Scaling up virus production without risking robustness

At CEVEC Pharmaceuticals

A rapid growth in the number of gene therapy products has led to an equal increase in the need for the industrial production of suitable viral vectors. Current production systems often lack scalability and robustness, however.

CEVEC Pharmaceuticals’ CAP-GT cell lines provide a novel platform for the industrial production of viral vectors. CAP-GT cell lines are fully documented and comply with regulatory requirements. Growing in serum-free suspension culture at high densities, the cells provide a fully scalable vector production platform with reproducible transfection and high-efficiency production of viral vectors.

The precise detection of GFP transfection efficiency is a crucial step during this process, but the methods available are often not sufficiently robust and are time and labor intensive. Thus, the GFP transfection efficiency at CEVEC is routinely determined using the NucleoCounter® NC-3000™ advanced image cytometer, which ensures reproducibility and fast screening of multiple samples.

Optimized workflow increases lentiviral titers

Flow showing that the NucleoCounter® NC-3000™ was used to determine the GFP Transfection Efficiency and optimize the virus culture system. The results are displayed as bar plots.

To optimize lentiviral vector titers in CAP-GT cells, CEVEC used a design of experiments approach to determine optimal cell density, and DNA and transfection reagent amounts. They also tested addition of lipid supplements and analyzed the influence on viral production. Using the optimized conditions, they achieved a ten-fold increase in lentiviral vector production. This high-density culture system is scalable to any current bioreactor format.

GFP transfection efficiency with the NucleoCounter® NC-3000™ – fast & easy

Handling a lot of samples requires reliable and fast quantification of cell numbers and GFP expression. With the bench-top NucleoCounter® NC-3000™, GFP fluorescence can be easily determined. In a few minutes, the NC-3000™ reports both transfection rate and cell death. All signals are automatically quantified, and results are displayed via tables, histograms and scatter plots.

With the NucleoCounter® NC-3000™, you get detailed and reproducible results fast, and you can perform detailed visual inspections of your cells.

See the full dataset in the PDF poster.

Images of the viable, dead, and GFP expressing cells (left) and GFP Transfection Efficiency Assay results in the plot manager of the NucleoView™ software (right).

Conclusion

Conditions for lentiviral production using the CAP-GT platform were optimized by a design of experiment approach in regard to cell density and DNA and PEI amount. Lipid supplementation significantly increased production of infectious LV particles. A scalable protocol without medium exchange step was developed using CD-FortiCHO medium. The optimization process resulted in significantly improved titers, also compared to adherent HEK293T cells. Using the NucleoCounter® NC-3000™, fast and reliable quantification of cell numbers and GFP expression for a large amount of samples was possible.

Authors

Martina Graßl, Kerstin Hein, Simon Fradin, Helmut Kewes and Nicole Faust.