Application Note

Radiance® Label-Free Monitoring Of AAV Transfection In HEK293 Cells Using Laser Force Cytology™

Source: Catalent
DNA gene patents and biotechnology

Gene therapy, which delivers DNA into a patient’s cells to replace or add genes that may be abnormal or missing, is delivering results in the clinic after 30 years of development. With the FDA approval of the second adeno-associated virus (AAV) based gene therapy treatment in May of 20191, it is truly an exciting time for the advanced therapeutics. AAV has proven to be a powerful tool for gene therapy purposes for its broad tropism, inability to replicate on its own in vivo, minimal immunogenicity, and ability to deliver effective and long-lasting results2. However, there are several challenges related to the development and manufacturing of AAV-based processes, including characterization, quantification, and downstream purification3,4. Manufacturing challenges in particular include the need for consistently high purity, potency, and safety for AAV products, all while maintaining acceptable large-scale manufacturing costs5. While there are several methods for AAV production, including transient transfection in human (HEK293) cells6, baculovirus driven production in insect (Sf9) cells7, or recombinant helper viruses such as HSV in mammalian cells8,9, the importance of precise and predictive analytics remains regardless of the production system. LumaCyte’s Radiance® instrument, which uses Laser Force Cytology™ (LFC) to measure the intrinsic biophysical and biochemical properties of single cells10,11, has the potential to improve the characterization of cellbased AAV transfection and production, improving the efficiency and accuracy of both processes and shortening development time. As a leader in the gene therapy field, Catalent Cell and Gene Therapy strives to incorporate the most advanced analytics in its processes in order to provide high quality, innovative solutions for its customers. In this technical note, collaborative efforts are described between Catalent and LumaCyte to compare AAV production with three different transfection reagents using both LFC and a digital droplet PCR (ddPCR) based viral genome titer assay. A strong correlation was generated between the LFC based measurements, which are available in near realtime (5 minutes analysis time per sample) and the ddPCR results which take significant time and labor, demonstrating the utility of LFC for rapid process monitoring. Additional applications of LFC throughout the AAV production process include adventitious agent monitoring to rapidly detect potential contamination as well as cell line characterization during process development and scale-up.


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