The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.

Altasciences Preclinical Seattle LLC Everett Washington USA

Asher Biotherapeutics Inc South San Francisco California USA

Beam Therapeutics Cambridge Massachusetts USA

BioAgilytix Laboratories Boston Massachusetts USA

BioAgilytix Laboratories Durham North Carolina USA

BioMarin Pharmaceutical Inc Novato California USA

Bristol Myers Squibb Princeton New Jersey USA

Charles River Labs Reno Nevada USA

Charles River Labs Senneville Québec Canada

Eli Lilly and Company Indianapolis Indiana USA

Eurofins Viracor BioPharma Services Inc Lenexa Kansas USA

Flowmetric Life Sciences Inc Spring House Pennsylvania USA

Genentech Inc South San Francisco California USA

GSK Collegeville Pennsylvania USA

Institute of Biotechnology Czech Academy of Sciences Prague Czech Republic

Janssen Research and Development LLC Spring House Pennsylvania USA

KCAS Bioanalytical and Biomarker Services Shawnee Kansas USA

Labcorp Drug Development Greenfield Indiana USA

Merck and Co Inc Rahway New Jersey USA

Pfizer Inc Groton Connecticut USA

PPD Clinical Research Thermo Fisher Scientific Richmond Virginia USA

Regeneron Pharmaceuticals Tarrytown New York USA

REGENXBIO Inc Rockville Maryland USA

Sanofi Ghent Belgium

TATAA Biocenter Gothenburg Sweden

Ultragenyx Pharmaceutical Inc Novato Calfornia USA

Zobrazit více v PubMed

FDA. What is Gene therapy? https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/what-gene-therapy . 25 July 2018. Accessed 25 June 2023.

Sayed N, Allawadhi P, Khurana A, Singh V, Navik U, Pasumarthi SK, et al. Gene therapy: comprehensive overview and therapeutic applications. Life Sci. 2022;294: 120375. https://doi.org/10.1016/j.lfs.2022.120375 . PubMed DOI

Jahangiri S, Rahimnejad M, Boroujeni NN, Ahmadi Z, Fath PM, Ahmadi S, et al. Viral and non-viral gene therapy using 3D (bio)printing. J Gene Med. 2022;24(12): e3458. https://doi.org/10.1002/jgm.3458 . PubMed DOI

American Society of Gene and Cell Therapy. Gene & Cell Therapy FAQs. 2021. https://asgct.org/education/more-resources/gene-and-cell-therapy-faqs . Accessed 27 June 2023.

Lin H, Cheng J, Mu W, Zhou J, Zhu L. Advances in universal CAR-T cell therapy. Front Immunol. 2021;12: 744823. https://doi.org/10.3389/fimmu.2021.744823 . PubMed DOI PMC

Labanieh L, Majzner RG, Mackall CL. Programming CAR-T cells to kill cancer. Nay Biomed Eng. 2018;6:377–91. https://doi.org/10.1038/s41551-018-0235-9 . DOI

Ma M, Balasubramanian N, Dodge R, Zhang Y. Challenges and opportunities in bioanalytical support for gene therapy medicinal product development. Bioanalysis. 2017;9(18):1423–30. https://doi.org/10.4155/bio-2017-0116 . PubMed DOI

Bernt KM, Ni S, Gaggar A, Li ZY, Shayakhmetov DM, Lieber A. The effect of sequestration by nontarget tissues on anti-tumor efficacy of systemically applied, conditionally replicating adenovirus vectors. Mol Ther. 2003;8(5):746–55. https://doi.org/10.1016/j.ymthe.2003.07.006 . PubMed DOI

Gonin P, Gaillard C. Gene transfer vector biodistribution: pivotal safety studies in clinical gene therapy development. Gene Ther. 2004;11(Suppl 1):S98–108. https://doi.org/10.1038/sj.gt.3302378 . PubMed DOI

US FDA. Guidance for industry: design and analysis of shedding studies for virus or bacteria-based gene therapy and oncolytic products. 2015. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/design-and-analysis-shedding-studies-virus-or-bacteria-based-gene-therapy-and-oncolytic-products . Accessed 10 Feb 2023.

