Purpose: Robust and fit-for-purpose method validation is crucial for the implementation of biomarker assays to show proof of biology in clinical trials. Because of their ease of use, limited sample volume and low hands-on time, specificity, automated Western Blot platforms like the WES from ProteinSimple, have recently moved into the spotlight for the assessment of biomarkers in clinical development. The Wes allows to detect biomarkers in a variety of matrices, including serum/plasma, as well as cell lysates derived from whole blood, PBMCs, exosomes, tissue biopsies or cell lines.
So far, only few validation strategies have been proposed to qualify biomarkers for clinical implementation by using a Western Blot-like platform. Hence, we propose a practical and efficient fit-for-purpose strategy for biomarker assay development and validation, which focusses on the intended use of the data. For this, we discuss the example of the relative quantitative validation of biomarker X in cell lysate. This assay is validated in a similar manner as a classical ligand-binding assay and allows to assess accuracy, precision, dynamic range, as well as the stability of the analyte.
In addition, we also discuss a lighter, semi-quantitative validation using the example of soluble biomarker Y in serum. The semi-quantitative validation focusses on precision but does not enable to access accuracy of the method. The data derived from this assay is numeric and expressed in term of sample characteristics, e.g. percent change from baseline.
Methods: We evaluated the applicability of Simple Western technique towards measuring biomarker X in whole blood lysate as well as soluble biomarker Y in serum. For biomarker Y, we included an immunoprecipitation (IP) step in order to improve sensitivity and to reduce matrix interference, which could hamper the separation of the proteins in the Wes Capillaries. Both types of assays are comprised of a common feasibility assessment, which is shown in the figure below, as well diverging validation steps:
Results: Assay parameters, their respective acceptance criteria and the results during the assay validation are given in tables below.
Conclusion: A crucial part of successful biomarker qualification to show proof of biology in clinical trials is robust assay validation. Herein, we could show the development and validation of two different biomarker assays with different levels of validation according to the assays respective purposes. Both assays show high sensitivity, specificity and reproducibility. In addition, the relative quantitative assay for biomarker X shows high accuracy comparable to our internal immuno-assay validation guidelines.
Hence, our approach provides a clear path forward towards developing and validating an assay on Simple Western technology, which can be broadly implemented in clinical trial settings.
Sabine Lennarz– Basel, Basel-Stadt, Switzerland
Lili Yu– Cambridge, Massachusetts
Agnieszka Kieloch– Basel, Basel-Stadt, Switzerland
Marie-Anne Valentin– Senior Investigator II, Novartis Pharma AG, Basel, Basel-Stadt, Switzerland