In our last blog we looked at why lab-scale lentiviral production is crucial to gene and cell therapy development and how do lentivirus characteristics impact purification strategies. Today we add to this, with commentary from Sujeong Yang and Ian Scanlon on the limitations of existing lab-scale purification approaches.
What are the limitations of existing lab-scale purification approaches?
Sujeong Yang: Ultracentrifugation and density gradient can both purify and concentrate LVVs. However, these have limitations including a lack of scalability, the need for high-powered centrifuges and special operational expertise. Additionally, the process is often time-consuming which limits throughput.
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, 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: 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.
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.
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.