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Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography
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  • Shriarjun Shastry,
  • WENNING CHU,
  • Eduardo Barbieri,
  • Paul Greback-Clarke,
  • William Smith,
  • Christopher Cummings,
  • Ariann Minzoni,
  • Jenifer Pancorbo,
  • Gary Gilleskie,
  • Kimberly Ritola,
  • Michael Daniele,
  • Stefano Menegatti
Shriarjun Shastry
NC State University
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WENNING CHU
NC State University
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Eduardo Barbieri
NC State University
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Paul Greback-Clarke
NC State University
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William Smith
NC State University
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Christopher Cummings
NC State University
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Ariann Minzoni
NC State University
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Jenifer Pancorbo
NC State University
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Gary Gilleskie
NC State University
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Kimberly Ritola
UNC
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Michael Daniele
NC State University
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Stefano Menegatti
NC State University

Corresponding Author:smenega@ncsu.edu

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Abstract

Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. The AAV purification pipeline, in particular, hinges on protein ligands for the affinity-based capture step: while featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product’s activity and stability; additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable – yet equally effective – affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, while (ii) enabling product elution under near-physiological conditions (pH 6.0) and (iii) granting extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades – AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 – corroborating the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (~1014 vp per mL) and product yields (~60-80%) on par with commercial adsorbents, and purified AAV2 from HEK293 and Sf9 cell lysates affording high recovery (up to 78%) and reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%) of the purified vectors.
19 May 2023Submitted to Biotechnology Journal
23 May 2023Assigned to Editor
23 May 2023Submission Checks Completed
23 May 2023Reviewer(s) Assigned
18 Jun 2023Review(s) Completed, Editorial Evaluation Pending
19 Jun 2023Editorial Decision: Revise Major
07 Jul 20231st Revision Received
11 Jul 2023Submission Checks Completed
11 Jul 2023Assigned to Editor
11 Jul 2023Reviewer(s) Assigned
23 Jul 2023Review(s) Completed, Editorial Evaluation Pending
02 Aug 2023Editorial Decision: Revise Minor
03 Aug 20232nd Revision Received
04 Aug 2023Submission Checks Completed
04 Aug 2023Assigned to Editor
04 Aug 2023Reviewer(s) Assigned
04 Aug 2023Review(s) Completed, Editorial Evaluation Pending
12 Sep 2023Editorial Decision: Revise Minor
12 Sep 20233rd Revision Received
13 Sep 2023Submission Checks Completed
13 Sep 2023Assigned to Editor
15 Sep 2023Review(s) Completed, Editorial Evaluation Pending
16 Sep 2023Editorial Decision: Accept