Accelerating Early Upstream Screening Activities for Viral Vector Therapies

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Biopharmaceuticals delivered by viral vectors (VVs) face distinctive obstacles during early upstream development. In October 2022, Andres Castillo (a portfolio manager at Sartorius) noted that drug makers set short development timelines to hasten therapies into the clinical evaluation. Doing so limits time for analyzing complex biointeractions, and studies for culture-media and VV screening are time- and resource-intensive. Castillo and Shanya Jiang (also a portfolio manager at Sartorius) explored how integrated technologies facilitate robust cell-line and VV screening.

The Presentation
Reflecting on the complexity of upstream development, Castillo said, “It is imperative to leverage systems that make initial data collection easier and more robust to facilitate scale-up.” Sartorius has established an ensemble of culture and bioanalytical solutions to help users perform comprehensive testing, evaluate data, and apply resulting insights to characterize critical quality attributes and process parameters (CQAs, CPPs).

Castillo highlighted the integrated nature of the portfolio. Developers can gather data quickly for multiple clones and adenoassociated virus (AAV) serotypes using Xell media and reagents and Ambr 15 microbioreactor systems, which are programmed to work with Modde and SIMCA data-analytics software. The Modde program helps users to establish experimental design space, apply design of experiments (DoE) approaches, and analyze multiple process parameters simultaneously. SIMCA tools enable multivariate data analysis, facilitating characterization.

Jiang demonstrated the importance of comprehensive screening and integrated bioanalytical solutions. In her first example, Xell (now part of Sartorius) evaluated production of different AAV serotypes in human embryonic kidney (HEK)293 cells. Scientists transfected two cell lines with plasmids for six AAV serotypes, then cultured the cells in Xell HEK ViP NX xeno-free, chemically defined media. For all tested serotypes, one cell line consistently generated higher viral titers than did the other. But even for the more productive clone, titers differed significantly by serotype. Thus, developers must screen both cell lines and AAV serotypes carefully to identify optimal culture conditions.

Developers likewise should assess multiple media formulations. Jiang noted that Xell also assessed AAV2 production in five HEK293 cell lines cultured in either HEK ViP NX media or a commercially available alternative. All cell lines generated significantly higher viral titers in Xell media than in the comparator. As Jiang explained, it is important to screen multiple cell lines and culture media to find the optimal HEK cell line and media formulation.

She described how scientists from the University of Nantes in France applied the Sartorius solutions to optimize an AAV production process. Ambr 15 systems were used to screen different cell lines, media types, transfection reagents, plasmid ratios, cell densities, and complexing periods. Data were modeled in a Modde DoE approach to rank tested parameters in terms of their criticality to viral titer. Based on those results, the team selected parameters for additional screening, which later helped to identify CPPs.

Oxgene (WuXi Advanced Therapies) used a similar approach to evaluate conditions for lentivirus (LV) production. Rather than screening parameters sequentially and then performing validation studies, the team performed two DoE runs to optimize and validate nine conditions in parallel. Oxgene reported a 50% reduction in the time required to validate a LV platform process. DoE modeling also enabled this team to increase infectious titers by 34-fold compared with what the initial process could achieve.

Jiang also highlighted work by scientists at GlaxoSmithKline to screen stable LV-producing cell lines. The study writers noted that Ambr–Modde integration helped them to select a clone with high functional titers while reducing development costs with experiments performed at 15-mL scale. The authors added that findings from the small-scale studies enabled scale-up to 50-L runs.

Questions and Answers
What characteristics do you seek out in a HEK cell culture media? Xeno-free, chemically defined media are good options that help to increase process reproducibility. Drug makers should consider whether a medium generates scalable results, comes in different formulations, and works effectively in a given culture format (e.g., fed-batch or perfusion mode). Ultimately, though, media selection requires comprehensive screening.

What are hallmarks of an effective scale-down model for cell culture? It must be a cost-effective tool for characterizing larger processes, and it must test parameters with reliable robustness. For instance, Ambr 15 systems can perform 24 to 48 experiments in parallel. They leverage automation to accelerate experiments and increase their reproducibility, and each microreactor’s geometry is designed to mimic that of larger vessels.

Find the full webinar online at