It’s not MAD, its Multi-Angled!

What is AAV?

In case you live under a rock, the Adeno Associated Virus (AAV) is the most popular viral vector currently being utilized in the development of gene therapies. It is so popular, there are already hundreds of clinical trials currently underway and multiple FDA-approved gene therapies that use AAV-based vectors.

In AAV drug development, there are multiple critical quality attributes (CQAs) for AAV drug products that relate to stability, safety, and efficacy. Two of these attributes are genome titer and capsid titer, both of which are related to efficacy and relate to each other by their ratio. Out of these three attributes, any two can be calculated from the third (Figure 1).

Figure 1. Three AAV titer attributes (genome, capsid, and % Full) and common assays to measure each below. qPCR/ddPCR = Quantitative or Digital Droplet Polymerase Chain Reaction. AUC = Analytical Ultra Centrifugation. SEC-MALS = Size Exclusion Chromatography Multi-Angle Light Scattering. ELISA = Enzyme-Linked Immunosorbent Assay. MADLS = Multi Angle Dynamic Light Scattering.

Advanced Advanced Light Scattering – The Zetasizer Ultra

That’s not a typo. The Zetasizer Ultra is the most advanced iteration of the Advanced Series of Zetasizer instruments. No offense intended towards the Zetasizer Lab and Pro, as they are also great instruments for dozens of other application types! However, for AAV characterization the Ultra stands alone.

In addition to size, thermal stability, and charge the Ultra has the unique ability to measure DLS at multiple angles (back, side, and forward) that propels it to the forefront of AAV light scattering characterization. Unsurprisingly, this multi-angled DLS technique is called MADLS, not to be confused with MALS which is a static light scattering measurement typically used as a SEC detector.

MADLS allows the Zetasizer both improved sizing resolution and accuracy, but also to account for fancy light scattering effects related to Mie-scattering of anisotropic scattering particles so that scattering intensity is properly assigned to each peak. Taken together, this allows the Ultra to accurately measure particle concentration which for viruses is equivalent to total capsid titer.

For starters, all you need is 20 microliters of a sample (do not worry, it’s non-destructive, you will get it back). To take a concentration measurement, all you have to do is measure a buffer blank, input the refractive index of your virus and buffer, plus viscosity into the brand new ZS Xplorer, a modernized, easy, beautiful, intuitive, and awesome software package.

Figure 2. The Zetasizer Ultra.

And finally, this assay is label-free, non-destructive, rapid, and comes in a small, compact, benchtop, workhorse, versatile instrument that fits anywhere in your lab.

Further reading