rAAV Purification via Affinity and Anion-Exchange Chromatography

The purification process is divided into distinct modular steps: depth filtration step, affinity chromatography step, anion-exchange chromatography step, and tangential flow filtration step. Each step targets specific contaminants or purification objectives, allowing the complex purification task to be managed through sequential, specialized operations rather than one extensive pre-treatment procedure

Depth filtration is performed as a preliminary step to remove cellular debris and large particles before chromatography steps. This preliminary action prevents clogging of chromatography columns and reduces the burden on subsequent purification steps, eliminating the need for extensive pre-treatment with detergents or nucleases

The purification process is designed as a continuous flow where the output of one step becomes the input of the next without interruption. Depth filtration feeds directly into affinity chromatography, which feeds into anion-exchange chromatography, and finally into tangential flow filtration. This continuous operation maintains productivity while achieving high purity through multiple steps

Affinity chromatography serves as an intermediary step that selectively captures AAV particles from the complex cell lysate mixture. This intermediate purification step concentrates the target particles and removes major contaminants before the subsequent anion-exchange and tangential flow filtration steps, making the overall process more efficient

The anion-exchange chromatography step specifically extracts and removes empty particles from the AAV preparation. By utilizing differences in surface charge between full and empty particles, this step separates the contaminating empty particles from the desired full particles, resolving the contamination issue introduced by the affinity chromatography step

The process utilizes changes in ionic strength and pH conditions across different chromatography steps to differentially bind and elute full versus empty particles. Anion-exchange chromatography employs salt gradients that selectively elute empty particles at lower salt concentrations while full particles require higher salt concentrations, achieving separation based on physical-chemical parameter differences

The purification platform is designed with universal chromatography steps that can process different AAV serotypes and production scales using the same methodology. The depth filtration, affinity chromatography, anion-exchange chromatography, and tangential flow filtration steps form a scalable platform that maintains consistent purification performance whether processing small research-scale samples or large clinical-grade batches

The process employs controlled parameter changes including pH adjustments, ionic strength gradients, and flow rate optimizations that can be precisely replicated across different scales. These parameter controls ensure reproducible separation and purification outcomes whether the process is run at pilot scale or full production scale, maintaining manufacturing precision while enabling scalability