Peptide ligands for the affinity purification of adeno-associated
viruses from HEK 293 cell lysates
Abstract
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. The current
AAV purification platform hinges on the capture step, for which several
affinity resins are commercially available. These adsorbents rely on
protein ligands – typically camelid antibodies – 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. Addressing 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 (K D ~ 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 (DBC 10%
> 10 13 vp per mL of resin) and product
yields (~50-80%) on par 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.