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.

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. The AAV purification pipeline, in particular, 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 – yet equally effective – 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, while (ii) enabling product elution under near-physiological conditions (pH 6.0) and (iii) granting extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades – AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 – corroborating 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 per mL) and product yields (~60-80%) on par with commercial adsorbents, and purified AAV2 from HEK293 and Sf9 cell lysates affording high recovery (up to 78%) and reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%) of the purified vectors.

Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands – typically camelid antibodies – that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (K D ~ 10 -5-10 -6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC 10% > 10 13 vp per mL of resin) and product yields (~50-80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50-80%), 80-to-400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.