(M0930-06-41) Comparison of Branched DNA (B-DNA) and Polymerase Chain Reaction (RT-qPCR) Method Validation for the Quantification of mRNA Therapeutics in Plasma

Purpose: The therapeutic use of mRNAs holds great promise for combating a wide range of diseases. The rapid development of biotechnology in recent years has made it possible to produce almost any functional proteins in the human body by using mRNA as a therapeutic agent ^[1]. There are many ongoing mRNA vaccine/therapeutic research pipelines. To better understand the mRNA’s efficacy and safety properties, especially for the pharmacokinetics (PK), toxicokinetic (TK), tissue distribution, and pharmacodynamics (PD). It is critical to quantify the level of mRNA in the system circulation and different tissues. RT-qPCR platform is widely used as a golden standard for the quantification of mRNA-based therapeutics. Recently, the B-DNA platform is emerging as a unique complement to the RT-qPCR platform. Here we compare the method validation performance on these two platforms for a mimic mRNA therapeutic bioanalysis (commercial LNP coated EGFP mRNA) in Sprague Dawley rat (SD rat) plasma. These evaluations can provide some hands-on experiences for researchers who plan to do therapeutic mRNA bioanalysis.

Methods: For RT-qPCR assay, we use a modified sample preparation method that can direct lysate the plasma without the enrichment of mRNA, then followed by a two-step Taqman RT-qPCR analysis. We use a modified protocol previously designed to measure endogenous mRNA for B-DNA assay. The workflow of the two methods is shown in Figure 1.

Results: We evaluate each method’s sensitivity, assay range, inter/intra accuracy and precision, endogenous mRNA interference, selectivity, and stability ^[2][3]. We found that with an optimized sample preparation procedure, the B-DNA method could achieve a comparable lower limit of quantification (2 pg/mL) with the RT-qPCR method (1 pg/mL) but with a much narrower assay range (2 pg-250pg/mL for B-DNA compared with RT-qPCR of 6 logs of linear range) (Figure 2). The B-DNA method shows much tighter back calculated %CV and %Bias (even for the LLOQ and ULOQ points) for accuracy and precision evaluation (Figure 3). For the B-DNA method, the total error (%CV plus |%Bias|)of quality control (QC) samples (ULOQ, HQC, MQC, LQC, LLOQ) are 2.8%, 4.4%, 6.5%, 6.7%, and 7.1%, respectively. In contrast, for RT-qPCR, the values are 19.6%, 22.5%, 19.5%, 30.2%, and 35.6%, respectively. Surprisingly, the RT-qPCR method could tolerate a higher level of endogenous mRNA interference (extracted from liver tissue in SD rat). By the way, this assessment is more informative for the bioanalysis of tissue samples, since the tissue samples will have much more endogenous mRNA than plasma. Both methods show comparable selectivity at different concentrations (ULOQ, HQC, and LLOQ) in different SD rat individual plasmas for both sex. For the RT-qPCR method, 100% ULOQ/HQC samples, 94% LLOQ samples passed the selectivity evaluation. As the B-DNA method, 100% ULOQ/HQC/LLOQ samples passed. And both methods show good stability for the test article at different conditions, including five freeze/thaw cycles, room temperature incubation for 6 hours, and 4℃ incubation for 24 hours. The |%Bias| between the stability sample and baseline samples are all less than 20%.

Conclusion: In summary, this comparison of both methods’ validation for the therapeutic mRNA quantification in plasma shows that the optimized B-DNA method could have comparable sensitivity, selectivity, and stability with the RT-qPCR method (without enrichment of mRNA). The B-DNA method is more robust than the RT-qPCR method (tighter %CV and %Bias). But the B-DNA method will have a narrower assay range and less tolerance to endogenous mRNA interference. And please note that the primer/probe design and optimization are critical for both methods. The proper design could improve the assay performance many.

References: [1]. Qin, Shugang, et al. "mRNA-based therapeutics: powerful and versatile tools to combat diseases." Signal transduction and targeted therapy 7.1 (2022): 166.
[2]. Wissel, Mark, et al. "Recommendations on qPCR/ddPCR assay validation by GCC." Bioanalysis 14.12 (2022): 853-863.
[3]. Bioanalytical Method Validation Guidance for Industry, FDA 2018.

Acknowledgements: The authors have no conflicts of interest to declare.

Figure 1: Flow chart for the experiment process by RT-qPCR method and branched DNA method for a mimic mRNA therapeutic (commercial LNP coated EGFP mRNA) quantification in plasma.

Figure 2: A. RT-qPCR amplification plot for EGFP mRNA quantification. X-axis is the qPCR cycle number; Y-axis is the ΔRn value. Curves indicate the standard sample from STD01 to STD07 (corresponding concentration shown in panel B); B. RT-qPCR method standard curve for EGFP mRNA qualification. X-axis from right to left indicates STD01-STD07 samples concentration (pg/mL). Y-axis is the qPCR cycle ct value; C. B-DNA method standard curve for EGFP mRNA qualification, X-axis from left to right indicates STD01-STD08 samples concentration (pg/mL). Y-axis is the relative light unit (RLU) value.

Figure 3. A, C. Shown RT-qPCR method %CV and %Bias for standard samples for each run, standard samples concentration are 1x106, 1x105, 1x104, 1x103, 1x102, 1x101, 1x100 pg/mL respectively. Lines indicate the average value of 3 runs, which also apply to other panels; B, D. Shown RT-qPCR method %CV and %Bias for quality control samples for each run. Quality control samples concentration are 1x106, 5x105, 5x103, 5x101, 1x100 pg/mL, respectively; E, G. Shown B-DNA method %CV and %Bias for standard samples for each run, standard samples concentration are 250, 212.5, 100, 40, 16, 8, 4, 2 pg/mL respectively; F, H. Shown B-DNA method %CV and %Bias for quality control samples for each run. Quality control samples concentration are 250, 200, 30, 6, 2 pg/mL, respectively.