(W1230-02-10) Qualification of a 48-plex Cytokine Olink Panel to Support Rare Disease and Pediatric Clinical Studies

Purpose: Measuring pathway-specific, physiologically relevant biomarkers is critical for successful development, testing, and approval of therapeutics. Accurate biomarker assessments produce key data for monitoring biological activity, therapeutic effect, and safety endpoints during clinical trials. However, selection of appropriate biomarkers for novel modalities and/or rare disease indications can be challenging due to the complexity of disease pathophysiology and the absence of historical data from previous clinical trials for these indications. Conventional biomarker assays typically measure single analytes and can require large sample volumes. For rare diseases and pediatric clinical studies with low sample volumes and a lack of established biomarkers, this presents a significant unmet need. To address this, we describe the qualification of a multiplex Olink method which simultaneously measures a panel of 48 cytokine biomarkers in human serum using extremely low sample volumes (1µL human serum). The assay demonstrated high levels of precision and accuracy. In this presentation, we describe common challenges for assessing the performance of high throughput multiplex panels and provide potential solutions to these including strategies for procuring individual sources for all analytes and handling large data sets for analysis.

Methods: Olink Target 48 Cytokine Panel kits were purchased from Olink (Boston, MA). Normal human serum samples were purchased from BioIVT (Westbury, NY). The method was conducted according to the manufacturer’s recommendations and qualified for its intended purpose. The Olink platform utilizes Proximity Extension Assay (PEA) technology that combines aspects of an immunoassay with polymerase chain reaction (PCR). Briefly, the incubation mix containing 48 forward and reverse probes is added to 96-well plate along with 1µL of sample, control, or calibrator. The plate is sealed and incubated at 4°C overnight. The next day, the extension mix is added to all wells and placed in a thermal cycler to begin the extension and pre-amplification of targets. Then the 96-well plate samples are added to detection mix, incubated with specific primers, and read on the Olink Signature Q100.

Results: The Olink Target 48 panel (CCL11, CCL13, CCL19, CCL2, CCL3, CCL4, CCL7, CCL8, CSF1, CSF2, CSF3, CXCL10, CXCL11, CXCL12, CXCL8, CXCL9, EGF, FLT3LG, HGF, IFNG, IL10, IL13, IL15, IL17A, IL17C, IL17F, IL18, IL1B, IL2, IL27, IL33, IL4, IL6, IL7, LTA, MMP1, MMP12, OLR1, OSM, TGFA, TNF, TNFSF10, TNFSF12, TSLP, VEGFA), was qualified in human serum. Precision was determined with 10 human serum samples tested in 3-4 replicate wells across a single run (intra-assay) and across 4 independent runs (inter-assay). Analytes showed detectable levels with acceptable intra-assay precision (CV ≤ 30%) for all but 5 out of 450 data points. Inter-assay precision was acceptable for all analytes for which there were detectible endogenous levels. Intra-assay accuracy was determined with 10 human serum samples spiked with calibrator cocktail. Accuracy was acceptable (± 30% bias) in all spiked donor samples for all but one analyte (IL17F), excluding analytes for which there was no detectible signal. In lieu of parallelism, which is difficult to perform with a high throughput panel, we assessed dilutional linearity of the 48 analytes. To assess dilutional linearity, samples were prepared by spiking 2 human serum samples with calibrator cocktail and serially diluting 2-fold in surrogate matrix. Of the 32 analytes for which there were detectable levels of analyte, 29 recovered well at 1:4 dilution, and 25 of 31 detectable analytes at 1:8 dilution recovered within the acceptable range. Lastly, freeze/thaw stability and PCR product stability were also assessed. Samples generally reported similar results following freeze/thaw at -20°C and following storage of PCR products at 4°C for one week.

Conclusion: A robust, high-throughput, multiplex cytokine panel was qualified in human serum with an input of 1µL of sample volume. This assay provides a much-needed tool that can be used for clinical trials with limited sample volumes and multiple potential biomarkers of interest. These types of large biomarker panels are important for understanding biological pathways, confirming therapeutic effect, and monitoring safety during clinical trials. This panel can be especially useful in rare disease studies where there is a lack of historical data to help streamline drug development and in pediatric studies where sample volumes can be scarce.

Acknowledgements: No conflict of interest to disclose