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?
We chatted with Sujeong Yang and Ian Scanlon, two viral vector purification experts at Astrea Bioseparations and discussed the importance of lab-scale (also known as small or technical scale) lentiviral vectors preparation.
Why is lab-scale lentiviral production crucial to gene and cell therapy development?
Sujeong Yang: 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 LVVs that can optimally deliver a GOI to the target cell. How efficiently the vector genome is packaged, or whether cell type-specific promoters are employed, can affect the efficiency of a LVV to deliver a GOI or “transduce” a target cell. 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 100-250 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.
How do lentivirus characteristics impact purification strategies?
Ian Scanlon: LVVs are different in several respects from many other viral vectors used in cell and gene therapy, they are considerably larger at 80–100 nm. This larger size is an advantage as genetic constructs up to 10 kb in size can be incorporate. However, its size is challenging with respect to purification. LVVs are too large to achieve sufficient capacity with traditional resin-based chromatography methods, so their purification requires the use of different types of adsorbents.
Additionally, LVV also tends to be more unstable than non-enveloped vectors. 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.
What are the major challenges for efficient lentiviral vector purification?
Sujeong Yang: The biggest challenge to LVV purification is low functional or physical recovery. To start with, LVVs are generally produced at low titers, compared to other viral vectors. In addition, as LVVs are fragile, minimizing processing time, and number of unit operations is essential for reducing LVV degradation during purification steps. So, if too many purification steps are used at the lab scale, it is probable that extremely small product quantities will be recovered. Moreover, this overall poor recovery results in oversized and expensive batches of LVVs productions.
Ian Scanlon: This low recovery also means that, rather than lose functional material, purity is sacrificed by limiting the number of process steps incorporated into a purification protocol.
Astrea Bioseparations is addressing this need through our development of an unprecedented and proprietary fiber-based technology, empowering therapeutic innovators with the tools they need to purify the quantities they desire.