Comparison of Branched DNA to RT-qPCR Technology for the Quantitation of mRNA from mRNA-LNP Drug Product in Human Serum.

Authors

Carl A Luongo, Ashley D Wright, Timothy L Lochmann, Sweilem B Al Rihani, Jean-Claude Marshall, Darshana Jani, Jason Pennucci, Jessica Ortiz

Published in

The AAPS journal. Volume 27. Issue 6. Pages 137. Sep 03, 2025. Epub Sep 03, 2025.

Abstract

Accurate quantitation of circulating messenger RNA (mRNA) is critical for the quantitation of lipid nanoparticle-formulated mRNA (mRNA-LNP) drug products. This study evaluated the concordance between branched DNA (bDNA) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays for quantifying mRNA in human serum from a phase 1 clinical trial. We compared analytical performance across bDNA and two RT-qPCR workflows-RNA purification and a simplified NP-40 detergent-based treatment. A total of 77 clinical serum samples were analyzed. Method performance was assessed using assay precision, accuracy, and total error, along with linear regression and Bland-Altman analyses to evaluate inter-platform concordance. Noncompartmental PK analysis was performed on a subset of samples from four subjects. Results showed that RT-qPCR methods yielded lower mRNA concentrations than bDNA, with a consistent negative bias more pronounced in NP-40-treated samples. The purification RT-qPCR method showed closer agreement with bDNA across the quantitative range (R² = 0.878) than NP-40 treated RT-qPCR (R² = 0.736). Despite quantitative differences, PK parameters derived from all methods were comparable, supporting RT-qPCR's suitability for clinical mRNA quantification. NP-40 treatment offered workflow efficiency and lower sample volume requirements, whereas mRNA purification had improved concordance with bDNA. These findings support the feasibility of adopting RT-qPCR as a viable alternative to bDNA method for mRNA quantification, with method selection guided by study phase, throughput needs, and available matrix volume. Cross-platform comparability ensures robust bioanalytical support for clinical development of mRNA drug candidates.

PMID:
40903636
Bibliographic data and abstract were imported from PubMed on 04 Sep 2025.

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