Choosing the Right PCR Strategy in Cell and Gene Therapy Development

Article summary

• PCR is used across the full CGT workflow, from early R&D and process development to clinical manufacturing and batch release.
• qPCR remains widely used, especially for higher-throughput, relative quantification tasks like many viral titer assays.
• dPCR offers greater precision and absolute quantification, making it especially useful for vector copy number, gene editing efficiency, and complex samples.
• PCR supports key safety and quality tests, including sterility, mycoplasma detection, RCL testing, and HLA typing.
• Newer approaches like direct-count PCR may expand capabilities further, but ddPCR is already well established across research, clinical, and manufacturing settings.

“PCR is integral to the development and manufacturing of cell and gene therapies, across a broad range of applications,” according to Chelsea Pratt, Ph.D., Biopharma Segment Marketing Manager at Bio-Rad Laboratories. Indeed, while quantitative PCR (qPCR) remains a mainstay in the analysis of cell and gene therapies (CGT), digital PCR (dPCR) has pushed the boundaries of what is possible in CGT development. Let’s take a how PCR is used throughout CGT development to detect, quantify, and characterize DNA and RNA sequences.

PCR supports the entire developmental process

CGT involve the transfer of genetic material into the patient, either directly (via viral gene therapy) or indirectly (via the transfer of genetically modified cells; i.e., cell therapy). A large part of CGT development is analyzing the success of this gene transfer. “In early research, PCR is used to evaluate gene targets, confirm editing efficiency, and assess early gene delivery techniques,” says Pawan Singh, Vice President, Molecular Biology, Thermo Fisher Scientific.

PCR also underlies the optimization of the CGT manufacturing process, also known as process development, according to Singh. “During process development, PCR becomes critical for establishing assays that support identity, purity, quality, and safety. In CGT therapies that use integrating viral vectors, PCR-based vector copy number assays are used to quantify how many copies of the therapeutic gene have integrated into each cell, helping define acceptable integration levels. PCR also supports safety assessments by amplifying DNA for downstream sequencing workflows that evaluate insertion sites or off-target editing events. In addition, PCR-based viral titer assays are used to confirm that the non-integrated vector has been sufficiently removed from the final product.”

Finally, PCR ensures consistency in clinical manufacturing during clinical trials and beyond. “As a therapy moves through clinical trials and toward licensure, PCR-based assays become essential for routine batch release, process monitoring, and safety testing. Vector copy number and viral titer assays are used to confirm consistency across batches.” Measuring clinical safety parameters, such as sterility and patient compatibility in the case of cell therapies, is equally important. As Singh notes, “PCR is also widely used for sterility-related testing, especially the rapid detection of mycoplasma contamination. Additional safety testing, such as replication-competent lentivirus (RCL) assays, can utilize PCR techniques. For allogeneic, off-the-shelf therapies, PCR-based human leukocyte antigen (HLA) typing can also play an important role in helping to characterize donor or cell material and support patient compatibility.”

The use of PCR in CGT development, from pre-investigational new drug (IND) submission research to biologics license applications and beyond, is a given. Less clear is the choice between PCR technologies such as conventional PCR, qPCR, and dPCR.

Developmental stage matters

One factor that dictates this choice is how far along CGTs are in development. As Singh points out, “Early on, researchers often use conventional PCR, followed by gel electrophoresis, to confirm whether a target sequence has been inserted, deleted, or modified as expected.” But later stages often demand more precision: “As programs mature, more quantitative PCR-based methods, such as digital PCR, are used to measure editing efficiency and the performance of different delivery strategies with increased precision,” says Singh.

Although qPCR is also a quantitative method—it is called quantitative PCR, after all—this method requires that a standard curve be developed. Thus, qPCR offers relative quantification that assumes high amplification efficiency.

Bio-Rad has developed a widely used oil-emulsion-based dPCR platform called Droplet Digital PCR (ddPCR). “ddPCR technology provides absolute quantification without the need for standard curves, improving accuracy and reproducibility because it relies on endpoint detection rather than amplification efficiency, making it more tolerant of inhibitors and assay-to-assay variability,” says Pratt. “It also makes ddPCR technology far better suited for multiplexing, where qPCR often struggles due to its dependance on amplification efficiency.”

Developmental needs matter, too

The higher resolution of dPCR means it can serve different needs, according to Singh: “dPCR excels when an application requires sensitivity, precision, and absolute quantification with the ability to distinguish between positive and negative cells. That makes it useful for assays such as vector copy number and gene editing efficiency. qPCR, by contrast, is best suited for higher throughput workflows that rely on relative quantification, such as many viral titer assays, where the required level of resolution is lower.”

“For CGT developers facing complex samples and tight performance requirements, ddPCR delivers high precision with fewer assumptions, supporting confident decision-making across development stages,” Pratt adds. Complex samples include those derived from challenging biological matrices (e.g., feces) that may be encountered in viral shedding studies, for example.

Pushing the boundaries

dPCR’s edge in sensitivity over qPCR enables applications in CGT development, such as the analysis of vector identity, structure, and organization: “One example is linkage analysis in both gene and cell therapy, where ddPCR assays can assess co-occurrence of neighboring genetic elements without amplifying long, difficult regions,” says Pratt. “ddPCR technology also supports highplex gene expression and biomarker panels, which are often limited in qPCR due to amplification efficiency constraints.” Such panels can measure transgene expression, off-target effects, and insertional mutagenesis, or facilitate patient selection for clinical trials. Pratt adds, “Lastly, the measurement of empty-full capsids for AAV is only possible with ddPCR technology, allowing measurements of both DNA and proteins simultaneously in droplets.”

A new technology called direct-count PCR has emerged that may further expand possibilities in the CGT space. This new method partitions samples into many more reactions than dPCR (~30 million to be exact), enabling true counting of DNA rather than the dPCR’s Poisson-based estimation.

Whatever the future holds, dPCR enjoys a solid foothold in the industry: “ddPCR platforms are widely adopted across research, clinical, and manufacturing environments,” says Pratt.