ddPCR™ Technology: Bringing New Power to the RNA Therapeutics Revolution

By Marwan Alsarraj, Biopharma Segment Manager, Digital Biology Group

The advent of RNA-based therapeutics has offered new hope for reaching previously undruggable targets. The ubiquitous role of RNA in physiology gives these therapies potential for a wide field of applications, from vaccines to personalized cancer treatments. It's a hot topic, with more than 18,000 publications on RNA therapeutics as of 2021. While many advancements have been made, the field is still new, so research is still focused on growth, establishment, and standardization to optimize processes that will make these drugs safer and more viable on a larger scale.

The intricacies of development and manufacturing are among the primary obstacles to the more widespread use of RNA biotherapeutics. Developing and testing these drugs demands unique and comprehensive RNA analyses in cells and therapeutics, many of which can't be adequately addressed using traditional qPCR assays. However, Droplet Digital™ PCR (ddPCR™) technology is well-suited to provide the precision and accuracy necessary for developers to be confident in their products without sacrificing workflow efficiency.

Here, I'll outline some of the strengths that set ddPCR technology apart in tackling the challenges of RNA therapeutic development and manufacturing.

Absolute quantification means more confidence from discovery to dosing.

Eliminating the need for calibration to a standard curve in the analysis is one primary differentiator of ddPCR technology over traditional qPCR. In addition, because the PCR-positive and PCR-negative droplets in a sample are individually counted in the ddPCR workflow, the number of target molecules initially present in the sample can be quantified through Poisson distribution analysis.

This absolute quantification makes all the difference for the development and validation of RNA therapeutics. Accurately quantifying RNAs and gene expression in cells at baseline is necessary to identify potential therapeutic targets. At the same time, re-evaluating these levels after treatment establishes a clear picture of a potential product's therapeutic effects.

Absolute quantification also enables precise determination of the ratio between drug species in a multivalent RNA therapeutic. For therapeutics in later stages of development, absolute quantification of RNA in the drug product gives developers greater confidence in analyzing biodistribution and accurately establishing safe and effective dosing. While qPCR assays generate estimated RNA counts, absolute quantification using ddPCR technology can offer the security of greater accuracy in creating and delivering RNA therapeutics.

Unparalleled sensitivity enables more comprehensive quality control.

Production of RNA biotherapeutics can present unique challenges in ensuring the quality and purity of the drug product. Here, the stakes of quality control are particularly high, as even minute levels of contamination or product inconsistency can compromise the safety and efficacy of a therapeutic. The ultrasensitive detection capabilities of ddPCR technology are thus well-suited to this type of analysis. With traditional qPCR, small quantities of target RNA can be obscured by high-copy templates and background noise—the massive sample fractionation of ddPCR increases the signal-to-noise ratio, enabling sensitive detection of rare target sequences.

This sensitivity enables precise detection and quantification of low copy numbers among samples, determination of poly(A) tail levels, and other measurements critical in RNA biotherapeutic development. For example, Pfizer/BioNTech used ddPCR for poly(A) tail analysis to manufacturing their COVID-19 mRNA vaccine. Highly sensitive ddPCR assays can also detect trace contamination by residual host cell DNA or adventitious agents such as mycoplasma, ensuring product safety and quality.

Increased performance in a simple workflow

The benefits of ddPCR in RNA therapeutic development don't compromise workflow efficiency or simplicity. Absolute quantification capabilities reduce the variability of results, meaning greater consistency from run to run throughout the development process. Additionally, users can run up to 96 individual samples per plate without reducing precision or accuracy, as each sample is partitioned into 20,000 individual PCR reactions. Each sample thus generates thousands of discrete measurements rather than just one individual measurement, as in the case of traditional qPCR. As a result, users can obtain deeper, more precise data from a single ddPCR assay than is possible with qPCR while still performing a comparable workflow.

Looking forward to increased adoption

Since its creation, ddPCR technology has demonstrated incredible utility in providing more sensitive and accurate detection and quantification of nucleic acid targets. These capabilities are invaluable to the RNA therapeutics revolution, helping to ensure that the field can reach its full potential. From identifying sequences of therapeutic interest to ensuring product safety at each step of manufacturing, ddPCR technology is helping developers overcome the unique challenges of developing and creating RNA biotherapeutics, bringing these revolutionary drugs closer to changing lives.

To find or design custom ddPCR assays for any RNA therapeutic, visit our website >