Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates.
Biotechnol Bioeng
; 120(8): 2283-2300, 2023 08.
Article
em En
| MEDLINE
| ID: mdl-37435968
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 (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/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.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Dependovirus
Tipo de estudo:
Risk_factors_studies
Limite:
Humans
Idioma:
En
Revista:
Biotechnol Bioeng
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos