Tailored analytical strategies for AAV aggregation: purpose‑driven method selection for enhanced product development
Cell & Gene Therapy Insights 2026; 12(2), 215–229
10.18609/cgti.2026.029
No single analytical method captures all AAV aggregation species across all conditions. A purpose-driven framework for selecting SEC-UV, SEC-FLD, DLS, and A4F – what each method detects, where each fails, and how to combine them for release testing, stability studies, and forced degradation.
Why SEC-UV is the GMP-compliant primary method for AAV release and stability testing, and what its column size limit means in practice
How SEC-FLD, DLS, and A4F each fill a distinct gap – distinguishing capsid vs DNA aggregates, detecting μm-scale species, and validating SEC recovery
A practical method selection strategy matched to study purpose: routine release, accelerated stability, forced degradation, and in-process hold studies
AAV1 stored at −80 °C for up to 12 months or handled at 25 °C for up to one month did not form aggregates large enough to challenge the SEC column. SEC-UV alone is sufficient as the analytical control strategy under these routine conditions.
At 40 °C, SEC %HMW plateaued while DLS continued to detect rising Z-average – revealing ongoing large-aggregate formation excluded by the SEC column frit. Under stress conditions, SEC results must be interpreted alongside DLS data.
SEC-UV detects both capsid-related and DNA-related HMW species via UV260/280 ratios. SEC-FLD provides complementary discrimination by responding only to protein fluorescence, enabling the two signals to be interpreted together.
A4F confirmed no significant aggregate loss due to SEC column filtration under typical storage conditions, validating SEC-UV as the primary method. A4F remains essential during method development and for characterising highly stressed samples.
AAV aggregation is a critical quality attribute impacting the safety, efficacy, and manufacturability of gene therapy products. AAV aggregation is a complex pathway; while multiple methods are available for aggregation analysis, there is no one‑size‑fits‑all solution, as different analytical methods are required to capture aggregate formation at different stages. This study evaluates multiple analytical techniques, including size exclusion chromatography (SEC) with UV and fluorescence (FLD) detectors, dynamic light scattering (DLS), and asymmetric flow field‑flow fractionation (A4F) to monitor aggregation in AAV1 samples under various storage and stress conditions. SEC‑UV demonstrated high sensitivity for detecting aggregates, establishing its suitability as a GMP‑compliant method for release and stability testing, particularly under typical frozen storage conditions or within normal holding and handling time at 25 °C, where significant aggregate formation was not observed. Additionally, SEC‑UV detected both capsid related and DNA related high molecular weight (HMW) species, characterizing broader size variants compared to fluorescence detection. Meanwhile, SEC‑FLD detects signals exclusively from proteins, enabling the distinction between capsid related and DNA related HMW species when analyzed in conjunction with SEC‑UV results. Under accelerated or stress conditions (e.g., 40 °C), DLS proved more effective in detecting large aggregates, complementing SEC data. A4F, although not GMP‑compliant, served as a valuable orthogonal technique for validating aggregate recovery and quantifying large‑size aggregates that might be excluded by SEC columns. These findings highlight SEC‑UV as a robust and reliable method for monitoring aggregation during product release and stability testing. Meanwhile, DLS and A4F play essential roles in elucidating degradation pathways and supporting analytical method development and product characterization as orthogonal techniques. The selection of analytical techniques should be carefully tailored to the specific objectives of the product development study to ensure effective monitoring of AAV aggregation and stability.