Expi293 cells were transferred to a 1 L shake flask at 2.5E6 viable cells/mL and 200 mL working volume. (pAdDeltaF6, pAAV-GFP, pAAV2/2, pAAV2/5, pAAV2/8, and pAAV2/9n) In each 1 L flask, 1.57 mg total DNA/10E6 cells were diluted in Opti-Plex. Complexation Buffer to 10 mL (5% culture volume) at a 1.5:1:1 (rAAV5, -8, and -9) or 5:1:0.31 molar plasmid ratio (rAAV2) of pRep2/CapX:pAdH:pEGFP. FectoVIR-AAV was added to plasmid mixtures at a 1.35 mg DNA/mL ratio, followed by mixing and a 30-minute complexation time. The mixtures were added to the cells and the flasks were placed in an incubator at 135 rpm, 37C, 80% RH, and 5% CO2 prior to being harvested at 72 h post-transfection Another 2x1 L flask of Expi293 cells was transfected with only the pEGFP plasmid.

Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, biolayer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalizing the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet AAVX biosensors across four rAAV serotypes (rAAV2, -5, -8, and -9) and applied in an upstream and downstream processing context. AAVX-BLI measured the capsid titer across a wide concentration range (1 × 10¹⁰-1 × 10¹² capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for the total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS CaptureSelect AAVX affinity chromatography, showing resin breakthrough dependence on the operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.