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\begin{document}

\title{Peptide ligands targeting the vesicular stomatitis virus G (VSV-G)
protein for the affinity purification of lentivirus particles}



\author[1]{Stefano Menegatti}%
\author[1]{Eduardo Barbieri}%
\author[1]{Gina N. Mollica}%
\author[1]{Brandyn Moore}%
\author[1]{Sobhana Alekhya Sripada}%
\author[1]{Shriarjun Shastry}%
\author[1]{Ryan Kilgore}%
\author[1]{Casee M. Loudermilk}%
\author[1]{Zachary H. Whitacre}%
\author[1]{Katie M. Kilgour}%
\author[2]{Elena Wuestenhagen}%
\author[2]{Annika Aldinger}%
\author[2]{Heiner Graalfs}%
\author[2]{Oliver Rammo}%
\author[2]{Michael Schulte}%
\author[3]{Thomas Johnson}%
\author[4]{Michael A. Daniele}%
\affil[1]{NC State University Department of Chemical and Biomolecular Engineering}%
\affil[2]{Merck KGaA}%
\affil[3]{University College London}%
\affil[4]{The University of North Carolina at Chapel Hill}%


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  \date{May 16, 2024}


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\begin{abstract}
The recent uptick in the approval of \emph{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 \emph{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\selectlanguage{ngerman}·10 \textsuperscript{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.%
\end{abstract}\selectlanguage{ngerman}%



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