Peptide ligands targeting the vesicular stomatitis virus G (VSV‐G) protein for the affinity purification of lentivirus particles

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.

Multiangle Light Scattering as a Lentivirus Purification Process Analytical Technology

“…Standard third-generation LVV particles were produced using the LV-MAX system as previously described . In short, plasmid pLenti6.3/V5-GW/EmGFP (Aldevron) was transformed into Stbl3 E.…”

“…Lentivirus Purification. A volume of 1 mL of GKEAAFAA-Poros and FEKISTAE-Poros resins 19 were packed into Tricorn 5/50 columns (Cytiva) and connected to the AKTA Avant FPLC system (Cytiva). Resins were equilibrated with 10 CV equilibration buffer (50 mM PIPES, 100 mM NaCl, pH 7.4), loaded with LVV CCF at a residence time of 1−3.5 min, washed, and eluted with a gradient of 50 mM PIPES, 0.65 M NaCl, pH 7.4 buffer.…”

“…To test the method's reproducibility, we purified LVVs loaded with a green fluorescent protein transgene from a HEK293 cell culture supernatant via affinity chromatography (LVV AC ) using an adsorbent developed by our team 19 and tested their biological (Samples A−E) and technical replicates (n = 5) in a random order over multiple days to investigate inter-sample variations. Figure 2A overlays the measurements of the Rayleigh ratio and R g obtained with Sample A, demonstrating repeatable, high-confidence measurements.…”

“…Lentiviral vectors (LVVs) are enveloped retroviruses (diameter ∼75 and 140 nm , ) that can deliver a genetic payload of up to 11 kilobases and integrate it into the genome of dividing and nondividing cells . Observed activity and limited stability of LVVs pose the need for innovative expression and purification technologies that can deliver high functional titer and purity. − LVV particle titer is typically estimated via real-time PCR (quantification of packaged RNA or other nucleic acids) and ELISA (p24 capsid protein quantification), while flow cytometry is used for potency measurements. , Analytical techniques of recent introduction include droplet digital PCR (ddPCR), surface-enhanced Raman spectroscopy, FFF-MALS, batch DLS, NTA for particle count, ,, and tunable resistive pulse sensing for single-particle isolation/characterization . In this study, we present the development of a rapid (25 min) analytical method that couples SEC with MALS and UV detectors to measure the size of LVVs purified from HEK293 cell harvests.…”

“…18−22 LVV particle titer is typically estimated via real-time PCR (quantification of packaged RNA or other nucleic acids) and ELISA (p24 capsid protein quantification), while flow cytometry is used for potency measurements. 19,22 Analytical techniques of recent introduction include droplet digital PCR (ddPCR), 23 surface-enhanced Raman spectroscopy, 24 FFF-MALS, batch DLS, NTA for particle count, 16,25,26 and tunable resistive pulse sensing for single-particle isolation/ characterization. 27 In this study, we present the development of a rapid (25 min) analytical method that couples SEC with MALS and UV detectors to measure the size of LVVs purified from HEK293 cell harvests.…”