Lipid nanoparticle-messenger RNA (LNP-mRNA) drug products are a growing class of drug modalities. The unique composition of these drug products requires multiple measurements to account for the different components of these drug modalities. Pharmacokinetic (PK) measurements include measurement of the encapsulated mRNA and components of the LNP in circulation to understand the effectiveness of the therapeutic mRNA. The PK measurements can utilize many different platforms including PCR. Current regulatory guidance documents for bioanalytical method validation are specific to ligand binding and chromatographic assay methods and difficult to interpret for use with molecular workflows. The purpose of this paper is to provide information on considerations for validation of regulated reverse transcription quantitative PCR (RT-qPCR) assays that are used to support the pharmacokinetic analysis of LNP-mRNA drug products.

ABX Biotech LLC Ambler Pennsylvania USA

Beam Therapeutics Inc Cambridge Massachusetts USA

BioAgilytix Laboratories Boston Massachusetts USA

BioAgilytix Laboratories Durham North Carolina USA

BioAgilytix Laboratories Hamburg Germany

Bristol Myers Squibb Lawrenceville New Jersey USA

Celerion Inc Lincoln NE USA

Charles River Laboratories Reno Nevada USA

Charles River Labs Senneville Quebec Canada

Eli Lilly and Company Indianapolis Indiana USA

Eurofins Viracor Biopharma Services LLC Lenexa Kansas USA

Genentech Inc South San Francisco California USA

Institute of Biotechnology Czech Academy of Sciences Prague Czechia

Johnson and Johnson Spring House Pennsylvania USA

Labcorp Harrogate UK

Merck and Co Inc Rahway New Jersey USA

Moderna Inc Cambridge Massachusetts USA

Moderna Inc Harwell Oxford UK

Northern Biomolecular Services Portage Michigan USA

Novartis Biomedical Research Cambridge Massachusetts USA

Pennsylvania Department of Health Exton Pennsylvania USA

PPD Clinical Research Thermo Fisher Scientific Richmond Virginia USA

QPS LLC Newark Delaware USA

Regeneron Pharmaceuticals Inc Tarrytown New York USA

Sanofi Ghent Belgium

TATAA Biocenter Gothenburg Sweden

Ultragenyx Pharmaceutical Inc Novato California USA

Unaffiliated Bangalore India

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Sparmann A, Vogel J. RNA-based medicine: from molecular mechanisms to therapy. EMBO J. 2023;42(21):e114760. https://doi.org/10.15252/embj.2023114760 . PubMed DOI PMC

Xu S, Yang K, Li R, Zhang L. mRNA vaccine era-mechanisms, drug platform and clinical prospection. Int J Mol Sci. 2020;21(18):6582. https://doi.org/10.3390/ijms21186582 . PubMed DOI PMC

Kon E, Ad-El N, Hazan-Halevy I, Stotsky-Oterin L, Peer D. Targeting cancer with mRNA-lipid nanoparticles: key considerations and future prospects. Nat Rev Clin Oncol. 2023;20(11):739–54. https://doi.org/10.1038/s41571-023-00811-9 . PubMed DOI

Sahin U, Karikó K, Türeci Ö. mRNA-based therapeutics—developing a new class of drugs. Nat Rev Drug Discov. 2014;13(10):759–80. PubMed DOI

Szabo GT, Mahiny AJ, Vlatkovic I. COVID-19 mRNA vaccines: platforms and current developments. Mol Ther. 2022;30(5):1850–68. https://doi.org/10.1016/j.ymthe.2022.02.016 . PubMed DOI PMC

Jia L, Qian SB. Therapeutic mRNA engineering from head to tail. Acc Chem Res. 2021;54(23):4272–82. https://doi.org/10.1021/acs.accounts.1c00541 . PubMed DOI

Cheng F, Wang Y, Bai Y, Liang Z, Mao Q, Liu D, et al. Research advances on the stability of mRNA vaccines. Viruses. 2023;15(3):668. https://doi.org/10.3390/v15030668 . PubMed DOI PMC

Qin S, Tang X, Chen Y, Chen K, Fan N, Xiao W, et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022;7(1):166. https://doi.org/10.1038/s41392-022-01007-w . PubMed DOI PMC

