The QbD approach is critical in developing and validating methods and the Quality and Development teams should work together since development phases to ensure the quality and robustness of the methods used in releasing the products. Critical attributes are evaluated and result in a list of common methods used in AAV Gene Therapy. The Quality control team plays a crucial role in supporting the Analytical Development to provide methods that meet the Regulatory Guidances, can be validated and produce strong data to support the clinical batched.

Potency is a critical quality attribute that can be difficult to measure via a robust analytical method.  CMC strategies for development, optimization, and implementation will be discussed. Case studies will be used to demonstrate method validation and transfer strategies, including method remediation, method health and performance monitoring, and reference standard and assay control implementation and bridging.

A critical aspect of drug development is ensuring lot-to-lot consistency for drug potency and safety profiles as the manufacturing process matures. The draft FDA Guidance “Potency Assurance for Cellular and Gene Therapy Products” (2023) provides recommendations for demonstrating potency in line with Phase 3 readiness expectations. This talk will discuss a potential strategy following this guidance, utilizing process understanding and analytical control.

This presentation details the development and qualification of a droplet digital PCR (ddPCR) assay for quantifying residual plasmids in adeno-associated virus (AAV) drug substances. The assay employs multiplex primers and probe targeting kanR and neoR sequences present on the manufacturing plasmids. Assay qualification results demonstrated compliance with ICH Q2 (R2), for linearity, repeatability, precision, and recovery. This assay supports testing of in-process sample and AAV drug substance.

Reference standards are a critical component for analytical method development, as they help ensure consistent method performance. Well-characterized gene therapy reference standards, specifically for adeno associated virus (AAV), have been difficult to reliably source. The United States Pharmacopeia (USP) has recently developed reference materials that support AAV stakeholders from raw material qualification through product release, including AAV standards for empty/full assessment, capsid titer, genomic titer, residual plasmid quantification, endonuclease activity, and plasmid topology.

Establishing the accuracy of a method for any new active substance is a challenge because de facto there is no established reference standard. The use of orthogonal methods (different measurement principle) helps to confirm a measured value is reliable. This talk will use some literature examples to discuss the challenges.

As the application of viral gene therapy matures, the quantification and analysis of empty and full AAV particles is also maturing. Using an atomic force microscope (AFM), we are studying the difference in capsid surface chemistry to help delineate an additional method to separate full and empty capsids besides anion exchange chromatography. Potential structural differences that explain these surface chemistry changes will be explored.

Non-wild type AAVs are engineered AAVs that improve the targeting efficiency, safety and specificity for therapeutic applications. The behavior of modified AAVs in CE-based techniques was investigated using labchip, CE-UV, and other techniques. The study revealed the presence of temperature-dependent aggregate peaks that failed to migrate according to their expected size. These anomalous migration patterns were observed across varying conditions, suggesting the resulting electropherogram contained artifact peaks.

Several analytical methods were evaluated to determine the level of DNA encapsulation in rAAV8, rAAV9, and HU37. The methods included SV-AUC (Sedimentation Velocity-Analytical Ultracentrifugation), Mass Photometry, SEC-MALS (Size-Exclusion Chromatography with Multi-Angle Light Scattering), CDMS (Charge Detection Mass Spectrometry, Waters), and ES-DMA (Electrospray-Differential Mobility Analysis, Nano Engineering). The advantages and limitations of each method will be discussed.

Ultragenyx is developing an AAV9 gene therapy for Wilson Disease, UX701, to deliver a modified ATP7B gene. UX701 leverages Ultragenyx’s proprietary PCL platform to produce rAAV in a 2000 L bioreactor. In this presentation, we highlight (1) characterization of production conditions that revealed parameters that influence capsid packaging efficiency; (2) in-process intermediate hold stability study that offered insights into the stability of rAAV; and (3) characterization of our virus heat inactivation process.

• Evaluate whether current gene therapy trial designs are optimized to demonstrate durable clinical benefit and value to payers.
• Identify manufacturing challenges—including cost, scale, and consistency—that are slowing commercial success.
• Explore gene therapy business models that begin with the end in mind: reimbursement, access, and lifetime value.
• Unpack investor perceptions of gene therapy risk and how strategic clarity can reshape confidence and capital flow.​

This presentation outlines the development of AAV manufacturing processes and concurrent advancements in analytical methods. It highlights the crucial role of product characterization in ensuring analytical comparability and details a risk-based approach to demonstrate comparability through statistical analysis aligned with established acceptance criteria. These strategies are vital for maintaining consistent product quality, meeting regulatory standards, and supporting the effective development of AAV-based therapies.

High-quality rAAV products depend on rigorous assessment of genome integrity and sequence identity, from initial plasmid constructs to cGMP manufacturing. A long-read sequencing-based approach enables robust and adaptable characterization at multiple development stages, allowing early identification of plasmid irregularities and detection of problems with rAAV lots that could impact product safety or efficacy. This method supports proactive quality management throughout the development and manufacturing lifecycle.

Recombinant AAV vectors are a favored option for in vivo gene therapy, currently involved in hundreds of clinical trials aimed at treating various genetic diseases. In an effort to develop formulations using a model AAV9 encoding GFP, we explored its degradation pathways under specific stressed conditions. This presentation will offer insights into the mechanisms responsible for the degradation and potency loss of AAV9.

Adeno-associated virus (AAV) is widely used in gene therapy, but its degradation mechanisms remain unclear. This study identifies two degradation pathways using capillary gel electrophoresis: encapsidated DNA degradation at acidic pH and DNA ejection at basic pH. Furthermore, potency loss strongly correlates with encapsidated DNA degradation rather than genome titer or full capsid percentage. These findings highlight the importance of maintaining DNA integrity in AAV-based gene therapy development and manufacturing.

To determine the stability of AAV drug substance and drug product, a comprehensive panel of analytical methods should be established and validated. We have investigated several novel analytical techniques for monitoring the percentage of full capsids and the sizing purity to support formulation research stability studies. These methods were qualified on a fit-for-purpose basis following ICH guidelines with additional forced degradation studies performed to determine the stability indicating capability.

AAV2 is widely used for gene therapy products treating ocular diseases, which require formulations with high vector genome (vg) titers due to limited injection volume. However, AAV2 showed low thermal stability and high propensity to aggregate. In this study, we systematically evaluated the impact of formulation components including pH, stabilizers, and surfactants. We successfully identified a formulation design space for achieving stable high titer AAV2 formulations suitable for preclinical studies.