Cell & Gene Therapy Answers: Addressing Complex Analytical Needs with ddPCR/qPCR

Your source for answers to the complex challenges of cell and gene therapy development.

As the rate of cell and gene therapy development increases, Labcorp Drug Development has continued to invest in the technology, capacity and expertise globally to support the evolving and complex analytical needs of our clients.  We recently spoke with Hazel Staton, Lead Scientist, BioPharm CMC at Labcorp Drug Development, to learn about droplet digital PCR (ddPCR), how it compares to qPCR, and where the technology can support work in Chemistry, Manufacturing and Control (CMC), preclinical biodistribution and a range of clinical applications (shedding, tracking, adventitious agent detection and the testing of replication competent viruses) to advance complex cell and gene therapies.

For those learning about this topic, can you discuss the differences between ddPCR and qPCR?

Hear from Hazel

Quantitative PCR (qPCR) is already a very powerful, robust and sensitive molecular tool that is used frequently in the industry to quantify DNA and RNA sequences to support the development of C&GT molecules. While the implementation of ddPCR is relatively new, the concept of droplet PCR was first described in the early 1990s which improved on the qPCR technology by using a more statistical based analysis endpoint. Later in the decade, the dilution and partition methods were also improved and optimized, becoming what we know as ddPCR today.

Droplet digital PCR (ddPCR) refined qPCR technology by partitioning the sample and reaction into many thousands of droplets, each of which perform their own PCR reaction instead of having single qPCR reactions. Each droplet is then “counted” by a reader that detects the increase in fluorescence and assigns a binary code, signifying the presence or absence of nucleic acid. ddPCR uses the Poisson statistical method to determine total DNA content of a target sequence, rather than back calculating against standard curve, which is used in traditional qPCR.

What is important to know about qPCR and ddPCR performance?

Hear from Hazel

As compared to qPCR methods, ddPCR brings improved precision and accuracy without the need for subjective and variable standard curves that can lead to PCR bias. This added precision and accuracy is a very good fit for genomic titer CMC assays or copy number variation type assays, but is also now being used to support the preclinical and clinical development of cell and gene therapy molecules.  The reactions in ddPCR also have more tolerance to potential inhibitors due to the method of partitioning. In addition, there is an increased chance of detecting rare mutations in the background of wild type DNA or detection of low copy numbers of target DNA in complex clinical matrices.   

While it is generally thought that ddPCR is more sensitive than qPCR, our experience is that a well-developed and validated qPCR assay, which can detect and quantify target nucleic acids up to 5x10e⁸, can be just as sensitive. In comparison, ddPCR has a slightly smaller dynamic range of ~1x10e5, so care should be taken when samples are diluted prior to ddPCR testing to preserve the precision and accuracy of this method.

Which applications are best suited for ddPCR?

Hear from Hazel

CMC is one of the main areas where ddPCR is beneficial for determining the quantity and titer of cell and gene therapy molecules that are being manufactured, also allowing the ability to monitor for batch-to-batch variations and changes to the manufacturing process. Regulatory scrutiny is driving the demand for very accurate and precise assays to show that preclinical species and patients are being dosed with the correct concentration.

In the clinical phase, ddPCR is beneficial for complex gene- and cell-based therapies given the requirement to monitor for a variety of different aspects related to the molecule as well as the manufacture of the therapy within a clinical setting. This can include analyzing patient samples for vector shedding or monitoring for any replication competent retro or lentiviruses used in the manufacture of CAR T-cells. With the expected low levels of these targets and inhibitory matrices such as saliva, urine or stool, ddPCR is considered a good fit for this type of analysis.

What about using ddPCR during to support preclinical efforts?

Hear from Hazel

In the preclinical biodistribution studies, used to support the IND-enabling package for cell and gene therapy molecules, ddPCR has potentially limited applicability given the small dynamic range of the assay, so care needs to be taken when diluting unknown/highly concentrated samples prior to analysis.  Also, the inherent variability observed between organs, animals and dose groups following extraction and sample analysis would suggest the increased precision and accuracy of the ddPCR will add little benefit to outcome and conclusion of the preclinical biodistribution studies. In this case, a well-designed and validated qPCR assay would likely be more suitable.

If we needed a very accurate relative quantification of the target compared to a housekeeping gene—especially for a multiplex assay—or if the vector is being developed to deliver multiple therapeutic genes and either a silencing gene or gene editing function, then ddPCR could be a good fit.

Do you follow regulatory guidance to determine which assay is most appropriate?

At Labcorp Drug Development, we apply our extensive expertise with qPCR and ddPCR assays to design suitable development and validation studies. The analytical requirements are dependent on the stage of development of cell and gene therapies as well as the context of use. However, the core development and validation parameters remain unchanged regardless of designing a ddPCR or a qPCR assay.

Regulatory guidance is still under development for these key analytical tools in the context of developing cell and gene therapy molecules, despite the increased scrutiny from regulatory agencies. To address this gap, we are currently engaging external working groups to develop industry-wide guidance in addition to evaluating the recent BMV guidance focused on qPCR and ddPCR to best understand its applicability to our clients’ programs.

Any thoughts on upcoming changes in analytical methods to support the development of cell and gene therapies?

Hear from Hazel

We’ve seen that well-developed and validated qPCR is still frequently used in the clinical environment, but we believe that ddPCR will have a growing role in this space due to its ability to potentially overcome inhibitors and increase the chances of picking up low-level copy numbers.

We will continue to evolve our analytical methods that support these molecules as the industry progresses. For example, as AAV technology or gene therapy advances with molecules designed to deliver multiple therapeutic genes, mRNA knockdown or gene editing, ddPCR will be used more frequently. It’s a better fit for more multiplex applications as it has no PCR bias, which can impact qPCR assays. ddPCR might even be used in conjunction with other powerful molecular tools such as next generation sequencing to provide more information.

Finally, as CAR T-cell products continue to develop and implement gene editing in an attempt to improve safety and efficacy, ddPCR will continue to play a critical role in supporting the CMC and clinical studies, where more powerful and appropriate analytics are needed.  

Learn how Labcorp Drug Development is investing in key technologies and additional capacity to support preclinical and clinical cell and gene therapy development by visiting drugdevelopment.labcorp.com/cgt