I had the great privilege to serve as a contributing author to the Critical Path Institute (C-Path)’s “Points to Consider Document” on Scientific and Regulatory Considerations for the Analytical Validation of Assays Used in the Qualification of Biomarkers in Biological Matrices released this past June, which contains a detailed description of necessary approaches that can be applied to most analytical situations that will be encountered in fluid-based biomarker qualification. As biomarkers are increasingly used as drug development tools (DDTs) for diagnosing diseases, identifying therapeutic disease targets, patient monitoring/stratification, and more, their weight as decision-drivers in the development process grows heavier. To ensure decisions based on biomarker data are sound, it is important to establish that the assay procedure and resulting measurements are suitable for the biomarker’s intended purpose. Therefore, proper analytical validation is needed to sufficiently understand the assay’s performance characteristics and to precisely specify its capability to establish the target biomarker as a qualified DDT.

Because there is no one clear regulatory standard for such evaluation, the Points to Consider document was designed to establish consensus on the expectations for the validation of assays used in the regulatory qualification of fluid biomarkers. It represents collective input from a diverse working group of experts, and we utilized the latest scientific peer-reviewed literature as well as public expert opinion in developing this resource. The document focuses on considerations for the analytical elements directly relevant to the biomarker of interest and its Context of Use (COU), or the way the biomarker will be used for regulatory decision-making in the development process, embracing a fit-for-purpose (FFP) approach to validation. Below I have outlined several of the key considerations discussed:

1. Considerations for Biomarker Assay Design
Defining the COU of the biomarker is the most important prerequisite for determining the assay rigor and technology to be used in the biomarker qualification process. Critical drug development decisions will be made based upon the qualified biomarker, and therefore the assay must be able to generate data robust, sensitive, specific, and selective enough to effectively evaluate the biomarker for its intended purpose.

Part of establishing COU is defining the intended use population, and here it is important to remember that “biology matters”. Biomarkers are endogenous molecules, and their levels can fluctuate with variability in physiology, disease biology, pathology, treatment administered, and individual factors like patient age, diet, and exercise levels. The biomarker’s reference interval (or reference range) can be influenced by these endogenous and environmental factors and understanding who the patient population is will help determine the performance characteristics for the assay. For example, the reference range may be different for a diseased population vs. the reference range for healthy subjects.

Patient population is but one of several practical considerations to be assessed when defining COU; other questions to be asked include: where and how are the samples being collected? What is the testing volume? Must we account for different and/or improved assay performance over the course of the program?

The answers to these questions provide an early foundation on which to design an assay format with the appropriate balance of rigor, as well as to select the most optimal detection technology. Platform selection will be primarily driven by the nature of the biomarker being measured (protein, lipid, etc.) and the sensitivity and selectivity requirements for the assay.

2. Establishing Analytical Performance Requirements
Analytical validation must confirm that the performance characteristics of the assay are suitable and reliable for its intended purpose. The Points to Consider document outlines seven key parameters to be considered in validating a biomarker assay’s performance: accuracy (relative), analytical measurement range (AMR), parallelism, precision, selectivity, specificity, and stability.

Each parameter should be assessed relative to the biomarker’s COU, to help ensure that the clinical requirements of the biomarker are translated into an assay that is fit for those purposes. For example, an exploratory biomarker may only need a minimally validated assay in order to generate enough information about the biomarker’s potential usefulness, but biomarker assays used to derive data for confirmatory and clinical study sample analysis must be qualified by thorough analytical validation.

3. Setting Assay Validation Acceptance Criteria
The variability of each biomarker’s physiological behavior brings with it challenges in determining acceptance criteria for biomarker assay validation. This is where FFP criteria comes heavily into focus. As stated by Lee et al. (2006), an assay is considered fit-for-purpose if it is capable of discriminating changes that are statistically significant from the intra- and inter-subject variation associated with the biomarker. If such discrimination is not possible with the assay, then the biomarker assay is not suitable for the intended purpose.

We pose an example in the paper that an assay with 40% TAE may be adequate for statistically detecting a desired treatment effect in a clinical trial for a certain acceptable sample size, but the same assay may not be suitable for a clinical trial involving a different study population with much greater physiological variability.

The FFP process is used to develop an assay that accomplishes what is necessary and relevant for the biomarker’s COU, directly relating to and supporting it. Properly implemented, FFP is an iterative process where data continually informs further development and refinement of the assay to ensure the best drug development and patient management decisions can be made and are in line with the ultimate study goals.

This shared consensus on bioanalytical assay validation in support of clinical biomarker qualification encourages further progression of drug development efforts in the biomarker field. If you have a large molecule biomarker project in need of bioanalytical support, contact our scientists to learn how we can expedite your therapeutic product to market.