As demand for cell and gene therapy continues to increase, so too does the demand for viral vectors and plasmid DNA development. Gene and cell therapies utilize a variety of viral vectors for gene transfer, including adeno-associated viruses (AAV), lentiviruses, retroviruses, herpes viruses, adenoviruses, and others. Among them, AAV-mediated gene therapy is garnering the most interest due to its safety profile since infection with the vector is not pathogenic, and AAV cannot replicate on its own. The AAV viral vectors, as well as the messenger RNAs produced from plasmid DNA template, are the critical intermediates that produce and carry the chimeric antigen receptor (CAR) to T-cells enabling CAR-T therapies.
For more information about digital PCR applications for cell and gene therapy workflows, explore the QualTrak qPCR & dPCR interactive ecosystem.
Controlling the concentration of a potential therapeutic is difficult since viral particles are produced in living cells and then purified. The process of generating and purifying viral vectors is also susceptible to contamination by host cell DNA, mycoplasma, and other contaminants. Viral titer must be measured accurately, precisely, and consistently to ensure correct formulation. Harmful adventitious agents (virus, bacteria, and fungi) and contaminant DNA must be detected and screened out with the highest sensitivity to ensure product quality and safety.
While real-time PCR remains the gold-standard tool for the quantitation of therapeutic viral vectors such as AAV used in cell and gene therapies, digital PCR (dPCR) offers significant advantages for quantification of target DNA molecules in a range of analytical assays necessary for viral vector production and characterization.
Unlike real-time PCR, dPCR can provide an absolute count of nucleic acids, enabling the precise quantification of AAV vectors, bacterial contaminants, and residual host cell DNA. No reference standard is required, improving precision and removing a source of variation. The variability in the standard curve used for real-time PCR is often due, in part, to the different architectures of the relevant DNA species: circular, supercoiled, linear DNA, or viral particles. dPCR is also less sensitive to contaminants that affect amplification, including those present in solutions used during the development of AAV-mediated gene therapies.
Applied Biosystems Absolute Q Viral Titer dPCR assays enable easy and accurate quantification of viral vectors. The assays can be run individually or multiplexed using a custom assay with your target gene of interest to measure concentration and evaluate quality for biopharma and gene therapy research.
Absolute Q dPCR assays are:
We guarantee the performance of all of our predesigned Absolute Q assays for dPCR experiments. Our application‑specific portfolio of assays enables you to obtain the highest quality and performance available. These assays are designed and verified using up-to-date annotations and gold-standard Applied Biosystems TaqMan chemistry.
If an Absolute Q Digital PCR Assay does not perform according to conformance documentation, we will replace it at no cost or credit your account.*
Figure 1. Quantification of AAV and CMV viral vector dilution series on the QuantStudio Absolute Q dPCR System.
Figure 2. Quantification of AAV and CMV viral vector dilution series across multiple digital PCR platforms using the Absolute Q AAV and Absolute Q CMV dPCR Assays.
Unlike other dPCR methods that rely on stochastic processes to generate micro-reactions by droplet formation, microfluidic array plate (MAP) technology facilitates automated sample distribution consistently into over 20,000 microchambers and utilizes >99% of the bulk reaction to minimize sample loss. Here we demonstrate how the increased robustness and consistency possible with MAP technology improves AAV viral titer quantification by performing comparison experiments using the QuantStudio Absolute Q Digital PCR system, as well as a comparison performed using a droplet digital PCR instrument.
Quantification of genomic copies (GC/mL) is important for characterizing rAAV. While quantitative PCR (qPCR) is considered the gold standard method for determining the genomic titer, this approach requires the use of standard curves and is thus more susceptible to variation from run to run. dPCR provides significant advantages for analytical assays necessary for viral vector production and characterization without the need for a standard curve. dPCR is also less sensitive to contaminants that affect amplification, including those present in solutions used during the development of AAV-mediated gene therapies.
Can’t find the assay you’re looking for? Contact us and our experts can help design a digital PCR assay formulated for success based on your target sequence of interest. Or submit the primer/probe sequences of your own design.
Absolute Q DNA Digital PCR Master Mix (5X) is optimized for use with the QuantStudio Absolute Q Digital PCR System and Absolute Q digital PCR assays in a simple workflow with minimal processing steps. The 5X formulation enables analysis of higher sample volume and delivers accurate quantification of DNA targets without using a standard curve.
Key product features:
Absolute Q 1-step RT-dPCR Master Mix (4X) is optimized for use with the QuantStudio Absolute Q Digital PCR System. The mix is designed as a single‑tube, one‑step formulation for fast, simple, and reproducible RNA quantification, requiring minimal upstream processing steps.
Key product features:
For Research Use Only. Not for use in diagnostic procedures.