Barbieri, Eduardo;
Mollica, Gina N;
Moore, Brandyn D;
Sripada, Sobhana A;
Shastry, Shriarjun;
Kilgore, Ryan E;
Loudermilk, Casee M;
... Menegatti1, Stefano; + view all
(2023)
Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles.
Biotechnology and Bioengineering
10.1002/bit.28594.
(In press).
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Abstract
The recent uptick in the approval of ex vivo cell therapies highlights 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 into 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 3·10⁹ TU per mL of resin, or 5·10¹¹ vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 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.
| Type: | Article |
|---|---|
| Title: | Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1002/bit.28594 |
| Publisher version: | https://doi.org/10.1002/bit.28594 |
| Language: | English |
| Additional information: | © 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, 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: | affinity chromatography, cell therapy, lentivirus, peptide ligands, vesicular stomatitis virus G protein. |
| 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/10181150 |
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