Lentiviral vectors hold great promise for engineering autologous cells into therapies and stem cells into differentiated cells for regenerative medicine, but scale-up concerns have limited interest from biopharmaceutical developers.
Unlike the adeno-associated viruses used for in vivo gene therapy, “No credible counterpart is truly available for lentiviruses used for ex vivo cell therapy,” Stefano Menegatti, PhD, associate professor, department of chemical and biomolecular engineering, North Carolina State University, and founder of LigaTrap, tells GEN.
That may change soon. In collaboration with Merck Life Science KGaA, Menegatti and colleagues developed an affinity-based capture technology that produces what they say is an “efficient, affordable, and safe affinity ligand for LV purification.”
The outcome of this joint effort is “a clear sign that LV purification can be updated towards unmet customer needs in terms of process economics and robustness,” says Oliver Rammo, technical director R&D, novel modalities, chromatography R&D, Merck Life Science KGaA.
Enhanced LV Purification
The highest-yielding ligand they identified was GKEAAFAA. It has a binding capacity of 3×109 transducing units per milliliter (TU/mL) of resin (equivalent to more than 1011 vp/mL). It produced a 60 to 70 percent yield of transducing LV particles, a more than 200-fold reduction in host cell proteins from that of the feed material, and the ability to withstand caustic cleaning.
Menegatti’s team, including first author Eduardo Barbieri, PhD, scientist at LigaTrap, targeted the enveloped vesicular stomatitis virus G protein (VSV-G)-pseudo lentiviruses (LVs). Both experimental and in silico discovery methods identified promising 8-mer peptides against VSV-G.
Their selection process identified ligands with high selectivity and tunable affinity, resulting in “a unique combination of high binding capacity, clearance of impurities, yield of transducing vectors, and lifetime,” the scientists wrote in a recent paper.
Scalability and speed
The primary benefits of these affinity ligands appear to be scalability and speed. Compared to alternative methods, these ligands feature “slightly higher binding capacity at half the residence time, enable mild elution conditions, and are more stable and durable,” says Menegatti, adding that’s because these peptides are produced synthetically and thus lack the risk of biological contaminants associated with recombinant approaches.
Additionally, they maintain their binding capacity and selectivity through at least 50 cycles and can withstand harsh cleaning-in-place practices composed of 0.5 M sodium hydroxide and 30 minutes contact time. Menegatti estimates production costs at scale of approximately $60 per gram, or roughly $8,000 per liter.
The next steps include expanding this research to lentiviral vectors loaded with a variety of genetic payloads and to additional chromatographic substrates, such as monoliths and membranes.