Executive Summary

Scaling adeno-associated virus (AAV) production from bench-scale to 200L+ suspension bioreactors introduces significant fluid dynamics challenges, primarily mechanical shear stress. This force can damage both producer cells and viral capsids, leading to reduced vector titers, an increased ratio of empty to full capsids, and potential alterations to vector integrity. Mitigating these effects requires a data-driven process development strategy focused on bioreactor geometry, impeller design, agitation rates, and media formulation. A tailored approach ensures that the final vector product maintains the quality and potency attributes established during small-scale runs, supporting an accelerated path to IND.

The Physics of Scaling AAV Production

Transitioning an AAV production process from a 50L to a 200L or 500L+ single-use bioreactor is not a linear exercise. As the volume increases, maintaining homogenous suspension of producer cells and uniform distribution of oxygen and nutrients requires significantly more powerful agitation. This introduces hydrodynamic forces that can compromise process outcomes if not properly characterized and controlled.

The primary challenge is managing the tip speed of the bioreactor impeller. Higher tip speeds generate greater shear, particularly in the turbulent zones nearest the impeller blades. This physical stress directly impacts the viability and productivity of the producer cell line during the transfection and vector expression phases.

Biological Consequences of Uncontrolled Mechanical Stress

The integrity of a viral vector is fundamental to its clinical profile. Manufacturing processes that introduce uncontrolled variables can negatively affect the final product’s characteristics. For instance, research into AAV vector biology has shown that even subtle changes to the vector can have significant downstream consequences for in vivo behavior and potential toxicity (PMID: 33177182). Shear-induced damage can lead to a higher percentage of empty or partially filled capsids, reducing the specific activity of the vector lot and complicating dose determination.

Damaged capsids or the release of intracellular contents from lysed cells also introduce impurities that challenge downstream purification. A well-designed, scalable manufacturing process is a prerequisite for the long-term clinical success and predictable safety profile of a gene therapy candidate, a principle observed across different viral vector platforms (PMID: 16243818).

We started collaborating with UPenn Vector core in 2023 and the AAV vector which they manufactured laid a foundation for development of a gene therapy candidate which will enter soon preclinical studies. The key people from UPenn Vector Core joined Franklin Biolabs and our partnership transitioned without interruption from UPenn Vecor Core to Franklin Biolabs Research Vector Division. Franklin Biolabs is an integral collaborator in our AAV-vector based gene therapy candidate development, and we hope to continue the partnership for years to come.
— Biotech Partner

A Data-Driven Approach to Process Development

A standardized template for large-scale AAV manufacturing does not exist. Each combination of serotype, transgene, and producer cell line has a unique tolerance profile. Our approach is built on rigorous, small-scale process development studies to define the operational envelope before scaling.

• Impeller & Bioreactor Selection: We utilize bioreactors with specific impeller geometries designed to maximize mixing efficiency while minimizing high-shear zones.

• Agitation Strategy: A multi-stage agitation strategy is developed, using lower speeds during initial cell growth and post-transfection phases, and adjusting as required by metabolic indicators.

• Media Optimization: We assess and implement media formulations containing shear-protectant excipients that stabilize cell membranes without interfering with transfection or vector production.

• Scale-Down Modeling: Before committing to a large-scale run, we use scale-down models (2L-10L) that mimic the fluid dynamics of the target 200L+ bioreactor to de-risk the process and confirm parameters.

This methodical process development ensures a robust and reproducible manufacturing run. By controlling for mechanical forces, we protect vector integrity and maximize the yield of high-quality, potent AAV. This focus on CMC excellence is integral to our ability to help sponsors move candidates toward IND within an 18-24 month timeline. Since 2019, programs developed by our scientific leadership have achieved a 100% IND success rate, a testament to this rigorous approach, which continues at Franklin Biolabs following its 2024 launch.