Sergeeva OV, Koteliansky VE, Zatespin TS. mRNA-based therapeutics - advances and perspectives. Biochemistry (Mosc). 2016;81(7):709–22. https://doi.org/10.1134/S0006297916070075 . PubMed DOI

Reichmuth AM, Oberli MA, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine delivery using lipid nanoparticles. Ther Deliv. 2016;7(5):319–34. https://doi.org/10.4155/tde-2016-0006 . PubMed DOI PMC

Hassett KJ, Benenato KE, Jacquinet E, Lee A, Woods A, Yuzhakov O, et al. Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines. Mol Ther Nucleic Acids. 2019;15:1–11. https://doi.org/10.1016/j.omtn.2019.01.013 . PubMed DOI PMC

Vasileva O, Zaborova O, Shmykov B, Ivanov R, Reshetnikov V. Composition of lipid nanoparticles for targeted delivery: application to mRNA therapeutics. Front Pharmacol. 2024;15:1466337. https://doi.org/10.3389/fphar.2024.1466337 . PubMed DOI PMC

Blasi P, Giovagnoli S, Schoubben A, Ricci M, Rossi C. Solid lipid nanoparticles for targeted brain drug delivery. Adv Drug Deliv Rev. 2007;59(6):454–77. https://doi.org/10.1016/j.addr.2007.04.011 . PubMed DOI

Hou X, Zaks T, Langer R, et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078–94. PubMed DOI PMC

Atterwala H. Impact of pharmacometrics in advancing mRNA therapeutics and vaccines. Nucleic Acid Insights. 2024;1(6):235–42. https://doi.org/10.18609/nai.2024.030 . DOI

August A, Attarwala HZ, Himansu S, et al. A phase 1 trial of lipid-encapsulated mRNA encoding a monoclonal antibody with neutralizing activity against Chikungunya virus. Nat Med. 2021;27(12):2224–33. PubMed DOI PMC

Ortiz J, Brunner L, Ci L, Baek R, Jani D, Marshall JC, et al. 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. AAPS J. 2025;27(1):27. https://doi.org/10.1208/s12248-024-01002-9 . PubMed DOI

US FDA. Guidance for Industry. M10 Bioanalytical Method Validation and Study Sample Analysis (2022). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/m10-bioanalytical-method-validation . Accessed 2 Feb 2025.

Hays A, Wissel M, Colletti K, Soon R, Azadeh M, Smith J, et al. Recommendations for method development and validation of qPCR and dPCR assays in support of cell and gene therapy drug development. AAPS J. 2024;26(1):24. PubMed DOI

Liu A, Wang X. The pivotal role of chemical modifications in mRNA therapeutics. Front Cell Dev Biol. 2022;10:901510. https://doi.org/10.3389/fcell.2022.901510 . PubMed DOI PMC

Kauppinen S, Vester B, Wengel J. Locked nucleic acid (LNA): high affinity targeting of RNA for diagnostics and therapeutics. Drug Discov Today Technol. 2005;2(3):287–90. https://doi.org/10.1016/j.ddtec.2005.08.012 . PubMed DOI PMC

Adams G. A beginner’s guide to RT-PCR, qPCR and RT-qPCR. Biochem (Lond). 2020;42:48–53. https://doi.org/10.1042/bio20200034 . DOI

Muramatsu H, Lam K, Bajusz C, Laczko D, Kariko K, Schreiner P, et al. Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine. Mol Ther. 2022;30(5):1941–51. https://doi.org/10.1016/j.ymthe.2022.02.001 . PubMed DOI PMC

Fabre AL, Colotte M, Luis A, Tuffet S, Bonnet J. An efficient method for long-term room temperature storage of RNA. Eur J Hum Genet. 2014;22(3):379–85. https://doi.org/10.1038/ejhg.2013.145 . PubMed DOI

Zhao P, Hou X, Yan J, Du S, Xue Y, Li W, et al. Long-term storage of lipid-like nanoparticles for mRNA delivery. Bioact Mater. 2020;5(2):358–63. https://doi.org/10.1016/j.bioactmat.2020.03.001 . PubMed DOI PMC

Guelman S, Zhou Y, Brady A, Peng K. A fit-for-purpose method to measure circulating levels of the mRNA component of a liposomal-formulated individualized neoantigen-specific therapy for cancer. AAPS J. 2022;24(2):64. https://doi.org/10.1208/s12248-022-00709-x . PubMed DOI

