Faster, Scalable dPCR Method

Laura Mohr explains how plate-based dPCR can accelerate gene therapy manufacturing

In vivo gene therapies represent life-saving treatments for millions of people; but to realise the full potential, recombinant adeno-associated virus (rAAV) vector manufacturing must reach scales beyond what’s possible today. This creates an urgent need for new processes that improve the purity, potency and yield of rAAV vectors. A precise and reproducible vector genome titer quantification is crucial in viral vector production, as clinical dosing of rAAV depends on vector genome concentration.

Quantitative PCR (qPCR) is a widely used method for AAV quantitation thanks to its sensitivity and ease of use. When performing absolute quantitation with qPCR, DNA standards of known concentration are needed to plot a standard curve, which is then used to extrapolate the titer of the unknown sample. To ensure accuracy, these standards must be monitored for degradation and quantitated accurately using a secondary method, typically spectrophotometry. Additionally, choosing a proper quantification standard that amplifies similarly to the AAV vector is challenging.

Digital PCR (dPCR) provides an absolute count of nucleic acids, enabling the precise quantification of AAV vectors without the need for a standard curve, improving accuracy compared to qPCR. dPCR also remains unaffected by inhibitors, contaminants and adventitious agents co-purified with viral vectors that might otherwise affect amplification efficiency.

The method is based on partitioning a sample into thousands of individual reactions before PCR amplification. Each partition might contain variable copies of the target molecule. Each partition undergoes amplification to the end-point using target-specific primers and fluorescent probes or dye. Following amplification, the partitions are analysed for the presence (positive) or absence (negative) of the fluorescence signal. The ratio of positive to negative partitions is calculated, and the Poisson distribution is used to determine the absolute quantity of initial target DNA/RNA in the sample. Partitioning can be achieved in droplets based on oil-water emulsions, on a microfluidic chip, separation onto microarrays, or in qPCR-like microfluidic plates.

Droplet digital PCR (ddPCR) is the currently used technique, offering a complete AAV viral titer workflow solution with an established track record, assay availability, GMP-compliant software for data analysis, etc. However, the workflow is laborious and limited by throughput.

The Modern Approach

A recent development in the field has been the introduction of the QIAcuity nanoplate-based digital PCR system that closely resembles the easy sample handling of qPCR and generates the same level of accuracy and precision as the currently used ddPCR system but with much faster sample-to-result times (2h versus 7h) and higher overall throughput and scalability.

Nanoplates are available with approximately 26,000 partitions for applications where the target is rare and/or diluted or 8,500 partitions in a 96-well format for high-throughput use with a concentrated template. Since AAV is usually produced with titers at or above 1x1,012 genome copies/ml, the nanoplate with 8,500 partitions is a good fit for this application. Using a single QIAcuity eight-plate instrument, it’s possible to load 16 plates in an eight-hour shift for 1,536 wells. The instruments can detect fluorescent signals in five different channels, allowing even more multiplexing, which may be valuable in further qualifying AAV genome integrity. Existing, well-characterised qPCR assays can typically be transferred to dPCR on the nanoplate-based system with very little optimisation. The system is designed to meet the biomanufacturing and quality control needs with software to assist with US FDA 21 CFR Part 11 compliance required in a GMP setting, audit trails, user management, installation qualification/operational qualification (IQ/OQ) service and services such as rapid on-site response time.

The nanoplate-based system has the potential to play a major role in the development of new manufacturing processes by keeping pace with the analytical demands of iterative process refinements. By exploring integration with high-throughput liquid handlers and multi-condition bioreactors, biopharma companies can further shorten time-to-data for rAAV process development and accelerate the scale-up of gene therapy products.

Laura Mohr is with Qiagen

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