Application Note: Real-Time Live-Cell Analysis of Lentiviral Titer Determination

As efficient gene delivery vehicles, viral vectors play an important role in gene therapy and gene-modified somatic cell therapy products.1 Several viral vector platforms exist—including adenoviral vectors, adeno-associated vectors, and retroviral vectors (e.g. lentiviral vectors)—each with advantages and disadvantages for specific applications. Lentiviral vectors (LV) represent the most frequently used viral gene delivery platform for the ex vivo generation of chimeric antigen receptor (CAR)-T cells for cancer immunotherapies.2 

Functional infectious titer determinations of viral vector samples are critically important. Vector quality control is a significant bottleneck for viral vector process development and production. To expedite the upstream and downstream development of viral vector production, reliable and efficient assays for quantification are required. Therefore, a method to determine lentivirus infectious titers rapidly and accurately is needed for both process development and process optimization phases where a high number of samples are typically generated.

Established and beneficial methods for infectious viral titer measurements include qPCR and flow cytometry. However, both workflows lack high throughput and require significant manual handling. To address the bottlenecks of current analytical methods, we developed a novel, real-time, imaged-based approach to quantify the infectious titer of anti-CD19 CAR lentiviral vectors using the Incucyte® Live-Cell Analysis System.

This application note describes the utility of the Incucyte® Live-Cell Analysis System in combination with Incucyte® Fabfluor-488 Antibody Labeling and Incucyte® Cytotoxicity Dyes to enable kinetic quantification of functional lentiviral titers in live cells.

Key discussion topics include: 

  • A simple, rapid, non-invasive approach for high-throughput functional quantification of infectious titers 
  • How real-time, kinetic readouts and integrated software enable viable transduced cell tracking over time
  • Exploration of real-time titer insights, in-process monitoring, and lentiviral vector production optimizations 
  • Assay flexibility and applicability to determine optimal processing conditions for improved viral vector development 
  • And more…

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1 Hanna, E., Rémuzat, C., Auquier, P., and Toumi, M. 2016. Advanced therapy medicinal products: current and future perspectives. Journal of market access & health policy 4.

2 Rininger, J., Fennell, A., Schoukroun-Barnes, L., Peterson, C., and Speidel, J. 2019. Capacity analysis for viral vector manufacturing: Is there enough? BioProcess international 2019.

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