Bioanalytical Strategies for CGT Products: Selected Developments and Validation of qPCR/ddPCR Methodologies

Release time:

2026-04-03

With the rapid development of cell and gene therapy (CGT), dozens of CGT products have now been approved for market launch worldwide.

Bioanalytical Strategies for CGT Products: Selected Developments and Validation of qPCR/ddPCR Methodologies

With the rapid advancement of cell and gene therapy (CGT), dozens of CGT products have now been approved and launched globally. Concurrently, qPCR and ddPCR technologies are being increasingly employed in regulated bioanalytical assays, as more CROs take on bioanalytical services for sponsors’ clinical trials—activities that invariably require method development and validation. Quantitative PCR and digital PCR (qPCR/ddPCR) are highly reproducible, sensitive, and standardized nucleic acid–sequence (gene) quantification techniques widely used in the bioanalysis of CGT products, such as assessing viral exposure, distribution, and shedding in AAV-based gene therapies, and conducting pharmacokinetic (cellular) pharmacokinetic analyses for CAR-T cell–based gene therapies. In early preclinical and IND studies, analyses often rely on establishing methods for quantifying the copy number of target genes. However, regulatory guidance and standards for qPCR remain incomplete, while the next-generation ddPCR technology currently lacks clear national guidelines. In 2022, the Global CRO Council (GCC) published a white paper that provides consensus statements and recommendations to support harmonization of PCR-based assays across various validation parameters, standardize validation methodologies, and establish uniform standard operating procedures (SOPs).

Existing consensus validation items in the methodological development and validation of qPCR include:

■ Analytical sensitivity;

■ Specificity;

■ Selectivity;

■ Accuracy;

■ Intra-/inter-assay precision;

■ Lower limits of quantitation (LLOQ);

■ Range of quantitation.

In addition, some verification items can also be designed to be performed during the verification process, including:

■ Dilutional linearity;

■ Sample stability;

■ Reproducibility;

■ Robustness.

The following are less frequently used as verification items:

■ Parallelism;

■ Inhibition;

■ Reproanalysis of tested samples (Incurred sample reproducibility, ISR).

Most researchers develop qPCR/ddPCR methods based on currently published white papers and regulatory guidelines, although acceptance criteria may vary slightly—for example, assay accuracy for reference standards and quality control (QC) samples is typically ±30%, with PCR efficiency ranging from 90% to 110%. Some studies provide more detailed qPCR validation criteria: for precision and accuracy, at least 50% of QC samples at each concentration level must meet the specified limits, and across all QC samples, ≥67% must satisfy %CV ≤ 25% (with an upper limit of ≤45%) and %RE within ±25% (with an upper limit of ±45%). For QC sample stability, the acceptance criterion is that the %CV from replicate analyses of each sample does not exceed 30%, and that >67% of replicate (duplicate) measurements meet this requirement. Additionally, the %CV for QC samples should also be ≤30%, among other specifications. The most pressing issue at present is the lack of uniformity in the validation acceptance criteria adopted by method developers, which can pose regulatory risks in regulated bioanalytical reports.

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