Hays A, Durham J, Gullick B, Rudemiller N, Schneider T. Bioanalytical assay strategies and considerations for measuring cellular kinetics. Int J Mol Sci. 2022;24(1):695. https://doi.org/10.3390/ijms24010695 . PubMed DOI PMC

Yang TY, Doddareddy R. Considerations in the development and validation of real-time quantitative polymerase chain reaction and its application in regulated bioanalysis to characterize the cellular kinetics of CAR-T products in clinical studies. Bioanalysis. 2021;13(2):115–28. https://doi.org/10.4155/bio-2020-0221 . PubMed DOI

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–22. https://doi.org/10.1373/clinchem.2008.112797 . PubMed DOI

Huggett J, Whale AS, Spiegeaere WD, Trypsteen W, Abdel Nour A, Bae YK, et al. The digital MIQE Guidelines update: minimum information for publication of quantitative digital PCR experiments for 2020. Clin Chem. 2020;66(8):1012–29. https://doi.org/10.1093/clinchem/hvaa125 . PubMed DOI

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

Corsaro B, Yang TY, Murphy R, Sonderegger I, Exley A, Bertholet S, et al. 2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation ( Part 3 - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays). Bioanalysis. 2021;6:415–63. https://doi.org/10.4155/bio-2021-0007 . DOI

Hays A, Islam R, Matys K, Williams D. Best practices in qPCR and dPCR validation in regulated bioanalytical laboratories. AAPS J. 2022;24(2):36. https://doi.org/10.1208/s12248-022-00686-1 . PubMed DOI

Loo L, Harris S, Milton M, Meena A, Lembke W, Berisha F, et al. 2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization). Bioanalysis. 2021;14(11):737–93. https://doi.org/10.4155/bio-2022-0081 . DOI

Lauren A, Braun M, Pybre P, Cazzin C, Colletti K, Cox C, et al. Applying context of use to quantitative polymerase chain reaction method validation and analysis: a recommendation from the European Bioanalysis Forum. Bioanalysis. 2021;23:1723–9. https://doi.org/10.4155/bio-2021-0218 . DOI

Lauren A, Braun M, Cazzin C, Colletti K, Cox C, Dietz L, et al. Quantitative polymerase chain reaction in the bioanalytical laboratory and technical and scientific considerations for nonclinical and clinical assay characterization, validation and sample analysis. Bioanalysis. 2022;16:1085–93. https://doi.org/10.4155/bio-2022-0170 . DOI

Wissel M, Pourier M, Satterwhite C, Lin J, Islam R, et al. Recommendations on qPCR/ddPCR assay validation by GCC. Bioanalysis. 2022;12:853–63. https://doi.org/10.4155/bio-2022-0109 . DOI

Uchiyama A, Naritomi Y, Hashimoto Y, Hanada T, Watanabe K, Kitta K, et al. Understanding quantitative polymerase chain reaction bioanalysis issues before validation planning: Japan Bioanalysis Forum discussion group. Bioanalysis. 2022;21:1391–405. https://doi.org/10.4155/bio-2022-0190 . DOI

International Organization of Standardization. Biotechnology — Requirements for evaluating the performance of quantification methods for nucleic acid target sequences — qPCR and dPCR. (ISO Standard No. 20395:2019). https://www.iso.org/standard/67893.html .

US FDA. Guidance for Industry: Bioanalytical Method Validation. 2018. https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf .

European Medicines Agency. Guideline on Bioanalytical Method Validation. 2011. https://www.ema.europa.eu/en/bioanalytical-method-validation .

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 .

Bustin SA, Mueller R, Nolan T. Parameters for successful PCR primer design. Method Mol Biol. 2020;2065:5–22. https://doi.org/10.1007/978-1-4939-9833-3_2 . DOI

Latorra D, Arar K, Hurley JM. Design considerations and effects of LNA in PCR primers. Mol Cell Probes. 2003;17(5):253–9. https://doi.org/10.1016/s0890-8508(03)00062-8 . PubMed DOI

Peters IR, Helps CR, Hall EJ, Day MJ. Real-time RT-PCR: considerations for efficient and sensitive assay design. J Immunol Methods. 2004;286(1–2):203–17. https://doi.org/10.1016/j.jim.2004.01.003 . PubMed DOI

Lind K, Ståhlberg A, Zoric N, Kubista M. Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis. Biotechniques. 2006;40(3):315–9. https://doi.org/10.2144/000112101 . PubMed DOI

Sedlak RH, Kuypers J, Jerome KR. A multiplexed droplet digital PCR assay performs better than qPCR on inhibition prone samples. Diagn Microbiol Infect Dis. 2014;80(4):285–6. https://doi.org/10.1016/j.diagmicrobio.2014.09.004 . PubMed DOI

Verheul RC, van Deutekom JCT, Datson NA. Digital droplet PCR for the absolute quantification of exon skipping induced by antisense oligonucleotides in (pre-)clinical development for Duchenne muscular dystrophy. PLoS ONE. 2016;11(9): e0162467. https://doi.org/10.1371/journal.pone.0162467 . PubMed DOI PMC

US FDA. Guidance for Industry. S12 Nonclinical Biodistribution considerations for Gene Therapy Products. 2023. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/s12-nonclinical-biodistribution-considerations-gene-therapy-products .

