The manufacturing process is based on a transient co-transfection of human embryonic kidney (HEK293) cells. The process is divided into upstream and downstream operations:
Upstream Process:
1. A vial from the qualified HEK293 Working Cell Bank (WCB) is thawed and cells are expanded through a series of passages in cell culture flasks and then bioreactors.
2. At the target cell density, cells are co-transfected with three plasmids: a vector plasmid containing the RPE65 expression cassette, and two helper plasmids providing the necessary AAV and adenovirus helper functions for viral replication and packaging.
3. After a defined incubation period to allow for vector production, the cells and media are harvested.
Downstream Process:
1. Cells are lysed to release the viral particles.
2. The lysate is clarified via centrifugation and depth filtration to remove cell debris.
3. The clarified lysate is purified through a series of column chromatography steps, including affinity chromatography to capture AAV2 particles and ion-exchange chromatography for polishing and removal of impurities (e.g., empty capsids, host cell proteins).
4. The purified vector is concentrated and buffer-exchanged into the final formulation buffer using Tangential Flow Filtration (TFF).
5. The resulting Drug Substance is sterile filtered and stored frozen as bulk material. 
Process Controls: Critical process parameters (e.g., temperature, pH, cell density at transfection, plasmid ratios, chromatography flow rates) are strictly monitored and controlled.