Abstract
Lentiviral vectors (LVs) are used in cell and gene therapies due to their ability to transduce both dividing and non-dividing cells while carrying a relatively large genetic payload and providing long-term gene expression via gene integration. Current cultivation methods produce titers of 105–107 transduction unit (TU)/mL; thus, it is necessary to concentrate LVs as well as remove process- and product-related impurities. In this work, we used a packaging cell line WinPac-RD-HV for LV production to simplify upstream processing. A direct capture method based on ion-exchange chromatography and cellulose nanofibers for LV concentration and purification was developed. This novel scalable stationary phase provides a high surface area that is accessible to LV and, therefore, has potential for high-capacity operation compared to traditional bead-based supports. We were able to concentrate LVs 100-fold while achieving a two-log removal of host cell protein and maintaining up to a 90% yield of functional vector.
Highlights
The encouraging results coming from ongoing gene therapy clinical trials and recently approved therapies, such as Kymriah,[1] means there is a strong interest in processes for the scalable and cost-effective production and purification of viral vectors, which are considered to be a major roadblock to the commercialization of gene therapies.[2]
There are insufficient data on Lentiviral vectors (LVs) half-life and stability at 37C in the literature, especially LVs carrying other envelope proteins rather than vesicular stomatitis virus G protein (VSV-G);15–18 this is an element that needs to be addressed in the near future in order to develop efficient harvesting strategies
Higher total amounts of infective LV were obtained in harvest B (1.5 Â 109 transduction unit (TU)) than harvest A (2.4 Â 108 TU), which can be explained by the higher density of seeded cells and larger volumes of collected LCMs
Summary
The encouraging results coming from ongoing gene therapy clinical trials and recently approved therapies, such as Kymriah,[1] means there is a strong interest in processes for the scalable and cost-effective production and purification of viral vectors, which are considered to be a major roadblock to the commercialization of gene therapies.[2] Current pricing for the commercially available gene therapies starts at half a million dollars per treatment, the number of patients with access to these therapies is small. It is necessary to extensively concentrate LV preparations as well as remove process(e.g., serum proteins) and product-related impurities (e.g., noninfective vector), which can cause unwanted immune responses in patients.[6] Small-scale purification and concentration can be achieved by ultracentrifugation, but there are several disadvantages to this approach: the method is time consuming, there are limited scale-up possibilities, some impurities can be co-purified that elicit an immune response, and the success of the process is strongly dependent on well-trained operator’s skills. Alternative methods that can provide scalable production include tangential flow filtration (TFF) and chromatography
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