Zhang H, Korenkova V, Sjoback R, Svec D, Bjorkman J, Kruhoffer M, et al. Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples. PLoS One. 2014;9(11):e111644. https://doi.org/10.1371/journal.pone.0111644 . (eCollection 2014). PubMed DOI PMC

Malentacchi F, Pizzamiglio S, Verderio P, Pazzagli M, Orlando C, Ciniselli CM, et al. Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience. Clin Chem Lab Med. 2015;53(12):1935–42. https://doi.org/10.1515/cclm-2014-1161 . PubMed DOI

Shroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Bol Biol. 2006;7:3. https://doi.org/10.1186/1471-2199-7-3 . DOI

Fleige S, Pfaffl M. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006;27(2–3):126–39. https://doi.org/10.1016/j.mam.2005.12.003 . PubMed DOI

Hiramatsu K, Matsuda C, Masago K, Toriyama K, Sasaki E, Fujita Y, et al. Diagnostic utility of DNA integrity number as an indicator of sufficient DNA quality in next-generation sequencing-based genomic profiling. Am J Clin Pathol. 2023; aqad046. https://doi.org/10.1093/ajcp/aqad046 .

Stahlberg A, Kubista M, Pfaffl M. Comparison of reverse transcriptase in gene expression analysis. Clin Chem. 2004;50(9):1678–80. https://doi.org/10.1373/clinchem.2004.035469 . PubMed DOI

Stahlberg A, Hakansson J, Xian X, Semb H, Kubista K. Properties of the reverse transcription reaction in mRNA quantification. Clin Chem. 2004;50(3):509–15. https://doi.org/10.1373/clinchem.2003.026161 . PubMed DOI

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 .

European Medicines Agency. Follow-up of Patients Administered with Gene Therapy Medicinal Products. 2009. https://www.ema.europa.eu/en/follow-patients-administered-gene-therapy-medicinal-products .

Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonak J, Lind K, et al. The real-time polymerase chain reaction. Mol Aspects Med. 2006;27(2–3):95–125. https://doi.org/10.1016/j.mam.2005.12.007 . PubMed DOI

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. https://doi.org/10.1016/j.bdq.2015.01.005 . PubMed DOI PMC

CLSI. Evaluation of Linearity of Quantitative Measurement Procedures, 2

Ambruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev. 2008;29(Suppl 1):S49-52.

Forootan A, Sjoback R, Bjorkman J, Sjogreen 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;2017(12):1–6. https://doi.org/10.1016/j.bdq.2017.04.001.eCollection . DOI

CLSI. EP17: Protocols for Determination of Limits of Detection and Limits of Quantitation, Approved Guideline, 2nd edn. CLSI Guideline EP17-A2. Clinical and Laboratory Standards Institute; 2012.

Stahlberg A, Kubista M. The workflow of single-cell expression profiling using quantitative real-time PCR. Expert Rev Mol Diagn. 2014;14(3):323–31. https://doi.org/10.1586/14737159.2014.901154 . PubMed DOI PMC

Lou JJ, Mirsadraei L, Sanchez D, Wilson RW, Shabihkhani M, Lucey GM, et al. A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories. Clin Biochem. 2014;47(4–5):267–73. https://doi.org/10.1016/j.clinbiochem.2013.12.011 . PubMed DOI

Hartman C, Lennartz K, Ibrahim H, Coz A, Kasper Y, Lenz C, et al. Application note: stable 16-year storage of DNA purified with the QIAamp® DNA blood mini kit. https://www.qiagen.com/us/resources . October 2016. Accessed 24 July 2023.

Walther W, Schmeer M, Kobelt D, Baier R, Harder A, Walhorn V, Anselmetti D, Aumann J, Fichtner I, Schleef M. A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis. Hum Gene Ther Clin Dev. 2013;24:147–53. PubMed DOI

Seelenfreund E, Robinson WA, Amato CM, Tan AC, Kim J, Robinson SE. Long term storage of dry versus frozen RNA for next generation molecular studies. PLoS ONE. 2014;9: e111827. https://doi.org/10.1371/journal.pone.0111827 . PubMed DOI PMC