Robust, rapid SEC-MALS titer analysis of AAV particles using 3 μm, 550 Å monodisperse SEC media in bioinert column hardware

Importance of the Topic

Adeno associated viruses are critical vectors for gene therapy applications and require reliable measurement of particle titer and aggregation state. Accurate and precise quantitation of viral monomers and higher order aggregates ensures the safety and efficacy of gene therapy products in research and production environments.

Objectives and Study Overview

The study aims to demonstrate a robust rapid size exclusion chromatography coupled with multi angle light scattering method to quantify adeno associated virus titer and aggregates. The evaluation compares a 3 micron 550 angstrom monodisperse SEC media column in bioinert hardware against a conventional 5 micron column from another vendor through precision repeatability intermediate precision and accuracy testing across multiple analysts and sample conditions.

Methodology and Instrumentation

Size exclusion chromatography with multi angle light scattering detection was employed to separate and characterize an in house AAV5 sample. The stationary phase consisted of a monodisperse silica based media with diol hydrophilic coating packed in a 4.6 by 150 millimeter bioinert column hardware. A conventional 4.6 by 300 millimeter 5 micron 500 angstrom column was used for comparison. Data acquisition and analysis were performed with a light scattering software platform.

• Chromatographic system with bioinert flow path
• 3 micron 550 angstrom monodisperse SEC media column
• Conventional 5 micron 500 angstrom SEC column
• Multi angle light scattering detector
• Data analysis software

Key Results and Discussion

The monodisperse media column reduced analysis time and sample consumption by more than 50 percent while offering improved separation of dimer trimer and higher aggregates. Resolution of the dimer peak increased from 1.26 to 1.72 enabling clearer trimer detection. Plate counts for monomer peaks more than doubled compared to the conventional column. Precision metrics including percent coefficient of variation for molecular weight size and titer met acceptance criteria below 20 percent across repeatability intermediate and accuracy assessments. The rapid method maintained agreement within 70 to 130 percent of reference values across all measured attributes.

Benefits and Practical Applications

The enhanced throughput and reduced sample requirements make the method suitable for high volume quality control and process development laboratories. Simultaneous quantitation of total full and empty particles as well as aggregate profiling supports comprehensive vector characterization. The bioinert hardware and hydrophilic stationary phase minimize secondary interactions ensuring consistent performance lot to lot.

Future Trends and Opportunities

Continued advances in monodisperse stationary phases and miniaturized bioinert hardware promise further reductions in analysis time and sample volume. Integration of inline detectors such as refractive index or UV absorbance can extend the characterization of viral vectors. Automation and high throughput platforms will drive broader adoption in gene therapy manufacturing and release testing.

Conclusion

The rapid SEC MALS method with a 3 micron monodisperse column provides a robust high resolution platform for accurate AAV titer and aggregate analysis. Reduced runtime and sample consumption coupled with improved repeatability and resolution support efficient vector characterization in both research and production settings.