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Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles
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  • Stefano Menegatti,
  • Eduardo Barbieri,
  • Gina N. Mollica,
  • Brandyn Moore,
  • Sobhana Alekhya Sripada,
  • Shriarjun Shastry,
  • Ryan Kilgore,
  • Casee M. Loudermilk,
  • Zachary H. Whitacre,
  • Katie M. Kilgour,
  • Elena Wuestenhagen,
  • Annika Aldinger,
  • Heiner Graalfs,
  • Oliver Rammo,
  • Michael Schulte,
  • Thomas Johnson,
  • Michael A. Daniele
Stefano Menegatti
NC State University Department of Chemical and Biomolecular Engineering

Corresponding Author:[email protected]

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Eduardo Barbieri
NC State University Department of Chemical and Biomolecular Engineering
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Gina N. Mollica
NC State University Department of Chemical and Biomolecular Engineering
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Brandyn Moore
NC State University Department of Chemical and Biomolecular Engineering
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Sobhana Alekhya Sripada
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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Ryan Kilgore
NC State University Department of Chemical and Biomolecular Engineering
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Casee M. Loudermilk
NC State University Department of Chemical and Biomolecular Engineering
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Zachary H. Whitacre
NC State University Department of Chemical and Biomolecular Engineering
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Katie M. Kilgour
NC State University Department of Chemical and Biomolecular Engineering
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Elena Wuestenhagen
Merck KGaA
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Annika Aldinger
Merck KGaA
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Heiner Graalfs
Merck KGaA
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Oliver Rammo
Merck KGaA
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Michael Schulte
Merck KGaA
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Thomas Johnson
University College London
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Michael A. Daniele
The University of North Carolina at Chapel Hill
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Abstract

The recent uptick in the approval of ex vivo cell therapies highlight the relevance of Lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification – currently reliant on filtration and anion-exchange or size-exclusion chromatography – suffers from long process times and low yield of transducing particles, which translate in high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50-60% of viral genomes; 40-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·10 9 TU per mL of resin at the residence time of 1 min) and clearance of host cell proteins (up to 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.
24 Aug 2023Submitted to Biotechnology and Bioengineering
24 Aug 2023Submission Checks Completed
24 Aug 2023Assigned to Editor
24 Aug 2023Review(s) Completed, Editorial Evaluation Pending
07 Sep 2023Reviewer(s) Assigned
18 Oct 20231st Revision Received
18 Oct 2023Submission Checks Completed
18 Oct 2023Assigned to Editor
18 Oct 2023Review(s) Completed, Editorial Evaluation Pending
18 Oct 2023Reviewer(s) Assigned
26 Oct 2023Editorial Decision: Accept