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Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates
  • +10
  • Stefano Menegatti,
  • Wenning Chu,
  • Shriarjun Shastry,
  • Eduardo Barbieri,
  • Raphael Prodromou,
  • Paul Greback-Clarke,
  • Will Smith,
  • Brandyn Moore,
  • Ryan Kilgore,
  • Christopher Cummings,
  • Jennifer Pancorbo,
  • Gary Gilleskie,
  • Michael A. Daniele
Stefano Menegatti
NC State University Department of Chemical and Biomolecular Engineering

Corresponding Author:smenega@ncsu.edu

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Wenning Chu
NC State University Department of Chemical and Biomolecular Engineering
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Shriarjun Shastry
NC State University Department of Chemical and Biomolecular Engineering
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Eduardo Barbieri
NC State University Department of Chemical and Biomolecular Engineering
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Raphael Prodromou
NC State University Department of Chemical and Biomolecular Engineering
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Paul Greback-Clarke
Biomanufacturing Training and Education Center (BTEC
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Will Smith
Biomanufacturing Training and Education Center (BTEC
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Brandyn Moore
NC State University Department of Chemical and Biomolecular Engineering
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Ryan Kilgore
NC State University Department of Chemical and Biomolecular Engineering
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Christopher Cummings
Biomanufacturing Training and Education Center (BTEC
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Jennifer Pancorbo
Biomanufacturing Training and Education Center (BTEC
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Gary Gilleskie
Biomanufacturing Training and Education Center (BTEC
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Michael A. Daniele
North Carolina Viral Vector Initiative in Research and Learning (NC-VVIRAL
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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.
22 Feb 2023Submitted to Biotechnology and Bioengineering
22 Feb 2023Submission Checks Completed
22 Feb 2023Assigned to Editor
22 Feb 2023Review(s) Completed, Editorial Evaluation Pending
25 Feb 2023Reviewer(s) Assigned
25 May 2023Editorial Decision: Revise Minor
16 Jun 20231st Revision Received
16 Jun 2023Submission Checks Completed
16 Jun 2023Assigned to Editor
16 Jun 2023Review(s) Completed, Editorial Evaluation Pending
22 Jun 2023Reviewer(s) Assigned
30 Jun 2023Editorial Decision: Accept