Why should you consider SEC to analyze AAV aggregates?
In the field of adeno-associated viruses (AAV) analysis, understanding the critical attribute of aggregation is of utmost importance for therapeutic safety and efficacy.
This blog post will highlight why Size Exclusion Chromatography (SEC) columns combined with UHPLC-FLD-UV analysis is the appropriate technique for analyzing your AAV aggregates.
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Separation of AAV from aggregates by size
The success of AAV-based gene therapies depends on the purity and homogeneity of the AAV vector populations, so monitoring AAV aggregation is essential for quality assurance.
To identify and distinguish aggregates, you need well-resolved separation power; the stationary-phase particle pores are crucial in this respect.
For AAV molecules about 20-25 nm (200-250 Å), the average pore size of your column should ideally range from 50-60 nm (500-600 Å).
Importance of efficient short SEC columns
The efficiency of SEC columns plays a vital role in achieving fast and accurate results.
Short columns can offer you abundant time-saving advantages; however, they must maintain high efficiency to ensure reliable data. The Thermo Scientific SurePac Bio 550 SEC MDi HPLC column is packed with 3-µm monodisperse particles, enabling the utilization of short columns without compromising efficiency.
This technology allows for the implementation of fast methods without sacrificing the quality and reliability of the analysis.
AAV samples do not come in abundance
AAV samples are often limited in quantity and concentrations. It is essential to use them judiciously and avoid unnecessary large-volume sample volume injections onto your column.
- When large volume injection is required, for instance when SEC is coupled to MALS (Multi-Angle Light Scattering) detection, the SurePac Bio 550 SEC MDi column can accommodate large volume injections, without the resolution of the critical pair.
Moreover, your results must be reliable from the first injection without conditioning.
- The diol-chemistry of the MDi particles and the inert hardware prevent unwanted secondary interactions that could cause sample loss in the initial injections.
Figure 1. Separation of AAV sample with injection volume ranging from 4 to 50 µL.
Sensitive detection with UHPLC-FLD-UV methods
Sensitive detection is crucial for accurately assessing your AAV samples, since AAV samples are limited in quantity.
- Fluorescence detection offers a highly sensitive detection down to the femtogram level while conserving sample volume. By utilizing fluorescence detection, researchers can achieve reliable results while minimizing the amount of AAV sample consumed.
- UV detection, particularly with double wavelength analysis, presents an excellent technique for assessing AAV samples. This approach not only allows for the detection of AAV aggregation at low nano-gram levels, but also provides valuable information on the efficacy based on empty versus full capsid ratios.
Figure 2. Separation of AAV monomer and High Molecular Weight Species (HMWS) using both UV and FLD detection. Black trace represents the UV 280 nm signal, blue trace represents the UV 260 nm signal, and pink trace represents the FLD signal.
Stay up to date on analytical methods for bioseparations
AAV aggregation analysis by SEC requires the right column to preserve precious samples and achieve sensitive detection.
By optimizing sample utilization and implementing efficient short columns, you can streamline fast analyses while maintaining data integrity.
Ready to learn more? Watch our webinar: Optimizing AAV Analysis with 3 μm Monodisperse SEC Columns
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