(M1430-01-06) Bridging of a Duplex to Singleplex bDNA Method to Quantitate mRNA from LNP-mRNA-ABC in Human Serum

Purpose: To optimize, develop, validate and bridge a singleplex branched DNA (bDNA) method to quantitate mRNA from lipid nanoparticle (LNP) encapsulated mRNA-ABC (LNP-mRNA-ABC) with a duplex bDNA method to improve method performance for support of ongoing clinical studies. LNP-mRNA-ABC contains two unique mRNA sequences (mRNA-1 and mRNA-2) encapsulated in a LNP. A duplex bDNA assay to measure mRNA-1 and mRNA-2 from a single sample was developed and validated to measure mRNA-1 and 2 in clinical study subject samples. The duplex method was initially validated to conserve sample volume as study population includes pediatric patients with limited blood draw volumes. During sample testing with the duplex method, it was determined that circulating levels of mRNA-1 and mRNA-2 differed significantly, resulting in retesting samples at higher dilutions to bring them into the range for mRNA-2 quantitation, but no retest for mRNA-1 was required. Additionally, the technical complexity of the duplex method made it difficult to test more than one plate at once. The bead based platform required complex inversions for wash and rinse steps. mRNA2 required much further dilution in the standard curve than mRNA1. The combination of the technical difficulty and further dilution is believed to have contributed to the higher variability in the assay, which ultimately contributed to high failure rates. Consequently, the method was transitioned to singleplex assays for mRNA-1 and mRNA-2. The singleplex methods were validated to target the duplex quantitation ranges, precision, and accuracy. As the new methods were to be used to support ongoing clinical studies, the performance of the methods was compared to the performance of the duplex method and a concordance of results was assessed to ensure comparability during sample analysis.

Methods: The duplex bDNA method utilized the QuantiGene™ Plex assay Kit, which included, capture beads, a customized probe set (capture extenders, label extenders, and blocking probes) that hybridize to the mRNA-1 and mRNA-2 and utilize branched DNA technology to amplify the signal is measured using the Bioplex system. The Singleplex bDNA method was validated for mRNA1 and mRNA2, but for the purpose of this presentation only mRNA1 is discussed. The method validation utilized the QuantiGene™ Singleplex Kit. This similarly uses a customized probe set (capture extenders, label extenders, and blocking probes), but utilized the bottom of a plate well for the capture surface. The probe set is specifically designed to hybridize to the target mRNA sequence (drug product) and amplifying the technology with branch DNA technology. Signal was measured using a TECAN SPARK Plate Reader Because of volume limitations, samples from study participants could not be used to bridge the methods. Consequently, mock samples were prepared by spiking LNP-mRNA-ABC drug product into human serum at various expected concentrations within the curve range to mimic actual study samples.

Results: The Singleplex bDNA PK assay was developed/validated for mRNA-1 with a similar range of standard curve, QCs and sensitivity (LLOQ) as the original and previously validated duplex assay. Bridging and concordance was assessed by testing 23 mock samples in both the singleplex and the duplex methods and the relative percent difference was calculated between the concentration determined from the two sets of samples. Out of the 23 result pairs, 100% were within 35 relative percent difference (RPD), and 91% of the pairs were within 30 RPD. With this result the 2 separate assays showed acceptable concordance.

Conclusion: The concordance of the Singleplex to the duplex assay was established enabling seamless support of sample analysis for a critical clinical study. Ongoing use of the singlex assay in sample testing has demonstrated a lower failure rate and reduced need for sample retesting mitigating issues with sample volume constraints. Additionally, after implementation of the Singleplex has allowed for more rapid testing of the samples with expedited data delivery as the throughput of the method has increased.