(T0930-02-07) Comparison of Various Methods to Quantitate mRNA from LNP-mRNA-ABC in Human Serum

Purpose: To compare multiple assay platforms (duplex branched DNA (bDNA), single-plex bDNA, and duplex RT-qPCR) used to quantify mRNA-1 and mRNA-2 from lipid nanoparticle (LNP) encapsulated mRNA-ABC (LNP-mRNA-ABC) in a phase 1 clinical study. Background - LNP-mRNA-ABC is the dosed therapeutic drug product that contains two unique mRNA sequences (mRNA-1 and mRNA-2) that are LNP-encapsulated. To support the mRNA pharmacokinetics (PK) in a phase 1 clinical study, a duplex bDNA assay was developed and validated to measure both mRNA-1 and mRNA-2 in human serum samples. The duplex method is a bead-based platform that lacks robustness due to a complex procedure and has a long read time for data extraction. Due to the technical complexity of the duplex method, high assay failure rate, and failed incurred sample reanalysis (ISR) for reproducibility assessment, the method was not robust, high-throughput, and efficient enough to meet critical study timelines. Consequently, two singleplex bDNA assays for mRNA-1 and mRNA-2 were developed and validated to replace the duplex bDNA assay in the phase 1 clinical study. To ensure a smooth transition between the assays, the performance of the methods was compared to the performance of the duplex method and a concordance of results from mock samples was assessed to ensure comparability during sample analysis. The separate singleplex methods showed improved passing rates over the duplex assays and demonstrated comparability, but the assays also had ISR issues. Because of the ISR failure, a duplex rtqPCR assay with an analyte extraction was developed and validated with the intent to replace the single-plex bDNA assays. In addition, concordance between the methods was performed in order to bridge the different platforms.

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 and is measured using the Bioplex system. The singleplex bDNA methods were validated for mRNA-1 and mRNA-2. Both assays utilized the QuantiGene™ Singleplex Kit. These assays use a customized probe set (capture extenders, label extenders, and blocking probes) that are specific to each mRNA’s target sequences, and are captured on the bottom of a plate well. The probe set hybridizes to the target mRNA sequence (drug product) and amplifies the signal which was measured using a TECAN SPARK Reader. The RT-qPCR duplex assay consists of an extraction method from serum samples using the KingFisher-Plex purification system, followed by RT-qPCR of the extracted samples. Primer/probe sets designed for mRNA-1 and mRNA-2 sequences were utilized on the QuantStudio 6 Flex Real-Time PCR System to detect and quantify the mRNA. 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. The duplex rtqPCR assay was successfully developed and validated to have a similar sensitivity with a more expanded curve range than the singleplex bDNA assays for mRNA-1 and mRNA-2. Concordance (for duplex bDNA to singleplex bDNA) was assessed by testing 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. For the concordance 100% of the samples were within 35 relative percent difference (%rpd). With this result the 2 separate assays showed acceptable concordance. Corncordance (for singleplex bDNA to rtqPCR) was assessed by similarly testing mock samples in the rtPCR and singleplex bDNA methods. Relative percent difference was calculated between the results for the samples between the methods. For the concordance 93% of the samples were within 35 % RPD. This demonstrates the concordance between the rtqPCR and singleplex bDNA.

Conclusion: The concordance of the Singleplex to the duplex assay was established enabling seamless support of sample analysis for a critical clinical study. Additionally, the singleplex assays during sample testing demonstrated a lower failure rate and reduced the number of repeat analysis. The RT-qPCR assay did meet concordance, but due to the throughput, cost and technical failure rate the singleplex bDNA assays continue to support the study. Although ISR issues were not resolved in both bDNA platforms, it will also be an issue if assessed in the RT-qPCR platform. Implementation of the singleplex assays will continue to meet the clinical study needs and support the remainder of the program. The discussion for the need for ISR for mRNA measurement is ongoing.