AAV production and harvest – Batch A AAV2, AAV5, and AAV8 batch A were generated by transient transfection of HEK293 cells (Expi293F Inducible Cells, Thermo Fisher Scientific, Waltham, MA) in non-baffled glass shake flasks in FreeStyle 293 Expression Medium (Thermo Fisher Scientific). AAV production processes were inoculated at a cell density of 3x10^5 viable cells/mL. Upon achieving a cell density of 1.3x10^6 viable cells/mL, transfection was carried out using a two-plasmid system (PlasmidFactory, Bielefeld, Germany), with 1 mg/mL DNA per 10^6 viable cells and FectoVIR-AAV (Polyplus, Illkirch, France) as a transfection reagent in a 1:1 ratio. AAV vectors were harvested 72 h after transfection. For cell lysis, the cell broth was treated with Tween 20 (Sigma-Aldrich, Darmstadt, Germany), Denarase (c-Lecta, Leipzig, Germany), and MgCl2 at final concentrations of 0.5%, 10 U/mL, and 2 mM, respectively, and incubated at 37°C for 1 h. Subsequently, the cell lysate was centrifuged at 800g for 5 min. AAV production and harvest: Batch B AAV2, AAV5, and AAV8 Batch B were generated by transient transfection of HEK293 cells (Expi293F Cells, Thermo Fisher Scientific) in 2 L bioreactor scale in HEK ViP NB Medium (Sartorius Xell, Schloß Holte-Stukenbrock, Germany). Cells were seeded at a cell density of 3x10^5 cells/mL on day 0 in a preculture/N-1 bioreactor. On day 3, AAV production processes were inoculated at a cell density of 2x10^6 viable cells/mL. Transient transfection was performed 24 h after seeding using a two-plasmid system (PlasmidFactory), with 1 mg/mL DNA per 10^6 viable cells and FectoVIR-AAV as a transfection reagent in a 1:1 ratio. For cell lysis 72 h after transfection, the culture was continuously stirred (1 h, 37°C) after addition of a Tergitol TMN-100x-based lysis buffer (20 mM MgCl2, 500 mM Tris, Tergitol TMN 1% [pH 7.5]) and Benzonase (both Sigma-Aldrich) at a final concentration of 25 U/mL. Subsequently, the cell lysate was centrifuged at 4,000g for 30 min

Adeno-associated virus (AAV) vectors are among the most prominent viral vectors for in vivo gene therapy, and their investigation and development using high-throughput techniques have gained increasing interest. However, sample throughput remains a bottleneck in most analytical assays. In this study, we compared commonly used analytical methods for AAV genome titer, capsid titer, and transducing titer determination with advanced methods using AAV2, AAV5, and AAV8 as representative examples. For the determination of genomic titers, we evaluated the suitability of qPCR and four different digital PCR methods and assessed the respective advantages and limitations of each method. We found that both ELISA and bio-layer interferometry provide comparable capsid titers, with bio-layer interferometry reducing the workload and having a 2.8-fold higher linear measurement range. Determination of the transducing titer demonstrated that live-cell analysis required less manual effort compared with flow cytometry. Both techniques had a similar linear range of detection, and no statistically significant differences in transducing titers were observed. This study demonstrated that the use of advanced analytical methods provides faster and more robust results while simultaneously increasing sample throughput and reducing active bench work time.