Developing a process of lentivirus purification from recombinant fluids using peptide affinity ligands

Lentiviral vectors (LVVs) are emerging as an enabling tool in gene and cell therapies, yet the toolkit for purifying them at scale is still immature. A pivoting moment in LVV isolation technology was marked by the introduction of affinity ligands for LVVs pseudo-typed with Vesicular Stomatitis Virus G (VSV-G) protein. Camelid antibody ligands were initially discovered and utilized to functionalize a resin with a capacity of 10¹⁴ LVV particles per liter (vp/L). Shortly thereafter, our team introduced VSV-G-targeting peptides and assessed their application as ligands for purifying LVVs from HEK293 cell harvests. In this study, we utilized these peptides to develop novel affinity resins and-first in this field-affinity membranes with optimal binding capacity, productivity, and removal of host cell contaminants. To that end, we evaluated resins of different material, particle and pore size, and functional density, as well as membranes with different fiber morphology, porosity, and ligand distribution. The lead peptide-functionalized resin and membrane featured high capacity (5 × 10⁹ and 1.2 × 10⁹ transducing LVV units per mL of adsorbent, TU/mL) and productivity (2.9 × 10⁹ and 1.7 × 10⁹ TU/mL min) and afforded a substantial enrichment of cell-transducing LVVs and reduction of contaminants (110-170-fold) in the eluates. Finally, we demonstrated an LVV purification process in four steps: clarification and nuclease treatment, affinity capture in bind-and-elute mode, polishing in flow-through mode, and ultra/dia-filtration and sterile filtration. The processes afforded yields of 33%-46%, a residual HCP level below 5 ng/mL, and productivity of 1.25-1.5 × 10¹⁴ active LVV particles per hour and liter of adsorbent.