In this talk, we aim at discussing challenges and opportunities to accelerate process development of AAV-gene therapies, by leveraging concepts of scale-down models and high-throughput experimentation

Adeno-associated viruses (AAVs) are currently one of the viral vectors of choice for gene therapy. High titers are necessary to ensure the desired outcome, therefore process optimization has a strong focus on yield improvement. The Upstream tested conditions for enhancing titers through ideal process conditions, the Downstream worked on keeping titers high whilst removing impurities and concentrating the vectors. Together, we established a process feasible for manufacturing at large scales.

The biopharmaceutical industry is looking at the concepts of intensification and integration to create more efficient processes. In this presentation, we will provide an overview of the use of perfusion bioreactors to integrate AAV harvesting and clarification processes. Continuous chromatography will then be presented as a tool for AAV capture. Finally, the two examples will be examined for their potential to link upstream and downstream processes.

Recombinant adeno-associated viral vectors (rAAV) are extensively used in gene therapy to introduce a genetic modification, and rAAV have been proven to be extremely effective and safe modalities for in vivo applications. The manufacturing of rAAV often relies on transient transfection of plasmids, and transfection reagents are used to convey plasmids through the plasma membrane. The selection of the best transfection reagents and the optimization of the transfection parameters is therefore key to achieving improved bioprocess productivity, consistency, and scalability. Our quality-by-design approach indicates that FectoVIR AAV (Polyplus) is a very efficient tool for GMP-compliant rAAV production.

Partial AAV capsids are heterogeneous and difficult to separate from full AAV capsids. We demonstrate unparalleled separation of empty, partial, and full capsids with single- and multi-column anion exchange displacement chromatography.

Developed a mechanistic anion exchange chromatography column model to expedite late phase process development for empty and full capsid separation. Modeling the transport of a large molecule, such as rAAV, in chromatography resin is very challenging and was overcome using an advance general rate model, which included variable surface diffusion.

Scalable methods of full capsid enrichment have often come at the cost of product recovery, thereby increasing the COGS. Here we would like to show that understanding of the AEX chromatography design space led to the identification of weak partitioning mode as an alternative to bind-and-elute. Results show that a full capsid ratio of >80% and a product yield >50% can be robustly achieved when utilizing this method of chromatography.

Oxford Biomedica (OXB) is a pioneer in developing products based on viral vectors. To meet the forecast vector demand for gene and cell therapies, OXB has evolved strategies to develop the next-generation manufacturing processes to yield higher vector quantities, suitable product quality attributes, and acceptable cost of goods in order to advance development of a diverse product portfolio including Lenti, Adeno and AAV products which currently present significant challenges.

mRNA is emerging as a new class of drug that has the potential to play a role in protein replacement therapies. Arcturus has established mRNA/LNP technology platform and applied to rare diseases treatments.

The use of liposomes and lipid nanoparticles (LNPs) as efficient vehicles for the transport and delivery of APIs is impressively being demonstrated in the ongoing COVID-19 pandemic. Current mRNA-based vaccines are formulated in LNPs to protect the cargo from environmental conditions and allow uptake in cells. Still, current formulations suffer from some drawbacks, like storage stability and rather low transfection efficiencies. I present a novel and scalable strategy for production, purification, and use of very special lipids from extremophilic Archaea for mRNA delivery. Using these lipids in formulations, the transfection efficiency of mRNA could be enhanced more than 80-fold.

State-of-the-art recombinant protein production with microbials is conducted in fed-batch cultivation. Switching to continuous processing would impose small-footprint manufacturing and increase the space-time yield compared to fed-batch processing. Still, industry and research struggle in achieving long-term microbial cultivations with stable productivity. Within this presentation, I will address reasons for fluctuating productivity in continuous processes, and discuss opportunities and how to overcome these challenges.