A deep dive into OriginPro for the analysis of high-resolution DGE-AUC data

In this free on-demand SelectScience^® webinar, join Dr. Shawn Sternisha, senior field applications scientist at Beckman Coulter Life Sciences, and Easwar Lyer, product manager at OriginLab, as they introduce DGE AUC – an orthogonal technique that can be conducted in the same Optima AUC instrument as SV-AUC.

AUC has been a benefit to the field of biophysical characterization for nearly a century. Sedimentation velocity (SV) AUC has emerged as a gold standard for quantifying the loading fraction of gene therapy delivery particles such as adeno-associated viruses (AAV) and liposomes. However, boundary analysis of SV-AUC data requires specialized software such as Sedfit or UltraScan. DGE-AUC offers improved sensitivity and sample consumption, along with dramatically simplified analysis. The webinar will also demonstrate a simple, expert analysis of DGE-AUC data in OriginPro* software.

Key learning objectives

• Learn about the advantages of DGE-AUC for your characterization suite
• Understand how to start running DGE-AUC experiments on viral vectors like AAV and adenovirus (AdV)
• Discover rapid and optimal workflows for analyzing DGE-AUC data in OriginPro including importing, peak fitting, calculations, and more

Read on for highlights from the live Q&A session, or watch the webinar on demand, at a time that suits you.

How do you process batch data?

EL: If you are looking for a simple peak identification and integration, you can use the Quick Peaks App. But if you are looking for curve fitting and fitting across multiple data sets with multiple peaks, the Deconvolution App can do that.

Can you normalize the X-axis to the density of the particle?

SS: In short, the answer is yes. This can be done computationally. I'm aware of a paper from Alan Minton in the '90s that looked into this using sector-shaped cell pieces so, that would have to be accounted for. And then experimentally as well, since the Optima AUC does have the Rayleigh interference detection module, you could convert fringes to a refractive index to establish that gradient profile. Lastly, if you know the buoyant density of your given species, perhaps from preparative work, you can use those fractions for the refractometer, enabling you to establish ‘goalposts’. This means if all things are held equal, you can expect consistent positioning for these species. So, quantifying the gradient like you mentioned, would be super useful. It's just something we haven't done yet.

Is DGE ultracentrifugation better in separating exosomes from other extracellular microvesicles in a culture media or in serum compared to conventional ultracentrifugation?

SS: In the Optima AUC, DGE is strictly meant to be a tool for characterization or analysis. This means you’re not able to recover samples after spinning them. Further, you’re working with pretty small volumes so, for something like exosome prep, you would definitely want to stick with ultracentrifugation. Although, I do think there are some analytical applications for DGE in that field as well.

Is it safe to use CsCl? So, isolation purification in DGE-AUC.

SS: I have not seen anyone publish anything on cesium chloride (CsCl) for exosome prep. That's not to say you can't do it. I know they do tend to be relatively fragile, though, so I would probably stick with the standard approaches that have been developed using density gradients, like iodixanol or sucrose. But there's also iohexol and several other ones that you can use that are a bit more gentle on the samples.

*Third-party analysis software, including UltraScan, Sedfit, and OriginPro has not been validated by Beckman Coulter Life Sciences for use with the Analytical Ultracentrifuge. Beckman does not endorse any third-party analyses software. Beckman Coulter Life Sciences warranty and/or performance guarantee that may be applicable or are provided by Beckman for Analytical Ultracentrifuge do not apply to any third-party software.

You can watch the full webinar here>>

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