The Breadth of Biomarkers and Their Assays

Keywords::

• biomarkers
• cytometry
• diagnostic
• gene expression
• immunoassay
• LC–MS
• PD
• prognostic

We are pleased to be guest editors for two Special Focus Issues of Bioanalysis on ‘Bioanalysis of biomarkers’. Our intention going in was to highlight the variety of challenges that measurement of biomarkers poses and the breadth of technological solutions being applied. In reaching out to peers and the scientific community, we were delighted to see the extensive response from not only those working with technology typically seen in the pages of this journal (e.g., immunoassays and LC–MS) but also from those using an array of other technologies (e.g., gene expression profiling and cytometry). Indeed, the extent of the response, which included editorials, commentaries and research articles, was so great that we expanded the topic over two dedicated issues that together highlight the science, challenges and technological advances in biomarker analysis.

Biomarkers are commonly defined as characteristics that are objectively measured and evaluated as indicators of normal biologic processes, pathogenic processes or pharmacologic responses to a therapeutic intervention [1]. From a disease perspective, biomarkers are utilized for diagnostic and prognostic purposes, to differentiate stages of disease, predict therapeutic response and to monitor treatment compliance. During drug development, biomarkers are heavily leveraged to demonstrate that the drug reached its intended target and resulted in anticipated biological and PD effects; to build PK-PD models that inform dosing paradigms; to monitor safety; and to select and stratify patients. Additionally, a small but growing area of research utilizes biomarkers as indicators of potential drug induction or inhibition that correlate with potentially dangerous drug–drug interactions. To further expand the concept of biomarkers, one of our guest editors (L Stevenson) has written an intriguing editorial on how biomarkers can also inform immunogenicity by positing that neutralizing antibody assays are essentially biomarker assays for the presence of neutralizing antibodies [2].

In order to address the breadth of biomarker questions being posed, measurement of a wide array of analytes (e.g., proteins, lipids, nucleic acids) is required. When the simultaneous measurement of multiple analytes (e.g., gene expression profiling, multiplexed protein measurements) is required, complexity increases. Furthermore, an understanding of the underlying biology and longitudinal biological variability is necessary in order to determine what constitutes clinically meaningful change, which then dictates assay performance requirements. A great deal of discussion has already occurred regarding the challenges of reliably measuring biomarkers, with emphasis to date focusing on ligand-binding assay (LBA) and LC–MS technology platforms. Some widely recognized challenges in the measurement of biomarkers include: lack of ‘true’ reference standards and the questionable relevance of purified/recombinant reference material to the endogenous analyte being measured; identification of appropriate QCs; how to determine LLOQ; analyte heterogeneity; and matrix effects. In another editorial in this Special Focus Issue, R King addresses this last topic and calls for more collaboration across the scientific community to tackle this issue [3].

To meet the variety of challenges associated with the measurement of biomarkers, a diverse array of technology solutions have been explored and deployed. While the specific platforms utilized may differ from lab to lab, and more than one platform may be viable for a given biomarker application, the final selection of technology platform must support the intended use of biomarker data, as highlighted in the Commentary article by P Bennett [4]. Technologies currently used in the biopharmaceutical industry include mass spectrometry, LBAs, flow cytometry, gene expression profiling and imaging approaches. Innovations in all of these technologies have led to improved bioanalytical performance and/or enabled the measurement of novel or previously intractable analytes. To highlight this, the two consecutive Bioanalysis issues dedicated to biomarkers provide case studies that illustrate such innovations. DL Chappell et al. report a novel immunoaffinity (IA) LC–MS/MS assay to measure BNP_1–32, with high sensitivity and the ability to differentiate from total BNP [5]. This assay promises significant utility in clinical trials of drug candidates for heart failure. This report follows a recent themed issue in Bioanalysis dedicated to immunoaffinity LC-MS/MS that illustrated the growing application of this emerging technique [6]. This issue also features the development of an ultrasensitive LBA for IL-17AA and IL-17AF isoforms as presented by A Baruch and colleagues [7]. Their method is applied in rheumatoid arthritis and multiple sclerosis samples to measure the concentrations of both isoforms in the sub-pg/ml and single digit pg/ml range, highlighting the ability of such techniques to measure low abundance soluble protein biomarkers. In the LBA space, SK Fischer et al. address the importance of thoroughly evaluating the performance of commercial kits [8]. Finally, Z Qi et al. present their evaluation of three novel gene expression methodologies along with traditional qPCR [9]. They found that qNPA, a quantitative nuclease-protection assay, is a powerful and improved methodology for detecting gene expression in FFPE tissue with potential for cancer biomarker development.

The rapidly evolving and expanding use of biomarkers in drug development has generated discussions on ‘to what extent’ and ‘how to’ validate assays during drug discovery/development for the purpose of internal company decision-making as compared with that needed to support regulatory approvals. In cases where the challenges and technology solutions do not fit historical drug assay validations, these questions become even more difficult to answer. It is generally understood, though, that a common-sense, scientifically grounded assay qualification/validation approach designed to address the questions being asked is appropriate for internal company decision-making. In this context, K Cowan et al. describe their experience with protein biomarker qualification and validation using commercial LBAs [10]. However, as the differences between therapeutic drug and biomarker assays are now becoming better understood within the regulatory community, it is clear that existing bioanalytical method validation guidance will not work well for biomarkers in the regulated space. To date, only the US FDA has drafted bioanalytical method validation guidance that includes any recommendations related to biomarkers. This preliminary framework relies heavily on the perspective of drug assays, and is not one specifically created for biomarkers [11,12]. Herein, P Timmerman provides a commentary on the various aspects of this validation issue [13]. Before health authorities can write meaningful regulations, the scientific community must continue to share its experiences to create a community of understanding; one that the health authorities can learn from prior to establishing guidance. We are pleased to be bringing forth the articles in these two issues that exemplify the type of essential knowledge sharing that is required for significant progress to be made. The issue also includes the first of three White Papers to be published following the WRIB annual meeting held in April this year, titled ‘2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV) (Part 1 – small molecules, peptides and small molecule biomarkers by LCMS)’ [14].

Here, we have highlighted content found in this first issue dedicated to biomarkers and together look forward to additional content on biomarker assay innovation, which will appear in the second edition. We hope that readers will take this opportunity to learn about the variety of technologies being employed for biomarker assays beyond their own field of interest. We anticipate that this examination of how challenges and problems have been overcome will help engender new understanding and lead to the generation of new ideas and applications for measuring biomarkers in the future.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.