Has lentiviral purification been left behind? Current technologies for lab-scale lentivirus feedstock preparation are inefficient and not fit-for-purpose. Why have lentiviral vector developers not benefited from standardized purification processes emerging from other viral vector fields?
Here, Sujeong Yang and Ian Scanlon, two viral vector purification experts at Astrea Bioseparations discuss the importance of lab-scale (also known as small or technical scale) lentiviral vectors preparation, review common industry practices, and share their experiences.
Why is lab-scale lentiviral production crucial to gene and cell therapy development?
How do lentivirus characteristics impact purification strategies?
Sujeong Yang: Adoptive cell therapies such as chimeric antigen receptor T cell therapies, (CAR-T cell therapies) and next-generation versions including CAR-NK, CAR-M, and other engineered T cell receptor treatments require genetic modification of the cells involved. Lentiviral vectors (LVVs) are the preferred vehicle employed for delivering the “gene-of-interest” (GOI) because they can often infect cells that other commonly used viruses, such as adeno-associated viruses (AAVs), cannot. Developers of adoptive cell therapies are looking for lentiviral vectors that can optimally deliver a GOI to the target cell, to efficiently express the transgene and produce the expected biological effect. There are several factors that will affect the efficiency of a LVV to deliver a GOI or “transduce” a target cell, for example how efficiently the vector genome is packaged or whether cell type-specific promoters are employed. Optimization of LVV design requires the analysis of numerous discrete small LVV batches, produced at small scale in small tissue culture flasks, shake flasks or 100250 mL mini bioreactors that can be operated in parallel to generate sufficient material. After the development of the LVV construct, lab scale LVV production is also required to provide material for cell line development and pre-clinical studies to determine thresholds for dosing efficacy, biodistribution, pharmacokinetics and safety studies. Ultimately these studies are used to enable investigational new drug (IND) filing that will allow the LVV therapy to be tested in clinical trials.
Ian Scanlon: LVVs are different in several respects from many other viral vectors used in cell and gene therapy. Whereas AAV capsids, at only 25 nm in diameter, are comparatively similar in scale to large proteins, LVVs are considerably larger at 80–100 nm. The larger size of LVV is an advantage as genetic constructs up to 10 kb in size can be incorporate. However, its size also poses a particular challenge with respect to purification. While standard resin-based separation methods can be used to purify AAVs, LVVs are too large to achieve sufficient capacity with these traditional chromatography methods. The size of LVVs is a fundamental difference; they are so much larger that their purification requires the use of different types of adsorbents. Additionally, LVV also tends to be more unstable than non-enveloped viruses such as AAVs. The LVV envelope plays an important role in vector functionality, including transduction and tropism. But this functionality is negatively impacted by a variety of processing conditions, such as changes in temperature, pH, and ionic strength of bioprocessing fluids. Sujeong Yang: At the vector design stage, LVVs are generally produced via transfection of human embryonic kidney (HEK) 293 cells using plasmid DNA under adherent cell-culture conditions. The presence of plasmid DNA complicates purification strategies as the negative surface charge, at working pH, of the LVV is similar to the nucleic acid impurities. Host cell proteins are another major contaminant that can cause issues, due to undesired immunogenic effects. The LVV envelope is comprised of the host cell membrane, and the similarity of the LVV and these contaminants can cause further difficulties for achieving a pure LVV product.