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P48

Optimisation of a high throughput residual DNA Picogreen assay for analysing lysate samples from AAV production without GFP interference. 

F Chowdhury(1) W Li(1) N Sweeney(1)

1:Cell Therapy Catapult

Adeno-associated viruses (AAV) are commonly used gene therapy vectors. Production of AAV happens within a producer cell line. During the harvest stage, nuclease treatment is carried out to digest residual DNA. Regulatory guidelines recommends that residual DNA should not be above 10ng/dose within administered drugs. Assays used to measure this are PicoGreen® and residual DNA qPCR. GFP is commonly used as a reporter gene in AAV process development work. During such AAV production, the producer cell line is transfected with GFP plasmid, which leads to the expression and presence of GFP protein in lysate. This causes an interference with the measurement using the picogreen dye due to them sharing similar emission wavelengths. This results in an inaccurate reading for the residual DNA concentration.  

 

We have developed and tested a streamline workflow to apply the WAKO purification kit for the purification of DNA in lysate samples prior to the picogreen assay. The WAKO kit purifies DNA through first solubilizing the proteins and lipids within the samples, and then precipitating the DNA with glycogen. It demonstrated good repeatability with a CV<20%, with some evidence of linearity and accuracy. The lack of signal from the purified samples in the absence of the picogreen dye suggests that GFP interference has been completely removed by the WAKO kit. Hence, the WAKO purification kit allows the measurement of residual DNA concentration from lysate samples using the picogreen assay free of GFP interference, which facilitates the high-throughput optimisation of nucleic acid removal strategies. 

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