Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.