The world today has seen an exponential surge in gene therapies, highlighting the importance of adeno-associated virus (AAV) vectors for therapeutic applications. AAV became the vector of choice for gene therapy companies looking to treat monogenetic disorders such as hemophilia or spinal muscular atrophy (SMA), with the ultimate aim to be the vector of choice for high-prevalence diseases. Contract Development and Manufacturing Organizations (CDMOs), such as 3PBIOVIAN, are at the forefront of this revolution. However, they face a daunting task to achieve a perfect blend of productivity, quality, and cost-effectiveness in AAV production. Understanding the risk factors in the production process and exploring ways to de-risk AAV production for CDMOs is crucial.
Efficient AAV Vector Production: A Necessity For CDMOs
Frequently, it is not the lack of innovative gene therapies that impedes the progress of regenerative medicine. The real challenge lies in the production aspect – scaling up the manufacturing process to meet the therapeutic demand is as important as the science behind the therapy itself.
The impact of these manufacturing challenges is reflected in the costs of these treatments. For AAV-based gene therapies, the bottleneck often lies in generating sufficient, high-quality AAV vectors at a low cost. AAV vectors produced for early clinical studies were made using scaled-up research processes with relatively limited regulatory scrutiny, and clinical studies were essentially “proof of principle”. However, with over 170 different AAVs in 1200 ongoing clinical trials and six licensed AAV vectors (Zolgensma, Elevidys, Upstaza, Hemgenix, Roctavian, and Luxturna), we are beyond the
“proof of principle” stage, and these products are now subject to increased levels of scrutiny regarding product purity, process consistency, control, and safety. At the same time, it is concerning for whole life-changing gene therapy field that the market withdrawal of approved Glybera in 2020, and the recent (February 2025) halt of the global rollout of Beqvez, were driven due to very high costs, competing therapies (e.g. Hemgenix), difficulty in reimbursement programs and thus low legible patient number lead to commercial unsustainability.
With complex products such as AAV vectors, the regulators recognize that only a limited level of product characterization can be performed. The manufacturing process defines the process to a large degree, hence the increased focus on chemistry, manufacturing and controls (CMC) for these products. 3PBIOVIAN approached the challenge by developing a serotype agnostic AAV production platform, called AAVion®, evaluating four commercially available cell lines from major suppliers. The AAVion® platform process was forged by building our own 3PBIOVIAN plasmids that are available off-the-shelf, with the possibility to modify with us, in-house plasmid manufacturing, thorough screening of all materials and optimal bioreactor, transfection and purification conditions as well as choice of robust manufacturing equipment that reliably scales. The meticulous comparison of four commercially accessible Producer Cell Lines (PCLs) was an integral part of the journey to proficiency, where we found enormous variations in cell robustness and titers achieved.
To de-risk and simplify the manufacturing process for our clients, 3PBIOVIAN has ensured that all DNA vectors (GOI, RepCap, and Helper) are readily available with a fast and adaptable sequence modification route. These free-to-operate DNA backbones are coupled with in-house high-quality plasmid production. 3PBIOVIAN’s de-risking strategy is further fortified by avoiding solutions and technologies that necessitate individual royalties, facilitating straightforward technology transfer.
Quality By Design (QbD) Approach: AAV Process De-Risking At Its Best
Building the AAV production process design space through Design-of-Experiments (DoE) ensures optimal choice of parameters, comprehensive process understanding, and confidence in scaling. As products enter the clinical phase, process consistency and reproducibility are becoming increasingly important. 3PBIOVIAN has a robust scheme to build design space in the upstream and partially downstream parts of the process. Process understanding defined by DoE will translate into improved process yields, vector purity, and, crucially, the potency of AAV products. Consequently, well- understood critical quality attributes (CQAs) of the vectors, along with identification and control of the critical process parameters (CPPs) that can impact the vector quality and help minimize the overall risk of failure, which is very costly.
The Need For Cost-Effectiveness In AAV Production
The significant expense associated with AAV production is a critical factor that may impede the widespread AAV success. Numerous diseases, which have a high prevalence and are not life-threatening, could potentially be cured with the use of AAV. However, for this to become a reality, the production costs of AAV must be kept at an affordable level for families with average incomes. Government subsidies would not be applicable in these conditions and, generally, are not sustainable as long-term solutions to make gene therapy common. At 3PBIOVIAN, exploring innovative solutions that optimize costs without sacrificing quality is crucial. One notable example is the revamped midstream-downstream AAV process, shortened to two days to improve efficiency and, in addition, enhance product yield and impurity clearance.
Exploring Ways To Improve Purity, Potency, And Stability
The purity and potency of viral vectors are crucial for effectual gene therapies and are heavily impacted by buffers applied in the entire process. The upstream process development affects not only viral vector productivity but also the quality of the vector in terms of the levels of full and empty capsids. While it may be possible to standardize certain upstream elements, such as base media and cell expansion processes, the transfection remains critical – particularly with regard to the amount and ratio of plasmid used or duration of complex formation and dilution level. These parameters must be characterized for each set of plasmids, as differences in promotors and other regulatory elements choice, sizes of plasmids, and Gene of Interests (GOIs) can significantly affect outcomes.
