Case Study: Custom MSD Biomarker Development for 9 Analytes
Background
Biomarker assessment is a critical component of pharmaceutical drug development, providing insight into how a therapeutic candidate affects biological systems and whether it is achieving its intended mechanism of action. Biomarkers can be selected through a review of published scientific literature or identified experimentally through biomarker analysis of plasma and other biological matrices collected during clinical trials.
To effectively analyze a large set of possible biomarker candidates, multiplex platforms such as those provided by Meso Scale Discovery (MSD) offer an efficient workflow and sensitive results.
Scope of Work
A sponsor engaged Emery Pharma to perform custom biomarker development and multiplex assay evaluation for nine biomarkers as part of a Phase 1b clinical trial investigating a novel oncology therapeutic. Although the biomarker program in this study was exploratory in nature, the sponsor requested a comprehensive assessment of assay performance characteristics, namely, analyte concentration determination, calibrator parallelism, minimum required dilution (MRD) & matrix effect to determine the suitability of the biomarkers in a multiplex format.
Target | MSD format |
C-reactive protein (CRP) | U-plex |
IP-10 | U-plex |
IL-18 | U-plex |
IL-32 | R-plex |
IL-36γ | R-plex |
MIP-1α | U-plex |
MIP-1β | U-plex |
S100A8 | R-plex |
PD-L1 | U-plex |
MSD’s U-PLEX platform is specifically designed to enable custom multiplex assay development through flexible mixing and matching of available targets. Six of the nine biomarkers were available in the U-PLEX format. However, three targets: IL-32, IL-36γ, and S100A8, were only available through MSD’s R-PLEX product line, which had not previously been evaluated for multiplex compatibility. A key objective of this study was therefore to assess whether these R-PLEX assays could be successfully integrated into a multiplex biomarker panel.
Key Findings
Target Analyte Concentration Determination
Biomarkers are often present at substantially different concentrations in clinical samples. Identifying an appropriate sample dilution is therefore essential to ensure that the greatest number of analytes fall within the assay’s quantifiable range.
To determine the optimal dilution factor, pooled normal human plasma was evaluated at dilutions ranging from 1:2 to 1:64. It was found that even at the highest dilution (1:64) the signal from CRP was saturating the MSD instrument and required a much higher dilution. Due to the holistic nature of a multiplex assay, this meant that CRP must be split off as a separate assay and could not be multiplexed.
For the remaining eight analytes, a 1:4 dilution provided the optimal balance between minimizing matrix interference and maintaining accurate analyte quantitation within the multiplex assay.
Calibrator Parallelism
A critical aspect of multiplex assay development is demonstrating calibrator parallelism when analytes are measured individually versus in combination. To evaluate multiplex compatibility of the three R-PLEX targets, standard curves for each analyte were generated both independently and within the multiplex panel. The resulting curves were overlaid to determine parallelism.
IL-32 demonstrated strong parallelism, with singleplex and multiplex standard curves closely overlapping throughout most of the quantifiable range.
The standard curves for IL-36γ (shown below) and S100A8 (not shown) exhibited greater divergence at lower concentrations, suggesting the possibility of minor non-specific interactions under multiplex conditions. However, the observed differences remained within acceptable limits for multiplex immunoassays and were comparable to performance characteristics commonly observed in commercially available MSD multiplex kits.
Matrix Effect and Minimum Required Dilution (MRD)
Matrix effect testing was performed to evaluate whether components within the plasma matrix interfered with analyte detection and to establish the minimum required dilution (MRD) necessary for reliable quantitation.
Twelve individual human plasma samples were analyzed across a range of dilution factors. Calculated concentrations were compared across dilutions for each analyte. In the absence of matrix effects, analyte concentrations are expected to remain relatively consistent regardless of dilution factor.
Several biomarkers produced signals that were too low for a complete matrix effect assessment. However, analytes such as IP-10 generated sufficient signal for evaluation. None of the twelve plasma samples tested exhibited meaningful matrix interference for IP-10.
For PD-L1, there was likely matrix effect, with half of the samples analyzed exhibiting significant signal suppression.
Target | Possible Matrix Effect (signal difference > 20%): Samples with Matrix Effect / Total Samples |
IP-10 | 0/12 |
IL-18 | 2/12 |
IL-32 | NA |
IL-36γ | NA |
MIP-1α | NA |
MIP-1β | 3/12 |
S100A8 | NA |
PD-L1 | 6/12 |
Conclusion
Emery Pharma was able to assess the compatibility of nine different targets and determine that eight could be used in a multiplex format. While IL-32 single vs multiplex standard curves showed more divergence than IL-36γ and S100A8, the level of divergence did not exceed what is typically seen in multiplex MSD assays. The signals for a number of targets were too low to assess matrix effect. That said, except for PD-L1, none of the other targets exhibited noticeable matrix interference.
These findings provided the sponsor with valuable biomarker feasibility data to support ongoing clinical development of their oncology therapeutic and informed the selection of biomarkers for future studies.
