Akta avant

Genes encoding heavy chains and light chain were inserted separately into pcDNA3.4 and amplified in E. coli DH5α. PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen) was used for low endotoxin plasmid preparation. Monoclonal antibodies were transiently expressed by co-transfecting ExpiCHO-S cells (ThermoFisher) with heavy chain and light chain plasmids using an ExpiCHO™ Expression System (Gibco). Cell culture was harvested after an 8–14 day of incubation at 37 °C with humidified atmosphere of 8% CO₂ with shaking. Full-length IgG was obtained by affinity purification utilizing a Protein A chromatography column (GE Healthcare) in AKTA avant (Cytiva). For long-term storage, antibodies were kept in a solution containing 10 mM Histidine-HCl, 9% trehalose, and 0.01% polysorbate 80.

An AAV6 cell paste slurry was generated using standard cell culture techniques and stored at −80°C. Capto AVB resin was sourced from Cytiva. C0SP filters were obtained from Millipore‐Sigma. Sartopore 2 XLG, 0.8/0.2um filters were obtained from Sartorius Stedim Biotech GmbH (Gottingen, Germany).
For detergent lysis, the AAV6 cell paste was diluted to a 10% (w/w) slurry with 20 mM Tris, 200 mM NaCl, 2 mM MgCl2, pH 7.5. A 10% (w/w) detergent stock solution was spiked into cell slurry to bring the final detergent concentration to 0.5% (v/w). Benzonase was spiked to 10 U/ml into cell slurry mixture and mixed slowly at room temperature for 1 h.
AAV purification was performed using a Capto AVB affinity column. Purification was controlled by Unicorn 7.0 software on an AKTA Avant purchased from Cytiva. A C0SP POD filter was flushed with 20 mM Tris, 500 mM NaCl, 2 mM MgCl2, pH 7.5. 10% AAV cell lysate was clarified with C0SP followed by Sartopore 2 XLG, 0.8/0.2um filter. The Capto AVB column was equilibrated with 20 mM Tris, 200 mM NaCl, 2 mM MgCl2, pH 7.5 for 3 column volumes. Clarified cell lysate was loaded with residence time of 6 min. The column was re‐equilibrated with 20 mM Tris, 200 mM NaCl, 2 mM MgCl2, pH 7.5 for 4 CV. Product was eluted from column with 50 mM citric acid, 500 mM NaCl, 2 mM MgCl2, pH 2.5. Eluate pH was adjusted to pH 8.0 with 1 M Tris/HCl, pH 8.0.

The aliquots from the depth filtration experiments above were used for the Protein A lifetime studies. For each condition, 100 cycles were performed. The studies were conducted using a 1 ml HiTrap column prepacked with MabSelect Sure LX and the AKTA Avant (Cytiva) chromatography system. Eluates were collected every cycle and neutralized with 200 μl 2 M Tris‐Base. Precipitation was observed in some loading material and was removed by centrifugation at 500 rpm for 5 min. All feeds were clarified using a 0.2 μm syringe filter before loading.