Over the past decade, adeno-associated virus (AAV)-based therapies have demonstrated breakthrough clinical outcomes, offering the possibility of one-time treatments for previously intractable genetic disorders, including neurological, myopathic, ophthalmic, and hematologic conditions.1 More recently, promising results have emerged from an AAV-mediated therapy designed to restore hearing in children and adults with otoferlin-associated deafness.2 To date, eight AAV-based gene therapies have received FDA approval, and more than 200 additional clinical trials are ongoing.3,4 These successes reflect the intrinsic properties of AAV vectors, which make them one of the most widely studied viral delivery systems for gene therapy.
AAV Is a Versatile Gene Therapy Platform
Among the various viral vectors used for in vivo gene therapy, AAVs have emerged as the preferred platform due to their favorable safety profile, and non-pathogenic nature. A key advantage of AAV lies in its serotype diversity, which enables engagement with a broad range of cellular receptors across diverse tissue types.3 For example, AAV9 and engineered variants have recently demonstrated the ability to cross the blood-brain barrier, expanding therapeutic potential for central nervous system disorders.5 Vector engineering further enhances these properties: during production, the therapeutic payload can be packaged into different capsid serotypes, enabling precise modulation of tropism and tissue specificity. Together, these features provide AAV vectors with a high degree of adaptability and broad applicability across a wide spectrum of therapeutic applications.
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Importance of Quality Control During AAV Manufacturing
As scientific interest in AAV-based therapies has grown, regulatory frameworks have evolved to ensure product safety, efficacy, and consistency. Accurate quantification of viral genomes and precise characterization of capsid composition and impurities are now central to product quality control and are therefore defined as Critical Quality Attributes (CQAs) by the FDA.6 As a result, fully validated analytical solutions that measure genome titer, capsid ratios, and product purity can support regulatory compliance, ensuring that AAV therapies meet stringent quality and safety standards.
Recombinant adeno-associated virus (rAAV) manufacturing for gene therapy is a complex multistep process in which host cells are co-transfected with plasmids encoding the therapeutic genome, the replication/packaging functions, and the capsid proteins, often in the presence of helper virus plasmids that enable AAV replication. During assembly and purification, this process routinely yields a heterogeneous population of particles, including fully packaged vectors, partially filled capsids, and empty capsids. In addition, a subset of these particles may also encapsulate host-derived or fragmented DNA (Figure 1).7 The overall composition of the particle population is directly associated with product safety as only capsids that contain the intended therapeutic genome can deliver the desired payload, and impurities (empty or partially full capsids and packaged host/fragmented DNA) directly affect potency and may trigger an immune response and other adverse effects. For example, a substantial presence of such impurities can negatively impact transduction efficiency and thus reduce the efficacy of AAV products. Thorough assessment and monitoring of the ratio of empty to full (“empty-full”) capsids is therefore a critical quality control parameter to ensure product quality and safety.
Analytics That Determine Gene Therapy Efficacy
A range of orthogonal analytical methods is commonly employed for capsid titer quantification.7 Analytical ultracentrifugation (AUC) is widely regarded as the reference method for determining empty, partially full and full capsids because it separates particles on the basis of weight with high resolution and reproducibility. However, AUC requires relatively large, purified samples and does not directly indicate whether encapsulated nucleic acid corresponds to the intended therapeutic sequence or contaminant DNA. Transmission electron microscopy (TEM) can perform capsid titer quantification using smaller samples than AUC, but is similarly labor-intensive and time-consuming, while resulting in lower resolution capsid titer analysis. Enzyme-linked immunosorbent assay (ELISA), often used to measure capsid titer, can be combined with qPCR or digital PCR (dPCR) to determine the capsid content ratio. This is achieved by combining the capsid and genome titer measurements independently provided by ELISA and qPCR/dPCR, respectively (ELISA + qPCR/dPCR). This approach, while commonly used, is time-consuming and suffers from cumulative error propagation due to the reliance on two separate assays, making it less suitable for regulated manufacturing processes (typically requiring <10% CV).
