(304d) Adeno-Associated Virus Purification from HEK293 Lysates Using Peptide-Based Affinity Adsorbents | AIChE

(304d) Adeno-Associated Virus Purification from HEK293 Lysates Using Peptide-Based Affinity Adsorbents


Menegatti, S., North Carolina State University
Cell and gene therapies (CGTs) are at the forefront of innovation to treat rare degenerative & dysregulation diseases, immunological disorders, and genetic defects diseases, such as A1AT deficiency. Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. To support the manufacturing of AAV vectors, we have developed innovative peptide-based affinity ligands to capture a broad range of Adeno-Associated Virus serotypes (AAVs) from HEK293 cell lysate. The current AAV purification platform replies on the capture step, for which several affinity resins are commercially available, e.g., POROS™ CaptureSelect™ AAVX Affinity and AVB Sepharose HP Resins. These adsorbents rely on protein ligands – typically camelid single-domain antibody fragments – that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. To address these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (KD ~ 10-5-10-6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC10% > 1013 vp per mL of resin) and product yields (~50-80%) compared with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50-80%), 80-to-400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.