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Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography

Shastry, Shriarjun; Chu, Wenning; Barbieri, Eduardo; Greback‐Clarke, Paul; Smith, William K; Cummings, Christopher; Minzoni, Arianna; ... Menegatti, Stefano; + view all (2023) Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography. Biotechnology Journal 10.1002/biot.202300230. (In press). Green open access

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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. In particular, the AAV purification pipeline 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 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, (ii) enable product elution under near-physiological conditions (pH 6.0), and (iii) grant extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades – namely, AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. This corroborates 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 mL−1) and product yields (≈60%–80%) on par with commercial adsorbents, and purifies AAV2 from HEK293 and Sf9 cell lysates with high recovery (up to 78%), reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%).

Type: Article
Title: Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography
Open access status: An open access version is available from UCL Discovery
DOI: 10.1002/biot.202300230
Publisher version: https://doi.org/10.1002/biot.202300230
Language: English
Additional information: Copyright © 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, https://creativecommons.org/licenses/by/4.0/, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Keywords: Adeno-associated virus; affinity chromatography; gene therapy; peptide ligands; transduction activity
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10177969
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