Development of an online two-dimensional SEC-UV-RPLC-MS method for the multi-attribute characterization of adeno-associated virus products

Importance of the topic

Adeno-associated virus (AAV) vectors have become a cornerstone in gene therapy due to their safety profile, tissue targeting capabilities and relatively simple structure. Characterizing critical quality attributes (CQAs) of AAV products—such as capsid integrity, aggregation state, genome packaging and post-translational modifications—is essential for ensuring therapeutic efficacy and safety. Conventional one-dimensional methods often require multiple runs and complementary assays, leading to longer analysis times and increased sample consumption. An integrated multidimensional approach can streamline characterization efforts and accelerate development timelines.

Aims and overview of the study

This study presents the development of an online two-dimensional liquid chromatography method coupling size exclusion (SEC), reversed-phase (RPLC) and mass spectrometry (MS) detection to quantify multiple CQAs of AAV in a single injection. The goal was to establish a generic workflow compatible with various serotypes, capable of resolving aggregation, assessing VP1:VP2:VP3 composition and detecting chemical variants within intact capsids.

Methodology and instrumental setup

The first dimension employs SEC-UV to separate monomeric capsids from high-molecular-weight species (HMWS) on a SEPAX SRT SEC-500 column under phosphate buffer. A precise heart cut of the monomer peak is transferred via an Agilent 1290 Infinity 2D-LC system featuring active solvent modulation into the second dimension. The RPLC separation uses a UPLC Protein BEH C4 column (1.7 µm, 2.1 × 150 mm) at 60 °C with an acidic DFA-modified gradient. Eluting fractions are directed into an Agilent Q-TOF 6530 mass spectrometer operating in positive ESI mode for intact mass analysis. MS parameters include 350 °C gas temperature, 900–3200 m/z range and optimized voltages for protein detection.

Main results and discussion

• Aggregation analysis: SEC-UV heart cut revealed monomer content (~88 %) and two HMWS populations (~8 % and ~3 %).
• Capsid integrity: MS deconvolution confirmed VP1:VP2:VP3 ratios close to 1:2:10 across AAV2, AAV5, AAV6, AAV8 and AAV9 serotypes without method readjustment.
• Post-translational variants: High-resolution MS detected low-level acetylated and phosphorylated species within the VP monomers.
The approach demonstrated consistent retention behavior for different serotypes, validating its robustness and broad applicability.

Benefits and practical applications

• Consolidates four CQAs into a single run, reducing analysis time and sample usage.
• Facilitates rapid comparability studies during process development and batch release.
• Applicable to multiple AAV serotypes without method reoptimization.

Future trends and potential applications

Integration of ion mobility separation, higher-resolution MS and automated data processing could further enhance detection of low-abundance variants. Expanding the workflow to include genome titer analysis or coupling with capillary electrophoresis may provide a fully comprehensive multi-attribute platform for gene therapy vectors.

Conclusion

An online SEC-UV-RPLC-MS method was successfully developed for simultaneous assessment of aggregation, capsid protein ratios and chemical variants in AAV products. Its high throughput, generic applicability and detailed information output make it a valuable tool for supporting regulatory submissions and ensuring product quality in gene therapy manufacturing.

References

1. Aebischer M. et al. Development of online two-dimensional SEC-UV-RPLC-MS for AAV characterization. Int J Mol Sci. 2022;23(20):12332.
2. Aebischer M. et al. Multi-attribute monitoring of AAV products by 2D-LC-MS. Int J Mol Sci. 2023;24(10):8503.
3. Fekete S., Guillarme D. On-off mechanism of proteins in RPLC. Anal Chem. 2019;91:12954–12961.