Strict control during upscale to achieve optimal process outcomes is crucial as even prolonged large volume loading of DNA-transfection reagent mix, absent in downscale, influences productivity. When focusing on downstream operations, early production processes relied on ultra-centrifugation for removing host contaminants and critically separating empty and full capsids. 3PBIOVIAN’s AAVion® Platform has been designed to be scalable, which means utilizing an affinity chromatography process for product capture and ion exchange chromatography to enrich full capsids.
While still challenging, these approaches ensure operation across various feed volumes and are not regarded as operator dependent. Additionally, the affinity-based systems, which include low pH elution steps, also serve as a non-product virus reduction step, now required by regulators in AAV purification processes. Conceptually, AAV vector production is based on a platform process that is universally applicable for all serotypes and GOIs. However, in reality both forementioned factors significantly impact the levels of aggregation, productivity, the amount of full and empty capsids, and potency. 3PBIOVIAN has committed to build a sub-platform to prescreen in process and final formulation buffer through forced-destabilizing product, as well as isothermal stability analysis. These functionalities allow addressing downstream purification challenges and developing cross-serotype buffer compatibility solutions.
High-Resolution Analytics And In-Process Monitoring
Early in the development of AAVion® platform it became crucial to employ advanced analytical techniques to monitor the AAV production process and critical quality attributes (CQAs) in real time. It enabled early detection of deviations, allowing immediate corrective actions that reduced the risk of product loss. We applied innovative mass photometry for quick, high-resolution empty and full AAV detection. We developed robust methodologies to monitor AAV product aggregation, instant titer measurement also from harvest, and occurrence of AAV genome ejection due to unfavorable process conditions. By implementing these advanced monitoring techniques, we significantly reduced process variability, enhanced robustness, and gained better control over the critical quality attributes of the AAV product.
Leveraging Scalable Production Systems
The breakthrough lies in the ability to scale up AAV production while maintaining the product’s purity, potency, and safety. Manufacturing processes have evolved significantly in recent years to address the needs for later clinical and commercial supply, regulatory concerns around process consistency, and control. Today, production processes are primarily based on either transient transfection in HEK293 cells or transduction with baculovirus in insect Sf9 cells.
Process innovation supporting scalability and low production costs is also substantial in enabling stable AAV component expression in suspension PCLs. While all approaches have been used for large-scale vector production, product developers and CDMOs continue to optimize scalable platform processes, particularly those based on triple transfection of HEK293 cells due to low IP entry point, flexibility, and early academic process inheritance. Although early production processes relied on adherent approaches in cell factories, most vector producers have now transitioned to suspension platforms due to the clear scalability advantages and abundant accessibility of robust suspension cell lines.
From Transaction To Partnership: The Evolving Dynamics Between CDMOs, Clients, And Suppliers
In the intricate landscape of the biopharmaceutical industry, a critical evolution is underway: the transformation of CDMOs from being mere service providers to becoming strategic partners. These partnerships involve close collaboration with material and technology suppliers, leading to solid AAV production processes and faster client pipeline advancement. This allows clients to focus on their core competencies, while CDMOs handling development and manufacturing tasks, ultimately simplifying product route to commercialization. This transformative shift helps navigate the complexity of AAV production, keep pace with the technological advancements, and adapt to changing regulatory requirements. By evolving into true
partners, CDMOs can offer deeper collaboration and expertise, catering to the increasing expectations of their clients. Strategic collaboration to de-risk AAV production should also span toward technologically advanced companies. It can usher in tremendous possibilities, allowing access to cutting-edge technologies, advanced manufacturing solutions, novel materials, and facilitate the expertise knowledge transfer needed to navigate the complexities of AAV production.
Adherence To Regulatory Compliance
Overcoming regulatory compliance hurdles is an essential aspect of de-risking AAV productions. However, compliance is particularly challenging for AAVs as the vector, its characteristics, and production are not settled. Knowledge about AAV and its production process continues to dynamically evolve. The production of AAV results in a heterogeneous mixture of vector genomes, including truncated versions or PCLs DNA fragments and plasmids used for production, heterologous in terms of post-translation modifications capsids and vectors lacking a functional payload. In addition, only a fraction of the complete vectors can successfully infect the target cells. It is well recognized that there can be significant variability in the levels of full and empty capsids, and the potency of vectors between batches. This variability underscores the need for increased batch-to-batch consistency, which is why regulators emphasize the CMC elements of vector production, even for early-stage clinical materials.
Incorporating Robust Quality Management Systems In AAV Production
Robust quality management systems (QMS) hold the key to risk reduction in AAV production. The standardization and control provided by a well-implemented QMS empower CDMOs to maintain product quality, reduce errors and inefficiencies, and continuously improve processes.
Conclusion
The journey towards successful AAV production is filled with numerous risks and challenges. However, these risks can be mitigated by embracing advanced technologies, adhering to regulatory standards, having scalable and verified platform processes, and leveraging strategic collaborations.
The world has already witnessed the potential of AAV-based gene therapies, and CDMOs, with their expertise and capabilities, are the torch bearers leading the way. By strategically de-risking AAV production, CDMOs not only ensure the reliable supply of these life-changing therapies but also contribute to a future where these therapies are accessible and affordable for all.