not constraints, it would be ideal to include mass spec analyses to look at proteins not included in the SomaScan panel. While the platform currently measures around 5,000 proteins, Somalogic has said it aims to ultimately boost that number to around 20,000.

Uppsala, Sweden-based Olink offers the highest protein assay multiplex outside of Somalogic, with 1,161 assays currently available. The company plans to expand that to 3,000 assays by the end of the year, said Gustavo Salem, the company's chair. Olink's platform is based on its proximity extension assay (PEA) technology, which uses pairs of antibodies linked to DNA strands that, upon antibody binding, are brought into proximity and then extended by a DNA polymerase, creating a new sequence that can be used as a surrogate marker for the target protein.

Jochen Schwenk, director of plasma profiling at the Science for Life Laboratory at Sweden's Royal Institute of Technology, has used all three platforms and said that they each have their pros and cons.

"Both Somalogic and Olink have grown in popularity for plasma-based biomarkers studies," he said, noting that this has been driven by the speed with which data from these assays can be returned to researchers and the fact that results from the platforms have correlated with known genetic variants and GWAS results, providing confidence in their validity.

Mass spec, meanwhile, has proven very successful for studies of cell and tissue samples and is becoming increasingly suitable for work in plasma as methods like data-independent acquisition advance, Schwenk said.

Mass spec also allows researchers to better characterize protein post-translational modifications and protein-protein interactions and complexes. Such work is a fast growing part of proteomics as the importance of different protein forms and their behavior in concert with other proteins has gained prominence within the field.

For conventional biomarker discovery research, the Somalogic and Olink proposition of being able to analyze thousands of proteins in thousands of plasma or serum samples is a compelling one, though.

Sebastiani said she sees growing interest among her colleagues in such approaches.

NIH funding data bears that out in part. Olink has seen a steady rise in funding over the last five years. In 2015, a search of grants for the company name or its proximity-extension assay technology yielded 23 projects totaling $8.5 million in funding. In 2019, that was up to 43 projects and $18.9 million in funding.

On the other hand, the pattern for Somalogic has been flat to slightly down, with 21 projects totaling $17.2 million in funding in 2015 and 18 projects totaling $16 million in funding in 2019. The company also saw a dip in the years between, with projects and funding totaling 10 and $7.1 million, 14 and $7.5 million, and 18 and $10.4 million in 2016, 2017, and 2018, respectively.

This trend likely reflects what is perhaps the biggest obstacle to Somalogic carving out a larger share of the protein biomarker discovery market — the fact that the company does not offer fee-for-service assays.

Somalogic ended its fee-for-service business in 2017, moving to a collaboration-based model where it retains rights to any data generated on its platform, the goal being to focus on development of its own in-house diagnostics and health monitoring business.

This has made it difficult for some researchers to gain access to the SomaScan platform. For instance, Suhre noted that when Somalogic ended its fee-for-service business, his lab at Qatar was stuck with a system it couldn't use and eventually purchased an Olink platform to replace it.

He said that in principle the lab could now send samples to Somalogic under a collaboration agreement it has with the company but that this is not an option in many cases because the Qatar Biobank does not permit sending samples abroad.

"I can follow the business decision Somalogic made, but for academic research it was a drawback," he said. However, he added that it has had the benefit of encouraging competition, "as the Olink platform now has more users than it would have were Somalogic still available on a kit basis."

Sebastiani said that she has also found this to be a challenge.

"If your [research] problem is not of interest to them, you are out of luck," she said, though she noted that she believed the company had become more open to collaborations in the last year or so.

Matt Norkunas, Somalogic's chief financial officer, indicated a level of flexibility regarding how the company would offer access to SomaScan.

"In the near term, we don't have an intent to distribute boxes or allow others to run the technology themselves in a true fee-for-service or fee-for-product fashion," he said. "But, we know it's important for our technology to be used in the market, to allow others to play with the technology, to help us advance the quality of the technology, and also to make sure the world trusts that what we have is readable and that it works the way that we say it is going to work."

"We have discussions with every collaborator and say, 'OK, how much information are we going to exchange here and how much cash are we going to exchange. ' And it could be a mix of fee-for-service type samples with other collaborative kind of samples," he said. "So, we are flexible inside the parameters of some mix of collaboration and fee-for-service."

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