How do I collect multiple digital UV wavelengths in ASTRA® with FFF-MALS?
Introduction
Multiple UV wavelengths are often required for advanced characterization in FFF-MALS, enabling the analysis of protein conjugates, copolymers, as well as payload quantification for complex gene therapy vectors such as adeno-associated virus (AAV) or lipid nanoparticles (i.e., LNP-RNA). VISION™ 3.1 allows multiple UV signals to be collected simultaneously and digitally in ASTRA® 8 during your FFF-MALS experiments, replacing the use of analog UV signals which may limit you to just one wavelength.
Upgrading your FFF-MALS experiments to use digital UV is easy. Other than enabling the UV signals and selecting the settings in the UV method through VISION RUN as you would normally do, only the ASTRA method needs to be adjusted for collecting digital UV data as shown below. In no time, you will be analyzing critical quality attributes like particle concentration, capsid content, and quantifying aggregate content.
Enabling Digital UV Collection in ASTRA
Before using the Protein Conjugate Analysis method, it is important to validate your system on a non-conjugated protein or polymer to ensure your RI and UV signals give accurate molar mass data. This can be done with a standard such as BSA, where we would expect the monomer molar mass from both the RI and UV detectors to be the same. You can toggle between the RI and UV as the concentration source in the Configuration Window of your experiment to confirm that each source calculates the correct molar mass.
This will convert the ASTRA configuration to one that is compatible with digital UV collection from VISION RUN, as shown to the right. The "generic UV" configuration in ASTRA will now display "VISION UV" and collect digital signals from your Agilent UV in VISION.
Digital Data in ASTRA
With the “VISION UV” configuration in ASTRA, you don’t need to worry about analog UV settings anymore. ASTRA will conveniently record all the UV signals enabled in your VISION UV method, and all UV signals will be available in the resulting ASTRA data file as shown to the right with a DAD collecting 8 signals.
Keep in mind that ASTRA will still only use one UV signal as a concentration source in the basic molar mass and protein conjugate methods but the signal can be easily switched, for example between 280 nm and 260 nm wavelengths when analyzing samples. For the viral vector method, two UV signals can be selected. Reach out to us to learn more about characterizing viral vectors with FFF-MALS.
Conclusion
VISION 3.1 requires ASTRA 8.0.1 or higher and is compatible with both legacy Eclipse™ AF4 and DualTec™ instruments, as well as the latest Eclipse models. For more information about how to upgrade your existing system to VISION 3.1 and ASTRA 8, or for how to employ multiple UVs for AAV and LNP analyses, please contact us at info@wyatt.com.
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