Comparison of RT-qPCR With Branched DNA to Quantify a Lipid Nanoparticle-Encapsulated mRNA Therapeutic in Serum and Liver Tissue Samples From Nonclinical PK Studies.

While the branched DNA (bDNA) assay is an established bioanalytical method for measurement of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) pharmacokinetic parameters, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has been considered as an alternative platform. RT-qPCR and bDNA platforms were compared for sensitivity, specificity, correlation, and overall assay performance using serum and tissue samples from 2 nonclinical mouse studies of a therapeutic mRNA candidate, LNP-PAH-mRNA, which encodes for human phenylalanine hydroxylase enzyme. Pharmacokinetic parameter noncompartmental analysis was completed using Phoenix WinNonlin. The assays were compared using simple linear regression and Bland-Altman analyses. Sensitivity ranged from 0.05 to 6.40 ng/mL for the bDNA assays, from 0.00000761 to 7.61 ng/mL in serum, and from 0.000179 to 179 ng/g in tissue for the RT-qPCR assay. Inter-assay accuracy was within ± 10%, inter-assay precision was ≤ 10%, and the total error for both assays was ≤ 20%. RT-qPCR serum mRNA concentrations were 2- to fourfold lower compared with the bDNA assay, whereas tissue samples were comparable between assays. A linear relationship with - 0.37 to - 0.02 systematic bias demonstrated acceptable concordance. Bland-Altman plots demonstrated close equivalence, with a negative bias of < 0.5, and ≥ 95% of the data points were within the 95% limits of agreement. The comparison of the RT-qPCR with bDNA assay platforms for quantification of pharmacokinetic properties of an mRNA-LNP therapeutic has demonstrated acceptable concordance. This comparison reinforces the use of the RT-qPCR, a widely accessible strategy, as an alternative platform for the quantification of subsequent mRNA-LNP therapeutics.