Digital PCR: Effective application in cell and gene therapy manufacturing and beyond

Digital PCR (dPCR) is a breakthrough technology for nucleic acid detection and absolute quantification that involves partitioning samples into thousands of individual real-time PCR reactions. After end-point amplification, each partition is analyzed for the presence or absence of a fluorescent signal, and the absolute number of target molecules is calculated without the need for a standard curve.​

By partitioning samples, dPCR mitigates the effects of target competition and is more tolerant to PCR inhibitors than quantitative PCR, making it particularly well suited to rare sequence detection, copy number variation analysis, and the quantification of low-abundance targets in complex samples. ​In the context of cell and gene therapy, dPCR can play a pivotal role in several critical workflows, from identifying sequences of therapeutic interest to evaluating viral vector potency and detecting trace contamination by residual host cell DNA or adventitious agents such as Mycoplasma.​

Below, we delve into these applications in detail through a curated collection of explainer videos, insightful articles, and free-to-download resources. We also explore the unique features of Bio-Rad's Droplet Digital PCR (ddPCR) technology and share success stories from researchers who have already reaped the benefits of ddPCR in their work.​

Troubleshooting

Quality control

Good versus bad dPCR data

There are several dPCR instrument options on the market, but not all systems produce the same quality of results. Learn how to distinguish high-quality from low-quality dPCR data, explore potential causes underlying specific data issues, and discover the difference between Droplet Digital PCR and other Digital PCR Systems.

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Achieve accuracy and precision​

Whether evaluating transgene copy number or verifying the absence of microbial pathogens, discover how Bio-Rad's comprehensive range of GMP-ready ddPCR assays can effectively ensure the safety of your cell and gene therapy manufacturing process.​
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Mycoplasma detection​

Testing for Mycoplasma contamination in cell cultures is a crucial step in ensuring the safe production of cell-based vaccines, biologics, and gene therapies. If Mycoplasma is detected, the contaminated batch must be discarded and entire facilities may shut down for sterilization, leading to significant time and financial losses.

​​In the video below, learn more about the challenges of traditional Mycoplasma detection methods and discover how these can be overcome using probe-based ddPCR technology. Capable of detecting up to 112 Mycoplasma species, Bio-Rad’s Vericheck ddPCR Mycoplasma Detection Kit is the first ddPCR-based Mycoplasma testing solution that has been validated to meet European, U.S., and Japanese pharmacopeia requirements. Download the infographic to explore the complete Mycoplasma detection workflow and learn how the kit can be integrated with regulatory-compliant software to auto-gate results, eliminating the need for manual intervention and data interpretation.​

Video: Choosing a mycoplasma detection strategy​

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Infographic: Detecting Mycoplasma contamination​

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Residual host cell DNA detection ​

Residual host cell DNA contamination poses a common challenge in cell and gene therapy manufacturing, requiring meticulous monitoring for lot release. To ensure patient safety, the FDA recommends that manufacturers limit the presence of residual DNA to less than 100 pg per dose, with a DNA size below approximately 200 base pairs.​​The resources below explore the impact of residual DNA contamination and introduce Bio-Rad’s new ddPCR kits, designed specifically for the absolute quantification and sizing of HEK293 residual DNA. Learn how these kits can enable manufacturers to effectively comply with regulatory guidance requirements and minimize false-positive results while avoiding the need for lengthy DNA extraction or purification.​

eBook: Addressing the challenges of residual host cell DNA contamination​

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Infographic: Detecting HEK293 residual DNA contamination​​

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Viral titer determination

From optimizing transduction efficiency and evaluating potency to ensuring appropriate dosing, the precise quantification of viral titer is critical at every stage of cell and gene therapy development.​​

For several years, qPCR was the go-to technology for viral titer determination, but it is neither accurate nor precise enough for gene therapy applications. In contrast to qPCR, ddPCR is an endpoint PCR assay that delivers an absolute count of viral vector genome copies. In the video below, learn more about the advantages of ddPCR technology compared to qPCR for titer determination and see how it can effectively support many aspects of viral vector-based gene therapy development.​

SUCCESS STORY​

“ddPCR confers some major advantages for vector genome quantification, which will be important for the production and use of clinical AAV gene therapy vectors.” Martin Lock Director, Process Development Gene Therapy Program, University of Pennsylvania.​

Read this success story to learn how ddPCR can provide a highly precise method for quantifying AAV genomes, outperforming qPCR in terms of sensitivity, accuracy, and resistance to PCR inhibitors.​

Featured technology:

QX200™ Droplet Digital PCR System

RNA therapeutics quality control​

RNA therapeutics are a rapidly growing new category of drug that can be used to activate or inhibit the expression of protein targets in cells to produce a therapeutic effect. These therapies have huge potential and are poised to provide a rapid, cost-effective, and readily adaptable way to reach previously “undruggable” targets.

​​Despite many advancements, the field is still in its infancy and many challenges remain to be addressed to make RNA therapeutics safer and more viable on a larger scale. From identifying sequences of therapeutic interest and ensuring manufacturing safety to evaluating gene expression before and after treatment, explore how ddPCR technology can help developers effectively address these challenges and bring these revolutionary drugs closer to changing patient lives. ​

A revolution empowered by ddPCR™ ​

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A new approach to an ​old problem​

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RNA: The next therapeutic era​

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​QX ONE™ Droplet Digital™ PCR System​

Additional applications​

The utility of ddPCR extends far beyond cell and gene therapy, covering many different areas of biology. In the field of oncology, ddPCR provides a rapid and accurate tool for detecting an increasing range of cancers, assessing tumor heterogeneity, and identifying novel biomarkers. Moreover, its high sensitivity enables the identification of residual traces of cancer tissue, known as minimal residual disease (MRD), to assess the need for continued treatment and detect potential relapses. ​​

ddPCR's capacity to detect low-abundance targets in complex sample matrices is equally advantageous for environmental studies, such as wastewater-based epidemiology. Bio-Rad offers optimized ddPCR assays designed to detect numerous viruses and bacteria in wastewater, including SARS-CoV-2, norovirus, adenovirus, Legionella, Enterococcus, and emerging variants of concern.
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​ddPCR also plays a pivotal role in multiple stages of vaccine development and manufacturing, including identifying target antigens, determining dosage and potency, detecting impurities, and verifying vaccine-induced immunity.​​

To gain further insight into the application of ddPCR in these fields, download the resources provided below.​

Featured technology: 

QX600 Droplet Digital PCR System

The QX600 Droplet Digital PCR System (ddPCR™) offers advanced six-color multiplexing and absolute quantification of nucleic acid while delivering the best-in-class performance you have come to expect from Bio-Rad’s ddPCR systems.​

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QX ONE™ Droplet Digital™ PCR System​

The QX ONE Droplet Digital PCR System integrates droplet generation, thermal cycling, droplet reading, and analysis into a single automated precision platform that delivers superior precision and accuracy and absolute quantification.​

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Vericheck ddPCR Mycoplasma Detection Kit

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Vericheck ddPCR HEK293 Residual DNA Quantification Kit

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ddPCR Cell and Gene Therapy Assays​

The QX ONE Droplet Digital PCR System integrates droplet generation, thermal cycling, droplet Bio-Rad’s ddPCR Cell and Gene Therapy (CGT) Assays are custom ddPCR-optimized assays generated by the Cell and Gene Therapy Design Engine for your target gene of interest. ​reading, and analysis into a single automated precision platform that delivers superior precision and accuracy and absolute quantification.​