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.
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.
What are the limitations of existing lab-scale purification approaches? Sujeong Yang: Ultracentrifugation and density gradient ultracentrifugation can both purify and concentrate LVV and are commonly used for lab-scale LVV preparation. But these have several limitations including a lack of scalability, the need for high-powered centrifuges and special operational expertise, and the process is timeconsuming. Ultimately this means that throughput is limited. Ian Scanlon: There is no standard downstream purification process for these smaller volumes. Molecular weight cut off filters can be used with benchtop centrifuges to concentrate clarified feedstocks but have limited capability to remove impurities. The same considerations apply for tangential flow filtration, or TFF, but additionally the LVV is concentrated along with salts, proteins, and DNA, generally for extended periods due to the length of time required for concentration. Precipitation is sometimes used to selectively remove impurities. PEGylation of the vector can enable selective product concentration, for instance. The problem here is that, once again, adding any additional unit operations with LVVs typically reduces the final yield of product considerably.
Alternatively, ion exchange membrane adsorbers allow the purification process to be more aligned with chromatography steps commonly used at clinical scale LVV manufacturing. However, functional product recovery is compromised using high salt elution steps.
What would be the impact of improved lab-scale lentiviral vector purification on therapeutic development? Ian Scanlon: When comparing LVV discovery workflows to those applied in protein and antibody drug discovery, it is clear there is much room for improvement. The latter leverages high-throughput technologies with multi-well plates for rapid separation and analysis. It would be highly beneficial to truly transfer that approach to the LVV space. Sujeong Yang: Although there are upstream technologies that can work in parallel to produce vector targets, the capabilities of existing purification solutions restrict the throughput of LVV purification for use in preclinical studies, and therefore extends the time needed to obtain material representative of that required for clinical and commercial applications. A more efficient purification process would shorten the vector development time, so reducing costs, and allow therapies to progress faster to clinical testing.
How could a lab-scale chromatography solution facilitate lentiviral therapeutic development? Sujeong Yang: First and foremost, the process must be easy to implement with equipment generally available in the lab. Many LVV purification methods that can provide pure, highly functional material are not very practical. The process should also be adapted to reduce LVV degradation. For example, using mild elution conditions to avoid the degradation seen at high salt concentrations, and a short process time should boost LVV recovery. Increased product recovery would increase production efficiency and reduce costs, so reducing the pressure on the upstream process to increase volumes of viral feedstocks.
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White Paper: Has lentiviral purification been left behind?
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Ian Scanlon: It would also need to increase throughput so researchers could advance more targets and assets that are likely to succeed through the drug development process faster, ultimately providing a greater number of therapies in the hands of patients. A chromatography-based purification technology that could be deployed simply and easily, be readily scalable, and afford robust LVV purifications in a rapid manner would be the best solution. A simplified chromatography solution that does not require extensive training to use would lower the barriers to adoption of this type of purification technology in discovery and early development labs. After all, processes will need to be scaled up to generate clinical and commercial quantities, and at that time chromatography will be used. Starting with a scalable chromatographic purification from the outset will ensure that the same results are obtained in discovery as in the manufacturing, and less time will be required in process development. In the end, this will contribute to faster progress to clinical trials.
In summary: The discussion makes it apparent there is a pressing need to address current lab-scale purification issues. A more time-efficient process, that simultaneously increased LVV recovery would serve as an enabling technology for future LVV-based drug development. 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.
Contact us to learn more about our state-of-the-art purification solutions.
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