Droplet-based Digital PCR: An Advanced and Thorough Solution for Capsid Assessment
Traditional methods for measuring genome titers and distinguishing full from empty viral capsids often fall short in sensitivity and precision, creating a need for more efficient analytical approaches. As such, researchers and manufacturers have increasingly been adopting droplet-based digital PCR (e.g., ddPCR) as an advanced, precise solution for AAV characterization. ddPCR technology provides absolute nucleic acid quantification by partitioning each sample into thousands of nanoliter droplets, enabling detection of individual amplification events. Poisson statistics are then applied to determine the precise number of target molecules in a sample, eliminating the need for external standard curves.8
Beyond genome titer determination, recent assay developments compatible with ddPCR technology expand ddPCR capabilities into empty-full capsid determination in a single workflow by leveraging proximity-dependent oligonucleotide labeling of capsid proteins.9 In these assays, oligo-conjugated antibodies bind nearby epitopes on an AAV capsid. When two oligos are brought into proximity they are ligated to form an amplicon enabling both capsid and genome titer determination. Simultaneous ddPCR readouts—with one probe targeting the ligated capsid oligo and another targeting an AAV genome sequence—enable determination of droplets that contain capsid signal only (indicating capsids that lack therapeutic payload), genome signal only (free DNA or fragment), or both signals (full capsids).
This integrated approach provides a more accurate assessment of AAV capsid quality compared to traditional methods by enabling simultaneous determination of capsid titer, genome titer, and empty-full ratio from a single sample (Figure 2). Combining exceptional sensitivity with rapid turnaround time and low sample volume demand, ddPCR technology enables high-throughput characterization of AAV across a wide range of sample types offering a versatile, scalable solution for both process development and quality control in AAV manufacturing.
Accurate measurement of genome titer, capsid titer and the empty-full ratio is a critical requirement for AAV-based gene therapy products, reflecting vector quality, therapeutic efficiency, and product safety. Advancements in ddPCR technology and the development of complementary assays enable the simultaneous determination of AAV CQAs in a single assay, overcoming the need for separate testing and the labor- and time-intensive limitations of traditional methods that require high sample volume inputs. The advanced capabilities of ddPCR technology provide consistent and reliable capsid determination to streamline AAV characterization, supporting more efficient manufacturing workflows.
References:
- Zhao Q, Peng H, Ma Y, Yuan H, Jiang H. In vivo applications and toxicities of AAV-based gene therapies in rare diseases. Orphanet J Rare Dis. 2025;20(1):368. Published 2025 Jul 17. doi:10.1186/s13023-025-03893-z
- Qi J, Zhang L, Lu L, et al. AAV gene therapy for autosomal recessive deafness 9: a single-arm trial. Nat Med. 2025;31(9):2917-2926. doi:10.1038/s41591-025-03773-w
- Wang JH, Gessler DJ, Zhan W, Gallagher TL, Gao G. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduct Target Ther. 2024;9(1):78. Published 2024 Apr 3. doi:10.1038/s41392-024-01780-w
- Tsang J. First brain-delivered AAV therapy approved by FDA. Drug Discovery News. https://www.drugdiscoverynews.com/first-brain-delivered-aav-therapy-approved-by-fda-16214. Published February 3, 2025. Accessed January 28, 2026.
- Lee NK, Na DL, Kim JW, et al. Evaluation of AAV transduction efficiency via multiple delivery routes: Insights from peripheral and central nervous system analysis. Neuroscience. 2025;573:96-103. doi:10.1016/j.neuroscience.2025.03.026
- Proposed DRAFT Guidance for FDA Consideration: Testing of Adeno Associated Viral (AAV) Vector-Based Human Gene Therapy Products for Empty Capsids During Product Manufacture. US Food and Drug Administration. https://uploads-ssl.webflow.com/6414e4e38bc16c7a84b0da2c/649cc2686e4845fa67d14fc8_DHC_Proposed-DRAFT-Guidance-for-FDA-Consideration.pdf?. Published January, 2020. Accessed January 28, 2026.
- Kontogiannis T, Braybrook J, McElroy C, et al. Characterization of AAV vectors: A review of analytical techniques and critical quality attributes. Mol Ther Methods Clin Dev. 2024;32(3):101309. Published 2024 Jul 30. doi:10.1016/j.omtm.2024.101309
- A quality control revolution for droplet digital PCR. Nature. https://www.nature.com/articles/d42473-020-00123-x. Accessed January 28, 2026.
- Pratt C. Enhancing Gene Therapy With Precise AAV Capsid Analysis. https://www.technologynetworks.com/biopharma/webinars/enhancing-gene-therapy-with-precise-aav-capsid-analysis-396907. Technology Networks. Accessed January 28, 2026.
