BIOTECHNOLOGY
New solutions can enable scaling gene therapy manufacturing
DPCR METHOD FASTER, SCALABLE
Laura Mohr explains how plate-based dPCR can accelerate gene therapy manufacturing
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n 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. Tis 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
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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
Measuring viral titer in gene therapies
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. Te 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. Te 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.
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