Comparing digital and real-time PCR platforms for detecting residual iPSCs and virus-producing cells in manufacturing

Digital PCR (dPCR) enables absolute nucleic acid quantification and has been widely adopted for quality control (QC) applications in cell therapy manufacturing. Ensuring patient safety during cell therapy manufacturing requires reliable detection of trace residual cells, such as undifferentiated induced pluripotent stem cells (iPSCs) and virus-producing cells. This study compared qPCR (CFX96 Opus System, Bio-Rad) with two dPCR platforms-the QX200 Droplet Digital PCR System (Bio-Rad) and the QIAcuity Digital PCR System (QIAGEN). For QC evaluation, iPSCs mixed with differentiated cardiomyocytes (CMs) or neural progenitor cells (NPCs) were analyzed using TDGF1, OCT4, and NANOG, while virus-producing 293T cells in CAR-T preparations were targeted using gag and VSVG sequences within the lentiviral packaging plasmid. Mixed samples were serially diluted from 1:1 to 1:10⁶ to evaluate performance across a wide concentration range. Both dPCR platforms and qPCR showed comparable sensitivity and linearity across most dilution points. However, qPCR exhibited more frequent signal loss at low template concentrations. In contrast, dPCR showed reduced variability across dilution intervals, and lower coefficients of variation (CV), indicating more stable quantification at low target levels. Despite minor differences in absolute copy number, both dPCR systems demonstrated comparable analytical performance. These results indicate that, although overall sensitivity and linearity were similar between qPCR and dPCR, dPCR provides more consistent quantification across dilution ranges, supporting its suitability for detecting low-abundance residual cells in cell therapy manufacturing.