Dovile Gruzdyte

June 26, 2023

3 Min Read

Although gene therapies are associated with treating rare diseases, candidate therapies for prevalent-disease indications have overtaken rare-disease treatments in clinical trials, including phase 3 studies. That indicates a change in momentum for new gene therapies that will bring innovative drug products to large patient populations. In an April 2023 webinar, Dovile Gruzdyte, global product manager for cell-line development at Cytiva, discussed how evolving needs in gene-therapy bioprocessing are driving advancements in cell-line development.

Gruzdyte’s Presentation
Gene therapies for prevalent indications are approaching commercialization, which will exacerbate already high demand for production materials. Gruzdyte said that gene-therapy manufacturing processes currently lag behind scientific progress, with many companies still relying on steps and methods that were designed for monoclonal antibody (MAb) production. To match the impending demand for new therapies, developers will need to scale up their processes.

Gruzdyte said that Cytiva customers are facing many obstacles to producing and purifying viral vectors. However, improving downstream methods to recover more product will not resolve the gap in demand. That can be done only by improving the upstream processing.

Adenoassociated virus (AAV) vectors remain prominent for gene-therapy applications. Gruzdyte’s team sought to maximize the potential of AAV-production cell lines.

Several methods exist for AAV production, and each method has its advantages and disadvantages. For instance, many scientists use a triple transfection method based on three plasmids carrying a therapeutic gene of interest (GoI), genes from a helper virus necessary for AAV replication, and the AAV replication (rep) and capsid (cap) genes, respectively. That process is flexible, relatively expedient, and less expensive to start up than other production options. However, it is difficult to control and doesn’t support a robust, large-scale manufacturing process.

Developers also have tried to stably integrate the AAV rep and cap genes and the GoI at the start of AAV production by coinfection with adenovirus. However, such methods still pose a risk of viral contamination.

By using an alternative platform such as a baculovirus expression vector system (BEVS) with Spodoptera frugiperda (Sf9) insect cells, scientists have been able to increase AAV vector yields dramatically. However, Sf9 cells pose immunogenicity risks to patients and involve labor intensive manufacturing processes.

Gruzdyte said that in the future, the industry will shift to delivery- and cargo-free producer cell lines that have stably integrated the AAV rep and cap genes, sequences from a helper virus, and a GoI. Such a design will allow for straightforward manufacturing with a simple induction step to start AAV production.

Cytiva’s Elevecta cell-line development platform allows users to create stable and inducible producer cell lines for large-scale AAV manufacturing. Customers currently can choose between Cytiva’s proprietary CAP human cell line or HEK293 cells and then provide capsid genes and transgenes. Rep and cap genes already are integrated stably into the Elevecta Alpha cell line.

Cytiva then creates a polyclonal producer pool, followed by a single-cell cloning process, clone screening, and selection of the best-performing clone. The chosen clone is delivered as a research cell bank (RCB) so that the customer can use it to create a good manufacturing practice (GMP) cell bank.

Users can seed small bioreactors and then transfer their material to larger bioreactors, add induction agent, and begin AAV manufacturing. No transfection is required, and helper viruses aren’t needed. Gruzdyte said that by going from a producer pool, to a single-cell clone, to a fully optimized upstream process, users can increase production by nearly two orders of magnitude compared with standard triple-transfection methods.

Cytiva has performed scale-up studies on the Elevecta platform in a 200-L Pall Allegro stirred-tank reactor (STR). The data gathered showed an improvement in cell concentration and cell viability when comparing harvests from a 10-L and 200-L bioreactor. Titers between the two STRs were comparable, demonstrating the robustness of scale-up for upgrading to an Elevecta producer cell line.

Question and Answer
Does the Elevecta platform work with any AAV serotype and GoI? We can take any serotype and any GoI that can be integrated into an AAV, and we work with customers to optimize their upstream processes.

Find the full webinar online at www.bioprocessintl.com/category/webinars.

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