Comparative assessment of RT-dPCR and RT-qPCR for RNA quantification in IgG mRNA-lipid nanoparticle bioanalysis

mRNA-LNP therapeutics have revolutionized drug development across various therapeutic areas, necessitating accurate and robust RNA quantification in bioanalysis. While RT-qPCR is commonly used for RNA quantification, RT-dPCR has emerged as an alternative, with enhanced precision and robustness. Yet, a systematic comparison of these methodologies for mRNA-LNP bioanalysis remains absent.

Duplex RT-dPCR and RT-qPCR assays using identical primers and probes were developed to quantify LNP encapsulated IgG heavy chain (HC) and light chain (LC) RNA. Both assays analyzed RNA from 50 rat serum samples dosed with the mRNA-LNP compound, and results were statistically compared for measurement agreement and method reliability.

Both assays were qualified, meeting the pre-defined assay acceptance criteria. HC RNA quantification showed strong agreement between the two methods (Lin's CCC = 0.96). However, RT-qPCR significantly underestimated LC RNA levels compared to RT-dPCR, with a dPCR/qPCR ratio mean of 2.83, suggesting gene-dependent variability in quantification accuracy.

This study provides a systematic evaluation of RT-dPCR and RT-qPCR in RNA quantification, advancing our understanding of their application in mRNA-LNP bioanalysis. These findings underscore the critical need for thorough assay evaluation to ensure reliable mRNA measurements, and prevent potential inaccuracies that could lead to misleading results in drug development and regulatory decision-making.