Implementation of Novel Affinity Ligand for Lentiviral Vector Purification

Abstract

The use of viral vectors as therapeutic products for multiple applications such as vaccines, cancer treatment, or gene therapies, has been growing exponentially. Therefore, improved manufacturing processes are needed to cope with the high number of functional particles required for clinical trials and, eventually, commercialization. Affinity chromatography (AC) can be used to simplify purification processes and generate clinical-grade products with high titer and purity. However, one of the major challenges in the purification of Lentiviral vectors (LVs) using AC is to combine a highly specific ligand with a gentle elution condition assuring the preservation of vector biological activity. In this work, we report for the first time the implementation of an AC resin to specifically purify VSV-G pseudotyped LVs. After ligand screening, different critical process parameters were assessed and optimized. A dynamic capacity of 1 × 1011 total particles per mL of resin was determined and an average recovery yield of 45% was found for the small-scale purification process. The established AC robustness was confirmed by the performance of an intermediate scale providing an infectious particles yield of 54%, which demonstrates the scalability and reproducibility of the AC matrix. Overall, this work contributes to increasing downstream process efficiency by delivering a purification technology that enables high purity, scalability, and process intensification in a single step, contributing to time-to-market reduction.

Affinity and Pseudo-Affinity Membrane Chromatography for Viral Vector and Vaccine Purifications: A Review

“…Reports of various applications have been reviewed in previous publications [ 24 , 25 ]. A recent example is the generation of an agarose-based affinity column for the selective retention of lentiviral vectors, which were pseudotyped with a vesicular stomatitis virus glycoprotein (VSV-G) envelope [ 71 ]. In this study, the group performed a ligand screening, using surface plasmon resonance sensors to generate a scalable affinity column afterwards.…”

“…A different approach to broad platform applications could be a focus on individual viruses that are currently of high pharmaceutical relevance, such as the adeno viruses and AAV, retroviruses, herpes simplex viruses, or hepatitis viruses, to name a few. It would be highly desirable to promote the development of scalable affinity membranes for these viruses as has been done recently for lentiviral vectors using a bead-based resin [ 71 ]. A comparable approach was made for resin column systems for the purification of AAV [ 45 , 98 ] and could be feasible for membranes as well.…”

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

“…In another approach, Mekkaoui et al applied streptavidin‐functionalized magnetics beads for purification of LV particles displaying a cTag8 (Mekkaoui et al, 2018), obtaining yields >60%, and 3‐log and 2‐log reduction of host cell DNA and HCPs, respectively; however, yield reduced to 20% when the ligands were transferred onto monoliths. More recently, the team led by Peixoto utilized a library of single‐domain camelid antibodies (V H H) to identify ligands targeting the VSV‐G protein (Moreira et al, 2023; ThermoFisher, 2023). The ligands, now commercialized in a chromatographic resin format (CaptureSelect™ Lenti VSVG Affinity Matrix), provide a binding capacity of ~10 11 viral particles per mL of resin (vp/mL) and afford good product purity, while mandating energic elution conditions (0.8 M arginine) and withstanding mildly caustic CIP (10–25 mM aqueous NaOH) (Moreira et al, 2023).…”

“…More recently, the team led by Peixoto utilized a library of single‐domain camelid antibodies (V H H) to identify ligands targeting the VSV‐G protein (Moreira et al, 2023; ThermoFisher, 2023). The ligands, now commercialized in a chromatographic resin format (CaptureSelect™ Lenti VSVG Affinity Matrix), provide a binding capacity of ~10 11 viral particles per mL of resin (vp/mL) and afford good product purity, while mandating energic elution conditions (0.8 M arginine) and withstanding mildly caustic CIP (10–25 mM aqueous NaOH) (Moreira et al, 2023).…”