Optimisation of adeno-associated viral vector production using Design of Experiments

Abstract

Adeno-associated virus (AAV) vectors are widely used in in vivo gene therapy. The increasing popularity leads to demand for higher manufacturing capacities. The yield achieved with the most common production method, triple transfection, is depended on optimisation of various factors. Design of Experiments (DoE) aids efficient planning, implementation, and analysis of experiments. Here, triple transfection factors were optimised with DoE to maximise vector yield. Simultaneously, this was a proof-of-concept project to assess feasibility and potential of large-scale DoEs for wider adaption at Biovian. Transfections were done with mammalian suspension cells grown in shake flasks. Viral titers were measured with droplet digital PCR from crude lysates prepared 48 and 72 hours post-transfection (hpt). The 72 hpt results are reported here. By implementing a central composite orthogonal design of 29 runs, a 65% increase in yield was achieved compared to manufacturer-recommended conditions (2.76E+10 vg/ml vs 4.56E+10 vg/ml). Complexation volume (p < 0.0001) is the most significant factor increasing the yield. Under the model of titer, we could predict yields up to 5.47E+10 vg/ml with the following conditions: 0.77 µg of plasmid DNA/1E+6 cells, 1.2 µl of transfection reagent/µg of plasmid DNA, 1.15E+6 viable cells/ml, 10% complexation volume and 30 min complexation time. In conclusion, the first large-scale DoE trial at Biovian was successful. However, analysis of yields from 48 hpt is still needed to estimate the possible noise in 72 hpt results due to depletion of cellular nutrients. After that, a confirmatory experiment can be planned with optimised factors.