Bower J, Zimmer J, McCown S, Tabler E, Karnik S, Kar S, et al. Recommendations for the content and management of certificates of analysis for reference standards from the GCC for bioanalysis. Bioanalysis. 2021;13(8):609–19. PubMed DOI

Asaga S, Kuo C, Nguyen T, Terpenning M, Guiliano AE, Hoon DSB. Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer. Clin Chem. 2011;57(1):84–91. https://doi.org/10.1373/clinchem.2010.151845 . PubMed DOI

Bachofen C, Willoughby K, Zadoks R, Burr P, Mellor D, Russell GC. Direct RT-PCR from serum enables fast and cost-effective phylogenetic analysis of bovine viral diarrhoea virus. J Virol Methods. 2013;190(1–2):1–3. https://doi.org/10.1016/j.jviromet.2013.03.015 . PubMed DOI

Zhang X, Yang X, Zhang Y, Liu X, Zheng G, Yang Y, et al. Direct serum assay for cell-free Bmi-1 mRNA and its potential diagnostic and prognostic value for colorectal cancer. Clin Cancer Res. 2015;21(5):1225–33. https://doi.org/10.1158/1078-0432.CCR-14-1761 . PubMed DOI

Li L, He JA, Wang W, Xia Y, Song L, Chen ZH, et al. Development of a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for clinical Zika diagnosis. Int J Infect Dis. 2019;85:167–74. https://doi.org/10.1016/j.ijid.2019.06.007 . PubMed DOI

Wee SK, Sivalingam SP, Yap EPH. Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler. Genes. 2020;11(6):664. https://doi.org/10.3390/genes11060664 . PubMed DOI PMC

Tichopad A, Kitchen R, Riedmaier I, Becker C, Stahlberg A, Kibista M, et al. Design and optimization of reverse-transcription quantitative PCR experiments. Clin Chem. 2009;55(10):1816–23. https://doi.org/10.1373/clinchem.2009.126201 . PubMed DOI

Beall RF, Hollis A. Global clinical trial mobilization for COVID-19: higher, faster, stronger. Drug Discov Today. 2020;25(10):1801–6. https://doi.org/10.1016/j.drudis.2020.08.001 . PubMed DOI PMC

Padhi BK, Singh M, Huang N, Pelletier G. A PCR-based approach to assess genomic DNA contamination in RNA: application to rat RNA samples. Anal Biochem. 2016;494:49–51. https://doi.org/10.1016/j.ab.2015.10.012 . PubMed DOI

Laurell H, Iacovoni JS, Abot A, Svec D, Maoret JJ, Arnal JF, Kubista M. Correction of RT–qPCR data for genomic DNA-derived signals with ValidPrime. Nucleic Acids Res. 2012;40 (7). https://doi.org/10.1093/nar/gkr1259

Svec D, Tichopad A, Novosadvoa V, Pfaffl MW, Kubista M. How good is a PCR efficiency estimate: recommendations for precise and robust qPCR efficiency assessments. Biomol Detect Quantif. 2015;3:9–16. PubMed DOI PMC

Forootan A, Sjöback R, Björkman J, Sjögreen B, Linz L, Kubista M. Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR). Biomol Detect Quantif. 2017;12:1–6. https://doi.org/10.1016/j.bdq.2017.04.001 . PubMed DOI PMC

US FDA. Guidance for Industry: Long Term Follow-up After Administration of Human Gene Therapy Products (2020). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/long-term-follow-after-administration-human-gene-therapy-products . Accessed 28 Jan 2025.

Svec D, Andersson D, Pekny M, Sjoback R, Kubista M, Stahlberg A. Direct cell lysis for single-cell gene expression profiling. Front Oncol. 2013;3:274. https://doi.org/10.3389/fonc.2013.00274 . (eCollection 2013). PubMed DOI PMC

Ma H, Bell KN, Loker RN. qPCR and qRT-PCR analysis: Regulatory points to consider when conducting biodistribution and vector shedding studies. Mol Ther Methods Clin Dev. 2020;20:152–68. https://doi.org/10.1016/j.omtm.2020.11.007 . PubMed DOI PMC

Kelley M. Incurred sample reanalysis: it is just a matter of good scientific practice. Bioanalysis. 2011;3(9):931–2. https://doi.org/10.4155/bio.10.215 . PubMed DOI