BackgroundIn recent years, gene therapy drugs have been widely marketed, and their effectiveness and potential have been confirmed. Thus, increasing their production on an industrial scale is critical. Recombinant adeno-associated viruses (rAAVs) are optimal vectors for gene therapy applications, and the baculovirus expression vector system (BEVS), which is based on Sf9 cell culture, is a common tool for rAAV production.MethodsIn this work, an Sf9 cell fed-batch process was developed using shake flasks. In the laboratory-scale bioreactor, four processes were selected as the key factors when carrying out the orthogonal experiment. On the basis of the equal P/V principle and considering the problem posed by air bubbles, a pilot-scale level bioreactor process was established.ResultsHere, we describe a method in which a BEVS was used to produce rAAV vectors, with the cell density increasing to 22.8 × 106 cells/mL and the rAAV titre increasing to 20 × 1011 VG/mL upon adding feed material. By resolving the problems associated with high-density cell culture and air bubbles, this process was successfully scaled to a 50 L pilot-scale level.ConclusionsThis successful experiment not only provides a technological basis for further scale-up but also guarantees product capacity. We hope that this development process can provide reference data for studying cell culture-based drug production.