Advancing AAV Vector Manufacturing: From Persistent Bottlenecks to Data-Driven, Scalable Gene Therapy Platforms
Abstract
Adeno-associated virus (AAV) vectors have become the dominant delivery platform for in vivo gene therapies, underpinning multiple approved products and a rapidly expanding clinical pipeline. Despite this success, AAV manufacturing remains one of the most significant barriers to scalability, cost reduction, and global patient access. Drawing on recent literature and the comprehensive review Advancing AAV vector manufacturing: challenges, innovations, and future directions for gene therapy (Kowshik & Singh, 2025), this article critically examines the end-to-end AAV manufacturing workflow. Persistent upstream and downstream challenges, recent technological innovations, and the increasing role of quality-by-design (QbD), process characterization, and advanced analytics are discussed. Emerging trends—including continuous manufacturing, artificial intelligence (AI)–enabled process control, and next-generation capsid and producer cell platforms—are evaluated for their potential to reshape AAV development and commercialization.
1. The Expanding Role of AAV in Gene Therapy
AAV vectors have emerged as the preferred delivery system for in vivo gene therapies due to their favorable safety profile, broad tissue tropism, and durable transgene expression. Multiple AAV-based therapies have now achieved regulatory approval across ophthalmology, neuromuscular disease, and hematological indications, validating the platform clinically and commercially (Kowshik & Singh, 2025; Wang et al., 2024).
Despite this progress, manufacturing capacity has struggled to keep pace with clinical demand. High cost of goods (COGs), limited scalability, batch-to-batch variability, and challenges in controlling empty-to-full capsid ratios remain major constraints on widespread patient access (Srivastava et al., 2021; Kowshik & Singh, 2025).
2. Upstream Manufacturing: Productivity Versus Control
2.1 Cell Culture Platforms
Upstream AAV production is predominantly based on transient transfection of HEK293 cells in adherent or suspension culture. While adherent systems offer historical familiarity, they are labor-intensive, space-limited, and poorly scalable. In contrast, suspension cultures—particularly when combined with high-density perfusion—enable superior scalability, automation, and process control (Kowshik & Singh, 2025).
Recent advances in N-1 perfusion strategies and optimized feeding regimens have demonstrated sustained cell densities exceeding 30–40 × 10⁶ cells/mL while maintaining high viability, translating directly into improved volumetric productivity (Guan et al., 2022; Deng et al., 2025).
2.2 Genome Packaging Efficiency and Rep Engineering
Genome packaging efficiency is a central determinant of AAV product quality and potency. Suboptimal packaging leads to elevated empty capsid levels, complicating downstream purification and increasing immunogenic risk. The review by Kowshik and Singh (2025) highlights the increasing adoption of hybrid Rep constructs, which combine serotype-specific Rep sequences with AAV2 Rep domains to improve packaging efficiency across non-AAV2 serotypes.
Independent studies confirm that hybrid Rep strategies can increase full capsid content by two- to four-fold, improving dose consistency and reducing downstream burden (Mario et al., 2021; Rumachik et al., 2020).
2.3 Plasmid System Innovation
Traditional triple-plasmid transfection remains widely used but introduces variability in plasmid uptake and expression. To address this, dual-plasmid and single-plasmid systems have emerged as more robust alternatives. Single-plasmid systems consolidate all helper and transgene elements, reducing DNA requirements, batch variability, and residual impurities—features particularly attractive for GMP manufacturing (Kowshik & Singh, 2025; Yang et al., 2025).
3. Downstream Processing: From Purification to Enrichment
3.1 Harvest and Lysis
Downstream challenges often originate at harvest. Continuous harvest strategies using alternating tangential flow (ATF) filtration allow real-time vector recovery while maintaining upstream culture viability, significantly improving operational efficiency (Kowshik & Singh, 2025; Mendes et al., 2022).
Cell lysis strategies have also evolved toward detergent-assisted and high-salt methods, which are scalable and compatible with continuous workflows while preserving capsid integrity (Jungbauer & Wheelwright, 2025).
3.2 Affinity and Ion-Exchange Chromatography
Affinity chromatography remains the primary capture step for AAV vectors. However, traditional ligands are often serotype-restricted and require harsh elution conditions. Serotype-agnostic ligands such as CaptureSelect™ AAVX now enable broad serotype coverage with high recovery and resin durability (Mietzsch et al., 2024; Kowshik & Singh, 2025).
Separation of full and empty capsids increasingly relies on anion-exchange chromatography (AEX) rather than ultracentrifugation, which remains poorly scalable. Two-pass AEX strategies, membrane-based systems, and isocratic elution approaches routinely achieve >70–80% full capsid enrichment at manufacturing scale (Lavoie et al., 2023; Huato Hernández et al., 2024).
4. Formulation, Fill–Finish, and Cold-Chain Constraints
Formulation and fill–finish operations represent high-risk stages due to AAV sensitivity to aggregation, freeze–thaw stress, and container interactions. Advances in buffer systems, cryoprotectants, and automated closed fill–finish platforms have significantly improved product stability and contamination control, particularly for ultra-low temperature storage and distribution (Kowshik & Singh, 2025; Croyle et al., 2001).
5. Process Characterization and Validation as Strategic Enablers
Regulatory expectations have shifted toward rigorous, QbD-driven process understanding. Process characterization—through structured risk assessment, design-of-experiments (DOE), and validated scale-down models—is now central to defining proven acceptable ranges (PARs) and normal operating ranges (NORs) for critical process parameters (Kowshik & Singh, 2025; Alliance for Regenerative Medicine, 2021).
Beyond regulatory compliance, this approach accelerates tech transfer, reduces deviation rates, and enables continuous process improvement across the product lifecycle.
6. The Future: Continuous Manufacturing and AI-Enabled Control
The convergence of continuous manufacturing, advanced analytics, and AI represents a paradigm shift for AAV production. AI-driven process modeling is increasingly applied to predict transfection efficiency, capsid assembly, and chromatographic performance before physical execution, reducing development timelines and experimental burden (Suarez-Amaran et al., 2025).
As these technologies mature, AAV manufacturing may evolve into a modular, adaptive platform capable of supporting personalized and ultra-rare disease therapies at unprecedented speed and scale.
Conclusion
AAV manufacturing is transitioning from artisanal, batch-based processes toward industrialized, data-driven platforms. While challenges remain—particularly around cost, scalability, and analytical throughput—the integration of upstream innovations, advanced purification strategies, and rigorous process characterization is steadily closing the gap between clinical promise and commercial reality. For senior scientists and decision-makers, manufacturing excellence is no longer a downstream concern but a central determinant of gene therapy success.
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